Carbazole-based semicarbazones and hydrazones as multifunctional anti-Alzheimer agents

Journal of Biomolecular Structure and Dynamics

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

Carbazole-based semicarbazones and hydrazones

as multifunctional anti-Alzheimer agents

Kishan B. Patel, Dushyant V. Patel, Nirav R. Patel, Ashish M. Kanhed, Divya

M. Teli, Bhumi Gandhi, Bhavik S. Shah, Bharat N. Chaudhary, Navnit K.

Prajapati, Kirti V. Patel & Mange Ram Yadav

To cite this article: Kishan B. Patel, Dushyant V. Patel, Nirav R. Patel, Ashish M. Kanhed,

Divya M. Teli, Bhumi Gandhi, Bhavik S. Shah, Bharat N. Chaudhary, Navnit K. Prajapati, Kirti

V. Patel & Mange Ram Yadav (2021): Carbazole-based semicarbazones and hydrazones as

multifunctional anti-Alzheimer agents, Journal of Biomolecular Structure and Dynamics, DOI:

10.1080/07391102.2021.1942212

To link to this article: https://doi.org/10.1080/07391102.2021.1942212

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

https://doi.org/10.1080/07391102.2021.1942212

Carbazole-based semicarbazones and hydrazones as multifunctional

anti-Alzheimer agents

Kishan B. Patel^a, Dushyant V. Patel^a, Nirav R. Patel^a, Ashish M. Kanhed^b, Divya M. Teli^c, Bhumi Gandhi^a,

Bhavik S. Shah^a, Bharat N. Chaudhary^a, Navnit K. Prajapati^a, Kirti V. Patel^aand Mange Ram Yadav^a,d

b

^aFaculty of Pharmacy, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India; Shobhaben Pratapbhai Patel – School of

c

Pharmacy & Technology Management, SVKM’s NMIMS University, Mumbai, India; Department of Pharmaceutical Chemistry, L. M. College of

d

Pharmacy, Navrangpura, Gujarat, India; Centre of Research for Development, Parul University, Vadodara, Gujarat, India

Communicated by Ramaswamy H. Sarma

ABSTRACT

ARTICLE HISTORY

Received 22 December 2020

Accepted 7 June 2021

With the aim to combat a multi-faceted neurodegenerative Alzheimer’s disease (AD), a series of carba-

zole-based semicarbazide and hydrazide derivatives were designed, synthesized and assessed for their

cholinesterase (ChE) inhibitory, antioxidant and biometal chelating activity. Among them, (E)-2-((9-

ethyl-9H-carbazol-3-yl)methylene)-N-(pyridin-2-yl)hydrazinecarbothioamide (62) and (E)-2-((9-ethyl-9H-

carbazol-3-yl)methylene)-N-(5-chloropyridin-2-yl)hydrazinecarbothioamide (63) emerged as the premier

candidates with good ChE inhibitory activities (IC₅₀values of 1.37 mM and 1.18 mM for hAChE, IC₅₀val-

ues of 2.69 mM and 3.31 mM for EqBuChE, respectively). All the test compounds displayed excellent

antioxidant activity (reduction percentage of DPPH values for compounds (62) and (63) were 85.67%

and 84.49%, respectively at 100mM concentration). Compounds (62) and (63) conferred specific copper

ion chelating property in metal chelation study. Molecular docking studies of compounds (62) and

(63) indicate strong interactions within the active sites of both the ChE enzymes. Besides that, these

compounds also exhibited significant in silico drug-like pharmacokinetic properties. Thus, taken

together, they can serve as a starting point in the designing of multifunctional ligands in pursuit of

potential anti-AD agents that might further prevent the progression of ADs.

KEYWORDS

Acetylcholinesterase; butyr-

ylcholinesterase; metal

chelation; MTDL; carbazole;

Alzheimer’s disease

1. Introduction

Wang et al., 2019). Currently, there are only four FDA-approved

medications available to treat AD, of which three are acetyl-

cholinesterase (AChE) inhibitors (donepezil, galantamine and

rivastigmine) and the fourth one is N-methyl-^D-aspartate

(NMDA) receptor antagonist (memantine). These drugs can

relieve the symptoms of AD, but they are not able to reverse

the progression of the illness.

Deficits in the cholinergic neurons and transmission

located in the basal forebrain could severely affect many fac-

ets of cognition and behavior, including cortical and hippo-

campal memory processing. The low levels of ACh are due

to its excessive hydrolysis by cholinesterases (ChEs) present

in the brain. Therefore, inhibition of ChEs is considered to be

an effective strategy to increase ACh levels within the brain

(Greig et al., 2002; Hartmann et al., 2007; Jing et al., 2019).

Recent studies have indicated that AChE could play a key

role in deposition of amyloid plaques. However, in later

stages of AD, diminished levels of AChE and upsurged or

unaltered levels of BuChE have been reported (Holzgrabe et

al., 2007). This suggests that if AD patients are treated with

dual AChE and BuChE inhibitors it may lead to more

assured results.

Alzheimer’s disease (AD) is a neurodegenerative disease,

causing severe neuronal disruption in the hippocampus and

cerebral cortex regions of the brain. This is recognized by

impairment of the cognitive functions, usually leading to

deterioration in emotional control, social behavior or motiv-

ation (Goedert & Spillantini, 2006). Globally, over 50 million

people are currently affected by AD and the toll is expected

to rise upto 150 million by 2050 (Patel, Patel, Kanhed, Teli,

Patel, Gandhi, et al., 2020; World Alzheimer Report, 2019).

The neuropathological hallmarks of the AD brain are low

levels of acetylcholine (ACh) (Talesa, 2001), diffused extracellu-

lar amyloid plaques caused by self or biometal-mediated

b-amyloid (Ab) aggregation, which are frequently surrounded

by damaged neurons (Selkoe, 2003), and hyperphosphorylated

intracellular neurofibrillary tangles (Maccioni et al., 2010). These

well-recognized pathological hallmarks are often accompanied

by high levels of oxidative stress generated by reactive oxygen

species (ROS) and biometal dyshomeostasis (Greenough et al.,

2013). Therefore, to combat a multifactorial disease like AD,

development of multi-target-directed ligands (MTDLs) is con-

sidered a promising drug discovery approach (Bajda et al.,

Under normal physiological conditions, the production of

2011; Oset-Gasque & Marco-Contelles, 2018; Patel et al., 2019; ROS and reactive nitrogen species (RNS) is counter balanced

CONTACT Mange Ram Yadav

mryadav11@yahoo.co.in

Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2021.1942212.

ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2

K. B. PATEL ET AL.

by the antioxidant activities of various defensive mechanisms interesting biological activities (Gomes et al., 2014; Mathew

€

et al., 2018; Onkol et al., 2013; Prathima et al., 2012; Sens et

al., 2018) (Figure 1(B)). Thiosemicarbazone group is reported

to be an indispensable pharmacophore due to its antioxidant

and metal chelating properties.

that include several antioxidants and enzymes. The imbalance

between the ROS generation or reduction in its metabolic

destruction will shift the equilibrium of the cellular redox bal-

ance to an oxidative imbalance, leading thereby to the over-

production of ROS (Poljuha et al., 2015). The change in the

redox transition metal balance, particularly of copper, iron and

zinc ions are crucial. Binding of these metal ions to Ab causes

dyshomeostasis that leads to an apparent state of Cu defi-

ciency in the intracellular region and an extracellular rise in Cu

and Zn levels. The metal dyshomeostasis causes production of

oxidative stress by dysfunction of various important cuproen-

zymes and antioxidants such as superoxide dismutase 1

(SOD1) resulting in the dysfunction of neuronal cells, which in

turn reduces the synaptic transmission.

The interesting biological activities associated with the

carbazole scaffold, and thiosemicarbazone and acyl hydrazide

moieties motivated us to merge these biologically active

pharmacophoric groups into a single entity to obtain mole-

cules with multiple beneficial effects for the treatment of AD

(Figure 2). Herein, we report the synthesis of carbazole-based

thiosemicarbazone and acyl hydrazone derivatives and their

biological assessment for possible anti-AD activities.

3. Results and discussion

Based on these reports and extending our ongoing research

work, we report herein designing, synthesis and biological

evaluation (ChE inhibition, antioxidant and metal chelation

studies) of a series of novel carbazole-based substituted thiose-

micarbazide and hydrazide derivatives as potential anti-AD

agents. The binding modes and interactions of the compounds

with the target proteins were determined by performing

molecular docking and molecular dynamics simulation studies.

The most promising compounds were then screened in silico

for their virtual ADMET properties.

3.1. Chemistry

The designed carbazole-based thiosemicarbazone and acyl

hydrazone derivatives were divided into a set of two series.

In series-I, the carbazole scaffold was linked to the substi-

tuted thiosemicarbazide moiety (compounds 54–65) and

semicarbazide moiety (compound 66), while in series-II, it

was linked to the acyl hydrazide moiety (compounds 67–73).

Syntheses of these designed compounds are depicted in

Schemes 1–3.

The required semicarbazide and thiosemicarbazide inter-

mediates were synthesized as shown in Scheme 1.

Substituted benzylamines (11–15) were reacted with carbon

disulfide and methyl iodide in the presence of triethylamine

to obtain methyl carbamodithioate intermediates, which

were reacted further with hydrazine hydrate by using metha-

nol as the solvent to give the desired thiosemicarbazide

intermediates (31–35) (He et al., 2019; Pervez et al., 2017).

Similarly, substituted anilines (16–22) were reacted with car-

bon disulfide in the presence of N,N-dimethylformamide

(DMF) and sodium hydroxide, followed by reaction with

hydrazine hydrate to offer the desired thisemicarbazides

(36–42). The semicarbazide intermediate (43) was synthe-

sized from 4-chloroaniline (23) in two-step procedure includ-

ing acylation reaction followed by hydrazinolysis. 4-

Chloroaniline (23) was acylated by phenyl chloroformate in

the presence of sodium carbonate in acetone, followed by

reaction with hydrazine hydrate to offer the desired semicar-

bazide intermediate (43).

The required acyl hydrazide intermediates (44–50) were

synthesized as depicted in Scheme 2. Substituted carboxylic

acids (24–30) were converted to their respective methyl

esters by the Fischer esterification method, and then these

esters were converted to the corresponding acyl hydrazides

(44–50) by refluxing them in methanol with excess hydrazine

hydrate.

The designed carbazole-based thiosemicarbazone, semi-

carbazone and acyl hydrazone derivatives (52–66) were syn-

thesized as depicted in Scheme 3. Ethylation of carbazole

(51) was carried out in the presence of aqueous NaOH solu-

tion by using ethyl bromide to obtain N-ethylcarbazole (52)

2. Rationale of designing

The mono-targeted anti-AD agents, even though having high

binding affinity and selectivity for a specific target, might not

be that much effective to combat AD having a complex eti-

ology. In contrast to a mono-targeted ligand, an MTDL with

well-balanced affinities for multiple targets can offer add-

itional advantages. So, development of MTDLs for AD is con-

sidered as the most promising drug discovery approach.

Carbazole is reported to be a structural component of

many natural compounds possessing many valuable medi-

cinal properties which could be helpful in AD treatment.

Carbazole derivatives have been reported to possess ChEs

inhibitory (Fang et al., 2016), Ab aggregation inhibitory (Yang

et al., 2012), ROS scavenging activities and the ability to pro-

tect neurons against oxidative damage (Naik et al., 2010).

Numerous carbazole-based hybrid molecules including carba-

zole-stilbene hybrids (1) (Patel, Patel, Kanhed, Teli, Patel,

Joshi, et al., 2020), carbazole-aminoadmantane hybrids (2)

(Makhaeva et al., 2018), sulfonamide derivatives of carbazole

(3) (Bertini et al., 2017), carbazole-tacrine hybrids (4)

(Thiratmatrakul et al., 2014) and carbazole-quinoline deriva-

tives (5) (Choubdar et al., 2019) (Figure 1(A)) have been

reported as potential therapeutic agents for treatment of AD.

A wide range of pharmacological activities exhibited by the

carbazole derivatives makes carbazole a privileged scaffold in

the discovery of new anti-AD agents.

Thiosemicarbazides and acyl hydrazides have received

great attention among medicinal chemists as important

chemical functionalities due to their wide spectrum of bio-

logical activity. Various substituted thiosemicarbazide and in high yield. Vilsmeier–Haack formylation reaction of com-

acyl hydrazide derivatives have been identified to possess pound (52) using DMF, and phosphoryl chloride afforded

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

3

Figure 1. Some reported biologically active (A) carbazole-based compounds (1–5) and (B) thiosemicarbazone and hydrazide-based compounds (6–10).

Figure 2. Design aspects of carbazole-based thiosemicarbazone and acyl hydrazone derivatives.

4

K. B. PATEL ET AL.

Scheme 1. Synthetic route for the intermediates (31–43). Reagents and conditions: For compounds (31–35) (i) carbon disulfide, triethylamine, methanol, 20–30 ^ꢀC,

2–3 h, methyl iodide in methanol at –10 ^ꢀC; (ii) hydrazine hydrate, methanol, reflux, 2–4 h. For compounds (36–42) (i) carbon disulfide, sodium hydroxide, dimethyl-

formamide, 20–30 ^ꢀC, 2–3 h; (ii) hydrazine hydrate, 70 ^ꢀC, 1–2 h. For compound (43) (i) acetone, potassium carbonate, phenyl chloroformate, sodium bicarbonate,

20–30 ^ꢀC, 3 h; (ii) hydrazine hydrate, dioxane, reflux, 4 h.

Scheme 2. Synthetic route for hydrazide intermediates (44–50). Reagents and conditions: (i) conc. sulfuric acid, methanol, reflux, 6 h; (ii) hydrazine hydrate, metha-

nol, reflux, 2–4 h.

mono-formylated intermediate 9-ethyl-3-formylcarbazole The benzyl derivative (54) exhibited balanced inhibitory

activity on both the ChEs (IC₅₀values of 2.21 and 2.33 mM for

hAChE and EqBuChE, respectively). Introduction of small lipo-

philic electron-withdrawing fluoro group (in compound 57)

and electron-donating methyl group (in compound 58) led

to a moderate increment in hAChE (1.64- and 1.38-fold,

respectively) and EqBuChE (1.31-fold for compound 58)

inhibitory activities, whereas introduction of methoxy group

(compound 55) and chloro group (compound 56) at the

para position of the benzyl ring resulted into a modest

reduction in the inhibitory activities. Replacement of the

benzyl ring with phenyl ring (compound 59) led to a 2.27-

fold improvement in the inhibitory activity of hAChE (IC₅₀

value of 0.97 mM) and a 1.20-fold increase in the inhibitory

activity of EqBuChE (IC₅₀value of 1.93 mM). Substitution of

the phenyl ring with a methoxyl group at its para position

(compound 60) or replacing the phenyl ring with other het-

erocyclic rings i.e. pyridine (compound 61), substituted pyri-

dines (compounds 62 and 63), pyrimidine (compound 64) or

pyrazine (compound 65) had a detrimental effect, leading to

moderate decrease in ChEs inhibitory activities. Substituting

the thiosemicarbazone linker with semicarbazone (compound

(53). Compound (53) was condensed with thiosemicarbazide

intermediates (31–42) and semicarbazide intermediate (43)

in the presence of acetic acid and methanol to yield the thi-

osemicarbazone derivatives (54–72) and semicarbazone

derivative (73), respectively. Similarly, carbazole-based acyl

hydrazone derivatives were obtained by condensation of the

aldehyde (53) with hydrazide intermediates (44–50) by

refluxing in methanol in the presence of catalytic amounts of

acetic acid.

3.2. Biological evaluation

3.2.1. In vitro ChE inhibition studies

The in vitro ChEs inhibitory activity of the synthesized com-

pounds was determined by Ellman’s assay, and protocol of

the study was same as described in our previous reports

(Kanhed et al., 2015, Kanhed, Patel, Patel, et al., 2020; Sinha

et al., 2015). Donepezil and tacrine were taken as the stand-

ards for Ellman’s assay. The calculated IC₅₀values and select-

ivity indices of the test compounds for ChEs are reported in

Tables 1 and 2. In series-I, thiosemicarbazones (54–65)

showed good ChEs inhibitory activity in micromolar ranges.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

5

Scheme 3. Synthetic route for the designed compounds (54–73). Reagents and conditions: (i) Ethyl iodide, sodium hydroxide, dimethyl sulfoxide, RT, 4–5 h; (ii)

phosphoryl chloride, DMF, chloroform, reflux, approx. 12 h; (iii) acetic acid, methanol, reflux, 4–5 h.

66) offered good hAChE and moderate EqBuChE inhibitory respectively, that were near to that of ascorbic acid used as

activities (IC₅₀value of 1.64 mM and 5.57 mM, respectively).

In series-II, acyl hydrazone derivatives (67–73) demon-

a reference compound (IC₅₀value of 9.01 mM). Donepezil and

tacrine were observed to be devoid of any notable radical

strated good hAChE inhibitory and moderate EqBuChE inhibi- scavenging activity with IC₅₀values greater than 500 lM

tory activities in micromolar ranges (Table 2). The

phenylacetic acid hydrazone derivative (67) showed good

ChEs inhibitory activity (IC₅₀values of 1.0 mM for hAChE and

2.07 mM for EqBuChE). Introduction of a 4-methoxyl group

(compound 68) or 4-chloro group (compound 69) on the

phenyl ring led to a drastic decrease in EqBuChE inhibitory

activity. Replacement of the phenylacetic hydrazide moiety

with benzhydrazide moiety (compound 70) did not affect

the ChEs inhibitory activities. Incorporation of substituted

benzhydrazide moiety (compounds 71 and 72) and picoli-

noyl hydrazide moiety (compound 73) had a detrimental

effect, leading to a decrease in ChEs inhibitory activity.

(Table 1).

3.2.3. Metal chelation study

Dyshomeostasis of redox transition metals, particularly cop-

per, iron and zinc, and their excessive deposition in the brain

have been correlated with AD etiology (Savelieff et al., 2013).

Thus, the use of metal ion chelators has been proposed as a

possible therapy for a disease like AD. The potential of the

test compounds to form chelates with biometals was deter-

mined by means of UV–vis spectroscopy (Wang et al., 2015).

Compounds (61 and 62) exhibited maximum absorption

peaks at 342 and 359 nm respectively, in methanol (as shown

in Figure 3). When Cu^2þion solution was added to the solu-

tion of the test compounds, a sharp decrease in the heights

3.2.2. DPPH radical scavenging activity

The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging of the absorbance peaks were observed, indicative of the

assay was used to evaluate the antioxidant activity of the interactions between the metal ions and the test compounds

synthesized compounds (Kedare & Singh, 2011). It was deter- to form ligand–Cu^þ2complexes. Insignificant changes in the

mined by the ability of the selected compounds to quench position and height of absorption peaks were observed

the purple-colored DPPHꢁ radical to a yellow colored stable when AlCl₃, CaCl₂, FeCl₃or FeSO₄were added into the test

DPPHH molecule and the corresponding free radical-scaveng- compounds’ solutions. This suggests that the test com-

ing potential of the test compounds was calculated by the pounds had poor chelating abilities for these ions (Al^þ3

,

decline in absorbance of the test solutions at 517 nm. The Ca^2þ, Fe^2þand Fe^3þ). The test compounds were also eval-

free radical scavenging activity was calculated as a reduction uated for their binding potential to other key biometal ions,

percentage (RP) of DPPH. Among the tested compounds, such as Zn^2þand Mg^2þwhere these compounds (61 and

compounds (61 and 62) exhibited excellent free radical scav- 62) showed negligible binding affinities for these metals. 8-

enging activity with IC₅₀values of 9.91 and 10.52 mM, Hydroxyquinoline (8-HQ) was used as a reference compound

6

K. B. PATEL ET AL.

Table 1. In vitro inhibitory activity on hAChE and EqBuChE, and DPPH radical scavenging activities of compounds (54–66).

IC50 SEM (mM)

RP of DPPHd %

Inhibition at 100 mM

(IC50 SEM (mM))

Compd

R

hAChE^a

EqBuChE^b

2.33 0.65

SI^c

54

2.21 0.78

1.05

24.29 1.93 (>100)

20.6 1.37 (>100)

21.54 1.23 (>100)

26.65 1.11 (>100)

25.61 1.98 (>100)

55

56

57

58

2.20 0.61

2.63 0.20

1.35 0.87

1.60 0.31

4.68 1.03

4.47 0.42

2.19 0.98

1.78 0.19

2.12

1.69

1.62

1.11

59

60

61

62

63

0.97 0.59

1.59 0.69

1.37 0.39

1.18 1.19

1.30 0.49

1.93 0.68

1.64 1.09

2.69 0.40

3.31 1.12

4.09 0.44

1.99

1.03

1.95

2.79

3.13

83.44 1.66

(35.29 1.43)

10.57 1.29 (>100)

85.67 2.09 (9.91 1.12)

84.49 1.87

(10.52 1.43)

83.76 1.51

(40.92 1.39)

64

65

66

1.98 0.64

1.43 0.88

1.64 0.42

5.79 1.17

2.88 0.98

5.57 0.91

2.91

2.00

3.39

nd

15.66 0.93 (>100)

Donepezil

Tacrine

Ascorbic acid

0.04 0.01

0.05 0.01

nd

1.87 0.08

0.007 0.00

nd

81.3

0.14

nd

5.4 1.11 (>500)

17.2 1.19 (>500)

98.26 1.88 (9.01 1.33)

IC₅₀, half maximal inhibitory concentration (mean SEM of 3 experiments).

^aAChE from human erythrocytes.

^bBuChE from equine serum.

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

^dRP of DPPH (%) ¼ Reduction percentage of DPPH at 100 mM.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

7

Table 2. In vitro inhibitory activity on hAChE and EqBuChE, and DPPH radical scavenging activities of compounds (67–73).

IC50 SEM (mM)

Compd

67

R

hAChE^a

EqBuChE^b

SI^c

RP of DPPHd % inhibition at 100 mM (IC50 SEM (mM))

14.01 1.33 (>100)

1.00 0.43

1.11 0.59

2.04 0.61

1.10 0.33

1.94 0.71

2.04 0.69

2.03

68

69

70

71

3.59 0.83

23.52 1.33

2.11 0.81

4.36 0.71

3.22

11.52

1.91

7.1 1.13 (>100)

10.88 1.25 (>100)

10.72 1.13 (>100)

10.80 1.39 (>100)

2.24

72

73

1.64 0.51

1.88 0.47

5.51 1.23

3.35

1.23 1.09 (>100)

19.52 1.28

10.34

nd

IC₅₀, half maximal inhibitory concentration (mean SEM of 3 experiments).

^aAChE from human erythrocytes.

^bBuChE from equine serum.

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

^dRP of DPPH (%) ¼ Reduction percentage of DPPH at 100 mM.

for validation of the assay protocol. A significant alteration in study. Tacrine formed a firm complex with the AChE by mak-

the height and position of absorption peaks was observed ing hydrogen bond between the protonated nitrogen of

when the solutions of the metal ions and the HQ solution acridine ring of tacrine and C ¼ O of His440 (hAChE His447).

were mixed indicating the ability of HQ to form metal–HQ The aromatic ring of tacrine was observed to be sandwiched

chelates non-selectively.

between Trp84 and Phe330 (hAChE Trp86 and Tyr337). In

donepezil, the 1-benzyl moiety of donepezil was observed to

be stabilized in the active site by Trp84 and Phe330 residues

(hAChE Trp86 and Tyr337). The dihydroinden-1-one group of

donepezil was stabilized by Trp279 (hAChE Trp286) residue

through hydrophobic interactions. The 6-methoxy group

exhibited H-bonding interaction with Trp279 (hAChE Trp286).

Tacrine and donepezil, used as standards in biological test-

ing, were also considered for docking with BuChE. The aro-

matic ring of tacrine stabilized the ligand–receptor complex

by p–p interactions with Trp82 of BuChE. H-bonding

between amino N-H of tacrine and C ¼ O of His438 imparted

The above finding asserted the selective Cu^2þion chela-

tion potential of the test compounds (61 and 62). Selectivity

of these compounds for the copper ions is a crucial factor in

the designing of biometal chelating agents to escape bind-

ing with other important biometal ions, the imbalance of

which could drive to undesired adverse effects.

3.3. Computational studies

3.3.1. Docking studies of compounds (61 and 62)

with ChEs

To gain insight into the interactions of the reference com- further stability to the complex. While in case of donepezil,

pounds and test compounds within the active sites of ChEs, the benzyl group was found to be stabilized by Trp82. The

docking studies were performed using Schrodinger Suite dihydroinden-1-one group stabilized the ligand–receptor

€

(Glide version 5.5 (2009) Schrodinger, LLC, New York, NY). complex by means of hydrophobic interactions with Ser198,

The 3D-crystal structures of TcAChE (PDB code: 2CKM) and Trp231 and Phe398. The interaction view of 61 within the

hBuChE (PDB code: 4BDS) retrieved from RCSB Protein Data active site of AChE is represented in Figure 4(A,B) (Patel,

Bank (www.rcsb.org/pdb/home/home.do.) were used for this Patel, Kanhed, Teli, Patel, Gandhi, et al., 2020). The active site

8

K. B. PATEL ET AL.

Figure 3. Biometal chelation study of compounds (61, 62) and HQ. The UV–vis absorption spectra of (A) compound (61), (B) compound (62) and (C) 8-HQ at

25 mM concentration alone and in presence of AlCl₃(25 mM), CaCl₂(25 mM), CuSO₄(25 mM), FeCl₃(25 mM), FeSO₄(25 mM), MgSO₄(25 mM) and ZnCl₂(25 mM) in

methanol at room temperature.

of AChE consists of an oxyanion hole, an acyl-binding pocket, structure of BuChE, Trp82 amino acid residue is conserved,

catalytic active site (CAS) and peripheral anionic site (PAS).

The key amino acid residues responsible for binding are

Trp84 and Phe330 in the CAS, and Trp279 in the PAS. The

docking study shows that compound (61) is fitting intimately

inside the groove formed by Trp84, Tyr121 and Phe330

amino acid residues. It was positioned along the active site

gorge similar to donepezil, starting from the active site

amino acid residue Trp84 to the peripheral site amino acid

residue Trp279. The pyridine ring of compound (61) was

sandwiched between Trp84 and Phe330 residues of CAS. It

showed p–p stacking interactions with Trp84 (3.9 Å) and

Phe330 (3.6 Å). The tricyclic carbazole ring was positioned

near the Trp279 residue of CAS. Binding mode of the com-

pound (61) within the active site of BuChE is depicted in

Figure 4(C,D). Precise analysis of interaction of 61 in the

active site of BuChE showed that the key hydrogen bonding

interaction observed experimentally for the tacrine moiety

with His438 residue was maintained for compound (61). In

addition, nitrogen atom of the pyridine ring formed a hydro-

gen bond with Trp-82 (2.8 Å). The -NH of semicarbazone

group showed hydrogen bonding with His438 (2.0 Å). The tri-

cyclic carbazole ring showed T-shape edge-to-edge p–p

but Phe330 present in AChE is replaced by Ala328 in BuChE.

Absence of phenylalanine residue influences the affinity of

compounds for BuChE.

Similarly, the interaction views of 62 within the active

sites of AChE and BuChE are represented in Figure 5.

Compound (62) fits compactly inside the groove formed by

amino acid residues Trp84, Try121 and Phe330. 5-

Chloropyridine ring of compound (62) was oriented near

Trp84 and Phe330 residues of CAS. The tricyclic carbazole

ring was positioned near Trp279 residue of CAS. In case of

BuChE, the tricyclic carbazole ring of compound (62) showed

T-shaped edge-to-edge p–p stacking interactions with

Trp231 (4.8 and 4.9 Å) and Phe329 (5.3 and 5.5 Å). 5-

Chloropyridine ring was oriented parallel to Trp82 residue in

CAS. The -NH of semicarbazone group was observed to form

a hydrogen bond with His438 (2.4 Å).

3.3.2. Molecular dynamics simulation studies

To determine the conformational stability and promising

molecular interactions of compounds (61) and (62) in the

active sites of ChEs, time-dependent molecular dynamics

stacking interactions with Trp231 (4.8 and 4.9 Å). In the (MD) stability analysis was performed. The MD simulations

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

9

Figure 4. Docking poses of compound (61) with TcAChE (PDB ID: 2CKM) and hBuChE (PDB ID: 4BDS). (A, C) Binding modes of 61 within the active sites of TcAChE

and hBuChE, respectively. Ligand is shown as green sticks. Hydrogen bonds and p–p interactions are shown as yellow and green dashed lines, respectively. (B, D)

2D-ligand interaction diagrams of 61 with TcAChE and hBuChE, respectively.

were performed for a period of 50 ns using GROMACS2020.1. with compound (61) docked in the receptor active site,

showed RMSF-P within 0.35 nm (Figure 6(C)). Apart from

these observations, SASA analysis also supported the stability

of the protein–ligand complex (Figure 6(D)). The short-range

electrostatic (Coul-SR) and van der Waals/hydrophobic (LJ-SR)

interaction energies of the protein–ligand complex sug-

gested potential electrostatic and hydrophobic interactions.

The average values of Coul-SR and LJ-SR were observed to

be –57.14 1.90 kJ/mol and –182.43 2.10 kJ/mol respect-

ively, suggesting that the contribution of hydrophobic inter-

actions was higher than the electrostatic interactions in

stabilizing the protein–ligand complex.

In a similar way, the MD analysis of compound (61)

docked in the BuChE active site was also carried out. The

average RMSD-P was 0.156 nm (range of 0.10–0.218 nm)

(Figure 7(A)) and the RMSD-L was found in the range of

0.14–0.43 nm (Figure 7(B)). Including terminal and loop resi-

dues, the observed RMSF-P for all the residues was below

0.25 nm (Figure 7(C)). SASA analysis also supported the stabil-

ity of the protein–ligand complex (Figure 7(D)).The short-

range electrostatic (Coul-SR energy value of –76.14 2.9 kJ/

mol) and van der Waals/hydrophobic (LJ-SR energy value of

–172.43 2.7 kJ/mol) interaction energies explained promis-

ing electrostatic and hydrophobic interactions between the

protein and the ligand. The obtained values suggested that

the role of hydrophobic interactions was much more than

The study was performed using the docked pose of the

ligand–receptor complex as the reference frame. The con-

formational changes and dynamic behavior in complexes

were analyzed by various computational parameters like

root-mean-square deviation (RMSD)-P, RMSD-L, root-mean-

square fluctuation (RMSF)-P (P ¼ protein, L ¼ ligand) and solv-

ent accessible surface area (SASA) for protein while having

ligand in the receptor active site are shown in Figures 6–9.

The protein RMSD is determined mainly to understand

the extent of movement of receptor atoms or groups while

having the ligand inside the receptor active site. This type of

analysis is indicative of the stability of conformation of the

receptor structure and deviation over a period of time. The

average RMSD-P for AChE in complexation with the docked

compound (61) was 0.1386 nm (range of 0.08–0.253) (Figure

6(A)). This suggests that over a period of time the protein

having compound (61) in the active site was quite stable.

The average RMSD-L was 0.3497 nm (range of 0.2–0.48 nm)

which strongly indicated the stability of the protein–ligand

complex without a major deviation in orientation of the

compound in the receptor active site over the period of MD

analysis (Figure 6(B)). RMSF indicates the integrity of the

structure and mobility of the residues in the protein struc-

ture. It shows the flexibility of each amino acid residue of

the protein upon binding to the ligand. Here, all the residues

of the protein structure including loop and terminal residues,

10

K. B. PATEL ET AL.

Figure 5. Docking poses of compound (62) with TcAChE (PDB ID: 2CKM) and hBuChE (PDB ID: 4BDS). (A, C) Binding modes of 62 within the active sites of TcAChE

and hBuChE, respectively. Ligand is shown as green sticks. Hydrogen bonds and p–p interactions are shown as yellow and green dashed lines, respectively. (B, D)

2D-ligand interaction diagrams of 62 with TcAChE and hBuChE, respectively.

Figure 6. Molecular dynamics simulation of compound (61) with AChE as a function of time. (A) RMSD-P, (B) RMSD-L, (C) RMSF-P, and (D) SASA for AChE with com-

pound (61).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

11

Figure 7. Molecular dynamics simulation of compound (61) with BuChE as a function of time. (A) RMSD-P, (B) RMSD-L, (C) RMSF-P and (D) SASA plots for BuChE

with compound (61).

Figure 8. Molecular dynamics simulation of compound (62) with AChE as a function of time. (A) RMSD-P, (B) RMSD-L, (C) RMSF-P and (D) SASA plots for AChE with

compound (62).

the electrostatic interactions in stabilizing the ligand within in the active site (Figure 8(A)). RMSD-L was observed to be

the protein.

0.134 nm with a range of 0.08–0.214 nm (Figure 8(B)). Here,

Similar MD simulation studies for ChEs with compound the observed overall RMSF-P value was below 0.45 nm sup-

(62) were also performed. For compound (62) with AChE, porting the stability of the protein while having the ligand in

the average RMSD-P of 0.151 nm (range of 0.08–0.223 nm) the receptor active cavity (Figure 8(C)). Apart from these

suggested high stability of the receptor with compound (62) observations, SASA analysis also supported the stability of

12

K. B. PATEL ET AL.

Figure 9. Molecular dynamics simulation of compound (62) with BuChE as a function of time. (A) RMSD-P, (B) RMSD-L, (C) RMSF-P and (D) SASA plots for BuChE

with compound (62).

the protein–ligand complex (Figure 8(D)). Here again, the

short-range electrostatic (Coul-SR energy value of

–61.34 1.6 kJ/mol) and van der Waals/hydrophobic (LJ-SR

energy of –206.73 2.4 kJ/mol) interaction energies explained

strong interactions between the protein and the ligand.

The RMSD-P value for BuChE-compound (62) complex

was 0.148 nm (range 0.12–0.18 nm) explaining high stability

of the protein (Figure 9(A)). The average RMSD-L was

0.237 nm (range of 0.1–0.46 nm) (Figure 9(B)) and RMSF-P

was less than 0.35 nm including loop and terminal residues

(Figure 9(C)). Here also the hydrophobic interaction contribu-

tion was found to be higher than the electrostatic interac-

tions (found Coul-SR energy and LJ-SR energy values were

–66.14 2 kJ/mol and –192.46 1.8 kJ/mol, respectively).

As shown in Table 3, compounds (61 and 62) obeyed all

of the Lipinski’s rule-of-five parameters in the given permis-

sible ranges. Veber et al. introduced two additional key

parameters viz. number of rotatable bonds (NRB) and topo-

logical polar surface area (TPSA) (Veber et al., 2002). NRB

describes the conformational flexibility of the molecules. For

better oral bioavailability and drug-likeness, a molecule

should have lesser than 8 rotatable bonds or 7 atoms in a

linear chain outside the ring. TPSA is a measure of polarity. It

is a major determinant of passive diffusion of a molecule

through biological membranes and thus, it enables the pre-

diction of drug absorption, including bioavailability, blood–-

brain barrier (BBB) permeation and intestinal absorption.

Lenz and coworker have reported an average TPSA value to

be less than 90 Å²for a central nervous system (CNS) active

drug (Pajouhesh & Lenz, 2005). The average value for mar-

keted CNS drugs is 40.5 Å²(range 4.63–108 Å²). Both the

compounds (61 and 62) have NRB values of six and TPSA

values of ꢂ56.5 Å². QPCaco-2 value correlates the oral

absorption of a drug predicting its gut–blood barrier perme-

ability. Values higher than 500 predict significant oral absorp-

tion, which was attained by both the test compounds.

Significant quantum of oral bioavailability of the test com-

pounds is further supported by the predicted human oral

absorption percent (% HOA) values. The n-octanol-water par-

tition coefficient (QPlogP_o/w), brain/blood partition coefficient

(QPlogBB), CNS and apparent MDCK (Madin-Darby canine

kidney) cell permeability (QPPMDCK) collectively predict the

potential of a ligand to cross the BBB. Compounds (61 and

62) are predicted to have borderline CNS penetration as they

possess CNS values of zero, and QPlogBB values of –0.276

and –0.113, respectively. QPPMDCK value is a good criterion

3.3.3. In silico prediction of physicochemical and pharma-

cokinetics parameters

Determination of pharmacokinetics properties of the lead

molecules is an important step in the drug discovery process

to prevent the drug attrition in the later stages of clinical tri-

als due to inadequate absorption, distribution, metabolism,

excretion, and toxicity (ADMET) profile. Application of com-

puter-based tools in prediction of ADMET indicators is

attracting considerable attention to screen out such outliers

in the very early stages of the drug discovery process. Here,

the ADMET parameters for the most promising compounds

(61 and 62) along with the standard drugs donepezil and

tacrine were assessed using QikProp module of Schrodinger

Suite (Table 3), and pkCSM web server (http://structure.bioc.

cam.ac.uk/pkcsm) (Table 4).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

13

Table 3. ADMET indicators of compounds (61 and 62), donepezil and tacrine predicted by QikProp.

Parameters

Limits

Compd (61)

Compd (62)

Donepezil

Tacrine

MW

HBD

HBA

130–725

0–6

2–20

373.475

2

407.92

2

5

379.498

0

5.5

198.267

1.5

5

2

NRB

0–8

6

1

PSA

7–200

–2 to 6.5

500–2000

300–1000

0–2

56.493

5.002

1220.936

713.155

0

56.502

5.496

1265.381

737.436

0

46.234

4.242

1248.451

681.675

0

33.825

2.536

701.299

425.06

0

QPlogP_o/w

Volume

SASA

ReFG

Rule of five (Violation)

CNS

0–1

–

1

0

1

0

1

0

1

QPPMDCK

QPPCaco

QPlogBB

QPlogS

QPlogK_hsa

% HOA

#star

–

2955.75

2565.936

–0.276

–6.753

0.673

100

7323.718

2562.007

–0.113

–7.495

0.788

100

589.289

1070.771

0.223

–4.059

0.516

100

1602.036

2965.755

0.047

–3.036

0.049

100

–3 to 1.2

–6.5 to 0.5

–1.5 to 1.5

0–100

0–5

2

1

0

MW, molecular weight; HBD, hydrogen-bond donor atoms; HBA, hydrogen-bond acceptor atoms; NRB, number of rotatable bonds; PSA, polar surface area;

QPlogP_o/w, predicted octanol/water partition coefficient; SASA, total solvent accessible surface area; ReFG, reactive functional groups; QPPMDCK, predicted appar-

ent MDCK cell permeability in nm/s; QPPCaco, caco-2 cell permeability in nm/s; QPlogBB, brain/blood partition coefficient; QPlogS, predicted aqueous solubility;

QPlogK_hsa, binding to human serum albumin; % HOA, human oral absorption on 0-100% scale; #star, number of parameters which fall outside the 95% range of

similar values for the known drugs.

Table 4. ADMET descriptors determined by pkCSM tool.

Predicted values

Sr. no.

Property

Model name

Water solubility (log mol/L)

61

62

1

2

3

4

5

6

7

8

Absorption

–4.916

1.21

94.176

–2.727

Yes

0.606

0.056

0.082

–1.783

No

–5.282

1.163

92.346

–2.723

Yes

0.526

0.031

0.063

–1.648

No

Caco2 permeability (log Papp in 10^–6cm/s)

Human intestinal absorption (% absorbed)

Skin permeability (log K_p)

P-glycoprotein substrate

P-glycoprotein I inhibitor

P-glycoprotein II inhibitor

Human VDss (log L/kg)

Distribution

Metabolism

9

Human fraction unbound (Fu)

BBB permeability (log BB)

CNS permeability (log PS)

CYP2D6 substrate

CYP3A4 substrate

CYP1A2 inhibitor

CYP2C19 inhibitor

CYP2C9 inhibitor

CYP2D6 inhibitor

CYP3A4 inhibitor

Total clearance (log mL/min/kg)

Renal OCT2 substrate

AMES toxicity

Human max. tolerated dose (log mg/kg/day)

hERG I inhibitor

hERG II inhibitor

Oral rat acute toxicity (LD₅₀, mol/kg)

Oral rat chronic toxicity (LOAEL) (log mg/kg_bw/day)

Skin sensitization

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

Yes

No

Yes

–0.174

No

Yes

–0.128

No

Yes

–0.118

No

Excretion

Toxicity

No

–0.195

No

Yes

2.326

0.895

No

Yes

2.294

0.97

No

Minnow toxicity (log mM)

0.392

0.204

for the prediction of BBB permeation. A QPPMDCK value Compounds (61 and 62) showed values of #star within the

greater than 500 is considered as good, and the test com- limit, thus they could be taken as drug-like compounds.

pounds displayed significantly high values for this parameter.

pkCSM tool was employed to predict some additional

QPlogK_hsapredicts the binding of test molecules to the ADMET properties. Some of these descriptors are mentioned

human serum albumin. The test compounds (61 and 62) in Table 4. Compounds (61 and 62) are predicted to have

show adherence to the recommended values. The descriptor good absorption values suggesting high chances of crossing

‘#star’ annotates the number of parameters having values the gut by passive diffusion. The water solubility, Caco2 per-

falling outside the 95% range of the same values for the meability, human intestinal absorption and skin permeability

reported drugs. A larger number of #star suggests that a values for the test compounds were observed to be in

molecule is less druglike than molecules with lesser #stars. acceptable ranges. The test ligands (61 and 62) are predicted

14

K. B. PATEL ET AL.

Figure 10. SOM prediction using SMARTCyp web server for compounds (61 and 62). The top three sites of metabolism were highlighted in colored circles at

respective positions in their chemical structures. Tables show the list of top 10 metabolic sites of the lead molecules based on their scores and energies.

to have high VDss (volume of distribution at steady-state)

(values of 4.036 and 3.357 L/kg, respectively). BBB and CNS

permeability values of the test compounds were also accept-

able when compared with the standard values. The results

indicated that these compounds were likely to cross these

barriers. Metabolism of any drug molecule depends mainly

on the action of the cytochrome (CYP) class of enzymes. The

lead molecules (61 and 62) were predicted to be the sub-

strates for CYP3A4 suggesting possible metabolism of these

compounds with the action of CYP3A4. They were also pre-

dicted to be the inhibitors of CYP1A2, CYP2C19, CYP2C9 and

CYP3A4 which could cause interference with the metabolism

of other drugs metabolized by these isoforms. The predicted

excretion values also supported drug-like behavior of the

test compounds based on the total clearance and interaction

with renal organic cation transporter 2 (OCT2). The test

ligands were found to be non-substrates of OCT2 predicting

their proper renal clearance. Compounds (61 and 62) were

predicted to have maximum tolerated doses (human) of

3.3.4. Site of metabolism prediction by SMARTCyp

The test compounds (61 and 62) were predicted to be

metabolized by CYP3A4 isoform. Common sites of metabol-

ism (SOM) of the test ligands were predicted using the

SMARTCyp web server (https://smartcyp.sund.ku.dk/mol_to_

som). For the validation of SMARTCyp predictions, verapamil,

a substrate of CYP3A4 was used as its metabolic products,

was well reported. The predicted SOM of verapamil by

SMARTCyp was in accordance with the literature (Sun et al.,

2004). As predicted by SMARTCyp (Figure 10), the primary

SOM in compounds (61 and 62) is S.1 of thiocarbonyl group

of semicarbazone as it ranked first with the least scores and

energies. The secondary predicted SOM are C.20 and C.21

sites on the carbazole ring of compound (61 and 62)

respectively, while the tertiary predicted SOM are the C.23

and C.24 methylene carbons of ethyl group attached to the

carbazole ring of compounds (61) and (62), respectively.

0.638 and 0.762 mg/kg/day, respectively. The AMES test pre- 4. Conclusion

dicts the non-mutagenic and non-carcinogenic nature of

In conclusion, a series of carbazole-based thiosemicarbazone,

compound (61), whereas compound (62) was predicted to

have possible mutagenic and/or carcinogenic properties.

Furthermore, the lead molecules were found to be non-

inhibitors of human Ether-a-Go-go Related Gene-I (hERG-I)

and hERG-II channels. The minnow toxicity was found to be

higher than –0.3 log mM which predicted that the test

ligands were acutely nontoxic. All these results together indi-

cate that the lead molecules (61 and 62) have drug-like

pharmacokinetics profiles, which would increase their bio-

logical significance.

semicarbazone and hydrazone derivatives were designed,

synthesized and biologically evaluated for their ChE inhibi-

tory, antioxidant and biometal chelating properties. In vitro

study showed that the synthesized compounds possessed

ChE inhibitory activity with IC₅₀values ranging between 0.9

and 2.6 mM for hAChE and 1.6 and 23.32 mM for EqBuChE.

Among them, compounds (61 and 62) showed good hAChE

inhibitory activities (IC₅₀values of 1.37 and 1.18 mM, respect-

ively) while exhibited moderate EqBuChE inhibitory activities

(IC₅₀values of 2.69 and 3.31 mM, respectively). These com-

pounds also exhibited excellent antioxidant activity.

Compounds (61 and 62) showed selective copper metal che-

lating properties without affecting other biometals.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

15

Molecular dynamics simulation and molecular docking stud-

ies indicated close interactions of the most promising com-

pounds in the active sites of ChEs. In addition, compounds

(61 and 62) were predicted to have in silico drug-like ADMET

properties. Findings of the present study suggest compounds

(61 and 62) as promising leads for further development as

balanced MTDLs to be used as anti-AD agents.

5.1.1.1. N-Benzylhydrazinecarbothioamide (31). The inter-

mediate (31) was synthesized as per the general Method A

from benzylamine (11) (1.0 g, 9.33 mmol) as white solid (He

et al., 2019). (Yield: 60%); m.p. 126–128 ^ꢀC (lit m.p.

129–130 ^ꢀC); IR (KBr, cm^ꢃ1): 3328, 3027, 2917, 1618, 1225,

1085; MS (m/z): 182.0 [M þ H]^þ.

5.1.1.2.

N-(4-Methoxybenzyl)hydrazinecarbothioamide

(32). The intermediate (32) was synthesized as per the gen-

eral Method A from 4-methoxybenzylamine (12) (1.0 g,

7.29 mmol) as white solid (Pervez et al., 2017). (Yield: 62%);

m.p. 112–114 ^ꢀC; IR (KBr, cm^ꢃ1): 3332, 3036, 2914, 1616,

1248, 1029; MS (m/z): 212.0 [M þ H]^þ.

5. Experimental

General: All the required reagents and solvents for the experi-

ments were procured from commercial suppliers such as

Sigma Aldrich and Spectrochem. The solvents and chemicals

were purified whenever required by basic laboratory techni-

ques prior to use. The silica gel 60 GF₂₅₄(Merck) TLC plates

having a thickness of 0.25 mm were used to observe the pro-

gress of the reaction by visualizing in a UV (254nm) chamber

and/or exposure to various staining reagents. Melting points of

the synthesized intermediates and final compounds were

recorded in a glass capillary using a VEEGO melting point

apparatus having a silicone oil bath and a temperature reso-

lution of 0.1^ꢀC. The IR spectra were obtained using FTIR spec-

trophotometer [Bruker ALPHA-T (Germany)] using KBr pellets.

5.1.1.3. N-(4-Chlorobenzyl)hydrazinecarbothioamide (33).

The intermediate (33) was synthesized as per the general

Method A from 4-chlorobenzylamine (13) (1.0 g, 7.06 mmol)

as white solid (Pervez et al., 2018). (Yield: 62%); m.p.

178–180 ^ꢀC; IR (KBr, cm^ꢃ1): 3317, 2982, 2926, 1615, 1238,

1083, 809; MS (m/z): 216.0 [M]^þ, 218.0 [M þ 2]^þ.

5.1.1.4. N-(4-Fluorobenzyl)hydrazinecarbothioamide (34).

The intermediate (34) was synthesized as per the general

Method A from 4-fluorobenzylamine (14) (1.0 g, 8.0 mmol) as

white solid (Pervez et al., 2017). (Yield: 63%); m.p.

138–140 ^ꢀC; IR (KBr, cm^ꢃ1): 3329, 3043, 2920, 1617, 1223,

1075; MS (m/z): 200.0 [M þ H]^þ.

1

The H NMR spectra were acquired on a Bruker 400MHz spec-

trometer in deuterated solvents. The chemical shift (d) values

are expressed in parts per million (ppm) in relation to tetrame-

thylsilane (TMS) as internal standard. The pattern of the peaks

is expressed as s (singlet), d (doublet), dd (doublet of doublet),

t (triplet), q (quartet) and m (multiplet). The mass spectrometer

of Thermo Fisher with electrospray ionization (ESI) ion source

was used for the recording of mass spectra.

5.1.1.5. N-(4-Methylbenzyl)hydrazinecarbothioamide (35).

The intermediate (35) was prepared according to the general

Method A from 4-methylbenzylamine (15) (1.0 g, 8.26 mmol)

as white solid (Pervez et al., 2017). (Yield: 65%); m.p.

154–156 ^ꢀC; IR (KBr, cm^ꢃ1): 3322, 2918, 1616, 1270, 1075; MS

(m/z): 195.75 [M]^þ.

5.1. Chemistry

For intermediates (36–42) (Method B): To a stirred solu-

tion of substituted aniline (16–22) (1.0 equiv.) and NaOH (1.2

equiv.) in DMF (10 mL), a solution of carbon disulfide (1.2

equiv.) in DMF (5 mL) was gradually added and the resulting

mixture was stirred for 3 h at RT. After complete consump-

tion of starting material as checked by TLC, hydrazine

hydrate (3.0 equiv.) was added to the above reaction mixture

and it was heated to 70 ^ꢀC for 1 h. After completion of the

reaction as checked by TLC, the reaction mixture was

allowed to cool to RT and diluted with cold water. The solid

precipitates so obtained were filtered, washed with cold

water and dried under reduced pressure to get the inter-

mediate compounds (36–42).

5.1.1. General method for the synthesis of 4-substituted

thiosemicarbazide intermediates (31–42)

For intermediates (31–35) (Method A): To a stirred solution

of substituted benzylamines (11–15) (1 equiv.) and triethyl-

amine (1.5 equiv.) in methanol (10 mL), a solution of carbon

disulfide (1.2 equiv.) in methanol (5 mL) was added gradually.

The resulting mixture was allowed to stir for 2–3 h at room

temperature (RT). To it, methyl iodide (1 equiv.) in methanol

(5 mL) was slowly added keeping temperature of the reaction

mixture at –10 ^ꢀC. After complete addition, the reaction mix-

ture was stirred for additional 2 h at RT. After completion of

the reaction as checked by TLC, the reaction mixture was

concentrated under reduced pressure and partitioned

between 30 mL of ether and 0.2 N HCl solution. The ethereal

layer was washed with cold water and saturated with NaCl

solution, dried using anhydrous MgSO₄, filtered and evapo-

rated under vacuum to obtain white solid mass. The

obtained solid mass was further charged to a solution of

hydrazine hydrate (3 equiv.) in methanol (15 mL). The result-

ing reaction mixture was refluxed for 4 h. After completion of

5.1.1.6. N-Phenylhydrazinecarbothioamide (36). The inter-

mediate (36) was synthesized as per the general Method B

from aniline (16) (1.0 g, 10.75 mmol) as white solid (Zhang et

al., 2020). (Yield: 63%); m.p. 142–144 ^ꢀC (lit m.p. 144–145 ^ꢀC);

IR (KBr, cm^ꢃ1): 3304, 3107, 1638, 1287, 1070.

the reaction as confirmed by TLC, the reaction mixture was 5.1.1.7.

N-(4-Methoxyphenyl)hydrazinecarbothioamide

allowed to attain RT. The solid so precipitated were collected (37). The intermediate (37) was synthesized as per the gen-

by filtration and washed with cold methanol to obtain the eral Method B from 4-methoxyaniline (17) (1.0 g, 8.13 mmol)

desired intermediates (31–35).

as white solid (Zhang et al., 2020). (Yield: 70%); m.p.

16

K. B. PATEL ET AL.

162–164 ^ꢀC (lit m.p. 165–166 ^ꢀC); IR (KBr, cm^ꢃ1): 3317, 3013,

2965, 1637, 1244, 1033.

5.1.3. General procedure for the synthesis of acyl hydra-

zide intermediates (44–50)

Method C. To a stirred solution of substituted benzoic acid/

substituted phenylacetic acids/nicotinic acid (1 equiv.) in

methanol (20 mL), conc. sulfuric acid in a catalytic amount

was added. The resulting mixture was refluxed for 6 h. After

completion of the reaction (confirmed by TLC), the mixture

was concentrated to a volume of ꢂ10 mL and partitioned

between chloroform (30 mL) and water. The organic phase

was washed with water, dried over anhydrous sodium sulfate

and evaporated under reduced pressure to obtain liquid

product. The resulting liquid was further added to a solution

of hydrazine hydrate (3 equiv.) in methanol (20 mL). The

reaction mixture was heated up to 65 ^ꢀC for 4 h. After com-

pletion of the reaction as checked by TLC, the reaction mix-

ture was allowed to cool to RT and the solid precipitates so

obtained were filtered and washed with cold methanol to

obtain the desired acyl hydrazides (44–50).

5.1.1.8. N-(Pyridin-2-yl)hydrazinecarbothioamide (38). The

intermediate (38) was synthesized as per the general Method

B from 2-aminopyridine (18) (1.0 g, 10.63 mmol) as white

solid (Aljahdali et al., 2014). (Yield: 65%); m.p. 188–190 ^ꢀC (lit

m.p. 193–194 ^ꢀC); IR (KBr, cm^ꢃ1): 3241, 3029, 1607, 1207,

1008; MS (m/z): 169.0 [M þ H]^þ.

5.1.1.9. N-(5-Chloropyridin-2-yl)hydrazinecarbothioamide

(39). The intermediate (39) was synthesized as per the gen-

eral Method B from 2-amino-5-chloropyridine (19) (1.0 g,

7.81 mmol) as white solid (Sriram et al., 2009). (Yield: 63%);

m.p. 112–114 ^ꢀC; IR (KBr, cm^ꢃ1): 3243, 2997, 1609, 1203,

1014, 820; MS (m/z): 203.0 [M]^þ, 205.0 [M þ 2]^þ.

5.1.1.10. N-(5-Methylpyridin-2-yl)hydrazinecarbothioamide

(40). The intermediate (40) was synthesized as per the gen-

eral Method B from 2-amino-5-methylpyridine (20) (1.0 g,

9.25 mmol) as white solid. (Yield: 65%); m.p. 153–155 ^ꢀC; IR

(KBr, cm^ꢃ1): 3252, 3006, 1617, 1238, 1017; MS (m/z):

183.0 [M þ H]^þ.

5.1.3.1. 2-Phenylacetohydrazide (44). The intermediate (44)

was synthesized as per the general Method C from 2-phenyl-

acetic acid (24) (1.0 g, 7.36 mmol) as white solid (Zimin et al.,

2018). (Yield: 75%); m.p. 112–114 ^ꢀC (lit m.p. 116–118 ^ꢀC); IR

(KBr, cm^ꢃ1): 3293, 3030, 1645, 1143.

5.1.1.11. N-(Pyrimidin-2-yl)hydrazinecarbothioamide (41).

The intermediate (41) was synthesized as per the general

Method B from 2-aminopyrimidine (21) (1.0 g, 10.52 mmol) as

white solid (El-Masry, 2000). (Yield: 67%); m.p. 168–170 ^ꢀC (lit

m.p. 172 ^ꢀC); IR (KBr, cm^ꢃ1): 3303, 3109, 1637, 1288, 1069.

5.1.3.2. 2-(4-Methoxyphenyl)acetohydrazide (45). The inter-

mediate (45) was synthesized as per the general Method C

from 2-(4-methoxyphenyl)acetic acid (25) (1.0 g, 7.35 mmol)

as white solid (Hanif et al., 2012). (Yield: 75%); m.p.

128–130 ^ꢀC (lit m.p. 126–127 ^ꢀC); IR (KBr, cm^ꢃ1): 3341, 3030,

2957, 1619, 1107.

5.1.1.12. N-(Pyrazin-2-yl)hydrazinecarbothioamide (42).

The intermediate (42) was synthesized as per the general

Method B from pyrazin-2-amine (22) (1.0 g, 10.52 mmol) as

white solid. (Yield: 64%); m.p. 148–150^ꢀ; IR (KBr, cm^ꢃ1): 3242,

3030, 1608, 1206, 1007.

5.1.3.3. 2-(4-Chlorophenyl)acetohydrazide (46). The inter-

mediate (46) was synthesized as per the general Method C

from 2-(4-chlorophenyl)acetic acid (26) (1.0 g, 7.35 mmol) as

white solid (Li et al., 2015). (Yield: 77%); m.p. 167–169 ^ꢀC (lit

m.p. 170–172 ^ꢀC); IR (KBr, cm^ꢃ1): 3298, 3041, 1657, 1132, 850.

5.1.3.4. Benzohydrazide (47). The intermediate (47) was

synthesized as per the general Method C from benzoic acid

(27) (1.0 g, 7.35 mmol) as white solid (Saha et al., 2010).

(Yield: 80%); m.p. 107–109 ^ꢀC (lit m.p. 110–113 ^ꢀC); IR (KBr,

cm^ꢃ1): 3298, 3020, 1661, 1119.

5.1.2. N-(4-Chlorophenyl)hydrazinecarboxamide (43)

To a cold stirring solution of 4-chloroaniline (1.0 equiv.) and

potassium carbonate (2.0 equiv.) in acetone (20 mL) at

0–5 ^ꢀC, a solution of phenyl chloroformate (1.1 equiv.) in

acetone (10 mL) was added gradually. After complete add-

ition, the resulting reaction mixture was stirred at room tem-

perature for additional 2 h. After completion of the reaction,

the reaction mixture was concentrated in vacuo, quenched

with 10% NaHCO₃solution and diluted with cold water. The

solid precipitates obtained were filtered and dried. The

obtained solids were added to solution of hydrazine hydrate

(3 equiv.) in 1,4-dioxane. The reaction mixture was heated to

100 ^ꢀC for 4 h. After completion of the reaction as checked

by TLC, the reaction mixture was allowed to cool to RT. The

resulting precipitates were filtered and washed with ether to

obtain the titled compound as white solid (Beukers et al.,

2003). (Yield 75.2%); m.p. 188–190 ^ꢀC (lit m.p. 190–192 ^ꢀC); IR

(KBr, cm^ꢃ1): 3334, 3230, 1670, 1142, 815.

5.1.3.5. 4-Fluorobenzohydrazide (48). The intermediate (48)

was synthesized as per the general Method C from 4-fluoro-

benzoic acid (28) (1.0 g, 6.49 mmol) as white solid (Murty et

al., 2016). (Yield: 79%); m.p. 162–164 ^ꢀC (lit m.p. 161–163 ^ꢀC);

IR (KBr, cm^ꢃ1): 3302, 3017, 1664, 1119.

5.1.3.6. 4-Methylbenzohydrazide (49). The intermediate

(49) was synthesized as per the general Method C from 4-

methylbenzoic acid (29) (1.0 g, 6.66 mmol) as white solid

(Saha et al., 2010). (Yield: 74%); m.p. 112–114 ^ꢀC (lit m.p.

116–117 ^ꢀC); IR (KBr, cm^ꢃ1): 3304, 3023, 1662, 1120.

5.1.3.7. Nicotinohydrazide (50). The intermediate (50) was

synthesized as per the general Method C from nicotinic acid

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

17

€

(30) (1.0 g, 8.12 mmol) as white solid (Elif Ozturkkan Ozbek et

al., 2020). (Yield: 70%); m.p. 158–160 ^ꢀC (lit m.p. 161–163 ^ꢀC);

IR (KBr, cm^ꢃ1): 3297, 3019, 1662, 1120.

5.1.6.1. (E)-N-Benzyl-2-((9-ethyl-9H-carbazol-3-yl)methyle-

ne)hydrazinecarbothioamide (54). Compound (54) was syn-

thesized as per the general Method

D

from the

intermediates (53) (1.0 g, 4.46 mmol) and (31) (0.89 g,

4.93 mmol) as a white solid. (Yield: 75%); m.p. 128–129 ^ꢀC; IR

(KBr, cm^ꢃ1): 3359, 3058, 2973, 1595, 1231, 1074; ¹H NMR

(CDCl₃): d 9.30 (s, 1H, -NHCSNH), 8.28 (s, 1H, ArH), 8.09 (d,

J ¼ 7.5 Hz, 1H, ArH), 7.96 (s, 1H, -CHN), 7.80 (t, J ¼ 6.0 Hz, 1H,

-NHCSNH), 7.76–7.74 (dd, J ¼ 8.5 Hz, 2.0 Hz, 1H, ArH),

7.51–7.36 (m, 7H, ArH), 7.33–7.24 (m, 2H, ArH), 5.03 (d,

J ¼ 6.0 Hz, 2H, -NHCH₂), 4.39–4.34 (q, J ¼ 7.5 Hz, 2H,

-NCH₂CH₃), 1.43 (t, J ¼ 7.5 Hz, 3H, -NCH₂CH₃); MS (m/z):

387.0 [M þ 1]^þ.

5.1.4. 9-Ethyl-9H-carbazole (52)

To stirring solution of carbazole (51) (1.0 g, 6 mmol) in DMSO

(15 mL), aqueous sodium hydroxide (0.48 g in 2 mL) solution

was added, and the resulting reaction mixture was stirred for

further 10 min. Ethyl iodide (0.6 mL, 7.06 mmol) was added in

a dropwise manner to the above reaction mixture and it was

allowed to stir at RT. TLC was used to monitor the progress

of the reaction where after completion of the reaction, the

reaction mixture was diluted with ice cold water and the

solid so precipitated was collected by filtration, rinsed with

cold water and dried in vacuum to yield the compound (52)

as off white solid (Patel, Patel, Kanhed, Teli, Patel, Joshi, et

al., 2020). (Yield: 1.07 g, 94%); m.p. 64–66 ^ꢀC (lit m.p.

64–66 ^ꢀC); IR (KBr, cm^ꢃ1): 3049, 2978, 2869, 1596, 1018, 753,

700; MS (m/z): 196.3 [M þ 1]^þ.

5.1.6.2. (E)-N-(4-Methoxybenzyl)-2-((9-ethyl-9H-carbazol-3-

yl)methylene)hydrazine carbothioamide (55). Compound

(55) was synthesized as per the general Method D from the

intermediates (53) (1.0 g, 4.46 mmol) and (32) (1.04 g,

4.93 mmol) as a white solid. (yield: 81%); m.p. 180–181 ^ꢀC; IR

(KBr, cm^ꢃ1): 3297, 3058, 2988, 1592, 1232, 1064; ¹H NMR

(CDCl₃): d 9.28 (s, 1H, -NHCSNH), 8.26 (d, J ¼ 1.5 Hz, 1H, ArH),

8.09 (d, J ¼ 8.0 Hz, 1H, ArH), 7.96 (s, 1H, -CHN), 7.75–7.71 (m,

1H, -NHCSNH; m, 1H, ArH), 7.51–7.47 (m, 1H, ArH), 7.42–7.36

(m, 4H, ArH), 7.27–7.24 (m, 1H, ArH), 6.93–6.90 (m, 2H, ArH),

4.94 (d, J ¼ 6.0 Hz, 2H, -NHCH₂), 4.38–4.34 (q, J ¼ 7.5 Hz, 2H,

-NCH₂CH₃), 3.80 (s, 3H, -OCH₃), 1.43 (t, J ¼ 7.5 Hz, 3H,

-NCH₂CH₃); MS (m/z): 417.0 [M þ 1]^þ.

5.1.5. 9-Ethyl-9H-carbazole-3-carbaldehyde (53)

Phosphoryl chloride (0.94 mL, 10.24 mmol) was added drop-

wise to an ice cooled stirred solution of ethyl carbazole (52)

(2.0 g, 10.24 mmol) and dimethylformamide (0.76 mL,

10.24 mmol) in chloroform (20 mL) over a period of 10 min.

After complete addition, the resulting reaction mixture was

heated to reflux for 12–14 h. After completion of the reaction

as checked by TLC, the reaction mixture was cooled to RT

and then slowly diluted with ice cold water. The product was

extracted using chloroform and washed with brine. The col-

lected organic phase was washed with water, dried using

anhydrous magnesium sulfate and concentrated under

reduced pressure. The crude product so obtained was puri-

fied using column chromatography (eluent: 10% ethyl acet-

ate in n-hexane) to afford the compound (53) as yellowish

white solid (Patel, Patel, Kanhed, Teli, Patel, Joshi, et al.,

2020). Yield 84%; m.p. 85–87 ^ꢀC (lit m.p. 84–86 ^ꢀC); IR (KBr,

cm^ꢃ1): 3049, 2971, 2822, 2743, 1679, 1588, 620; ¹H NMR

(CDCl₃): d 10.12 (s, 1H, CHO), 8.64 (d, J ¼ 2.0 Hz, 1H, ArH),

8.20–8.18 (m, 1H, ArH), 8.05–8.03 (m, 1H, ArH), 7.58–7.37 (m,

4H, ArH), 4.43 (q, J ¼ 7.2 Hz, 2H, NCH₂CH₃), 1.50 (t, J ¼ 7.2 Hz,

3H, NCH₂CH₃); MS (m/z): 224.0 [M þ 1]^þ.

5.1.6.3. (E)-N-(4-Chlorobenzyl)-2-((9-ethyl-9H-carbazol-3-

yl)methylene)hydrazinecarbo thioamide (56). Compound

(56) was synthesized as per the general Method D from the

intermediates (53) (1.0 g, 4.46 mmol) and (33) (1.05 g,

4.93 mmol) as a white solid. (Yield: 79%); m.p. 208–210 ^ꢀC; IR

(KBr, cm^ꢃ1): 3347, 2977, 1596, 1228, 1093, 801; ¹H NMR

(CDCl₃): d 9.31 (s, 1H, -NHCSNH), 8.28 (d, J ¼ 1.5 Hz, 1H, ArH),

8.09 (d, J ¼ 8.0 Hz, 1H, ArH), 7.97 (s, 1H, -CHN), 7.80 (t,

J ¼ 6.0 Hz, 1H, -NHCSNH), 7.76–7.74 (dd, J ¼ 8.5 Hz, 1.5 Hz, 1H,

ArH), 7.51–7.48 (m, 1H, ArH), 7.43–7.32 (m, 5H, ArH),

7.28–7.25 (m, 2H, ArH), 4.99 (d, J ¼ 6.0 Hz, 2H, -NHCH₂),

4.39–4.34 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.43 (t, J ¼ 7.5 Hz, 3H,

-NCH₂CH₃); ¹³C NMR (CDCl₃): d 177.4, 144.8, 141.3, 140.4,

136.4, 133.4, 129.1, 128.8, 126.4, 124.8, 123.9, 123.2, 122.7,

120.7, 120.6, 119.7, 108.9, 108.8, 47.4, 37.7, 13.8; MS (m/z):

421.0 [M]^þ, 423.0 [M þ 2]^þ.

5.1.6. General procedure for the synthesis of carbazole-

based thiosemicarbazones (54–65), semicarbazone

(66) and hydrazones (67–73)

Method D: A stirring reaction mixture of 9-ethyl-9H-carba-

zole-3-carbaldehyde (53) (0.25 g, 1.12 mmol) and appropiate

thiosemicarbazides (31–42), semicarbazide (43) or acyl hydra-

zides (44–50) (1.1 equiv.) with acetic acid (0.1 mL) in metha-

nol (20 mL) was heated to reflux for 4–5 h. After completion

of reaction, the reaction mixture was allowed to cool to RT.

After standing for 2 h, the crystalline solid so formed was col-

5.1.6.4. (E)-N-(4-Fluorobenzyl)-2-((9-ethyl-9H-carbazol-3-

yl)methylene)hydrazinecarbo thioamide (57). Compound

(57) was synthesized as per the general Method D from the

intermediates (53) (1.0 g, 4.46 mmol) and (34) (0.98 g,

4.93 mmol) as a white solid. (Yield: 80%); m.p. 195–197 ^ꢀC; IR

1

(KBr, cm^ꢃ1): 3354, 3062, 3002, 1598, 1215, 1091, 941; H NMR

(CDCl₃): d 9.42 (s, 1H, -NHCSNH), 8.28 (d, J ¼ 1.5 Hz, 1H, ArH),

8.09 (d, J ¼ 8.0 Hz, 1H, ArH), 7.98 (s, 1H, -CHN), 7.78 (t,

J ¼ 6.0 Hz, 1H, -NHCSNH), 7.76–7.74 (dd, J ¼ 8.5 Hz, 1.5 Hz, 1H,

ArH), 7.51–7.48 (m, 1H, ArH), 7.43–7.37 (m, 4H, ArH),

lected by vaccum filtration and rinsed with methanol to 7.28–7.25 (m, 1H, ArH), 7.08-7.04 (m, 2H, ArH), 4.99 (d,

obtain the desired compounds (54–73).

J ¼ 6.0 Hz, 2H, -NHCH₂), 4.38-4.34 (q, J ¼ 7.5 Hz, 2H,

18

K. B. PATEL ET AL.

-NHCH₂CH₃), 1.43 (t, J ¼ 7.5 Hz, 3H, -NHCH₂CH₃); MS (m/z): 4.40–4.36 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.45 (t, J ¼ 7.5 Hz, 3H,

405.0 [M þ 1]^þ.

-NCH₂CH₃); ¹³C NMR (CDCl₃): d 173.7, 151.7, 148.0, 144.9,

141.5, 140.4, 137.7, 126.4, 125.1, 123.5, 123.3, 122.7, 121.1,

120.8, 120.3, 119.7, 118.1, 115.4, 108.9, 37.8, 13.8; MS (m/z):

374.0 [M þ 1]^þ.

5.1.6.5. (E)-N-(4-Methylbenzyl)-2-((9-ethyl-9H-carbazol-3-

yl)methylene)hydrazinecarbo thioamide (58). Compound

(58) was synthesized as per the general Method D from the

intermediates (53) (1.0 g, 4.46 mmol) and (35) (0.82 g, 5.1.6.9. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)-N-(5-

4.93 mmol) as a white solid. (Yield: 84%). m.p. 210–212 ^ꢀC; IR chloropyridin-2-yl)hydrazine

carbothioamide

(62).

(KBr, cm^ꢃ1): 3361, 3054, 3003, 1594, 1233, 1123; ¹H NMR Compound (62) was synthesized as per the general Method

(CDCl₃): d 9.13 (s, 1H, -NHCSNH), 8.27 (d, J ¼ 1.5 Hz, 1H, ArH), D from the intermediates (53) (1.0 g, 4.46 mmol) and (39)

8.09 (d, J ¼ 7.5 Hz, 1H, ArH), 7.94 (s, 1H, -CHN), 7.76–7.74 (m, (0.99 g, 4.93 mmol) as a white solid. (Yield: 81%); m.p.

1H, -NHCSNH; m, 1H, ArH), 7.51–7.48 (m, 1H, ArH), 7.43–7.33 204–206 ^ꢀC; IR (KBr, cm^ꢃ1): 3311, 3050, 2974, 1597, 1236,

1

(m, 4H, ArH), 7.28–7.24 (m, 2H, ArH), 7.19 (d, J ¼ 8.0 Hz, 1H, 1119, 753; H NMR (CDCl₃): d 9.90 (s, 1H, -NHCSNH), 9.20 (s,

ArH), 4.97 (d, J ¼ 5.5 Hz, 2H, -NHCH₂), 4.39–4.34 (q, J ¼ 7.5 Hz, 1H, -NHCSNH), 8.97 (d, J ¼ 9.0 Hz, 1H, ArH), 8.36 (s, 1H, ArH),

2H, -NCH₂CH₃), 2.36 (s, 3H, ArCH₃), 1.43 (t, J ¼ 7.5 Hz, 3H, 8.33 (d, J ¼ 3 Hz, 1H, ArH), 8.16–8.14 (dd, J ¼ 9.0 Hz, 1.0 Hz,

-NCH₂CH₃); ¹³C NMR (CDCl₃): d 177.2, 144.4, 141.3, 140.4, 1H, ArH), 8.01 (s, 1H, -CHN), 7.90 (d, J ¼ 8.5 Hz, 1H, ArH),

137.3, 134.7, 129.4, 127.8, 126.3, 124.8, 124.0, 123.2, 122.7, 7.72–7.70 (dd, J ¼ 9.0 Hz, J ¼ 3.0 Hz, 1H, ArH), 7.53–7.50 (m,

120.7, 120.6, 119.6, 108.8, 108.8, 48.1, 37.7, 21.1, 13.8; MS (m/ 1H, ArH), 7.45–7.42 (m, 2H, ArH), 7.31–7.25 (m, 1H, ArH),

z): 401.0 [M þ 1]^þ.

4.41–4.37 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.46 (t, J ¼ 7.5 Hz, 3H,

-NCH₂CH₃); ¹³C NMR (CDCl₃): d 173.4, 150.0, 146.6, 145.3,

141.6, 140.4, 137.2, 127.3, 126.4, 125.0, 123.4, 123.3, 122.7,

121.2, 120.7, 119.8, 115.9, 109.0, 108.9, 37.8, 13.8; MS (m/z):

408 [M]^þ, 410 [M þ 2]^þ.

5.1.6.6. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)-N-phe-

nylhydrazinecarbothioamide (59). Compound (59) was syn-

thesized as per the general Method

intermediates (53) (1.0 g, 4.46 mmol) and (36) (0.82 g,

4.93 mmol) as a white solid. (Yield: 85%). m.p. 207–209 ^ꢀC; IR 5.1.6.10. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)-N-(5-

(KBr, cm^ꢃ1): 3242, 3047, 2977, 1596, 1202, 1073; ¹H NMR methylpyridin-2-yl)hydra-

zinecarbothioamide (63).

D

from the

(CDCl₃): d 9.97 (s, 1H, -NHCSNH), 9.28 (s, 1H, -NHCSNH), 8.34 Compound (63) was synthesized as per the general Method

(s, 1H, ArH), 8.13 (d, J ¼ 7.5 Hz, 1H, ArH), 8.09 (s, 1H, -CHN), D from the intermediates (53) (1.0 g, 4.46 mmol) and (40)

7.84–7.82 (dd, J ¼ 7.5 Hz, 2.0 Hz, 1H, ArH), 7.71 (d, J ¼ 7.5 Hz, (0.89 g, 4.93 mmol) as a white solid. (Yield: 79%); m.p.

2H, ArH), 7.52–7.40 (m, 5H, ArH), 7.29–7.25 (m, 2H, ArH), 173–176 ^ꢀC; IR (KBr, cm^ꢃ1): 3278, 3019, 2973, 1594, 1234,

4.40–4.35 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.45 (t, J ¼ 7.5 Hz, 3H, 1070; ¹H NMR (CDCl₃): d 9.86 (s, 1H, -NHCSNH), 9.26 (s, 1H,

-NCH₂CH₃); MS (m/z): 373.0 [M þ 1]^þ.

-NHCSNH), 8.76 (d, J ¼ 8.0 Hz, 1H, ArH), 8.39 (d, J ¼ 2.0 Hz, 1H,

ArH), 8.21 (d, J ¼ 2.0 Hz, 1H, ArH), 8.16 (d, J ¼ 8.0 Hz, 1H, ArH),

8.00 (s, 1H, -CHN), 7.93–7.91 (m, 1H, ArH), 7.59–7.57 (m, 1H,

ArH), 7.52–7.49 (m, 1H, ArH), 7.44–7.41 (m, 2H, ArH),

7.30–7.25 (m, 1H, ArH), 4.41–4.37 (q, J ¼ 7.5 Hz, 2H,

-NCH₂CH₃), 2.10 (s, 3H, ArCH₃), 1.46 (t, J ¼ 7.5 Hz, 3H,

-NCH₂CH₃); MS (m/z): 388.0 [M þ 1]^þ.

5.1.6.7. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)-N-(4-

methoxyphenyl)hydrazine carbothioamide (60). Compound

(60) was synthesized as per the general Method D from the

intermediates (53) (1.0 g, 4.46 mmol) and (37) (0.97 g,

4.93 mmol) as a white solid. (Yield: 82%); m.p. 223–225 ^ꢀC; IR

(KBr, cm^ꢃ1): 3331, 3043, 2977, 1595, 1236, 1067; ¹H NMR

(CDCl₃): d 9.20 (s, 1H, -NHCSNH), 9.10 (s, 1H, -NHCSNH), 8.36 5.1.6.11.

(E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)-N-

(d, J ¼ 1.5 Hz, 1H, ArH), 8.13 (d, J ¼ 8 Hz, 1H, ArH), 8.00 (s, 1H, (pyrimidin-2-yl)hydrazine carbothioamide (64). Compound

-CHN), 7.84–7.82 (m, 1H, ArH), 7.54–7.49 (m, 3H, ArH), (64) was synthesized as per the general Method D from the

7.45–7.42 (m, 2H, ArH), 7.29–7.25 (m, 1H, ArH), 6.96–6.94 (m, intermediates (53) (1.0 g, 4.46 mmol) and (41) (0.83 g,

2H, ArH), 4.41–4.37 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 3.84 (s, 3H, 4.93 mmol) as a white solid. (Yield: 78%); m.p. 87–89 ^ꢀC; IR

1

-OCH₃), 1.46 (t, J ¼ 7.5 Hz, 3H, -NCH₂CH₃); MS (m/z): (KBr, cm^ꢃ1): 3055, 2972, 1587, 1233, 1143; H NMR (CDCl₃): d

403.0 [M þ 1]^þ.

10.08 (s, 1H, -NHCSNH), 8.60 (s, 1H, -NHCSNH), 8.14 (d, 1H,

-CHN, 1H, ArH), 8.01–7.99 (m, 1H, ArH), 7.54–7.51 (m, 2H,

ArH), 7.47–7.44 (m, 4H, ArH), 7.33–7.30 (m, 2H, ArH),

4.41–4.37 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.46 (t, J ¼ 7.5 Hz, 3H,

-NCH₂CH₃); MS (m/z) 375.4 [M þ 1]^þ.

5.1.6.8. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)-N-(pyri-

din-2-yl)hydrazinecarbo thioamide (61). Compound (61)

was synthesized as per the general Method D from the inter-

mediates (53) (1.0 g, 4.46 mmol) and (38) (0.82 g, 4.93 mmol)

as a white solid. (Yield: 83%); m.p. 198–201 ^ꢀC; IR (KBr, cm^ꢃ1): 5.1.6.12.

(E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)-N-

3228, 3041, 2968, 1602, 1236, 1144; ¹H NMR (CDCl₃): d 9.91 (pyrazin-2-yl)hydrazinecarbo thioamide (65). Compound

(s, 1H, -NHCSNH), 9.35 (s, 1H, -NHCSNH), 8.95 (d, J ¼ 8.0 Hz, (65) was synthesized as per the general Method D from the

1H, ArH), 8.44–8.37 (m, 2H, ArH), 8.15 (d, J ¼ 7.5 Hz, 1H, ArH), intermediates (53) (1.0 g, 4.46 mmol) and (42) (0.83 g,

8.02 (s, 1H, -CHN), 7.92–7.90 (dd, J ¼ 8.5 Hz, 1.5 Hz, 1H, ArH), 4.93 mmol) as a white solid. (Yield: 81%); m.p. 88–90 ^ꢀC; IR

7.78–7.75 (m, 1H, ArH), 7.52–7.49 (m, 1H, ArH), 7.44–7.40 (m, (KBr, cm^ꢃ1): 3054, 2971, 1586, 1233, 1143; H NMR (CDCl₃): d

1

2H, ArH), 7.30–7.25 (m, 1H, ArH), 7.13–7.10 (m, 1H, ArH), 10.08 (s, 1H, -NHCSNH), 8.59 (s, 1H, -NHCSNH), 8.15–8.13 (m,

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

19

1H, -CHN, 1H, ArH), 8.01–7.99 (m, 1H, ArH), 7.54–7.51 (m, 2H, J ¼ 7.5 Hz, 3H, -NCH₂CH₃); MS (m/z): 390.0 [M]^þ,

ArH), 7.47–7.44 (m, 4H, ArH), 7.33–7.30 (m, 2H, ArH), 392.0 [M þ 2]^þ.

4.40–4.36 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.45 (t, J ¼ 7.5 Hz, 3H,

-NCH₂CH₃); MS (m/z): 374.0 [M]^þ.

5.1.6.17. (E)-N⁰-((9-Ethyl-9H-carbazol-3-yl)methylene)ben-

zohydrazide (70). Compound (70) was synthesized as per

5.1.6.13. (E)-N-(4-Chlorophenyl)-2-((9-ethyl-9H-carbazol-3- the general Method D from the intermediates (53) (1.0 g,

yl)methylene)hydrazine carboxamide (66). Compound (66) 4.46 mmol) and (47) (0.67 g, 4.93 mmol) as a white solid.

was synthesized as per the general Method D from the inter- (Yield: 77%); m.p. 229–231 ^ꢀC; IR (KBr, cm^ꢃ1): 3177, 3015,

mediates (53) (1.0 g, 4.46 mmol) and (43) (0.91 g, 4.93 mmol) 2978, 1639, 1599, 1143; ¹H NMR (CDCl₃): d 9.13 (s, 1H,

as a white solid. (Yield: 75%); m.p. 251–253 ^ꢀC; IR (KBr, cm^ꢃ1): -NHCO), 8.51 (s, 1H, ArH), 8.41 (s, 1H, -CHN), 8.11 (d,

3322, 3061, 2973, 1672, 1593, 1133, 742; ¹H NMR (CDCl₃): d J ¼ 8.0 Hz, 1H, ArH), 7.94 (d, J ¼ 8.5 Hz, 1H, ArH), 7.88 (d,

8.32 (s, 1H, -NHCONH), 8.20 (s, 1H, -NHCONH), 8.14 (d, J ¼ 7.5 Hz, 1H, ArH), 7.57–7.54 (m, 1H, ArH), 7.51–7.47 (m, 4H,

J ¼ 7.5 Hz, 1H, ArH), 7.91 (s, 1H, -CHN), 7.90 (d, 1H, ArH), ArH), 7.42–7.40 (m, 2H, ArH), 7.27–7.25 (m, 1H, ArH),

7.83–7.81 (m, 1H, ArH), 7.56–7.50 (m, 3H, ArH), 7.44 (d, 2H, 4.39–4.35 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.44 (t, J ¼ 7.5 Hz, 3H,

ArH), 7.32–7.25 (m, 3H, ArH), 4.42–4.38 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃); ¹³C NMR (DMSO-d₆): d 162.8, 148.9, 140.5, 139.9,

-NCH₂CH₃), 1.46 (t, J ¼ 7.5 Hz, 3H, -NCH₂CH₃); ¹³C NMR 133.6, 131.4, 128.3, 127.4, 126.1, 125.2, 124.5, 122.2, 122.0,

(DMSO-d₆): d 153.0, 142.2, 140.2, 139.9, 138.2, 128.1, 126.0, 120.5, 120.0, 119.2, 109.4, 109.3, 37.01, 13.6; MS (m/z):

125.7, 125.2, 124.8, 122.2, 122.1, 121.1, 120.5, 119.7, 119.1, 342.0 [M þ 1]^þ.

109.3, 109.1, 37.01, 13.6; MS (m/z): 391.0 [M]^þ,

393.0 [M þ 2]^þ.

5.1.6.18. (E)-N⁰-((9-Ethyl-9H-carbazol-3-yl)methylene)-4-flu-

orobenzohydrazide (71). Compound (71) was synthesized

5.1.6.14.

(E)-N⁰-((9-Ethyl-9H-carbazol-3-yl)methylene)-2- as per the general Method D from the intermediates (53)

phenylacetohydrazide (67). Compound (67) was synthe- (1.0 g, 4.46 mmol) and (48) (0.75 g, 4.93 mmol) as a white

sized as per the general Method D from the intermediates solid. (Yield: 82%); m.p. 232–235 ^ꢀC; IR (KBr, cm^ꢃ1): 3186,

1

(53) (1.0 g, 4.46 mmol) and (44) (0.73 g, 4.93 mmol) as a white 3016, 2978, 1644, 1599, 1150; H NMR (CDCl₃): d 9.06 (s, 1H,

solid. (Yield: 80%). m.p. 237–239 ^ꢀC; IR (KBr, cm^ꢃ1): 3170, -NHCO), 8.50 (s, 1H, ArH), 8.41 (s, 1H, -CHN), 8.12 (d,

1

3021, 2931, 1651, 1597, 1149; H NMR (CDCl₃): d 8.67 (s, 1H, J ¼ 7.0 Hz, 1H, ArH), 7.91 (s, 2H, ArH), 7.51–7.48 (m, 1H, ArH),

-NHCO), 8.31 (d, J ¼ 1.5 Hz, 1H, ArH), 8.13 (d, J ¼ 7.5 Hz, 1H, 7.43–7.40 (m, 2H, ArH), 7.28–7.24 (m, 3H, ArH), 7.19–7.16 (m,

ArH), 7.88 (s, 1H, -CHN), 7.87–7.85 (m, 1H, ArH), 7.52–7.41 (m, 1H, ArH), 4.40–4.35 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 1.44 (t,

5H, ArH), 7.37–7.25 (m, 4H, ArH), 4.42–4.37 (q, J ¼ 7.5 Hz, 2H, J ¼ 7.5 Hz, 3H, -NCH₂CH₃); MS (m/z): 360.0 [M þ 1]^þ.

-NCH₂CH₃), 4.16 (s, 2H, -COCH₂), 1.46 (t, J ¼ 7.5 Hz, 3H,

-NCH₂CH₃); MS (m/z): 356.0 [M þ 1]^þ.

5.1.6.19.

(E)-N⁰-((9-Ethyl-9H-carbazol-3-yl)methylene)-4-

methylbenzohydrazide (72). Compound (72) was synthe-

5.1.6.15. (E)-N⁰-((9-Ethyl-9H-carbazol-3-yl)methylene)-2-(4- sized as per the general Method D from the intermediates

methoxyphenyl)aceto hydrazide (68). Compound (68) was (53) (1.0 g, 4.46 mmol) and (49) (0.73 g, 4.93 mmol) as a white

synthesized as per the general Method D from the intermedi- solid. (Yield: 85%); m.p. 252–254 ^ꢀC; IR (KBr, cm^ꢃ1): 3181,

1

ates (53) (1.0 g, 4.46 mmol) and (45) (0.88 g, 4.93 mmol) as a 3030, 2979, 1640, 1554, 1145; H NMR (CDCl₃): d 9.04 (s, 1H,

white solid. (Yield: 79%); m.p. 214–217 ^ꢀC; IR (KBr, cm^ꢃ1): -NHCO), 8.51 (s, 1H, ArH), 8.40 (s, 1H, -CHN), 8.12 (d,

1

3170, 3017, 2970, 1645, 1598, 1263, 1151; H NMR (CDCl₃): d J ¼ 8.0 Hz 1H, ArH), 7.93 (d, J ¼ 9.0 Hz 1H, ArH), 7.78 (d,

8.80 (s, 1H, -NHCO), 8.32 (d, J ¼ 1.5 Hz, 1H, ArH), 8.13 (d, J ¼ 7.0 Hz, 1H, ArH), 7.50–7.47 (m, 1H, ArH), 7.43–7.40 (m, 3H,

J ¼ 7.5 Hz, 1H, ArH), 7.89 (s, 1H, -CHN), 7.88–7.86 (dd, ArH), 7.30–7.25 (m, 3H, ArH), 4.40-4.36 (q, J ¼ 7.0 Hz, 2H,

J ¼ 1.5 Hz, 8.5 Hz, 1H, ArH), 7.52–7.49 (m, 1H, ArH), 7.43 (d, -NCH₂CH₃), 2.43 (s, 3H, ArCH₃), 1.45 (t, J ¼ 7.0 Hz, 3H,

J ¼ 8 Hz, 2H, ArH), 7.35 (d, J ¼ 8.5 Hz, 2H, ArH), 7.29–7.25 (m, -NCH₂CH₃); ¹³C NMR (DMSO-d₆): d 162.6, 148.6, 141.4, 140.5,

3H, ArH), 4.42–4.37 (q, J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 4.10 (s, 2H, 139.9, 130.7, 128.8, 127.4, 126.1, 125.2, 124.4, 122.2, 122.0,

-COCH₂), 3.77 (s, 3H, -OCH₃), 1.46 (t, J ¼ 7.5 Hz, 3H, 120.5, 119.9, 119.2, 109.7, 109.4, 109.3, 37.05, 20.9, 13.6; MS

-NCH₂CH₃); MS (m/z): 386.0 [M þ 1]^þ.

(m/z): 356.0 [M þ 1]^þ.

5.1.6.16. (E)-N⁰-((9-Ethyl-9H-carbazol-3-yl)methylene)-2-(4- 5.1.6.20. (E)-N⁰-((9-Ethyl-9H-carbazol-3-yl)methylene)picoli-

chlorophenyl)aceto hydrazide (69). Compound (69) was noylhydrazide (73). Compound (73) was synthesized as per

synthesized as per the general Method D from the intermedi- the general Method D from the intermediates (53) (1.0 g,

ates (53) (1.0 g, 4.46 mmol) and (46) (0.90 g, 4.93 mmol) as a 4.46 mmol) and (50) (0.74 g, 4.93 mmol) as a white solid.

white solid. (Yield: 83%); m.p. 239–241 ^ꢀC; IR (KBr, cm^ꢃ1): (Yield: 76%); m.p. 212–216 ^ꢀC; IR (KBr, cm^ꢃ1): 3178, 3009,

3311, 3106, 2967, 1672, 1599, 1129; ¹H NMR (CDCl₃): d 8.64 2978, 1637, 1597, 1148; ¹H NMR (CDCl₃): d 9.90 (s, 1H,

(s, 1H, -NHCO), 8.31 (d, J ¼ 1.5 Hz, 1H, ArH), 8.12 (d, J ¼ 7.5 Hz, -NHCO), 8.77–8.73 (m, 1H, ArH), 8.42 (s, 1H, -CHN), 8.26–8.23

1H, ArH), 7.89 (s, 1H, -CHN), 7.86–7.84 (dd, J ¼ 8.5 Hz, 1.5 Hz, (m, 1H, ArH), 8.08–8.03 (m, 2H, ArH), 7.90–7.88 (m, 1H, ArH),

1H, ArH), 7.52–7.49 (m, 1H, ArH), 7.45–7.43 (m, 2H, ArH), 7.36 7.47–7.32 (m, 5H, ArH), 7.25–7.20 (m, 1H, ArH), 4.36–4.28 (m,

(d, J ¼ 8.5 Hz, 2H, ArH), 7.31–7.25 (m, 3H, ArH), 4.42–4.38 (q, 2H, -NCH₂CH₃), 1.42–1.37 (m, 3H, -NCH₂CH₃); MS (m/z):

J ¼ 7.5 Hz, 2H, -NCH₂CH₃), 4.13 (s, 2H, -COCH₂), 1.46 (t, 343.0 [M þ 1]^þ.

20

K. B. PATEL ET AL.

structures of 61 and 62 were built with the Build module

within Maestro, and conformational search was carried out

using OPLS force field at physiological pH with the LigPrep

5.2. Biology

5.2.1. In vitro ChE inhibition studies

The potential of the test compounds to inhibit ChEs was

assessed using Ellman’s method as reported in our earlier

publications (Kanhed, Patel, Patel, et al., 2020; Patel, Patel,

Kanhed, Teli, Patel, Gandhi, et al., 2020). Tacrine and donepe-

zil were used as the standard drugs for the study. The

absorbance values at different concentrations of the test

compounds and the standard drugs were used to calculate

the IC₅₀values of the respective compounds.

€

module of Schrodinger. Docking studies were conducted in

extra-precision (XP) mode where extensive conformational

search is carried out to identify the suitable binding con-

formation which can bind within the receptor active site.

5.3.2. Molecular dynamics simulation

The MD analyses were carried out for 50 ns time duration by

using GROMACS 2020.1 tool (GROMACS 2020). MD protocol

was followed as reported previously by our group (Kanhed,

Patel, Patel, et al., 2020).

5.2.2. DPPH radical scavenging activity

The potential of the test compounds to scavenge the DPPH

radical was evaluated by DPPH spectrophotometric assay as

described in our previous reports (Patel et al., 2019, Patel,

5.3.3. In silico prediction of ADMET properties

Patel, Kanhed, Teli, Patel, Gandhi, et al., 2020). The quantifica- Prediction of in silico ADMET properties was performed using

tion of free radical scavenging activity of the test compounds the QikProp module of Schrodinger Suite and pkCSM web

was done in the form of reduction percentage (RP) of DPPH tool. The ligand structures built for the docking studies were

calculated by the equation RP ¼ 100[(A₀ꢃ A_C)/A₀], where A₀used for the prediction of the different ADMET descriptors.

is the absorbance of DPPH without sample and A_Cis the The significant descriptors examined are presented in

absorbance after adding sample with concentration C. Here, this report.

ascorbic acid was taken as a positive control (Kedare &

Singh, 2011).

5.3.4. Prediction of site of metabolism by SMARTCyp

SMARTCyp, a SOM predicting software released by Rydberg

5.2.3. Metal chelation study

The tendency of the selected test compounds to form che- CYP450 with precalculated density functional theory (DFT)

lates with the biometals was evaluated using UV spectropho- activation energies (Rydberg, Gloriam,

Olsen, 2010;

et al. contains a database of compounds metabolized by

&

tometry as detailed in our earlier report (Patel, Patel, Kanhed, Rydberg, Gloriam, Zaretzki et al., 2010). To rank the SOM,

Teli, Patel, Joshi, et al., 2020). The UV absorption spectra of SMARTCyp uses database having SMiles ARbitrary Target

the test compounds (61 and 62) and 8-hydroxyquinoline Specification-based fragments in combination with an acces-

(reference compound) were recorded alone and in the pres- sibility descriptor. The test ligands were uploaded as SMILES

ence of CuSO₄, FeCl₃, FeSO₄, ZnCl₂, AlCl₃, CaCl₂or MgSO₄strings format representing the molecule. The obtained

with wavelengths ranging from 200 to 600 nm after incubat- results included the molecular structure and an atom ranking

ing the solutions for 30 min. in methanol at room tempera- table for each ligand. Three top-ranked atoms were high-

ture. Final concentration of the test compounds and the lighted in the structure as well as in the table, as shown in

reference compound was 25 mM.

Figure 5.

5.3. Computational studies

Disclosures statement

5.3.1. Docking studies of compounds (61 and 62) The authors declare that there are no conflicts of interest.

with ChEs

Docking studies were performed using Glide module of

Funding

€

Schrodinger Suite (Schrodinger 2009, LLC, New York, NY).

Kishan B. Patel is thankful to the AICTE, New Delhi for awarding PG

Scholarship (GPAT) [ID-1-3526872437]. Dushyant V. Patel greatly

acknowledges the DST-INSPIRE, New Delhi for awarding Research

Fellowship (IF-150660).

The protein structures of AChE (PDB code: 2CKM, 1B41) and

BuChE (PDB code: 4BDS) were obtained from the RCSB

Protein Data Bank and checked for structural correctness

with Protein Preparation Wizard of Schrodinger using OPLS

force field. The grids were generated on the active sites of

the respective protein structures. The generated grids were

validated by redocking of the knocked out co-crystallised

Author contributions

ligands into the active sites of the grids. The redocked M.R.Y. conceptualized the whole study. K.B.P., D.V.P. and

ligands showed similar interactions as shown by the original N.R.P. carried out synthesis and data collection. A.M.K.

co-crystallized ligands. The grids were validated by compar- planned and executed the computational studies. B.G. and

ing the RMSD values of the redocked ligands with the co- B.S.S. assisted in synthesis and data collection. K.V.P.

crystallized ligands present in 2CKM and 4BDS and the val- designed the biological studies. B.N.C. and K.B.P. performed

ues obtained were 0.40 and 0.26 A, respectively. The 3D biological studies and data collection. N.K.P. and D.M.T.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS

21

activity. Journal of Neurochemistry, 100(5), 1421–1429. https://doi.org/

10.1111/j.1471-4159.2006.04347.x

He, Z., Qiao, H., Yang, F., Zhou, W., Gong, Y., Zhang, X., Wang, H., Zhao,

B., Ma, L., Liu, H. m., & Zhao, W. (2019). Novel thiosemicarbazone

derivatives containing indole fragment as potent and selective anti-

cancer agent. European Journal of Medicinal Chemistry, 184, 112–349.

helped in the synthesis and data interpretation. K.B.P. and

D.V.P. drafted the manuscript. All authors reviewed and

approved the final version of the manuscript.

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Article Doi

Carbazole-based semicarbazones and hydrazones as multifunctional anti-Alzheimer agents

DOI: 10.1080/07391102.2021.1942212

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