Synthesis, biological activity and structure activity relationship studies of novel conazole analogues via conventional, microwave and ultrasound mediated techniques

DOI: 10.1016/j.bioorg.2018.07.036

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Bioorganic Chemistry p. 55 - 70 (2018)

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Authors:

Mermer, Arif

Demirbas, Neslihan

Demirbas, Ahmet

Colak, Nesrin

Ayaz, Faik Ahmet

Alagumuthu, Manikandan

Arumugam, Sivakumar

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Article abstract of DOI:10.1016/j.bioorg.2018.07.036

1,2,4-Triazole derivatives containing a piperazine nucleus (4a-d and 10) were prepared starting from 1-(2-methoxyphenyl)piperazine or ethyl 4-(4-amino-2-fluorophenyl)piperazine-1-carboxylate via several steps. The synthesis of fifteen compounds (7a-l and 13a-c), which can be considered as new analogues of azole class antifungals was performed starting from 1,2,4-triazoles (4a-d and 10) via three steps containing the condensation with 2-bromo-1-(4-chlorophenyl)ethanone, reduction of carbonyl group to alcohol and alkylation of OH group, respectively. All the reactions were examined under conventional, ultrasound and microwave irradiation conditions as green chemistry techniques, and optimum conditions were defined. The newly synthesized compounds were screened for their biological potentials including antimicrobial, antioxidant, antiurease and anti α-glucosidase activities and promising results were obtained. The enzyme inhibitory potentials of these compounds were further validated through molecular docking.

Full text of DOI:10.1016/j.bioorg.2018.07.036

Bioorganic Chemistry 81 (2018) 55–70

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Synthesis, biological activity and structure activity relationship studies of

novel conazole analogues via conventional, microwave and ultrasound

mediated techniques

a,⁎

Arif Mermer , Neslihan Demirbas , Ahmet Demirbas , Nesrin Colak , Faik Ahmet Ayaz ,

Manikandan Alagumuthu , Sivakumar Arumugam

Department of Chemistry, Karadeniz Technical University, Trabzon, Turkey

Department of Biology, Karadeniz Technical University, Trabzon, Turkey

Department of Biotechnology, School of Bio-Sciences and Technology, VIT, Vellore 632014, India

A R T I C L E I N F O

A B S T R A C T

Keywords:

α-Glucosidase activity

Antimicrobial

Antioxidants

Conazole

Microwave irradiation

Molecular docking

Ultrasound sonication

Urease activity

1,2,4-Triazole derivatives containing a piperazine nucleus (4a-d and 10) were prepared starting from 1-(2-

methoxyphenyl)piperazine or ethyl 4-(4-amino-2-ﬂuorophenyl)piperazine-1-carboxylate via several steps. The

synthesis of ﬁfteen compounds (7a-l and 13a-c), which can be considered as new analogues of azole class an-

tifungals was performed starting from 1,2,4-triazoles (4a-d and 10) via three steps containing the condensation

with 2-bromo-1-(4-chlorophenyl)ethanone, reduction of carbonyl group to alcohol and alkylation of OH group,

respectively. All the reactions were examined under conventional, ultrasound and microwave irradiation con-

ditions as green chemistry techniques, and optimum conditions were deﬁned. The newly synthesized compounds

were screened for their biological potentials including antimicrobial, antioxidant, antiurease and anti α-gluco-

sidase activities and promising results were obtained. The enzyme inhibitory potentials of these compounds were

further validated through molecular docking.

1. Introduction

agents (Fig. 1), such as Econazole, Miconazole, Itraconazole, Vor-

iconazole, and Ketoconazole have launched (see Fig. 1).

At present, although humans have been accustomed to ﬁght with

But as in all antimicrobial therapies, the overuse of azole class an-

tifungals has resulted in the development of severe resistance, which

signiﬁcantly reduced their eﬃcacy [7–13]. This has motivated the re-

searchers to design and synthesis of novel and eﬃcient drug candidates

with diﬀerent mode of action, more bioactivity and lowest side eﬀects

[14]. In the medicinal chemistry ﬁeld, homology modeling and phar-

macophore modeling techniques have been intensively used for design

and development of new antimicrobial drug candidates [15]. According

to these models depicted in Fig. 2 schematically, the fundamental azole

drugs should have at least three basic pharmacophore groups: (1)

imidazole or triazole unit (triazoles in recent years) for binding with

CYP51 active side through coordination to hemeiron; (2) a hydro-

phobic, preferably aromatic group near the azole ring; (3) a second

aromatic (preferably halogenated) region to increase the lipophilicity of

the molecule, because it is well known that the lipophilic character of a

bioactive molecule facilitates the penetration into the cell. Moreover,

certain active compounds contain an additional hydrophobic sub-

stituent, and the presence of an eOe atom in the structure is necessary

the invasion of pathogens with both inherent defenses and medical

protections, many pathogenic bacteria have been resistant to most of

synthetic or natural antibiotics caused by the excessive and prolonged

use of them, and this made microbial infections one of the foremost

health crises worldwide. Literature survey reveals that bacterial infec-

tions cause to hundreds of thousands of deaths annually and billions of

dollars in healthcare expenses emphasizing the urgent need to constant

push to discover and improve strategies to counter these threats [1–5].

Similarly, Although various antibacterial and antifungal drugs with

diﬀerent structures and mode of action have been marketed, their

clinical implementation have been limited by drug resistance, high risk

of toxicity, insuﬃciencies in their eﬃcacy and side eﬀects [6–8]. One of

the most common classes of antifungal agents, azoles play a leading role

for the treatment of invasive fungal infections, and act by inhibit the

synthesis of ergosterol, the bulk sterol in fungal membranes, by binding

to the heme cofactor located in the active site of the cytochrome P450

4α-demethylase (CYP51). Several of these novel azole antifungal

⁎

Corresponding author.

E-mail address: neslihan@ktu.edu.tr (N. Demirbas).

https://doi.org/10.1016/j.bioorg.2018.07.036

Received 15 June 2018; Received in revised form 21 July 2018; Accepted 31 July 2018

Available online 01 August 2018

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Fig. 1. Some known azole class antifungal drugs.

Fig. 2. General representation of the target compounds.

for interaction with target enzymes via the formation of H-bond

15,16].

In recent years, the application of microwave (MW) techniques for

monosaccharide absorption [28–31].

[

In light of these considerations, as the continuation of our ongoing

eﬀorts, we reported here the synthesis and biological activity screening

studies of novel analogues of azole class antifungals by conventional

method and green chemistry techniques.

organic synthesis has attracted considerable interest due to some su-

perior properties including the low reaction times, improved yields,

simpliﬁed work-up and designing energy-saving protocols [17]. More-

over, with the development of ‘green chemistry’, the focus has now

shifted to less cumbersome solvent-free methods, undergoing facile

reactions to provide high yields of pure products, thus eliminating or

minimizing the use of organic solvents [18–22]. Ultrasound mediated

procedures constitute another powerful technique used to accelerate

organic reactions. The noteworthy properties of sonication, which has

been accepted as an important technique for green chemistry proce-

dure, are formation of pure products in high yields, easier work up and

enhanced reaction rates [23–26].

Urease, a nickel containing binuclear enzyme, is a virulent factor for

human and animal infections containing many urinary and gastro-

intestinal tracts, hepatic diseaonomic loesses and gastric cancer [27]. α-

Glucosidase (EC3.2.1.20) is a crucial enzyme found in the brush border

surface of cells in the small intestine. During the digestion of food this

enzyme hydrolyzes carbohydrates and produces α-D-glucose, which is

absorbed in the blood stream, raises postprandial blood glucose levels

and brings about diabetes. Thus, for the control and prevention of

diabetes, a-glucosidase inhibitors are of particular interest as they can

help to reduce the carbohydrate digestion and subsequent

2. Result and discussion

2.1. Chemistry

In the present study, traditional and ecofriendly synthesis, anti-

microbial, enzyme inhibition and antioxidant activity screening studies

of novel analogues of azole class antifungals were intended (Table 1).

Synthetic routes leading to the formation of the targeted compounds

was presented in Schemes 1–3 and all compounds were presented

Table 2. Compounds 2, 3, 4, 8, 9 and 10 were synthesized following the

procedure reported by us earlier [27]. The synthesis of novel analogues

of azole class antifungals (7a-l and 13a-d) was carried out starting from

the corresponding 3H-1,2,4-triazoles (4a-d, 10) containing a piperazine

nucleus, via three steps. The alkylation of compounds 4a–d and 10 with

2-bromo-1-(4-chlorophenyl) ethanone in ethanol yielded compounds

5a–d. The comparison with a conventional heating showed that MW

irradiation and sonication decreased the reaction time from 27 h to

10 min and increased the yields from 76–90% to 87–94% (Table 2).

It is well known that mercapto- or oxo-triazoles can exist in (thi)

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

oxo-mercapto(hydroxy) tautomeric forms [32–35], due to this reason,

compounds 4a–d and 10 can, in principle, be alkylated at the sulfur or

nitrogen atom. Literature survey revealed that the SH proton at mer-

sonication techniques. The synthesis of compounds 7a–l was achieved

by treatment of compounds 6a–d with substituted benzyl chlorides,

namely 4-chloro-, 2,4-dichloro- and 2,6-dichlorobenzyl chlorides in the

presence of NaH under US and MW-irradiated procedures beside con-

ventional conditions. In order to improve the MW and ultrasound

conditions, the reaction leading to the formation of 7a was selected as a

model reaction (Table 3). After optimization of the conditions, the

synthesis of the remaining compounds 7b–l was carried out.

capto triazoles resonances at about 13–14 ppm in the H NMR spectra,

whereas NH proton of oxo-triazoles seen at about 9–12 ppm. Moreover,

in the FT IR spectra of oxo-triazoles, the absorption band of carbonyl

function appears as a sharp signal [32–35].

In the present study, the spectral data of compounds 4a-d and 10

compatible with the literature ﬁndings show that compounds 4a-c and

The formation of compounds 5a-d and 11a-b was conﬁrmed by

recording their FT-IR, ¹HNMR, CNMR, EI-MS spectra and elemental

10 present in mercapto form, while 4d is in keto form, predominantly.

−

As a result of this, compounds 4a-c and 10 were alkylated at the sulfur

atom, whereas 4d at nitrogen atom. The reduction of carbonyl group of

analysis results. A sharp peak at 1677–1721 cm in the FT IR spectra

conﬁrmed the presence of carbonyl group. The most prominent peak at

192.66–195.13 ppm in the ¹³C NMR spectra of compounds 5a-d and

11a-d conﬁrmed the presence of carbonyl carbon. Compounds 5a-d and

5a–d to alcohol generated compounds 6a–d. This conversion was

achieved using conventional heating, MW irradiation and ultrasound

Table 1

Molecular formula of all compounds.

(

continued on next page)

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Table 1 (continued)

(

continued on next page)

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Table 1 (continued)

11a-b were further reacted with NaBH

aﬀording the corresponding

2.2. Biological activity

reduction products (6a-d and 12a-b). The most important peak at

−

121–3319 cm

in FT-IR spectra of compounds 6a-d and 12a-b con-

2.2.1. Antimicrobial activity

ﬁrmed the presence of hydroxyl group. In C NMR spectra of com-

pounds 6a-d and 12a-d, the signal between 67.59 and 70.73 ppm was

attributed to CH carbon. In the H NMR spectra, the appearances of

eOH signals and eCH protons of these compounds between 5.71 and

.16 ppm and 4.85–5.90 ppm, respectively, supported the formation of

compounds 6a-d and 12a-b. The synthesis of compounds 7a-l and 13a-

d was performed by the reaction of compounds 6a-d with substituted

benzyl chlorides, namely 4-chloro-, 2,4-dichloro- and 2,6-di-

chlorobenzyl chlorides in the presence of NaH. In the FT IR spectra, the

most important evidence for the formation of compounds 7a-l and 13a-

All the newly synthesized compounds were screened for their anti-

microbial activity and the results obtained were presented in Table 4. It

is interesting to note that the newly synthesized compounds did not

exhibit activity on the test fungy, Candida albicans (Ca) and Sacchar-

omyces cerevisiae (Sc), except 7i, although they might be the analogues

of azole class antifungals; instead, most of the prepared compounds

could eﬀectively inhibit the growth of all the tested bacteria in vitro. As

shown in Table 4, compounds 5a-d and 6a-d displayed varying degrees

of inhibitory eﬀects on the growth of the selected Gram (+), Gram (−)

bacteria and micobacteria Mycobacterium smegmatis (Ms). In fact, the

inhibition properties of some of these compounds were better than

standard drugs ampicillin and streptomycin. The alkylation of eOH

group in compounds 6a-d leading to the formation of compounds 7a-l

caused to loss of activity on Ec, Yp and Ms. On the other hand, these

compounds (7a-l) displayed pretty good activity towards the remaining

test bacteria. Similarly, compounds 11a, 11b, 12a, 12b and 13a-d

d was the disappearance of the signal due to hydroxyl group. The ap-

pearance of CH

signal at 4.70–5.23 ppm in the H NMR spectra sup-

ported the formation of conazole derivatives. Furthermore, the mass

spectral data and elemental analysis results are in accordance with their

structures.

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

2 6 4 2 4

Scheme 1. Reaction and Conditions: i: suitable alkylisothiocyanate; ii: NaOH, EtOH; iii: NaOEt, EtOH, (2,4)-Cl C H COCH Br; iv: NaBH , EtOH; v: NaH, the cor-

responding halogenated benzyl chloride.

displayed good-moderate activity against the test bacteria with the MIC

values ranging from 3.9 to 62.5 μg/mL, except Ec, and Yp.

0.16 ± 0.02 IC50 value. The remaining compounds displayed not good

or no inhibitory eﬀect (Table 6).

Table 5 shows antioxidant capacity µM TE/g) values of 25 synthe-

sized novel compounds measured by DPPH, FRAP and CUPRAC assays.

The average highest AC values were determined for compounds 10

.2.3. α-Glucosidase inhibitory activity

All compounds were evaluated with regard to α-glucosidase in-

(

442.71 µM) followed by 11a (121.57 µM) and 9 (64.63 µM) or 8 for

DPPH, (6430.28 µM) followed by 10 (5092.85 µM) and 13a

3916.90 µM) for FRAP, 10 (1149.87 µM) followed by 11a (333.63 µM)

and 13a (321.08 µM) for CUPRAC (Table 5). Whereas the compounds 7 h

0.24 µM), 7e (939.68 µM) and 7c (49.80 µM) had the lowest AC values

hibition. Compounds 7a, 7c, 7d, 7f, 7h, 7i, 7k, 12a and 13c which may

be regarded as new analoques of triazole and conazole class antifungals,

exhibited good inhibitory eﬀect. 12a showed the best α-glucosidase

inhibitory activity compared to standard drug Acarbose (Table 6). 11a,

(

13a, 13b and 13d showed almost the same activity with acarbose. The

amongst (Table 5). No AC values were determined for the compounds 7e

for DPPH and 6a and 6d for CUPRAC assays, respectively.

other compounds displayed very week or no inhibition eﬀect.

2.2.4. Results of molecular docking studies

2.2.2. Urease inhibitory activity

Table 7 depicts the obtained free energy values from the docking

The newly prepared compounds were assayed for their in vitro ur-

studies. According to the results, maximum of the docked compounds

showed a fortunate molecular mechanistic values over α-Glucosidase

(PDB: 3CXN) than Urease (PDB: 3WY1). The obtained inhibitory con-

stant (ki) was used to determine the desired binding aﬃnity of the

ease inhibitory activity against Jack bean urease. The compounds 7a,

c, 11a, 11b, 12b showed promising antiurease activity. Among these

compounds, 12b showed the best inhibition eﬀect with the

Scheme 2. i: NaOEt, EtOH, (2,4)-Cl

2 6

H COCH

Br; ii: NaBH

, EtOH; iii: NaH, the corresponding halogenated benzyl chloride.

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Scheme 3. i: Phenylisothiocyanate; ii: NaOH, EtOH; iii: NaOEt, EtOH, (2,4-) Cl

6 4

COCH

Br; iv: NaBH

, EtOH; v: NaH, the corresponding halogenated benzyl

chloride.

present study compounds. In the initial assessments, we summated and

averaged the originally obtained individual molecular mechanistic va-

lues such as least binding energy, ligand eﬃciency and inhibitory

constant (ki). The average and sum of all docking molecular mechanics

values have given a clearcut idea about the activity selectivity of

compounds towards the α-Glucosidase (PDB: 3CXN) inhibition with an

average least binding energy value of −10.34 kcal/mol. Meanwhile,

the average binding energy value of Urease (PDB: 3WY1) was also

found as signiﬁcant (−9.63 kcal/mol). The other category, ligand ef-

ﬁciency, was remarkable (−0.30) against α-Glucosidase (PDB: 3CXN).

The next category, average inhibitory constant (ki), was as good as

168.62 nM against α-Glucosidase (PDB: 3CXN). This indicates the

possible bioactivity potentials of the compounds towards α-Glucosidase

(PDB: 3CXN). Individually, compound 6 was found as the best (average

binding energy −12.59 kcal/mol against both receptors) among all

compounds against both α-Glucosidase (PDB: 3CXN) and Urease (PDB:

3WY1). The overall consideration from molecular docking study is, all

these compounds can be actively inhibit both α-Glucosidase (PDB:

3CXN) and Urease (PDB: 3WY1) and also could establish a signiﬁcant

route of inhibition mechanism of these two enzymes.

Table 2

Time and yield data of compounds 5a-d, 6a-d, 7a-l and 11–13 using conventional. microwave irradiation and sonication techniques.

Comp.

Microwave Irradiation

Sonication

Conventional Method

Power (W)

Time (min)

Yield (%)

Power (kHz)

Time (min)

Yield (%)

Time (h)

Yield (%)

100

125

100

120

100

125

100

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Table 3

Table 5

Antioxidant capacity (AC) values of 25 synthesized novel compounds.

Optimization of the reaction conditions for compound 7a.

Entry

Solvent

Time (min)

Yield (%)

58^a

Entry

DPPH (µM TE/g)

FRAP (µM TE/g)

CUPRAC (µM TE/g)

270.77 ± 4.20^g

EtOH

THF

DCM

59.05 ± 0.07

57.24 ± 0.28

2184.02 ± 46.67

2184.43 ± 53.71

273.85 ± 8.61

277.88 ± 7.64

2734.35 ± 87.13^k

28.33 ± 0.35

122.09 ± 4.46^b

0.48 ± 0.01

1368.17 ± 44.37

1573.07 ± 30.45

1163.05 ± 48.67

2124.33 ± 50.30

15.69 ± 0.16ⁱ

n.d.^*

47.35 ± 0.38

218.67 ± 6.77

165.8 ± 5.99^c

n.d.

1.76 ± 0.06

46.05 ± 0.34

1044.78 ± 26.83

69.03 ± 5.21^a

2.40 ± 0.39

8.90 ± 0.92

1006.05 ± 80.28

1588.48 ± 22.33

1067.97 ± 60.38

162.03 ± 11.61

3.61 ±₀_.₃₇d

49.80 ±₆_.₇₄a

114.43 ± 2.73

208.25 ± 8.58

Conventional method.

Microwave method.

Ultrasound method.

n.d.

939.68 ± 53.45

48.90 ± 0.02ⁿ

1196.48 ± 33.70

1896.45 ± 60.38

1596.72 ± 35.77

1989.12 ± 58.97

6430.28 ± 121.85

2143.53 ± 58.89

103.85 ± 1.85^b

188.45 ± 10.20

231.83 ± 18.47

0.24 ± 0.01

44.36 ± 0.07^k

61.53 ± 0.05

63.48 ± 0.02

64.63 ± 0.05

.2.5. Molecular assessments of Receptor-Ligand interactions of α-

Glucosidase (PDB: 3CXN) than Urease (PDB: 3WY1)

The molecular interaction of α-Glucosidase (PDB: 3CXN) and Urease

PDB: 3WY1) with the proposed compounds was assessed to consider

318.52 ± 4.81

296.57 ± 3.42

1149.87 ± 50.98^k

442.71 ± 0.26

121.57 ± 0.13

5092.85 ± 30.54

2033.23 ± 22.72

2227.70 ± 48.16

(

333.63 ± 11.19

the drug eﬃcacy. From this, the most probable inhibition mechanism of

action of the present study compounds as well as the molecular inter-

action by means of hydrogen bonding and non-covalent bonding (π-π

interaction and π-cation interactions) was recognized. The best docked

pose were extracted down to have the interaction information of elec-

tron donor and acceptors of both ligands and receptors. Fig. 3 illustrates

the best docked pose of compound 6d against α-Glucosidase (PDB:

CXN) and 7l against Urease (PDB: 3WY1). While discussing about the

drug-target protein interactions, there are many external and internal

factors are involving in the interaction of them. These factor are

56.85 ± 0.23

63.46 ± 0.04

317.32 ± 1.32

3916.90 ± 64.22^l

321.08 ± 2.62^j

13a

13b

11.92 ± 0.52

1422.45 ± 58.61

1505.13 ± 13.76

1051.47 ± 95.78

277.88 ± 7.64

123.17 ± 2.71

8.19 ± 0.60

14.24 ± 0.14^h

121.2 ± 13.79^b

Values represent the mean ± SD of three determinations. An analysis of

variance (SPSS version 11.5. one-way ANOVA) was used for comparisons

among the means. Values with the same letter at superscript within a column

are not signiﬁcantly diﬀerent at P < 0.05.

* n.d.; not determined.

Table 4

Screening for the antimicrobial activity of compounds 5–13.

Entry

Microorganisms and Minimal Inhibition Concentration (μg/mL)

12.5

7.8

12.5

1.9

12.5

31.3

62.5

15.6

–

3.9

–

12.5

31.25

12.5

31.25

12.5

15.6

62.5

31.3

–

250

–

13.25

7.8

12.25

7.8

18.5

7.8

16.5

15.6

16.5

31.3

7.8

15.6

7.8

15.6

7.8

3.9

15.6

7.8

3.9

7.8

3.9

15.6

3.9

15.5

15.6

12.5

15.6

7.8

31.3

7.8

31.3

15.6

7.8

15.6

7.8

15.6

7.8

15.6

7.8

62.5

7.8

3.9

7.8

15.6

10.25

15.6

16.5

15.6

7.8

62.5

31.3

–

62.5

3.9

62.5

31.3

–

62.5

–

62.5

–

625

–

13.25

15.6

20.25

31.3

7.8

15.6

7.8

15.6

7.8

15.6

3.9

7.8

15.6

7.8

15.6

7.8

15.6

7.8

15.6

7.8

13.25

15.6

18.25

15.6

62.5

15.6

7.8

15.6

7.8

3.9

15.6

7.8

15.6

625

7.8

62.5

31.25

31.3

15.6

31.3

3.9

62.5

15.6

7.8

15.6

9.7

–

15.6

–

Amp.

Strep.

Flu.

9.7

–

7.8

–

Ec: Escherichia coli ATCC 35218. Yp: Yersinia pseudotuberculosis ATCC 911. Pa: Pseudomonas aeruginosa ATCC 10145. Sa: Staphylococcus aureus ATCC 25923. Ef:

Enterococcus faecalis ATCC 29212. Bc: Bacillus cereus 709 Roma. Ms: Mycobacterium smegmatis ATCC607. Sc: Saccharomyces cerevisiae RSKK 251. Ca: Candida albicans

ATCC 60193. Amp: ampicillin. Flu: ﬂuconazole. Strep.: streptomycin. –: no activity.

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Table 6

normally controlling the strength, duration, and diﬀerent types of the

drug-receptor interaction. Obviously, these factors are dictating the

strength of the interaction formed by a drug by constituting a complex

with its receptor that is the aﬃnity. The intermolecular forces between

drug and binding sites like Van der Waals forces (weak bonds and

transient reversible eﬀects), Hydrogen bonds (intermediate bonds and

transient reversible eﬀects), Covalent bonds (strong bonds and long-

lasting or irreversible eﬀects) and non-covalent bonding (involving in

the electromagnetic interactions among molecules).

In the interaction conﬁrmation, for instance, with α-Glucosidase, at

least one hydrogen bonding between Threonine, Phenylalanine and

Glycine by the present study compounds was anticipated. Because,

there is an unusual long β → α loop 4 exists in α-glucosidase which is

considered to be the responsible factor for the ﬁrm recognition of dis-

accharides that are owed to steric interference. Also, two amino acid

residues namely Thr203 and Phe297, (supported with Gly228), were

found to regulate the glycosidic linkage of the substrate at subsite +1.

As displayed in Fig. 3 here, there was a non-covalent interaction

Inhibitory activities of the synthesized compounds against Jack bean urease and

α-glucosidase.

α-Glucosidase inhibition activity

Urease inhibition activity

Comp. Code

IC50 (µM)

Comp. Code

IC50 (µM)

1280.45 ± 7.82

2584.56 ± 10.98

154.85 ± 5.61

1875.45 ± 7.11

234.46 ± 6.69

176.89 ± 8.63

88.74 ± 2.63

119.12 ± 4.12

122.88 ± 6.91

212.9 ± 4.21

316.75 ± 7.74

64.56 ± 1.46

25.48 ± 0.03

35.87 ± 0.05

0.24 ± 0.03

88.74 ± 0.08

0.28 ± 0.01

4.63 ± 0.02

96.75 ± 0.06

52.54 ± 0.09

48.75 ± 0.08

67.45 ± 0.04

(

golden cylindrical structure) between Phe397 (in the presence of

Phe297) and compound 6d against α-Glucosidase (PDB: 3CXN) was

found to have established. The expected α 1– > 4 linkage interruption,

perhaps, facilitated by this interaction. The spherical shaped structures

are indicating the hydrogen bond establishment due to the inter-ex-

change of electrons between the ligand and receptor. These H-bonds not

only ensuring the Ligand-Receptor interactions, but also the drug so-

lubility.

11a

11b

12a

12b

13a

13b

13c

13d

Thiourea

0.58 ± 0.02

0.49 ± 0.03

74.45 ± 0.03

0.16 ± 0.02

85.11 ± 0.05

441.98 ± 10.72

465.99 ± 6.36

107.8 ± 2.88

508.74 ± 5.63

400.74 ± 11.21

.3. Structure activity relationship (SAR)

Acarbose

0.12 ± 0.00

As preliminary, prior to develop these newly synthesized conazole

analogs as drugs, a SAR study was anlysed. Widely substituted com-

pounds were found with evenly poised electron donar and acceptors. To

Table 7

Molecular mechanistic values of molecular docking results.

Entry Binding energy

Ligand eﬃciency

3CXN

Inhibitory constant (ki) (nM)

CXN

3WY1

3CXN

3WY1

−12.68

−10.22

−10.70

−9.26

−8.09

−9.71

−10.53

−12.62

−9.15

−8.26

−10.48

−8.95

−9.04

−8.5

−10.44

−10.27

−9.54

−10.31

−9.94

−10.36

−7.52

−10.38

−10.41

−8.81

−9.1

−9.56

−9.02

−11.21

−9.84

−8.87

−10.25

−11.02

−12.67

−10.12

−9.24

−12.45

−9.94

−10.21

−9.28

−11.16

−11.08

−10.52

−12.63

−10.47

−12.84

−8.94

−11.49

−11.56

−9.12

−9.67

−9.74

−9.02

−10.25

−7.89

−0.34

−0.26

−0.31

−0.24

−0.21

−0.25

−0.31

−0.32

−0.20

−0.18

−0.22

−0.19

−0.18

−0.25

−0.24

−0.22

−0.24

−0.21

−0.22

−0.31

−0.33

−0.20

−0.22

80.21

−0.25

−0.24

−0.31

−0.26

−0.23

−0.28

−0.34

−0.46

−0.34

−0.24

−0.26

−0.28

−0.30

−0.25

−0.27

−0.32

−0.31

−0.42

−0.30

−0.48

−0.21

−0.36

−0.38

−0.28

−0.26

−0.27

−0.26

−0.32

−0.20

−8.63

−0.298

0.403

32.27

14.31

163.97

1170.0

76.29

97.95

884.16

24.86

124.56

918.21

38.49

8.49

0.608

26.48

178.94

0.744

42.15

30.84

68.79

6.65

19.07

565.14

196.07

879.88

20.94

273.55

237.38

590.91

22.29

29.79

101.0

27.8

51.37

25.38

3.05

24.44

23.35

347.08

214.94

203.66

483.81

116.74

1671.15

7586.033

261.5873

7.58

14.59

0.483

4.69

0.519

864.59

0.984

0.912

132.45

104.59

98.46

134.67

15.48

1058.12

4890.04

168.6221

−9.31

−8.62

−9.46

−6.52

−279.18

−9.6269

−0.21

−0.19

−0.13

−6.89

−0.23759

Sum

Average

−299.96

−10.3434

Note: 3CXN (PDB crystal structure of Urease); 3WY1 (PDB Crystal structure of alpha-glucosidase).

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Fig. 3. Interaction of compound 6d and 7l to α-Glucosidase (PDB: 3CXN) and Urease (PDB: 3WY1) respectively. The globular structures are indicating the binding

pockets of corresponding receptor proteins.

have a well-established bioactivity, the electronegativity and positivity,

obviously, plays an important role. In our observation, in the physical

structure of the conazole analoges, aromatic arene, benzene ring

bath, respectively. The Mass spectra were obtained on a Quattro LC-MS

(70 eV) Instrument.

(

phenyl), eCHN containing imine or iminyl, aldehyde containing al-

2.4.1. General method for the synthesis of compounds 5a-d and 11a,b

Method 1. The solution of compounds 4a-d and 10 (10 mmol) in

ethanol was stirred in the presence of sodium ethoxide at room tem-

perature for 15 h (the progress of the reaction was monitored by TLC).

Then, 2-bromo-1-(2,4-dichlorophenyl)ethanone (30 mmol) was added

into it and stirred for additional 10 h. After evaporating the solvent

under reduced pressure, a solid obtained. The crude product was re-

crystallized from an appropriate solvent to aﬀord the desired product.

Method 2. The solution of compounds 4a-dand 10 (1 mmol) and

sodium ethoxide (1 mmol) in ethanol (10 mL) was irradiated in closed

vessels at 90 °C, 100 W, for 1.5 h (for 5a-cand 11a,b) or 2 h (for 5d) (the

progress of the reaction was monitored by TLC). Then, 2-bromo-1-(2,4-

dichlorophenyl)etanone (3 mmol) was added into it and irradiated for

additional 1 h. The mixture was poured into ice-water and a solid ob-

tained. This crude product was collected by ﬁltration and recrystallized

from an appropriate solvent to aﬀord the desired product.

Method 3. The solution of compounds 4a-d and 10 (1 mmol) and

sodium ethoxide (1 mmol) in ethanol (10 mL) was sonicated for 4 min

(for 5a-c and 11a,b) or 6 min (for 5d) (the progress of the reaction was

monitored by TLC). Then, 2-bromo-1-(2,4-dichlorophenyl) etanone

(3 mmol) was added into it and sonicated for additional 6 min. The

mixture was poured into ice-water and a solid obtained. This crude

product was collected by ﬁltration and recrystallized from an appro-

priate solvent to aﬀord the desired product.

kanol/carbonyl are consistently available in all compounds. Mainly, it

was observed through the molecular docking studies, the aryl rings

were established to have pi-pi interactions with the amino acid residues

Phe397 and Tyr57. The eN available in the pyrrole ring was found as

the main electron donor to establish anticipated hydrogen bonds. As

illustrated in Fig. 3 (Top right, compound 6d), a bunch of hydrogen

bonds could be observed established with the association of the amino

acid residues Asn301, Lys225 and Val334. In fact, these compounds

were signiﬁcantly exhibited showed good bioactivity potentials against

all proposed activities in this study. While discussing the individual

drug eﬃcacy of present study compounds, we considered the ligand

eﬃciency of all individual compounds to deter mine the same. Ligand

eﬃciency could be considered to measure the binding energy per atom

of a drug candidate (ligand) to their corresponding pair partner (a re-

ceptor or an enzyme). Compounds 6d and 7l were found to have an

excellent ligand eﬃciency values as −0.46 and −0.48 respectively.

These ligand eﬃciency values are encouraging to do further drug va-

lidation studies for the compounds 6d and 7l.

.4. Experimental section

All the chemicals were purchased from FlukaChemie AG Buchs

Switzerland) and used without further puriﬁcation. Melting points of

(

the synthesized compounds were determined in open capillaries on a

Büchi B-540 melting point apparatus and are uncorrected. Reactions

were monitored by thin-layer chromatography (TLC) on silica gel 60

F254 aluminium sheets. The mobile phase was ethyl acetate: diethyl

ether (1:1), and detection was made using UV light. FT-IR spectra were

2.4.1.1. 1-(2,4-Dichlorophenyl)-2-[(5-{[4-(2-methoxyphenyl)piperazin-1-yl]

methyl}-4-phenyl -4H-1,2,4-triazol-3-yl)thio]ethanone (5a). Recrystallized

−1

from acetone:H

CH), 1696 (C = O), 1553 (C]N). H NMR (DMSO‑d

2CH ), 2.78 (s, 4H, 2CH ), 3.51 (s, 2H, CH ), 3.73 (s, 3H, OCH

2H, CH ), 6.81–6.92 (m, 4H, arH), 7.44–7.57 (m, 6H, arH), 7.82–7.84 (m,

2H, arH). C NMR (DMSO‑d

(CH ), 52.74 (2CH ), 55.76 (OCH

O (1:3). mp. 132–133 °C. FT-IR (υmax, cm ): 3065 (ar-

, δppm): 2.38 (s, 4H,

recorded using a Perkin Elmer 1600 series FTIR spectrometer. H NMR

), 4.73 (s,

and C NMR spectra were registered in DMSO‑d

on a BRUKER AVENE

II 400 MHz NMR Spectrometer (400.13 MHz for H and 100.62 MHz for

C). The chemical shifts are given in ppm relative to Me

, δppm): 42.42 (CH

2 2

), 50.36 (2CH ), 51.67

), arC: [112.39 (CH), 118.36 (CH),

Si as an in-

ternal reference, J values are given in Hz. Microwave and ultrasound

mediated syntheses were carried out using monomod CEM-Discover

microwave apparatus and Bandelin Sonorex Super RK102H ultrasonic

121.25 (CH), 122.87 (CH), 127.46 (CH), 128.00 (CH), 129.35 (CH),

129.96 (CH), 130.46 (CH), 131.99 (CH), 133.61 (CH), 135,87 (C), 137.18

(C), 141.53 (C), 149.72 (C), 150.42 (C), 152.39 (C)], 153.26 (triazole C-5),

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

68.69 (triazole C-3), 195.03 (C]O). EI MS m/z (%): 109.12 (22), 590.26

2.4.1.6. Ethyl 4-(2-chloro-4-{[(5-{[2-(4-chlorophenyl)-2-oxoethyl]thio}-4-

phenyl-4H-1,2,4-triazol-3-yl)methyl]amino}phenyl)piperazine-1-carboxylate

(

[M + Na] , 18), 606.24 ([M + K] , 100), 608.25 ([M + 2 + K] ,79).

(

11b). Recrystallized from methanol:H O (1:3). m.p. 112–113 °C. FT-IR

−1

.4.1.2. 2-[(4-Benzyl-5-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-

H-1,2,4-triazol-3-yl)thio]-1-(2,4-dichlorophenyl)ethanone (5b).

Recrystallized from acetone:H (1:3). mp. 92–93 °C. FT-IR (υmax

(υmax, cm ): 3285 (NH), 3066 (ar-CH), 1696 and 1686 (C]O). H NMR

(DMSO‑d , δppm): 1.18 (t, J = 16.0 Hz,3H, CH ), 2.50 (s, 2H, CH ), 2.76

(s, 4H, 2CH ), 3.46 (s, 2H, CH ), 4.03 (s, 2H, CH ), 4.16 (s, 2H, CH ), 4.87

(s, 2H, CH ), 6.26–6.47 (m, 2H, arH), 6.70–6.78 (m, 2H, arH), 7.46 (s, 2H,

arH), 7.58–7.63 (m, 4H, arH), 8.00–8.02 (m, 2H, arH), 8.39 (s, 1H, NH).

−1

cm ): 3061 (ar-CH), 1681 (C]O), 1587 (C]N). H NMR (DMSO‑d

δppm): 2.43 (s, 4H, 2CH

), 2.74 (s, 4H, 2CH

), 3.32 (s, 2H, CH

), 3.70 (s,

H, OCH

), 4.67 (s, 2H, CH ), 5.22 (s, 2H, CH

), 6.86 (s, 3H, arH),

C NMR (DMSO‑d

(DMSO + CH ), 44.39 (CH

61.22 (CH ), arC: [101.10 (CH), 108.49 (CH), 113.67 (CH), 117.32 (CH),

, δppm): 15.02 (CH

), 38.78 (CH

), 39.56–40.60

.12–7.31 (m, 5H, arH), 7.53 (s, 2H, arH), 7.70–7.78 (d, 2H, arH).

, δppm): 42.81 (CH ), 46.46 (CH ), 50.18 (2CH ), 52.31

), 53.08 (2CH ), 55.74 (OCH ), arC: [112.34 (CH), 118.31 (CH),

21.26 (CH), 122.89 (CH), 127.18 (CH), 127.77 (CH), 127.95 (CH),

), 50.87 (CH

), 51.47 (CH

), 56.34 (CH ),

NMR (DMSO‑d

(

118.91 (CH), 127.48 (CH), 128.22 (CH), 129.04 (CH), 129.38 (CH),

130.33 (CH), 130.46 (CH), 130.81 (CH), 133.18 (C), 134.47 (C), 139.10

(C), 142.79 (C), 143.22 (C), 145.51 (C)], 150.63 (triazole C-5), 154.19

(triazole C-3), 155.04 (C]O), 192.66 (C]O). EI MS m/z (%): 191.93

(38), 239.95 (56), 242.02 (64), 273.30 (47), 443.26 (68),613.35 (53),

28.14 (CH), 128.52 (CH), 129.05 (CH), 130.44 (CH), 131.91 (CH),

35.82 (C), 136.04 (C), 137.16 (C), 141.52 (C), 150.47 (C), 152.42 (C)],

53.33 (triazole C-5), 156.67 (triazole C-3), 195.13 (C]O). EI MS m/z

(

%): 205.15 (100), 360.51 (66), 447.52 (99), 448.52 (31), 582.23

[M + 1] , 37), 604.27 ([M + Na] , 61).

631.28 ([M + Na] , 100), 633.30 ([M + 1 + Na] , 38).

2.4.2. General Method for the synthesis of compounds 6a-d and 12a-b

.4.1.3. 1-(4-Chlorophenyl)-2-[(4-ethyl-5-{[4-(2-methoxyphenyl)

piperazin-1-yl]methyl}-4H-1,2,4-triazol-3-yl)thio]ethanone

5c). Recrystallized from acetone:H O (1:3). mp. 117–118 °C. FT-IR

Method 1. NaBH (50 mmol) was added the solution of the corre-

sponding compound 5a-dand 11a,b (10 mmol) in ethanol and the

mixture was stirred at room temperature for 24 h (the progress of the

reaction was monitored by TLC). After evaporating the solvent under

(

−1

(

max, cm ): 3072 (ar-CH), 1721 (C]O), 1582 (C]N). H NMR

DMSO‑d , δppm): 1.05 (t, J = 4.0 Hz, 3H, CH ), 2.37 (s, 4H, 2CH ),

.50 (s, 2H, CH ), 2.77 (s, 4H, 2CH ), 3.36 (s, 2H, CH ), 3.73 (s, 3H,

), 4.73 (s, 2H, CH ), 6.80–6.89 (m, 2H, arH), 7.45 (d, J = 8.0 Hz,

reduced pressure, a solid obtained. The crude product was re-

OCH

crystallized from an appropriate solvent to aﬀord the desired product.

Method 2. The solution of compound 5a-d and 11a,b (1 mmol) in

ethanol (10 mL) was irradiated in closed vessel in the presence of

H, arH), 7.56 (s, 2H, arH), 7.56 (s, 1H, arH). C NMR (DMSO‑d

), 42.68 (2CH ), 50.43 (2CH ), 52.22 (CH ), 53.20

), 55.77 (OCH ), arC: [112.37 (CH), 118.40 (CH), 121.26 (CH),

δppm): 15.30 (CH

NaBH (3 mmol) with pressure control (physical parameters were given

(

2CH

in Table 2) (the progress of the reaction was monitored by TLC). Then,

the mixture was poured into ice-water and a solid obtained. This crude

product was collected by ﬁltration and recrystallized from an appro-

priate solvent to aﬀord the desired product.

(

22.95 (CH), 127.96 (CH), 130.44 (CH), 131.89 (CH), 132.00 (C),

35.86 (C), 137.15 (C), 141.92 (2C)], 149.29 (triazole C-5), 152.44

triazole C-3), 195.13 (C]O). EI MS m/z (%): 520.22 ([M + 1]⁺, 100),

522.22 (68).

Method 3. The solution of compound 5a-d and 11a-b (1 mmol) in

ethanol (10 mL) was sonicated in the presence of NaBH (3 mmol) for

.4.1.4. 4-Benzyl-2-[2-(2,4-dichlorophenyl)-2-oxoethyl]-5-{[4-(2-

15 min (the progress of the reaction was monitored by TLC). Then, the

mixture was poured into ice-water and a solid obtained. This crude

product was collected by ﬁltration and recrystallized from an appro-

priate solvent to aﬀord the desired product.

methoxyphenyl) piperazin-1-yl]methyl}-2,4-dihydro-4H-1,2,4-triazol-3-one

(5d). Recrystallized from ethyl acetate. mp. 89–91 °C. FT-IR (υmax

−

cm ): 3092 (ar-CH), 1704 (C]O), 1583 (C]N). H NMR (DMSO‑d

δppm): 2.37 (s, 4H, 2CH ), 2.77 (s, 4H, 2CH ), 3.48 (s, 2H, CH

s, 3H, OCH ), 5.17 (s, 2H, CH ), 6.09 (s, 2H, CH ), 6.80–6.90 (m, 4H,

arH), 7.43 (d, J = 16.0 Hz, 3H, arH), 7.56 (d, J = 16.0 Hz, 5H, arH).

), 3.73

(

2.4.2.1. 1-(2,4-Dichlorophenyl)-2-[(5-{[4-(2-methoxyphenyl)piperazin-1-yl]

methyl}-4-phenyl -4H-1,2,4-triazol-3-yl)thio]ethanol (6a). Recrystallized

−1

C NMR (DMSO‑d

, δppm): 44.21 (CH

), 50.12 (2CH

), 53.01 (2CH

from acetone:H

(OH), 3072 (ar-CH), 1525 (C]N). H NMR (DMSO‑d

4H, 2CH ), 2.77 (s, 4H, 2CH ), 3.36 (s, 2H, CH ), 3.48 (s, 2H, CH

(s, 3H, OCH ), 5.30 (t, J = 12.0 Hz, 1H, CH), 6.09 (s, 1H, OH), 6.80–6.92

(m, 4H, arH), 7.41–7.57 (m, 8H, arH). C NMR (DMSO‑d

39.20–40.62 (DMSO + CH ), 45.82 (CH ), 50.36 (2CH ), 51.73 (CH

52.74 (CH ), 55.76 (OCH ), 67.59 (CH), arC: [112.38 (CH), 117.26 (CH),

118.35 (CH), 120.83 (CH), 121.25 (CH), 122.86 (CH), 127.63 (CH),

27.82 (CH), 129.04 (CH), 129.86 (CH), 129.90 (CH), 130.18 (CH),

O (1:3). m.p. 180–181 °C. FT-IR (υmax, cm ): 3186

3.36 (CH ), 55.74 (OCH

), 56.28 (CH

), arC: [112.36 (CH), 118.34

, δppm): 2.37 (s,

(

CH), 121.27 (CH), 122.87 (CH), 126.99 (CH), 127.55 (CH), 127.71

CH), 128.64 (CH), 128.89 (CH), 129.92 (CH), 130.30 (CH), 131.01

CH), 137.66 (C), 139.57 (C), 141.56 (C), 143.23 (C), 144.80 (C),

), 3.73

, δppm):

47.50 (C)], 152.43 (triazole C-5), 156.00 (triazole C-3). 195.03. EI MS

m/z (%): 588.31 ([M + Na] , 34), 590.32 ([M + 2 + Na]⁺, 35),

34.31 (100), 636.38 (70).

2.4.1.5. Ethyl-4-(4-{[(5-{[2-(2,4-dichlorophenyl)-2-oxoethyl]thio}-4-

133.06(C), 133.79 (C), 141.54 (C), 141.97 (C), 146.42 (C), 151.18 (C)],

phenyl-4H-1,2,4-triazol-3-yl)methyl] amino}-2-ﬂourophenyl)piperazin-1-

carboxylate (11a). Recrystallized from methanol:H (1:3). m.p.

08–109 °C. FT-IR (υm₁ax, cm ): 3325 (eNH), 3056 (ar-CH), 1677

C]O), 1588 (C]N). H NMR (DMSO‑d , δppm): 1.18 (t, J = 4.0 Hz,

), 3.16 (s, 2H, CH ), 3.36 (s, 2H, CH ), 3.45

), 4.05 (s, 2H, OCH ), 4.17 (s, 2H, CH ), 4.71 (s, 2H, CH ),

152.39 (triazole C-5), 153.09 (triazole C-3). EI MS m/z (%):447.58 (27),

592.28 ([M + Na] , 100), 594.22 ([M + 2 + Na] , 73), 595.23 (23).

−

(

2.4.2.2. 2-[(4-Benzyl-5-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-

4H-1,2,4-triazol-3-yl)thio]-1-(2,4-dichlorophenyl)ethanol

3H, CH

), 2.96 (s, 2H, CH

(

s, 2H, CH

(6b). Recrystallized from acetone:H O (1:3). m.p. 151–152 °C. FT-IR

−

.86–6.97 (m, 3H, arH), 7.04 (s, 1H, arH), 7.16 (s, 1H, arH), 7.26 (s,

H, arH), 7.43 (s, 2H, arH), 7.81–7.87 (m, 2H, arH), 8.82 (brs, 1H, NH).

(υmax, cm ): 3145 (OH), 3032 (ar-CH), 1587 (C]N). H NMR

(DMSO‑d , δppm): 2.46 (s, 4H, 2CH ), 2.78 (s, 4H, 2CH ), 3.36 (s,

2H, CH ), 3.61 (s, 2H, CH ), 3.74 (s, 3H, OCH ), 5.14–5.25 (m, 3H,

CH + CH), 6.15 (s, 1H, OH), 6.77–6.90 (m, 4H, arH), 7.12–7.25 (m,

2H, arH), 7.27–7.35 (m, 3H, arH), 7.41–7.60 (m, 3H, arH). C NMR

(DMSO‑d , δppm): 39.38–40.64 (DMSO + CH ), 41.23 (CH ), 47.37

(CH ), 50.20 (2CH ), 52.43 (CH ), 53.11 (CH ), 55.76 (OCH ), 67.80

C NMR (DMSO‑d

, δppm): 15.02 (CH

), 42.46 (CH

), 44.15 (CH

50.96 (CH ),51.49 (CH

), 57.34 (CH ), 61.27 (CH

), 92.23 (OCH

), arC:

[

114.39 (CH), 118.69 (CH), 121.98 (CH), 122.26 (CH), 127.88 (CH),

28.02 (CH), 128.20 (CH), 129.19 (CH), 129.33 (CH), 130.35 (CH),

30.50 (CH), 131.85 (C), 132.30 (2C), 134.36 (C), 145.29 (2C), 146.07

(

C)], 153.00 (triazole C-5), 155.05 (triazole C-3), 194.93 (C]O). EI MS

(CH), arC: [112.38 (CH), 118.33 (CH), 121.28 (CH), 122.89 (CH),

127.11 (CH), 127.29 (CH), 127.81 (CH), 128.07 (CH), 128.54 (CH),

m/z (%): 315.25 (30), 462.27 (100), 683.30 ([M + 2 + K] , 59).

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

128.82 (CH), 129.04 (CH), 129.91 (CH), 132.44(C), 133.05 (C), 136.25

(CH

2 2

), 64.33 (CH ), 70.73 (CH), arC: [101.11 (CH),108.49 (CH),

(

C), 140.28 (C), 141.55 (C), 151.32 (C)], 153.04 (triazole C-5), 156.49

113.68 (CH), 117.29 (CH), 118.93 (CH), 127.53 (CH), 128.30 (CH),

128.51 (2CH), 129.05 (CH), 130.26 (2CH), 132.25 (C), 133.21 (C),

143.16 (C), 145.39–145.50 (d, J = 11.0 Hz, C), 146.89 (C), 151.67 (C)],

triazole C-3). EI MS m/z (%):205.21 (42), 474.54 (36), 606.24

[M + Na] , 100), 608.32 ([M + 1 + Na] , 64).

54.07 (triazole C-5), 155.07 (triazole C-3). EI MS m/z (%): 445.20

.4.2.3. 1-(4-Chlorophenyl)-2-[(4-ethyl-5-{[4-(2-methoxyphenyl)

piperazin-1-yl]methyl}-4H-1,2,4-triazol-3-yl)thio]ethanol

6c). Recrystallized from acetone: H O (1:3). m.p. 142 °C. FT-IR (υmax

(30), 615.25 (39), 633.21 ([M + Na] , 100), 635.28 ([(M + 2)+Na]

38).

(

−

cm ): 3121 (OH), 3030 (ar-CH), 1585 (C]N). H NMR (DMSO‑d

δppm): 1.27 (s,3H, CH ), 2.51 (s, 4H, 2CH ), 2.93 (s, 4H, 2CH

H, CH ), 3.67 (s, 2H, CH ), 3.76 (s, 3H, OCH ), 3.97 (s, 2H, CH

s, 1H, CH), 6.16 (s, 1H, OH), 6.86–6.92 (d, J = 24.0 Hz, 4H, arH), 7.45

s, 1H, arH), 7.58 (d, J = 36.0 Hz, 2H, arH). ¹³C NMR (DMSO‑d

), 39.29 (CH ), 39.37–40.63 (DMSO + CH ), 50.49

), 53.22 (2CH ), 55.78 (OCH ), 67.79 (CH), arC:

112.38 (CH), 118.41 (CH), 121.27 (CH), 122.94 (CH), 127.83 (CH),

28.81 (CH), 129.93 (CH), 132.48 (C), 133.05 (C), 140.30 (C), 141.53

2.4.3. General Method for the synthesis of compounds 7a-l and 13a-d.

), 3.34 (s,

), 5.17

Method 1. NaH (10 mmol) was added the solution of the corre-

sponding compound 6 or 12a-b (10 mmol) in THF and the mixture was

reﬂuxed for 5 h. Then, the corresponding substituted benzylchloride

was added into it and the reﬂuxed for additional 9–10 h. After the

completion of the reaction, solvent was evaporated under reduced

pressure, and the obtained oily product was extracted with 15 mL of

(

δppm): 15.45 (CH

(

2CH

), 52.37 (CH

[

ethyl acetate three times in the presence of K

2 3

CO . The organic layer

was dried on Na SO . The oily product obtained on the removal of

(

2C)], 150.09 (triazole C-5), 152.71 (triazole C-3). EI MS m/z (%):

solvent under reduced pressure was puriﬁed by column chromato-

graphy (n-hexane/ethyl acetate) (3:1) on silica gel.

87.14 ([M] , 62), 488.17 ([M + 1] , 41), 510.25 ([M + Na]⁺, 100),

12.24 (48).

Method 2. NaH (10 mmol) was added the solution of the corre-

sponding compound 6 or 12a-b (10 mmol) in THF and the mixture was

irradiated 80 °C 100 Watt, 10 min. Then, the corresponding substituted

benzylchloride (30 mmol) was added into it and the reaction was con-

tinued for 45 min at 125 °C, 150 Watt. Solvent was evaporated under

reduced pressure, and the obtained oily product was extracted with

.4.2.4. 4-Benzyl-2-[2-(2,4-dichlorophenyl)-2-hydroxyethyl]-5-{[4-(2-

methoxyphenyl) piperazin-1-yl] methyl}-2,4-dihydro-3H-1,2,4-triazol-3-

one (6d). Recrystallized from ethyl acetate. m.p. 123–124 °C. FT-IR

−

(

max, cm ): 3285 (OH), 3063 (ar-CH), 1687 (C]O), 1579 (C]N). H

NMR (DMSO‑d , δppm): 2.38 (s, 4H, 2CH ), 2.76 (s, 4H, 2CH ), 3.29 (s,

H, CH )3.75 (s, 3H, OCH ), 4.26 (s, 1H, CH), 4.87 (s, 2H, CH ), 5.71

brs, 1H, OH), 6.80 (s, 1H, arH), 6.89–6.91 (d, J = 8.0 Hz, 2H, arH),

15 mL of ethyl acetate three times in the presence of K

2 3

CO . The or-

(

ganic layer was dried on Na SO . The oily product obtained on the

removal of solvent under reduced pressure was puriﬁed by column

chromatography (n-hexane/ethyl acetate) (3:1) on silica gel.

.12 (s, 1H, arH), 7.28 (s, 3H,arH), 7.34 (s, 5H, arH). C NMR

, δppm): 43.11 (CH ), 44.71 (CH ), 50.03 (2CH ), 51.93

), 52.90 (CH ),53.20 (CH ), 55.72 (OCH ), 56.35 (CH ), 70.20

CH), arC: [112.32 (CH), 118.31 (CH), 121.27 (CH), 122.87 (CH),

(

DMSO‑d

Method 3. NaH (10 mmol) was added the solution of the corre-

sponding compound 6 or 12a-b (10 mmol) in THF and the mixture was

sonicated 7–8 min. Then, the corresponding substituted benzylchloride

(30 mmol) was added into it and the reaction was continued for 8 min.

Solvent was evaporated under reduced pressure, and the obtained oily

product was extracted with 15 mL of ethyl acetate three times in the

27.33 (CH), 127.49 (CH), 127.55 (CH), 127.66 (CH), 128.02 (CH),

28.46 (CH), 128.51 (CH), 128.70 (CH), 132.31 (2C), 137.39 (C),

41.57 (C), 141.99 (C), 143.39 (C)], 152.43 (triazole C-5), 154.32

(

triazole C-3). EI MS m/z (%): 568.21 ([M + 1] , 56), 590.22

2 3 2 4

presence of K CO . The organic layer was dried on Na SO . The oily

(

[M + Na] , 43), 632.37 (100), 639.38 (78).

product obtained on the removal of solvent under reduced pressure was

puriﬁed by column chromatography (n-hexane/ethyl acetate) (3:1) on

silica gel.

.4.2.5. Ethyl-4-(4-{[(5-{[2-(2,4-dichlorophenyl)-2-hydroxyethyl]tio}-4-

phenyl-4H-1,2,4-triazol-3-yl) methyl]amino}-2-ﬂourophenyl)piperazin-1-

carboxylate (12a). Recrystallized from acetone:H (1:3). m.p.

41–142 °C. FT-IR (υmax, cm ): 3334 (NH + OH), 3061 (ar-CH),

2.4.3.1. 1-[(5-{[2-[(4-Chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)ethyl]

thio}-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]-4-(2-methoxyphenyl)

piperazine (7a). FT-IR (υmax, cm ): 3066 (ar-CH), 1526 (C]N). ¹H

−

−1

596 (C]N). H NMR (DMSO‑d

H, CH ), 2.89 (s, 2H, CH ), 3.35 (s, 2H, CH

), 5.14 (s, 2H, CH ), 5.90 (s, 1H, CH), 6.28 (brs, 1H, OH),

, δppm): 1.19 (s, 3H, CH

), 2.82 (s,

), 3.46 (s, 2H, CH

), 4.15

NMR (DMSO‑d

2H, CH ), 3.75 (s, 3H, OCH

CH ), 6.85 (d, J = 8.0 Hz, 2H, arH), 6.93 (d, J = 8.0 Hz, 2H, arH),

7.40–7.48 (m, 7H, arH), 7.52 (s, 1H, arH), 7.52–7.60 (m, 4H, arH).

NMR (DMSO‑d , δppm): 39.25–40.62 (CH ), 43.71 (CH ), 45.65

(2CH ), 47.96 (CH ), 52.45 (CH ), 55.78 (OCH ), 57.61 (CH ), 67.57

, δppm): 2.09 (s, 4H, 2CH

), 2.90 (s, 4H, 2CH

), 3.50 (s,

(

s, 2H, CH

), 4.76 (s, 3H, CH

+ CH), 5.20 (s, 2H,

.79 (s, 1H, arH), 6.95 (s, 1H, arH), 7.06 (s, 2H, arH), 7.14 (s, 1H, arH),

.45 (s, 3H, arH), 7.55 (s, 3H, arH), 8.96 (brs, 1H, NH). ¹³C NMR

(

DMSO‑d

, δppm): 15.26 (CH

), 43.52 (CH

), 44.15 (CH

), 50.48 (CH

1.52 (CH

), 57.46 (CH ), 61.32 (CH

), 69.23 (CH), 92.67 (OCH

), arC:

[

114.45 (CH), 118.61 (CH), 121.76 (CH), 122.26 (CH), 127.93 (CH),

28.42 (CH), 128.78 (CH), 129.29 (CH), 129.41 (CH), 130.39 (CH),

30.54 (CH), 131.88 (C), 132.41 (2C), 134.56 (C), 145.78 (2C), 146.17

(CH), arC: [112.37 (CH), 117.29 (CH), 117.91 (CH), 118.48 (CH),

121.25 (CH), 123.38 (CH), 127.82 (CH), 128.82 (CH), 128.97 (CH),

129.03 (CH), 129.07 (CH), 129.11 (2CH), 129.86 (CH), 129.92 (CH),

131.18 (C), 132.44 (C), 133.08 (CH), 135.55 (C), 136.73 (C), 137.18

(C), 140.33 (C), 143.11 (C), 146.34 (C)], 152.34 (triazole C-5), 153.98

(triazole C-3). EI MS m/z (%):581.10 (88), 583.11 (100), 585.12 (30),

(

C)], 153.19 (triazole C-5), 156.17 (triazole C-3), 195.03 (C]O). EI MS

m/z (%): 409.34 (38), 629.22 (37), 667.28 ([M + Na] , 100), 669.23

(

[(M + 2)+Na] ,76).

96.17 ([M + 1]⁺, 36).

.4.2.6. Ethyl 4-(2-chloro-4-{[(5-{[2-(4-chlorophenyl)-2-hydroxyethyl]

thio}-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]amino}phenyl)piperazine-1-

carboxylate (12b). Recrystallized from acetone:H (1:3). m.p.

32–133 °C. FT-IR (υmax, cm ): 3319 (NH + OH), 3063 (ar-CH),

2.4.3.2. 1-[(5-{[2-[(2,4-Dichlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

ethyl]thio}-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]-4-(2-methoxyphenyl)

piperazine (7b). FT-IR (υmax, cm ): 3068 (ar-CH), 1562 (C]N). ¹H

−

−1

678 (C]O), 1595 (C]N). H NMR (DMSO‑d

), 2.50 (s, 2H, CH ), 2.76 (s, 4H, 2CH ), 3.45 (H

s, 2H, CH ), 4.16 (s, 2H, CH ), 4.85 (1s, H, CH), 5.91 (s, 1H, OH),

, δppm): 1.18 (s, 3H,

NMR (DMSO‑d

2H, CH ), 3.73 (s, 3H, OCH

CH ), 6.83–6.91 (m, 4H, arH), 7.38–7.56 (m, 7H, arH), 7.62–7.65 (m,

4H, arH). C NMR (DMSO‑d

43.35 (CH ),46.03 (CH ), 50.16 (CH

(OCH ), 60.25 (CH ), 67.10 (CH), arC: [112.33 (CH), 122.89 (CH),

, δppm): 2.40 (s, 4H, 2CH

), 2.78 (s, 4H, 2CH

), 3.69 (s,

O + 2CH ), 4.04

), 4.80 (s, 3H, CH

+ CH), 5.03 (s, 2H,

(

.29–6.47 (m, 2H, arH), 6.72–6.78 (m, 2H, arH), 7.46 (d, J = 48.0 Hz,

H, arH), 8.34 (brs, 1H, NH). C NMR (DMSO‑d

, δppm): 39.27–40.63 (DMSO + CH

, δppm): 15.01 (CH

), 52.65 (CH ), 52.89 (CH ), 55.72

8.80 (CH

),44.01 (CH

), 50.87 (CH

), 51.47 (CH

), 56.35 (CH ), 61.25

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

27.55 (CH), 127.72 (CH), 127.97 (CH), 128.04 (CH), 128.58 (CH),

28.93 (CH), 129.12 (CH), 129.38 (CH), 129.45 (CH), 129.66 (CH),

29.72 (CH), 129.76 (C), 131.72 (CH), 133.30 (C), 133.55 (C), 133.68

7.60–7.68 (m, 4H, arH). ¹³C NMR (DMSO‑d

44.23 (CH ), 47.40 (CH ), 50.57 (CH

56.27 (CH ), 58.71 (CH ), 65.18 (CH), 67.81 (CH

, δppm): 41.45 (CH

), 53.14 (CH ), 55.69 (OCH

), arC: [ 112.28 (CH),

(

CH), 134.65 (C), 134.73 (C), 134.89 (C), 136.17 (C), 141.43 (C),

42.83 (C)], 152.36 (triazole C-5), 159.03 (triazole C-3). EI MS m/z

%):615.00 (72), 617.01 (100), 619.02 (51), 728.06 ([M + 1]⁺, 24),

118.32 (CH), 121.23 (CH), 127.30 (CH), 127.40 (CH), 127.55 (CH),

127.82 (CH), 127.86 (CH), 128.87 (CH), 129.04 (CH), 129.08 (CH),

129.26 (2CH), 131.80 (2CH), 132.27 (2C), 133.25 (C), 135.07 (C),

135.61 (C), 136.15 (2C), 142.97 (C), 144.38 (C)], 152.72 (triazole C-5),

30.07 (32).

55.44 (triazole C-3). EI MS m/z (%):106.07 (42), 584.22 (33), 742.14

2.4.3.3. 1-[(5-{[2-[(2,6-Dichlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

(69), 744.14 ([M + 1] , 100), 746.08 (51).

ethyl]thio}-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]-4-(2-methoxyphenyl)

piperazine (7c). FT-IR (υmax, cm ): 3063 (ar-CH), 1561 (C]N). ¹

NMR (DMSO‑d , δppm): 2.05 (s, 2H, CH ), 2.50 (s, 4H, 2CH ), 2.80 (s,

H, CH ), 3.30–3.45 (m, H O + CH ), 3.73 (s, 3H, OCH ), 4.91 (s, 3H,

CH + CH), 5.12 (s, 2H, CH ), 6.73–7.05 (m, 2H, arH), 7.20 (s, 1H,

arH), 7.43–7.51 (m, 5H, arH), 7.55–7.62 (m, 7H, arH). C NMR

DMSO‑d , δppm): 41.54 (CH ), 44.09 (CH ), 50.14 (CH ), 52.40 (CH ),

3.05 (CH ), 55.79 (OCH ), 66.03 (CH ), 67.57 (CH), 77.46 (CH ), arC:

112.39 (CH), 117.32 (CH), 120.89 (CH), 121.26 (CH), 127.83 (CH),

−1

2.4.3.7. 1-[(5-{[2-[(4-Chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)ethyl]

thio}-4-ethyl-4H-1,2,4-triazol-3-yl)methyl]-4-(2-methoxyphenyl)

−

piperazine (7 g). FT-IR (υmax, cm ): 3061 (ar-CH), 1589 (C]N). ¹H

NMR (DMSO‑d

J = 4.0 Hz, 4H, 2CH

(s, 3H ,OCH ), 4.90 (s, 2H, CH

6.86–6.95 (m, 2H, arH), 7.13 (d, J = 12.0 Hz, 3H, arH), 7.37–7.45

, δppm): 1.40 (t, J = 16.0 Hz, 3H, CH

), 2.79 (s, 4H, 2CH ), 3.01 (s, 4H, 2CH

), 5.98 (q, J = 36.0 Hz,1H, CH),

), 2.50 (t,

), 3.79

(

[

(m, 5H, arH), 7.54–7.62 (m, 2H, arH), 7.68–7.70 (m, 1H, arH).

NMR (DMSO‑d , δppm): 14.17 (CH ),41.55 (CH ), 42.66 (CH ), 49.86

(2CH ), 50.38 (2CH ), 53.18 (2CH ), 55.83 (OCH ), 69.83 (CH), arC:

27.90 (CH), 128.23 (CH), 128.84 (CH), 128.95 (CH), 129.04 (CH),

29.09 (CH), 129.12 (CH), 129.32 (CH), 129.35 (CH), 129.85 (CH),

30.20 (C), 130.28 (C), 131.93 (C), 132.37 (C), 132.73 (C), 133.04 (C),

33.09 (C), 140.31 (C), 140.43 (C)], 152.41 (triazole C-5), 155.68

[112.43 (CH), 118.53 (CH), 121.27 (CH), 123.16 (CH), 127.94 (CH),

128.41 (CH), 128.48 (C), 128.57 (CH), 128.92 (CH), 129.02 (CH),

131.95 (CH), 132.44 (CH), 132.82 (C), 133.31 (C), 135.60 (C), 136.89

(

triazole C-3). EI MS m/z (%):205.21 (100), 524.29 (29), 682.17 (32),

684.18 (45),710.19 (40).

(C), 141.31 (C), 142.69 (C)], 152.09 (triazole C-5), 154.52 (triazole C-

3). EI MS m/z (%):608.10 (100), 610.23 (76), 685.50 ([M + K] , 81),

2.4.3.4. 1-[(4-Benzyl-5-{[2-[(4-chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

771.63 (30).

ethyl]thio}-4H-1,2,4-triazol-3-yl)methyl]-4-(2-methoxyphenyl)piperazine

−

(

7d). FT-IR (υmax, cm ): 3063 (ar-CH), 1587 (C]N). H NMR

DMSO‑d , δppm): 2.50 (s, 4H, 2CH ) 2.80 (s, 4H, 2CH ), 3.40 (s,

H2O + CH ), 3.74 (s, 3H, OCH ), 4.75 (s, 4H, 2CH2), 4.91(t,

J = 12.0 Hz, 1H, CH), 4.93 (s, 2H, CH ), 6.84–7.02 (m, 3H, arH),

.27–7.30 (m, 4H, arH), 7.31–7.46 (m, 9H, arH). C NMR (DMSO‑d

), 47.41 (2CH ), 49.99 (CH ), 52.95 (CH ), 55.73

), 56.27 (CH ), 60.30 (CH ), 65.26 (CH), 67.77 (CH ), arC: [

2.4.3.8. 1-[(5-{[2-[(2,4-Dichlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

ethyl]thio}-4-ethyl-4H-1,2,4-triazol-3-yl)methyl]-4-(2-methoxyphenyl)

piperazine (7h). FT-IR (υmax, cm ): 3061 (ar-CH), 1563 (C]N). ¹H

−

NMR (DMSO‑d

2CH ), 2.94 (s, 4H, 2CH

(s, 3H ,OCH ), 4.91 (s, 2H, CH

5H, arH), 7.38–7.51 (m, 5H, arH). C NMR (DMSO‑d

(CH ),36.14 (CH ),41.38 (CH ), 50.49 (2CH ), 52.37 (CH

(2CH ),53.24 (CH ),55.76 (OCH ), 70.92 (CH), arC: [112.34 (CH),

, δppm): 1.23 (t, J = 12.0 Hz, 3H, CH

), 3.38 (s, 4H, 2CH ), 3.69 (s, 2H, CH

), 5.94 (s, 1H, CH), 6.89 (d, J = 24.0 Hz,

), 2.51 (s, 4H,

), 3.77

δppm): 45.03 (CH

(

OCH

, δppm):14.64

), 53.18

(

12.31 (CH), 118.34 (CH), 121.26 (C), 127.21 (CH), 127.54 (CH),

28.84 (2CH), 128.94 (CH), 129.02 (CH), 129.05 (CH), 129.09 (2CH),

31.16 (CH), 132.68 (CH), 133.04 (CH), 133.18 (2CH), 133.43 (C),

36.07 (C), 136.46 (C), 136.88 (C), 137.15 (C), 137.27 (C), 140.26

C)], 152.39 (triazole C-5), 154.36 (triazole C-3).EI MS m/z (%):

05.21(20), 488.32 (30), 644.26 (100), 646.26 (70), 710.23

(M + 1)⁺, 34).

115.67 (C), 118.42 (CH), 121.26 (CH), 122.94 (CH), 128.31 (CH),

128.40 (CH), 128.46 (C), 129.02 (CH), 128.92 (CH), 129.02 (CH),

132.21 (C), 135.86 (CH), 141.52 (C), 143.14 (C), 144.76 (C), 150.63

(C), 152.44 (C)], 152.67 (triazole C-5), 155.61 (triazole C-3). EI MS m/z

[

(%):195.04 (100), 369.36 (26), 633.21 (38), 719.36 ([(M + 1)+K]

0).

2.4.3.5. 1-[(4-Benzyl-5-{[2-[(2,4-dichlorobenzyl)oxy]-2-(2,4-

dichlorophenyl)ethyl]thio}-4H-1,2,4-triazol-3-yl)methyl]-4-(2-

methoxyphenyl)piperazine (7e). FT-IR (υmax, cm ): 3063 (ar-CH), 1561

2.4.3.9. 1-[(5-{[2-[(2,6-Dichlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

ethyl]thio}-4-ethyl-4H-1,2,4-triazol-3-yl) methyl]-4-(2-methoxyphenyl)

−1

(

C]N). H NMR (DMSO‑d

CH ), 3.41 (s, 4H, 2CH ), 3.75 (s, 3H, OCH

CH), 4.87 (s, 2H, CH ), 5.20 (s, 2H, CH ), 6.78–6.90 (m, 3H, arH),

.26–7.30 (m, 2H, arH), 7.32–7.40 (m, 4H, arH), 7.41–7.50 (m, 6H,

, δppm): 2.50 (s, 4H, 2CH

) 2.80 (s, 4H,

), 4.43 (t, J = 24.0 Hz, 1H,

piperazine (7i). FT-IR (υmax, cm ): 3068 (ar-CH), 1562 (C]N).

NMR (DMSO‑d , δppm): 1.27 (t, J = 12.0 Hz, 3H, CH ), 2.50 (s, 4H,

2CH ), 2.93 (s, 4H, 2CH ), 3.37 (s, 2H, CH ), 3.74–3.77 (m, 5H,

OCH + CH ), 4.89 (s, 4H, 2CH ), 5.19 (s, 1H, CH), 6.84–6.91 (m, 2H,

arH), 7.39–7.43 (m, 3H, arH), 7.51–7.54 (m, 5H, arH). C NMR

(DMSO‑d , δppm): 14.20 (CH ), 41.50 (2CH ), 42.69 (CH ), 43.99

(CH ), 47.50 (CH ), 49.89 (CH ), 50.35 (CH ), 53.17 (CH ), 55.76

(OCH ), 67.03 (CH), arC: [112.35 (CH), 118.48 (CH), 121.25 (CH),

arH). C NMR (DMSO‑d

, δppm): 39.37–40.63 (DMSO + CH

), 49.98 (CH ), 52.14 (CH ), 52.95 (CH

), 67.77 (CH), 81.07 (CH ), arC: [112.35 (CH), 118.37 (CH),

21.26 (CH), 127.28 (CH), 127.80 (2CH), 128.33 (CH), 128.80 (CH),

), 43.36

), 55.75

(

2CH

OCH

), 47.42 (CH

29.07 (CH), 129.30 (CH), 129.68 (CH), 129.91 (2CH), 132.40 (C),

33.04 (C), 133.33 (C), 134.68 (2CH), 134.73 (C), 134.89 (C), 136.16

123.04 (CH), 127.73 (CH), 127.80 (C), 128.12 (CH), 128.28 (CH),

128.78 (CH), 129.32 (CH), 129.94 (CH), 131.89 (C), 133.28 (C), 135.60

(C), 136.21 (C), 139.46 (C), 140.33 (C), 141.29 (C)], 152.44 (triazole C-

5), 154.24 (triazole C-3). EI MS m/z (%):205.18 (100), 460.27 (32),

650.16 (31), 652.17 (42), 682.14 ([M + 1]⁺, 40).

(

C), 140.28 (2C), 142.89 (C)], 152.40 (triazole C-5), 155.69 (triazole C-

). EI MS m/z (%):106.07 (43), 584.22 (33), 742.14 (70), 744.14

(

[M + 1] , 100), 746.08 (55).

2.4.3.6. 1-[(4-Benzyl-5-{[2-[(2,6-dichlorobenzyl)oxy]-2-(2,4-

2.4.3.10. 4-Benzyl-2-[2-[(4-chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

dichlorophenyl)ethyl]thio}-4H-1,2,4-triazol-3-yl)methyl]-4-(2-

ethyl]-5-{[4-(2-methoxyphenyl

1,2,4-triazol-3-one (7j). FT-IR (υmax, cm ): 3068–3028 (ar-CH), 1704

(C]O), 1562 (C]N). ¹H NMR (DMSO‑d

, δppm): 2.50 (s, 4H, 2CH ),

2.80 (s, 4H, 2CH ), 3.34 (s, 6H, 3CH ), 3.74 (s, 3H, OCH ), 4.70 (s, 3H,

CH + CH), 6.79–7.02 (m, 3H, arH), 7.28–7.33 (m, 3H, arH), 7.42–7.60

(m, 5H, arH), 7.62–7.72 (m, 5H, arH). C NMR (DMSO‑d , δppm):

piperazin-1-yl]methyl}-2,4-dihydro-3H-

−

−1

methoxyphenyl)piperazine (7f). FT-IR (υmax, cm ): 3066 (ar-CH), 1562

(

C]N). H NMR (DMSO‑d

CH ), 3.38 (s, 4H, 2CH ), 3.75 (s, 3H, OCH

CH), 4.84 (s, 2H, CH ), 5.23 (s, 2H, CH ), 6.75–6.91 (m, 3H, arH), 7.10

s, 2H, arH), 7.29–7.34 (m, 3H, arH), 7.46–7.58 (m, 3H, arH),

, δppm): 2.52 (s, 4H, 2CH

) 2.84 (s, 4H,

), 4.29 (t, J = 20.0 Hz, 1H,

(

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

3.45 (CH

), 45.64 (CH

), 58.35 (CH

), 50.00 (2CH

), 67.77 (CH), arC: [112.32 (CH), 118.34 (CH),

), 52.96 (2CH

), 55.73 (OCH

(NH), 3063 (ar CH), 1698 (C]O), 1558 (C]N). ¹H NMR (DMSO‑d

6.28 (CH

δppm): 1.35 (s, 3H, CH

), 2.08 (s, 4H, 2CH

), 2.50 (s, 2H, CH

), 2.94 (s,

21.26 (CH), 127.22 (CH), 127.27 (CH), 127.54 (CH), 127.83 (2CH),

28.13 (CH), 128.85 (CH), 128.90 (CH), 128.94 (CH), 129.10 (CH),

29.84 (CH), 129.90 (CH), 131.16 (CH), 133.44 (C), 135.15 (C), 136.47

4H, 2CH ), 4.04 (s, 2H, CH

), 4.48 (s, 1H, CH),4.76 (s, 4H, 2CH

7.34–7.38 (m, 2H, arH), 7.42 (s, 1H, arH), 7.42–7.48 (m, 6H, arH),

7.52–7.56 (m, 2H, arH), 7.56 (d, J = 4.0 Hz, 3H, arH), 11.86 (brs, 1H,

(

C), 137.16 (C), 137.28 (C), 139.66 (C), 140.27 (C), 141.40 (C)],

52.40 (triazole C-5), 155.43 (triazole C-3). EI MS m/z (%):205.34

100), 360.51 (75), 558.32 (22), 572.50 (55), 732.71 (16).

NH). ¹³C NMR (DMSO‑d

, δppm): 14.99 (CH ), 30.87 (CH

), 51.82 (CH ), 60.21 (CH ), 61.39 (CH

), 34.26

), 61.82

(CH

), 45.63 (CH

), 62.53 (CH

), 67.54 (CH), arC: [112.38 (CH), 121.45 (CH),

122.58 (C), 125.36 (CH), 127.57 (CH), 127.91 (CH), 128.42 (CH),

.4.3.11. 4-Benzyl-2-[2-[(2,4-dichlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

128.63 (CH), 128.84 (CH), 128.96 (CH), 129.02 (CH), 129.11 (CH),

129.89 (CH), 130.38 (CH), 131.18 (CH), 131.86 (C), 132.40 (C), 132.90

(C), 133.12 (C), 138.18–137.46 (C dc-c = 18.0 Hz) 140.27 (C), 142.65

(C), 151.90 (C), 153.43 (C)], 154.95 (triazole C-5), 157.40 (triazole C-

3), 197.27 (C]O). EI MS m/z (%):457.20 (100), 459.21 (76), 581.15

(45), 769.17 (26), 771.23 (28).

ethyl]-5-{[4-(2-methoxyphenyl) piperazin-1-yl]methyl}-2,4-dihydro-3H-

−1

,2,4-triazol-3-one (7k). FT-IR (υmax

cm ): 3061–3028 (ar-CH),

710 (C]O), 1587 (C]N). H NMR (DMSO‑d , δppm): 2.50 (s, 4H,

CH ), 2.82 (s, 2H, CH ), 3.44 (s, 4H, 2CH ), 3.74 (s, 3H, OCH ), 4.81

), 5.28 (d, J = 12.0 Hz,2H, CH ), 6.18 (s, 1H, CH),

.79–6.91 (m, 3H, arH), 7.14 (s,1H, arH), 7.27–7.43 (m, 3H, arH),

(

s, 4H, 2CH

.44–7.59 (m, 4H, arH) 7.60–7.67 (m, 4H, arH). C NMR (DMSO‑d

), 43.36 (CH ), 47.15 (CH ), 49.99

), 52.96 (CH ), 55.76 (OCH ), 67.91

CH), arC: [112.35 (CH), 118.37 (CH), 121.26 (CH), 127.28 (CH),

2.4.3.15. Ethyl

4-(4-{[(5-{[2-[(2,6-dichlorobenzyl)oxy]-2-(2,4-

δppm): 39.38–40.63 (DMSO + CH

dichlorophenyl)ethyl]thio}-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]amino}-

−1

(

2CH

), 51.10 (CH

), 52.14 (CH

2-ﬂuorophenyl)piperazine-1-carboxylate (13c). FT-IR (υmax, cm ): 3272

(NH), 3063 (ar-CH), 1693 (C]O), 1562 (C]N). ¹H NMR (DMSO‑d

27.53 (CH), 127.80 (CH), 127.97 (CH), 128.12 (CH), 128.33 (CH),

28.80 (CH), 129.07 (CH), 129.31 (CH), 129.68 (CH), 129.91 (CH),

32.41 (C), 133.05 (C), 133.33 (CH), 134.68 (C), 134.73 (C), 134.90

δppm): 1.18 (t, J = 4.0 Hz, 3H, CH

2CH ), 3.39 (s, 2H, CH ), 3.50 (s, 4H, 2CH

3H, CH

7.35–7.41 (m, 2H, arH), 7.42 (d, J = 8.0 Hz, 2H, arH), 7.46–7.53 (m,

), 2.08 (s, 2H, CH

), 2.89 (s, 4H,

2 2

), 4.07 (s, 2H, CH ), 4.89 (s,

+ CH), 6.95 (d, J = 8.0 Hz, 4H, arH), 7.24–7.27 (m, 2H, arH),

(

C), 136.16 (C), 139.67 (C), 140.29 (C), 141.23 (C)], 152.40 (triazole C-

), 155.69 (triazole C-3). EI MS m/z (%):125.09 (72), 169.01 (100),

4H, arH), 9.21 (brs, 1H, NH). ¹³CNMR (DMSO‑d

41.49 (2CH ), 43.73 (CH ), 50.53 (CH ), 50.95 (CH

8.71 (CH ), 61.26 (CH

, δppm): 15.00 (CH

57.33 (69), 581.21 (81), 583.22 (84), 769.23 (42), 771.23 (40).

), 53.17 (CH

), 67.52 (CH), arC: [106.88 (CH), 114.30 (CH),

.4.3.12. 4-Benzyl-2-[2-[(2,6-dichlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

118.46 (CH), 120.36 (CH), 122.12 (CH), 128.28 (CH), 128.44 (CH),

129.16 (2CH), 130.29 (CH), 130.68 (CH), 131.86 (CH), 132.15 (2CH),

132.90 (C), 133.29 (C), 135.60 (C), 136.06 (2C), 137.46 (C), 138.01

(C), 140.23 (C), 153.07 (C), 154.21 (C)], 155.05 (triazole C-5), 156.62

(triazole C-3), 206.50 (C]O). EI MS m/z (%):159.13 (45), 315.36 (47),

ethyl]-5-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-2,4-dihydro-3H-

−1

1,2,4-triazol-3-one (7l). FT-IR (υmax, cm ): 3068 (ar-CH), 1699 (C]

O), 1562 (C]N). H NMR (DMSO‑d

s, 4H, 2CH ), 3.30 (d, J = 4.0 Hz, 2H, CH

), 4.39 (s, 2H, CH ), 4.89 (s, 3H, CH

H, arH), 7.32 (d, J = 8.0 Hz, 3H, arH), 7.40–7.44 (m, 5H, arH),

, δppm): 2.43 (s, 4H, 2CH

), 3.36 (s, 2H, CH ), 3.74 (s,

+ CH), 7.25–7.27 (m,

), 2.79

(

H, OCH

462.26 (100), 529.34 (94), 683.39 (70), 805.15 ([M + 1] , 50).

.52–7.61 (m, 4H, arH). C NMR (DMSO‑d

3.45 (CH ), 44.21 (CH ), 48.66 (CH ), 50.10 (CH

3.70 (CH ), 55.73 (OCH ), 56.27 (CH

, δppm): 41.53 (CH

2.4.3.16. Ethyl

4-[4-({[5-({2-(4-chlorophenyl)-2-[(2,6-dichlorobenzyl)

), 52.98 (CH

oxy]ethyl}thio)-4-phenyl-4H-1,2,4-triazol-3-yl]methyl}amino)-2-

), 66.22 (CH), arC: [112.33

ﬂuorophenyl]piperazine-1-carboxylate (13d). FT-IR (υmax, cm⁻¹): 3250

(

CH), 118.35 (CH), 121.26 (CH), 122.91 (CH), 126.98 (CH), 127.44

CH), 127.55 (CH), 127.72 (CH), 127.97 (CH), 128.12 (CH), 128.22

CH), 128.63 (CH), 128.89 (CH), 129.33 (CH), 131.89 (CH), 133.30 (C),

(NH), 3045 (ar-CH), 1694 (C]O), 1515 (C]N). ¹H NMR (DMSO‑d

δppm): 1.18 (t, J = 12.0 Hz, 3H, CH

CH ), 2.78 (s, 4H, 2CH ), 3.37 (s, 2H, CH

J = 16.0 Hz,3H, CH + CH

6H, arH), 7.48–7.55 (m, 2H, arH), 7.55–7.57 (m, 2H, arH), 11.85 (s,

), 2.08 (s, 4H, 2CH

), 2.50 (s, 2H,

2 2

), 3.47 (s, 2H, CH ), 4.04(t,

34.00 (C), 134.14 (C), 134.63 (C), 134.75 (C), 135.61 (C), 136.21 (C),

), 7.38–7.42 (m, 5H, arH), 7.43–7.48 (m,

37.61 (C), 139.63 (C)], 152.41 (triazole C-5), 155.97 (triazole C-3). EI

MS m/z (%):696.11 (48), 698.12 (60), 726.05 (34), 728.00 ([M + 1]

1H, NH). C NMR (DMSO‑d

39.35–40.98 (DMSO + CH ), 44.07 (CH

6.34 (CH ), 61.22 (CH ), 64.36 (CH

, δppm): 15.01 (CH ), 38.82 (CH

0), 759.32 (100), 760.26 (53), 772.00 (35).

), 50.86 (CH ), 51.47 (CH

), 70.54 (CH), arC: [117.30 (CH),

.4.3.13. Ethyl 4-(4-{[(5-{[2-[(4-chlorobenzyl)oxy]-2-(2,4-dichlorophenyl)

125.37 (CH), 127.51 (CH), 127.54 (CH), 128.29 (CH), 128.33 (CH),

128.46 (CH), 128.51 (2CH), 128.93 (CH), 129.03 (CH), 129.75 (2CH),

130.25 (CH), 130.30 (CH), 131.45 (C), 133.26 (C), 141.97 (2C),

143.20–143.27 (d, J = 28.0 Hz, C), 145.34 (C), 145.42 (C), 145.53

(C), 146.86 (C)], 154.06 (triazole C-5), 155.49 (triazole C-3), 207.14

(C]O). EI MS m/z (%):195.04 (100), 196.17 (40), 369.36 (26), 633.21

(38), 719.36 (19).

ethyl]thio}-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]amino}-2-ﬂuorophenyl)

piperazine-1-carboxylate (13a). FT-IR (υmax, cm ): 3241 (NH), 3063 (ar-

−1

CH), 1697 (C]O), 1560 (C]N). H NMR (DMSO‑d

CH ), 2.49 (d, J = 4.0 Hz, 4H, 2CH ), 2.91 (s, 4H, 2CH

H, 2CH ), 4.53 (s, 2H, CH ), 4.81 (s, 3H, CH + CH), 7.42–7.47 (m, 2H,

arH), 7.53–7.55 (m, 3H, arH), 7.56–7.57 (m, 3H, arH), 7.57–7.63 (m, 4H,

, δppm): 1.35 (s, 3H,

), 4.04–4.07 (m,

arH), 7.65–7.67 (m, 3H, arH), 9.39 (s, 1H, NH). C NMR (DMSO‑d

δppm): 14.98 (CH ), 43.35 (CH ), 46.72 (CH ), 50.44 (CH ), 51.98 (CH

4.51 (CH ), 60.21 (CH ), 61.32 (CH ), 61.42 (CH ), 67.53 (CH), arC:

127.63 (CH), 127.86 (CH), 128.33 (CH), 128.58 (CH), 128.84 (CH),

3. Antimicrobial activity

[

The test microorganisms were obtained from the Hifzissihha

Institute of ReﬁkSaydam (Ankara, Turkey) and were as follows:

Escherichia coli (E. coli) ATCC35218, Yersinia pseudotuberculosis (Y.

pseudotuberculosis) ATCC911, Pseudomonas aeruginosa (P. aeruginosa)

ATCC43288, Enterococcus faecalis (E. faecalis) ATCC29212,

Staphylococcus aureus (S. aureus) ATCC25923, Bacillus cereus (B. cereus)

709 Roma, Mycobacterium smegmatis (M. smegmatis) ATCC607, Candida

albicans (C. albicans) ATCC60193 and Saccharomyces cerevisiae (S. cer-

evisia) RSKK 251. All the newly synthesized compounds were weighed

and dissolved in hexane to prepare extract stock solution of 20.000 µg/

milliliter (μg/mL). The antimicrobial eﬀects of the substances were

29.37 (CH), 129.67 (CH), 129.80 (2CH), 129.88 (CH), 130.44 (CH),

30.60 (CH), 130.71 (CH), 132.19 (C), 132.24 (C), 132.38 (C), 132.68 (C),

33.33 (2CH), 134.68 (C), 134.72–134.88 (d, J = 16.0 Hz, C), 139.28 (C),

40.22 (C), 152.24 (C)], 155.04 (triazole C-5), 157.52 (triazole C-3),

06.78 (C]O). EI MS m/z (%):395.07 (46), 547.20 (79), 526.03 (40),

15.06 (71), 617.00 (100), 805.08 (36), 807.09 ([M + K] , 24).

.4.3.14. Ethyl

4-(4-{[(5-{[2-[(2,4-dichlorobenzyl)oxy]-2-(2,4-

dichlorophenyl)ethyl]thio}-4-phenyl-4H-1,2,4-triazol-3-yl)methyl]amino}-

−1

2-ﬂuorophenyl)piperazine-1-carboxylate (13b). FT-IR (υmax, cm ): 3233

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

tested quantitatively in respective broth media by using double mi-

crodilution and the minimal inhibition concentration (MIC) values (μg/

mL) were determined. The antibacterial and antifungal assays were

performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI) at pH.7.3

and buﬀered Yeast Nitrogen Base (Difco, Detroit, MI) at pH 7.0, re-

spectively. The micro dilution test plates were incubated for 18–24 h at

v phenol and 0.005% w/v sodium nitroprusside) was added, vortexed

and then, 750 µL of alkali reagent (0.5% w/v NaOH and 0.1% v/v

NaOCl) was added and vortexed. This mixture was incubated for 15 min

more at 35 °C and optical density was measured at 625 nm against a

blank solution including distilled water instead of enzyme. For the

determination of the IC50 value of the extracts, activity assays were

conducted at ﬁve diﬀerent extract concentration and dose response

curve was generated. Thiourea was used as standard inhibitor.

5 °C. Brain Heart Infusion broth (BHI) (Difco, Detriot, MI) was used for

M. smegmatis, and incubated for 48–72 h at 35 °C [36]. Ampicillin

10 μg) and ﬂuconazole (5 μg) were used as standard antibacterial and

antifungal drugs, respectively. Dimethylsulphoxide with dilution of

:10 was used as solvent control. Only positive results were presented

in Table 4.

(

7. Molecular docking studies

To get an insight about the molecular level interaction between the

ligand (present study compounds) and to the receptor (drug target

molecule/protein), molecular docking was carried out by

Autodock4.2.6, Autodock Tools 1.5.6 and the Arguslab version 4.0.1.

The 3D crystal structures of target proteins were downloaded from the

protein data bank (PDB) (www.rcsb.org/pdb/). The X-ray crystal-

lography structures of the receptors with the PDB ID: 3CXN (Urease)

and PDB ID: 3WY1 (α-Glucosidase) was used to dock into their active

sites. All molecular docking strategies and their relevant aspects were

stated as we have published earlier with necessary modiﬁcations

wherever it needed [42–45]. To unveil the drug eﬃcacy of target

compounds, the statistical mechanical analysis was carried out using

the obtained binding aﬃnity values from the molecular docking study.

The lowest binding energy values along with the ligand eﬃciency and

the inhibitory constant (ki) values were extracted to consider the drug

eﬃcacy assessments. Ligand eﬃciency is used in drug discovery to

support in narrowing focus to candidate drugs with optimum combi-

nations of physicochemical and pharmacological potentials [46]. Mo-

lecular interaction (hydrogen bonding, π-π interaction, and π-cation

interaction) results were validated.

. Antioxidant activity

DPPH (2.2-diphenyl-1-picrylhydrazyl) radical scavenging activity:

The scavenging activity of diﬀerent chemicals was determined using the

free radical DPPH (2.2-diphenyl-1-picrylhydrazyl), as described by

Blois [37]. A 100-μL: chemical solution was mixed with 1 mL of freshly

prepared methanolic DPPH solution. The reaction mixture was in-

cubated for 30 min at room temperature in the dark and was then

measured at 520 nm. The activity was expressed as μmol Trolox

equivalent. FRAP (the ferric reducing ability of plasma): FRAP was

measured using the method described by Benzie & Strain [38] with

some modiﬁcation. To 100 μL of each sample was added 2900 μL

freshly prepared FRAP reagent containing 300 mmol/L acetate buﬀer

(

pH 3.6), 10 mmol/L TPTZ (2,4,6-tripyridyle-s-triazine) and 20 mmol/L

FeCl·6H O in proportions of 10:1:1 (v/v/v). The mixture was incubated

for 30 min at 37 °C and measured at 593 nm. The values were expressed

as μmol of Trolox/g CUPRAC (cupric ion reducing antioxidant capa-

city): CUPRAC was measured following the procedure described by

Apak et al. [39] with some modiﬁcation. Brieﬂy, 100 μL of each che-

mical solution was mixed with 900 μL bidistilled water, 1 mL acetate

buﬀer solution (1 mmol/L, pH: 7.0), 1 mL CuCl2 (10 mmol/L) and 1 mL

8. Conclusion

.5 mmol/L neocuproine to a ﬁnal volume of 4 mL. The reaction mix-

This study reports the conventional and successfully developed

microwave and ultrasound assisted synthesis of novel analogues of

azole class antifungals. The structures of new compounds were con-

ﬁrmed by FT-IR, ¹H NMR, C NMR, mass spectroscopy and elemental

analysis techniques. The antimicrobial, antioxidant, antiurease and α-

glucosidase activity screening studies were also performed in the study.

The antimicrobial screening suggests that compounds 5a-d and 6a-d

displayed varying degrees of inhibitory eﬀects on the growth of the

selected Gram (+), Gram (−) bacteria and micobacteria Mycobacterium

smegmatis (Ms). 8 of newly synthesized compounds, 7c, 7e, 7h, 8, 9, 10,

11a, 13a exhibited antioxidant capacity for DPPH, FRAP and/or

CUPRAC. Moreover, 7a, 7c, 11a, 11b and 12b showed urease inhibi-

tion ranging from 0.16 ± 0.02 to 96.75 ± 0.06 and compounds 7a,

7c, 7d, 7f, 7h, 7i, 7k, 12a and 13c which may be regarded as new

analoques of triazole and conazole class antifungals, exhibited good

inhibitory eﬀect at various IC50 values ranging from 64.56 ± 1.46 to

2584.56 ± 10.98. Based on the biological activity results, it is con-

cluded that these newly synthesized compounds can be regarded as new

drug candidates displaying antibacterial, antiurease, anti α-glucosidase

and/or antioxidant activity.

ture was then incubated in the dark for 30 min at room temperature and

the absorbance of the reaction mixture was measured at 450 nm against

a water blank. Trolox was used as the standard calibration curves and

the results were expressed as μmol Trolox equivalent per g.

. α-Glucosidase inhibition assay [40]

The enzyme and the substrate solution was prepared by dissolving

−

Saccharomyces cerevisiae α-glucosidase in 0.01 mol·l

phosphate buﬀer (pH 7). Diluted enzyme solution (10 mL), test samples

potassium

(

0.016–2 mL, in DMSO) and buﬀer solution (90 mL) were mixed in test

tubes. After pre-incubating for 20 min at 37 °C, PNPG (10 mL,

−

1.5 mg mL ) was added to start the enzymatic reaction measured by a

UV spectrophotometer. The increment of absorption at 405 nm is based

on the hydrolysis of PNPG. Controls without enzyme or without sub-

strate were included. The acarbose was used as reference and averages

of three replicates were presented. The inhibition percentage (%) was

calculated by the equation: [Abs. sample/Abs. blank] × 100 (%), where

Abs. blank represents the absorbance of the blank with the same vo-

lume DMSO. The IC50 value was determined as the concentration of

compound giving 50% inhibition of maximal activity.

9. Statistical analysis

. Urease inhibition assay [41]

Triplicate measurements (n = 3) were performed during the mea-

surements of antioxidant capacity (AC) of the compounds. The AC va-

lues were presented as mean ± pooled Standard deviation

(mean ± SD). For statistical evaluation, one-way ANOVA and

Duncan’s multiple range test for comparison among the means at sig-

niﬁcance level of P < 0.05 (IBM SPSS Statistics V22.0) were employed.

A statistical software package (XLSTAT version 2015.6) using ADDIN-

SOFT (Damremont, Paris, France) was also employed to perform prin-

cipal component analysis (PCA).

Urease activity was determined by using Weatherburn method. This

method is based on the determination of the amount of ammonia re-

leased from urea by the action of urease enzyme by using indo phenol

method that has an absorbance of 625 nm. Shortly, reaction mixture

contained 5 µL of urea solution (100 mM), 40 µL of Jack bean urease

and 5 µL of buﬀer (0.01 M KH

2 4

PO , 1 mM EDTA and 0.01 M LiCl; pH

8.2). After incubation at 35 °C 15 min, 750 µL of phenol reagent (1% w/

A. Mermer et al.

Bioorganic Chemistry 81 (2018) 55–70

Acknowledgements

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