Bioorganic Chemistry 101 (2020) 103956

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

Design, synthesis, mechanistic studies and in silico ADME predictions of T

benzimidazole derivatives as novel antifungal agents
Martha M. Morcossa, , El Shimaa M.N. Abdelhafezb, , Reham A. Ibrahemc,
⁎ ⁎

Hamdy M. Abdel-Rahmana,d, Mohamed Abdel-Azizb, Dalal A. Abou El-Ellae

a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, 62513 Beni-Suef, Egypt
b
Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
c
Department of Microbiology and Immunology, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
d
Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, 71526 Assiut, Egypt
e
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt

ARTICLE INFO ABSTRACT

InChIKeys: Herein, novel three series of benzimidazole scaffold bearing hydrazone, 1,2,4-triazole and 1,3,4-oxadiazole
DPPNEVOLBFKREL-MXAYSNPKSA-N moieties 1-3, 4a-j, 6a-c and 7 derivatives were designed, synthesized and evaluated for their antimicrobial
RLJULKKUVQJSKV-OYKKKHCWSA-N activity. The structures of the prepared compounds were assigned using different spectroscopic techniques such
BVFOVKOPLPVONA-MYKKPKGFSA-N as IR, 1H NMR, 13C NMR and elemental analyses. Compounds 3, 4a, 4e and 4f exhibited remarkable antifungal
JZZFSHWYBPFBMO-QRVIBDJDSA-N
activity against C. albicans and C. neoformans var. grubii with MIC values ranging from 4 to 16 μg/mL.
XMTCTWLYMUWJBV-FMCGGJTJSA-N
CIUBNLKSDHWNRM-UUYOSTAYSA-N
Furthermore, they were not cytotoxic against red blood cells and human embryonic kidney cells at concentration
ZSIJXQVSXPRDCG-XKZIYDEJSA-N up to 32 μg/mL. The study was expanded to forecast the mechanism of action of the prepared compounds and
FEHWFBQVECJEEZ-UUYOSTAYSA-N determine sterol quantitation method (SQM) by spectrophotometric assay. On the other hand, compound 4e
ZBDGZWCMRKHABS-YVNNLAQVSA-N showed the highest inhibitory activity against lanosterol 14α-demethylase (CYP51) with IC50 value = 0.19 μg/
KMSMQKLBYVCZSK-FMCGGJTJSA-N mL compared to fluconazole as reference IC50 value = 0.62 μg/mL. Also, compounds 4d and 4f exhibited mild to
HPAUFWVLUFUWFB-UHFFFAOYSA-N moderate antibacterial activity. Moreover, molecular docking of the active target compound 4e in active site of
HJSAHZOISANHHR-UHFFFAOYSA-N lanosterol 14α-demethylase (CYP51) revealed that docking scores and binding mode are comparable to that of
OKXVTZXULHQNMA-UHFFFAOYSA-N
co-crystallized ligand confirming their antifungal activity. In silico ADME prediction investigations also fore-
LAFZVLYFBAMBON-UHFFFAOYSA-N
casting the drug-like characters of these compounds.
GWPNRURGFXJQHM-UHFFFAOYSA-
NKeywords:
Benzimidazole
Antimicrobial activity
Molecular docking
ADME prediction

1. Introduction antibiotics: amphotericin B), 5. inhibition of nucleic acid synthesis,

antimetabolites (5-fluorocytosine) and 6. inhibition of squalene epox-
In the last decade, the fungal infections are considered the main idase (terbinafine and naftifine) [4,5].
cause of morbidity and mortality with high occurrence in im- Benzimidazole is a building block of heterocycles which plays an
munosuppressed individuals, especially in patients subject to cancer essential role in medicinal chemistry for discovery of novel therapeutic
chemotherapy or organ transplantation and patients with AIDS [1,2]. agents [6]. Benzimidazole is called ‘privileged structure’ due to their
Concerning mechanism of action for antifungal agents; they are divided wide recurrence in numerous bioactive compounds including: anti-
into six classes including: 1. inhibition of fungal ergosterol synthesis cancer [7–10], antimicrobial [11–14], antifungal [15–19], analgesic
(azoles are widely used for treatment of invasive fungal infections (IFIs) and anti-inflammatory [20,21], antiviral [22–24], anthelmintic
[3] such as ketoconazole, itraconazole, and fluconazole), 2. inhibition [25,26], antihypertensive [27–31], antitubercular [32–35], anti-oxi-
of glucan synthesis (caspofungin and echinocandins), 3. inhibition of dant [36–38], anti-histaminic [39] and antiulcer activities [40].
chitin synthesis (nikkomycin), 4. ergosterol disruptors (polyenes Currently, many antifungal drugs containing benzimidazole core are

⁎
Corresponding authors.
E-mail addresses: [email protected] (M.M. Morcoss), [email protected] (E.S.M.N. Abdelhafez).

https://doi.org/10.1016/j.bioorg.2020.103956
Received 8 March 2020; Received in revised form 17 May 2020; Accepted 18 May 2020
Available online 22 May 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

at δ 10.44 ppm attributed to COOH proton which is exchangeable with

D2O and seven aromatic protons at δ 6.76–8.45 ppm. Esterification of
compound 1 with methanol and concentrated sulfuric acid afforded the
corresponding methyl 2-(4-nitrophenyl)-1H-benzimidazole-5-carbox-
ylate 2. IR spectra of compound 2 revealed shifting of C]O group to
1691 cm−1 due to ester formation and disappearance of OH peak.
1
HNMR spectrum of compound 2 showed singlet peak at 3.87 ppm
related to CH3. Furthermore, reaction of the ester 2 with hydrazine
monohydrate afforded the corresponding hydrazide 3 [11]. IR spectra
of 3 exposed forked peak at 3439, 3421 cm−1 and 3306 cm−1 related to
NH2 and NH protons, respectively. 1H NMR spectra of 3 showed the
disappearance of the methyl protons signal and the appearance of
singlet signals at δ: 5.66 and 9.69 ppm assigned for NH2 and NH, re-
spectively (Scheme 1).
Moreover, the reaction of hydrazide 3 with the different aromatic
aldehyde afforded the corresponding hydrazones 4a-j [7,44]. IR spectra
of hydrazone characterized by disappearance of forked peak of NH2. 1H
Fig. 1. Some marketed antifungal drugs containing benzimidazole scaffold. NMR spectral data revealed one singlet assigned to N]CH proton of the
imine group at 8.50–8.86 ppm. Also, the 1H NMR spectra of eCONH
available in market such as: benomyl, carbendazim, fuberidazole and revealed a signal appeared as singlet at 11.86–12.14 ppm exchangeable
thiabendazole (Fig. 1). with D2O and disappearance of forked peak. 13C NMR spectra of com-
Ergosterol biosynthesis can be inhibited by azoles through inhibi- pounds 4a-j exhibited two distinctive peaks at δ: 161.15–164.25 related
tion of lanosterol 14α-demethylase, which is a main constituent of to (C]N) and 171.02–172.60 related to (C]O). On the other hand, the
fungal cytoplasmic membranes and regulate biological process of reaction of hydrazide 3 with appropriate isothiocyanate resulted in
membrane asymmetry, integrity and fluidity [41]. This inhibition takes substituted thiosemicarbazides derivatives 5a-c in a good yield. Addi-
place with aid of azole nucleophilic nitrogen heterocyclic ring in the tion of sodium hydroxide solution to compounds 5a-c afforded the
active site of lanosterol 14α-demethylase (CYP51). It is the sixth ligand corresponding triazoles 6a-c [4]. IR spectra for compounds 6a-c
of the heme ferric ion and the binding of the CYP51 polypeptide showed peaks at 3214–3361 cm−1 and 1501–1503 cm−1 related to NH
structure with the azole drug side chains [42]. triazole and C]S, respectively. 1H NMR spectra of compounds 6a-c
Literatures survey revealed that compounds with benzimidazole exhibited a singlet signal at δ = 14.03–14.20 ppm related to NH proton
moiety attached to hydrazone [43,44], 1,2,4-triazole [4,45] and 1,3,4- of triazole and the aromatic protons appeared at δ: 6.70–8.14 ppm
oxadiazole derivative [15] manifested antifungal activities against (Scheme 2). 13C NMR spectra of compounds 6a-c showed characteristic
Candida albicans (Fig. 2). The validated antifungal activity of these peaks at δ: 167.31–169.05 ppm related to (C]S).
compounds superintends our design of novel structures containing hy- On the other hand, the reaction of hydrazide 3 with carbon disulfide
drazone, triazole and oxadiazole derivatives linked to benzimidazole in ethanolic potassium hydroxide gave the corresponding 1,3,4-ox-
moiety in a single structure framework as illustrated by the structures in adiazole-s-thione 7 (Scheme 2). 1H NMR of compound 7 exhibited a
(Fig. 2) for the purpose of synergism or decreasing toxicity. The de- singlet signal at δ: 11.43 ppm attributed to NH proton of oxadiazole
signed structures are of different substituents either electron with- ring which is exchanged with D2O [47] (Scheme 2). 13C NMR spectrum
drawing group (EWG) or electron donating group (EDG) in order to of 7 characterized by peaks at δ: 161.18 corresponding to (C]S).
explore the activity and performing a reasonable structure activity re-
lationship study. 2.2. Biological evaluation
Additionally, screening the antimicrobial activity as antifungal for
the proposed compounds against C. albicans and C. neoformans var. 2.2.1. Antimicrobial activities
grubii and as antibacterial against five bacterial strains: Methicillin The antifungal and antibacterial screenings were carried out at CO-
Resistant S. aureus (MRSA) (Gram positive), E. coli, K. pneumoniae, A. ADD (The Community for Antimicrobial Drug Discovery), sponsored by
baumannii and P. aeruginosa (Gram negative) were carried out. the Wellcome Trust (UK) and The University of Queensland (Australia).
Moreover, the mechanism of action of the prepared compounds was All compounds were examined at 32 μg/mL for their antifungal activity
investigated. against two fungal species, C. neoformans and C. albicans, using fluco-
nazole as a positive control. The antibacterial activity against five
bacterial strains: Methicillin Resistant S. aureus (MRSA) (Gram posi-
2. Results and discussion tive), E. coli, K. pneumoniae, A. baumannii and P. aeruginosa (Gram ne-
gative) using ceftriaxone as a positive control. Besides, compounds
2.1. Chemistry exhibited significant growth inhibition % as antibacterial (20–60%) and
as antifungal (66–100%) at 32 μg/mL against of the tested bacterial and
The pathways supposed for synthesis of the purposed compounds 1- fungal species, respectively were assayed for their minimum inhibitory
3, 4a-j, 6a-c and 7 were shown in Schemes 1 and 2. The starting 2-(4- concentrations (MIC) and the results were illustrated in (Table 1).
nitrophenyl)-1H-benzimidazole-5-carboxylic acid 1 was synthesized by Results in (Table 1) revealed that compounds 3, 4a, 4e and 4f ex-
the condensation of 3,4-diaminobenzoic acid with 4-nitrobenzaldehyde hibited remarkable antifungal activity against C. albicans with MIC
and Na2S2O5 in DMF [46]. Also, synthesis of compound 1 was per- values ranging from 4 to 16 μg/mL. The results indicated that acid 1 or
formed using microwave heating which has the advantages of sig- ester 2 has weak antifungal activity. Converting to hydrazide 3 gave
nificantly reducing reaction time from several hours to few minutes compound with moderate antifungal activity with MIC value of 16 μg/
with improved yield, using limited amount of DMF solvent to minimize mL. Otherwise, the Schiff base derivatives 4a, 4e and 4f revealed the
hazards and pure product. IR spectra of compound 1 revealed the highest activity against C. albicans with MIC values = 4, 16 and 8 μg/
prescence of C]O at 1654 cm−1. In addition, a dragged peak char- mL, respectively.
acterized for OH of the COOH group was observed at 2653–3345 cm−1. The results revealed that the group of hydrazones 4a-j with the
Moreover, 1HNMR spectra of 1 revealed the presence of a singlet signal unsubstituted phenyl ring or electron withdrawing substituted are more

2
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

Fig. 2. Design of target compounds 4a-j, 6a-c and 7 were synthesized as antifungal agents.

effective than electron donating phenyl groups. Cyclization of hy- non-cytotoxic. Nevertheless, compound 4a has HC10 = 15.16 μg/mL
drazide 3 into their 1,2,4-triazoles 6a-c or 1,3,4-oxadiazole 7 is asso- (Table 1).
ciated to the decreasing in the antifungal activity.
On the other hand, all the screened compounds showed mild anti-
2.2.3. Sorbitol assay (Inhibition of fungal cell wall synthesis)
bacterial activity except compound 4f that showed moderate anti-
The sorbitol assay involved the determination of the MIC in the
bacterial activity against MRSA with MIC value = 32 μg/mL
presence and absence of 0.8 M sorbitol which act as an osmoprotective
agent. Compounds targeting the fungal cell wall possess a characteristic
feature that their antifungal effects are inverted in a medium containing
2.2.2. Cytotoxicity against human embryonic kidney cell line and hemolysis
an osmotic stabilizer like sorbitol. Cells protected with sorbitol can
of human red blood cells effects
grow in the presence of drugs that inhibit synthesis of cell wall, but the
This screening method was also performed at CO-ADD institution.
growth would be inhibited in absence of sorbitol. It was found that
Cytotoxicity against a human embryonic kidney cell line and hemolysis
compounds 4a, 4e and 4f MICs against C. albicans ATCC 90028 and
of human red blood cells were done to ensure the safety margin for the
clinical strains were identical (Table 2) either in the absence or pre-
potent compounds and the results were illustrated in (Table 1). Samples
sence of sorbitol. Similar MICs for samples with and without sorbitol
were classified cytotoxic when their concentration at 50% cytotoxicity
indicate that the cell wall is not the target for the tested compounds, but
and HC10 is concentration at 10% hemolysis exhibited higher than
these compounds may interact by another mechanism
32 μg/mL (CC50, HC10 ≤ 32 μg/mL).
From the obtained results, the tested compounds 3, 4a, 4d, 4e and
4f showed CC50 > 32 μg/mL against the human embryonic kidney cell 2.2.4. Ergosterol binding assay
line and exhibited HC10 > 32 μg/mL, so, they are considered safe and This test was used to investigate the ability of compounds 4a, 4e

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M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

Scheme 1. Synthesis of intermediates 1–3 and target compounds 4a-j. Reagent and condition: (a) DMF, Na2S2O5; (b) Conc. H2SO4, methanol; (c) NH2.NH2·H2O; (d)
different substituent aldehyde, few drops of CH3COOH.

and 4f to form complex with ergosterol in the fungal cell membrane ergosterol. This finding indicates that the mechanism of action of these
affecting its integrity and its function to control inflow and outflow compounds does not involve binding with ergosterol. On the contrary,
[48]. The ergosterol binding assay test showed that the tested com- the MIC values of Amphotericin B (is known to act by ergosterol
pounds 4a, 4e and 4f MICs were equal in medium with and without binding mechanism of cell membrane) increased about 64 times in the

Scheme 2. Synthesis of target compounds 6a-c and 7. Reagent and condition: (a) Different isothiocynates, ethanol; (b) NaOH; (c) KOH, CS2, ethanol, reflux 24 h.

4
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

Table 1
The antimicrobial activities (growth inhibition % at 32 μg/mL concentration, MIC, cytotoxicity and hemolysis) of the examined compounds.
Code Gram positive Gram negative bacteria Fungi Cytotoxicity &
bacteria hemolysis

MRSA E. coli K. pneumonia P. aeruginosa A. baumannii C. albicans C. neoformans var. CC50 HC10
grubii (μg/ (μg/
mL)d mL)e
GI%a MICb GI% MIC GI% MIC GI% MIC GI% MIC GI% MIC GI% MIC

c c c c c c
1 −4.87 NT 11.18 NT 5.43 NT 15.37 NT 8.67 NT 4.81 NT 6.43 NTc NTc NTc
2 −0.07 NTc 10.04 NTc 2.80 NTc 22.52 NTc 11.30 NTc 3.41 NTc 18.15 NTc NTc NTc
3 0.25 > 32 0.55 > 32 6.56 > 32 23.86 > 32 16.18 > 32 101.10 16 101.90 > 32 > 32 > 32
4a 29.44 > 32 −15.13 > 32 −9.99 > 32 11.75 > 32 10.86 > 32 99.15 4 104.1 16 > 32 15.16
4b − NTc −17.25 NTc 2.68 NTc 0.77 NTc −25.40 NTc 54.55 NTc 98.81 NTc NTc NTc
73.47
4c 22.28 NTc −12.70 NTc 4.88 NTc 20.35 NTc 2.60 NTc 38.02 NTc 32.86 NTc NTc NTc
4d 59.01 > 32 6.51 > 32 29.25 > 32 22.78 > 32 5.02 > 32 88.99 > 32 32.59 > 32 > 32 > 32
4e 8.27 > 32 −23.65 > 32 −16.03 > 32 34.08 > 32 −54.66 > 32 95.56 16 104.91 16 > 32 > 32
4f 64.69 32 −8.22 > 32 −1.12 > 32 21.69 > 32 −17.53 > 32 64.52 8 33.94 > 32 > 32 > 32
4g 28.30 NTc − 11.95 NTc 5.35 NTc 24.42 NTc −0.57 NTc 31.45 NTc −25.69 NTc NTc NTc
4h 20.84 NTc 25.51 NTc 27.67 NTc 46.59 NTc 22.60 NTc 48.70 NTc 21.30 NTc NTc NTc
4i 33.79 > 32 1.05 > 32 18.03 > 32 17.65 > 32 14.06 > 32 84.45 > 32 −41.47 > 32 NTc NTc
4j 42.68 > 32 −8.64 > 32 11.25 > 32 19.75 > 32 3.44 > 32 32.38 > 32 101.81 > 32 NTc NTc
6a 18.99 NTc 4.47 NTc 19.23 NTc 17.08 NTc 7.75 NTc 7.14 NTc 98.99 NTc NTc NTc
6b 18.46 NTc 8.02 NTc 21.58 NTc 19.38 NTc 25.37 NTc 74.04 NTc −9.61 NTc NTc NTc
6c 17.44 NTc 5.02 NTc 22.1 NTc 10.20 NTc 16.22 NTc 4.44 NTc 99.80 NTc NTc NTc
7 25.75 NTc 5.76 NTc 22.87 NTc 22.53 NTc 23.05 NTc 66.43 NTc −47.11 NTc NTc NTc

Ceftriaxone – 32 – 0.125 – 16 – 32 – 32 – NTc 0.125 – NTc – –

Fluconazole – NTc – NTc – NTc – NTc – NTc – – 8 – –

a
Growth inhibition %;
b
minimum inhibitory concentration;
c
not tested;
d
CC50 is the concentration at 50% cytotoxicity;
e
HC10 is the concentration at 10% hemolysis.

Table 2 biosynthesis in candida isolates (Susceptible to it) then complete in-

MICs (µg/ml) in the presence of sorbitol (0.8 M) and ergosterol (400 μg/ml) hibition in ergosterol biosynthesis at the higher concentrations was
against Candida albicans ATCC 90028 and clinical isolates. observed due to inactivation of lanosterol 14 α-demethylase. This in-
Strain Drug Sorbitol Ergosterol dicated fungistatic effect of ketoconazole.
Data in (Table 3) revealed that the tested compounds 4a, 4e and 4f
Absence Presence Absence Presence efficiently inhibit the ergosterol biosynthesis in C. albicans isolates ei-
ther clinical or standard strains. This was observed by the decrease of
C. albicans ATCC 4a 4 4 4 4
90028 4e 8 8 8 8 ergosterol percentage calculated upon increasing the concentrations of
4f 16 16 16 16 the tested compounds till complete absence of ergosterol. The compa-
Amphotericin B – – 0.5 64 tible results of tested compounds and ketoconazole suggested that these
C. albicans vaginal 4a 4 4 4 4 compounds act by inhibiting the biosynthesis of ergosterol in fungal cell
4e 8 8 8 8 membrane by binding to their cytochrome P-450 sterol 14α-demethy-
4f 16 16 16 16 lase like the reference drug (Ketoconazole).
Amphotericin B – – 0.5 64

C. albicans oral 4a 2 2 2 2
4e 4 4 4 4 2.2.6. Lanosterol 14α-demethylase (CYP51) inhibition activity
4f 8 8 8 8 Fluconazole is the most widely used antifungal drug which inhibit
Amphotericin B – – 1 128 lanosterol 14α-demethylase (CYP51) and ergosterol biosynthesis in
fungal cell membrane. This test was carried out at confirmatory diag-
nostic unit VACSERA – Egypt. Fluconazole was used as a reference drug
presence of ergosterol rather than in absence of it. This is attributed to
and the IC50 values (μg/mL) were displayed in (Table 4). Obtained
the binding of the control drug (Amphotericin B) to the free (added)
results revealed that 4e showed preferable activity (IC50 of 0.19 μg/mL)
ergosterol, so higher concentration of Amphotericin B is required to
than the reference drug, fluconazole (IC50 of 0.62 μg/mL). On the other
inhibit fungal growth and that was not happen towards the tested
hand, 4f showed moderate activity than 3 and 4a with IC50 = 2.72,
compounds (Table 2).
8.90 and 14.18 μg/mL, respectively. The structure activity relationship
exhibited that electron withdrawing group at position 4 of phenyl hy-
drazone ring showed better activity than unsubstituted derivatives 4a
2.2.5. The sterol quantitation method (SQM) by spectrophotometric assay.
or hydrazide 3.
The objective of this method was used to demonstrate if the tested
compounds 4a, 4e and 4f causing disorders in the ergosterol biosyn-
thetic enzymes (squalene epoxidase and lanosterol 14α-demethylase) 2.3. Molecular docking
leading to reduction in ergosterol content in a dose dependent manner.
This was done by using Ketoconazole (reference drug that is known to Molecular modeling study was performed through docking of the
inhibit the synthesis of ergosterol) at different concentration. most active compound 4e in the 14α-lanosterol demethylase, using C-
Results in (Table 3) showed that ketoconazole decreased ergosterol Docker protocol in Discovery Studio® 2.5 Software.

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M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

Table 3
Ergosterol percentage (%) in C. albicans isolates treated with different concentrations of tested compounds (µg/ml).
Drug conc. (µg/ml) C. albicans ATCC 90028 C. albicans vaginal C. albicans oral

a
4a 4e 4f Ket.* 4a 4e 4f Ket. 4a 4e 4f Ket.

0 1.4 1.4 1.5 1.4 1.4 1.4 1.5 1.6 1.6 1.7 1.7 1.7
1 0.9 0.8 1.1 1 0.5 1 1.2 1.2 0.8 0.9 1 1
4 0 0.08 0.3 0.4 0 0.3 0.5 0.5 0 0.1 0.4 0.7
8 0 0 0.1 0.1 0 0.02 0.1 0.1 0 0.05 0.2 0.2
16 0 0 0 0.05 0 0 0 0.06 0 0.01 0.01 0.09
32 0 0 0 0 0 0 0 0 0 0 0 0.001
64 0 0 0 0 0 0 0 0 0 0 0 0

a
*Ketoconazole.

Table 4 less orally active. Also, it has been proposed that the number of hy-
IC50 of compounds 3, 4a, 4e, 4f and fluconazole on CYP51. drogen bonding groups could be replaced with polar surface area (PSA)
Code (CYP51) IC50 ± SEM μg/ml as a factor and involved in calculation of percentage absorption (%ABS)
as it is inversely proportional to %ABS
3 8.90 ± 0.22
4a 14.18 ± 0.34 %ABS = 109 0.345 PSA
4e 0.19 ± 0.004
4f 2.72 ± 0.06 Compounds with PSA of less than 140 A2 and 10 or fewer rotatable
Fluconazole 0.62 ± 0.015
bonds should exhibit high oral bioavailability [53].
Herein, we used Pre-ADMET [54], Molinspiration [55], Molsoft
Molecular docking is in silico computer based algorithm used to [56], and SwissADME [57] softwares for forecasting the pharmacoki-
estimate two main terms; the first is to evaluate the appropriate pose netic parameters of the most active compounds.
(orientation & conformation) of the target compounds inside the The results narrative in (Table 5) exhibit that compounds stratify to
binding site in comparison to that of the X-ray crystallographic enzyme- Lipinski's rule, with MW range from 297.27 to 475.45 (< 500), LogP
substrate complex and the second is the computation of the docking values from 0.91 to 3.94 (< 5), HBD from 2 to 3 (< 5) and HBA from 5
scoring (C-docker energy), which is the evaluated protein ligand in- to 8 (< 10). So, they should theoretically manifest good oral absorption
teraction energy. The X-ray crystal structure of posaconazole bound to and differences in their bioactivity can‘t be assigned to to this property.
the 14α-lanosterol demethylase enzyme binding site (PDB: ID 5FSA) Besides, the compounds exhibited number of rotatable bonds values
active site was obtained from protein data bank at research collabora- from 4 to 9 (< 10) and topological PSA values of 129.62 and 143.65 A2
tion for Structural Bioinformatics (RSCB) protein database [PDB][15]. (< 140 A2) with consequent percentage oral absorption from 64.28 to
Compound 4e interacted with HEM as HB donor with Ser 507 as 59.45%, indicating good permeability, absorption and transport via
reported C-docker interaction energy = −53 in comparison to co- biological membranes.
crystallized ligand can interact with HEM by C-docker interaction en- Furthermore, Molsoft software was applied to appreciate the drug-
ergy = −70 (Figs. 3 and 4). likeness model score and solubility for compounds (Table 6). Absorp-
tion and distribution characteristics can be altered by aqueous solubi-
lity. In this circumstances, these compounds accomplished the specifi-
2.4. Computational analysis cations of solubility with values from 0.01 to 3.97 mg/L (> 0.0001 mg/
L); the more positive drug-likeness model scores, the more likely it is to
2.4.1. Prediction of physicochemical properties, pharmacokinetic and drug- be drug molecule. A positive model-score was anticipated for com-
likeness profile in silico pound 4j (0.16) while that for other compounds was negative (-0.06 to
In clinical trials of new drug elected is considered so complicated −0.61).
due to inappropriate ADME (absorption, distribution, metabolism and Additionally, in silico study of the subsequent pharmacokinetic
excretion) possessions, in addition to the costs of development a new parameters was fulfilled using Pre-ADMET software: Blood brain barrier
drug. Therefore, assessment of pharmacokinetic possessions of new partition coefficient (BBB), Caco2 (human colon adenocarcinoma)
drug elected is a vital step in the process of drug development that can permeability coefficient, inhibition of cytochrome P4502D6 (CYP2D6),
direct lead optimization efforts into recovered analogs [49]. Nowadays, MDCK (Madin-Darby canine kidney cells) permeability coefficient,
in silico ADME screens can be used to pick out the most promising Human intestinal absorption (HIA) and human plasma protein binding
compounds and minimize the risk of drug attrition in late stage [50]. (PPB). The outcomes of the predicted ADME parameters are shown in
There should be an equilibrium between pharmacodynamic and phar- (Table 7). The results from compounds exhibited medium CNS ab-
macokinetic properties to get desirable in vivo response. Also, predic- sorption range from 0.22 to 1.17 (≤0.1–2) except 4j showed low CNS
tion of volume of distribution, brain penetration, oral bioavailability absorption 0.08 (< 0.1); investigated compounds exhibited medium to
and clearance give more details about regimen and drug dose [51]. low cell permeability in Caco-2 and MDCK models range from 3.82 to
Throughout methods of virtual screening, a lot of parameters are stu- 15.88 nm/s and 0.05 to 15.76 nm/s, respectively. As well as, they were
died, such as partition coefficient, drug solubility S, cell permeability, non-inhibitors of CYP2D6 enzyme and thus may pretend no interactions
human intestinal absorption HIA, polar surface area PSA and drug with CYP2D6 inhibitors and/or inducers.
likeness score. An available orally drug elected in agreement with Li- Moreover, they manifested high human intestinal absorption values
pinski's rule if molecular weight is lower than 500, LogP is not more 91.06 to 93.87% (≤80%) indicating very well-absorbed compounds
than 5, number of hydrogen bond acceptor is less than 10 and number except 3 showed moderate absorption 78.82% (30–79%). The ex-
of hydrogen bond donor is less than 5 [52]. Number of rotatable bonds amined compounds were found to be highly bound to human plasma
is used to represent molecular flexibility which plays a vital role in oral proteins from 91.69 to 93.63% (≥90%), except 3 and 4j that were low
bioavailability; if the the molecule is flexible, that indicate the drug is bounded to plasma protein 69.01, 88.56%, respectively.

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M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

Fig. 3. 2D& 3D interacting mode of lead comp. (Cyan blue) in the active region of 14α-lanosterol demethylase (PDB code: 5FSA) and HEM with red (C-DOCKER
INTERACTION ENERGY = −70).

3. Conclusion 4. Experimental

Evaluation of antimicrobial activity of different three series of 4.1. Chemistry

benzimidazole bearing hydrazones 4a-j, 1,2,4-triazoles 6a-c and 1,3,4-
oxadiazole 7 were synthesized and identified with different spectro- Chemicals and solvents were purchased from the Aldrich Chemical
scopic techniques. The antifungal activity of the prepared compounds Company (Milwaukee, WI), Cornell Lab and El Nasr pharmaceutical
was evaluated. The most robust compounds 3, 4a, 4e and 4f revealed chemicals companies, Cairo, Egypt. Progress of the reaction was mon-
promising antifungal activity with MIC values 16, 4, 16 and 8 μg/mL, itored by TLC sheets precoated with UV fluorescent silica gel MERCK 60
respectively. They were safe without cytotoxicity against human em- F254 that were visualized by U.V. lamp. Melting points were determined
bryonic kidney cell line and don’t cause 10% red blood cell hemolysis at on Gallenkamp melting point apparatus and were uncorrected. IR
concentration up to 32 μg/mL. Compound 4e can be evaluated as a hit spectra were determined as KBr discs on Schimadzu FT-IR 8400S
for further screenings to acquire more active antifungal agents due to its spectrophotometer and expressed in wave number (υmax) cm−1 at
higher potency with IC50 value = 0.19 μg/mL to inhibit lanosterol 14α- Faculty of Pharmacy, Nahda University, Beni-Seuf, Egypt. 1H NMR
demethylase (CYP51) than fluconazole with IC50 value = 0.62 μg/mL. spectra were carried out on Burker 400 MHz spectrophotometer, at
Therefore, it can be concluded that compound 4e is the most potent Nuclear Magnetic Resonance Center, Faculty of Pharmacy, Beni Suef
compound that should be taken into consideration for further study. University and Mansoura University, Egypt, using TMS as internal
Regarding the antibacterial activity of compounds 4d and 4f showed standard and DMSO‑d6 as a solvent. Chemical shifts were recorded in
moderate activity. ppm on δ scale and coupling constants (J) in Hertz (HZ). Signals are

7
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

Fig. 4. 2D& 3D interacting mode of compound 4e (Purple) in the active region of 14α-lanosterol demethylase (PDB code: 5FSA) and HEM with red Showed HB donor
with Ser 507 as reported C-docker interaction energy = −53.

designed as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, Center, Faculty of Pharmacy, Beni Suef University and Mansoura
multiplet; brs, broad singlet. 13C NMR spectra were carried out on University, Egypt, using TMS as internal standard and chemical shifts
Burker at 100 MHz spectrophotometer, at Nuclear Magnetic Resonance were recorded in ppm on δ scale. Samples were dissolved in DMSO‑d6.

Table 5
Physicochemical and lipophilicity of the most active compounds using SwissADME & Molinspiration software.
Code Lipophilicity consensus log P Physicochemical properties

MWa g/mol Heavy atoms Aromatic heavy atoms Rot. bond H‐bond acc. H‐bond don. MRb TPSAc (A2) % ABSd

3 0.91 297.27 22 15 4 5 3 81.25 129.62 64.28

4a 2.93 385.38 29 21 6 5 2 111.52 115.96 69.00
4d 2.94 428.44 32 21 7 5 2 125.73 119.20 67.88
4e 3.46 419.82 30 21 6 5 2 116.53 115.96 69.00
4f 3.94 454.27 31 21 6 5 2 121.54 115.96 69.00
4i 2.94 415.40 31 21 7 6 2 118.01 125.19 65.81
4j 2.88 475.45 35 21 9 8 2 131.00 143.65 59.45

Abbreviation: aMW, molecular weight; bMR, molar refractivity; cTPSA, topological polar surface area; d%ABS: percentage of absorption.

8
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

Table 6 mixture was heated under reflux for 17 h. The reaction mixture was
Lipinski drug likeness of the most active compounds using Molsoft & cooled. The crude was then poured on distilled water (50.00 mL) and
SwissADME software. neutralized with Na2CO3 solution (5%). The formed precipitate was
Code Sa (mg/L) Drug likeness model Lipinski Bioavailability score filtered off and recrystallized from ethanol to afford compound 2.
score violations As Buff powder, 67% yield, m.p. 200–202 °C, IR (KBr, υmax cm−1):
3307 (NH), 3030 (CH aromatic), 2954 (CH aliphatic), 1691 (C]O),
3 3.97 −0.61 0 0.55
1605 (C]C), 1451 (C]N), 1316–1518 (NeO); 1HNMR (DMSO‑d6,
4a 0.05 −0.31 0 0.55
4d 0.03 −0.15 0 0.55 400 MHz, δ ppm): 3.87 (s, 3H, CH3), 6.70 (d, 2H, J = 8.30 Hz, Ar-H),
4e 0.01 −0.06 0 0.55 7.56 (d, 1H, J = 8.00 Hz, Ar-H), 7.78 (d, 1H, J = 8.00 Hz, Ar-H), 7.87
4f 0.00 −0.10 0 0.55 (d, 2H, J = 8.30 Hz, Ar-H), 8.45 (s, 1H, Ar-H), 13.60 (brs, 1H, NH
4i 0.03 −0.18 0 0.55
benzimidazole, D2O exchangeable).
4j 0.09 0.16 1 0.55

a
S: solubility. 4.1.3. Synthesis of 2-(4-nitrophenyl)-1H-benzimidazole-5-carbohydrazide
3
Table 7 A mixture of compound 2 (1 mmol, 0.30 g) and hydrazine mono-
ADME data of most active compounds calculated using preADMET software. hydrate (5.00 mL, 2 mmol) in ethanol (15.00 mL) were heated till reflux
for 7 h. The mixture was cooled and poured into iced water. The ob-
Code Pharmacokinetics
tained crude solid was filtered off, washed with water, dried, and re-
BBBa Caco-2b HIAc MDCKd PPBe CYP 2D6f crystallized from absolute ethanol to afford 3.
As Yellow powder, 60% yield, m.p. 216–218 °C, IR (KBr, υmax
3 0.10 3.82 78.82 15.76 69.01 No cm−1): 3439, 3421 (forked NH2), 3306 (NH), 3037 (CH aromatic),
4a 0.31 12.49 91.13 0.11 93.63 No
1609 (C]O), 1583 (C]C), 1427 (C]N) and 1296–1489 (NeO);
4d 0.37 9.35 91.88 0.06 90.87 No 1
4e 0.66 15.88 92.63 0.06 92.47 No HNMR (DMSO‑d6, 400 MHz, δ ppm): 5.66 (s, 2H, NH2, D2O ex-
4f 1.17 13.68 93.87 0.05 93.41 No changeable), 6.67(d, 2H, J = 8.30 Hz, Ar-H), 7.49 (d, 1H, J = 8.00 Hz,
4i 0.22 7.90 91.06 0.09 91.69 No Ar-H), 7.65 (d, 1H, J = 8.00 Hz, Ar-H), 7.86 (d, 2H, J = 8.30 Hz, Ar-H),
4j 0.08 12.27 91.26 0.06 88.56 No
8.01 (s, 1H, Ar-H), 9.69 (s, 1H, NH amide, D2O exchangeable), 12.64
a
BBB: blood brain barrier penetration;
(brs, 1H, NH benzimidazole, D2O exchangeable).
b
CACO-2: permeability through cells derived from human colon adeno-
carcinoma; 4.1.4. General procedure for synthesis of N′-(substituted benzylidene)-2-(4-
c
HIA: percentage human intestinal absorption; nitrophenyl)-1H-benzimidazole-5-carbohydrazide 4a-j
d
MDCK: permeability through Madin-Darby canine kidney cells; Equimolar quantities of compound 3 (2 mmol, 0.50 g) and appro-
e
PPB: plasma protein binding; priate different substituted benzaldehydes in 25 mL of absolute ethanol
f
CYP2D6: cytochrome P450 2D6. were refluxed for 6 h in the presence of catalytic amount of glacial
acetic acid. The resulting crude solid was filtered off and recrystallized
At the Regional Center for Mycology and Biotechnology has carried out from absolute ethanol.
the elemental analysis, Al-Azhar University, Cairo, Egypt. Synthesis was
done by using synthesis reactor monowave 50 (Anton Paar GmbH, 4.1.4.1. N′-Benzylidene-2-(4-nitrophenyl)-1H-benzimidazole-5-
Austria) (Serial No.82628529). carbohydrazide 4a. Yellowish green powder, 80% yield, m.p.
207–209 °C, IR (KBr, υmax cm−1): 3426, 3393 (NH benzimidazole,
4.1.1. Synthesis of 2-(4-nitrophenyl)-1H-benzimidazole-5-carboxylic acid NH hydrazone), 3073 (CH aromatic), 1621 (C]O), 1606 (C]C), 1455
1 (C]N), 1373–1502 (NeO); 1HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.70
4.1.1.1. By conventional method:. 3,4-Diaminobenzoic acid (2.0 mmol, (d, 2H, J = 8.00 Hz, Ar-2H), 7.45–7.50 (m, 4H, Ar-H), 7.62 (d, 1H,
0.3 g) was added to 4-nitrobenzaldehyde (2.0 mmol, 0.3 g) and sodium J = 8.00 Hz, Ar-H), 7.76 (d, 2H, J = 8.00 Hz, Ar-H), 7.88 (d, 2H,
metabisulfite Na2S2O5 (2.4 mmol, 0.5 g) in DMF (30 mL). The mixture J = 8.00 Hz, Ar-H), 7.95 (d, 1H, J = 8.00 Hz, Ar-H), 8.48 (s, 1H, CH]
was heated under reflux to 120 °C for 6–12 h, then poured into ice- NH), 11.90 (s, 1H, NH, D2O exchangeable), 12.82 (s, 1H, NH
water and the formed solid was filtered off and re-crystallized from benzimidazole, D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ
ethanol to afford 1. ppm):113.65, 116.48, 121.99, 126.37, 127.27, 128.28, 129.15, 129.79,
130.06, 130.98, 133.14, 134.69, 147.10, 151.39, 164.40, 168.14,
4.1.1.2. By microwave heating. 3,4-Diaminobenzoic acid (2.0 mmol, 172.76; Elemental analysis calculated for C21H15N5O3 (M.Wt.
0.3 g) was added to 4-nitrobenzaldehyde (2.0 mmol, 0.3 g) and 385.38): C, 65.45; H, 3.92; N, 18.17; Found C, 65.71; H, 4.13; N, 18.42.
sodium metabisulfite Na2S2O5 (2.4 mmol, 0.5 g) in DMF (6 mL). The
mixture was heated using microwave radiation 120 °C for 5 min with 4.1.4.2. N′-(4-Fluorobenzylidene)-2-(4-nitrophenyl)-1H-benzimidazole-5-
stirrer speed 600 rpm, then poured into ice-water and the formed solid carbohydrazide 4b. Brown powder, 60% yield, m.p. 243–245 °C, IR
was filtered off to afford 1. (KBr, υmax cm−1): 3441, 3385 (NH benzimidazole, NH hydrazone),
As Yellowish brown powder, 88% yield, m.p. 125–127 °C, IR (KBr, 3042 (CH aromatic), 1648 (C]O), 1605 (C]C), 1452 (C]N),
υmax cm−1): 3308 (NH), 3024 (CH aromatic), 2653 (broad band OH 1394–1508 (NeO); 1HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.70 (d,
acidic), 1654 (C]O), 1608 (C]C), 1467 (C]N) and 1317–1527 2H, J = 8.40 Hz, Ar-H), 7.31 (t, J = 8.80 Hz, 2H, Ar-H), 7.59 (d, 1H,
(NeO); 1HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.76 (d, 2H, J = 8.30 Hz, J = 8.40 Hz, Ar-H), 7.76–7.80 (m, 3H, Ar-H), 7.89 (d, 2H, J = 8.40 Hz,
Ar-H), 7.67 (d, 1H, J = 8.00 Hz, Ar-H), 7.92 (d, 1H, J = 8.00 Hz, Ar-H), Ar-H), 8.11 (s, 1H, Ar-H), 8.50 (s, 1H, CH]NH), 11.87 (s, 1H, NH, D2O
8.16 (d, 2H, J = 8.30 Hz, Ar-H), 8.45 (s, 1H, Ar-H), 10.44 (s, 1H, exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): 113.36, 114.08,
COOH, D2O exchangeable). 116.26, 116.80, 118.26, 122.18, 126.85, 128.07, 128.79, 129.30,
129.51, 131.74, 146.43, 151.55, 155.21, 162.31, 173.36; Elemental
4.1.2. Synthesis of methyl 2-(4-nitrophenyl)-1H-benzimidazole-5- analysis calculated for C21H14FN5O3 (M.Wt. 403.37): C, 62.53; H, 3.50;
carboxylate 2 N, 17.36; Found C, 62.82; H, 3.75; N, 17.53.
To a solution of compound 1 (1.77 mmol, 0.50 g) in methanol
(50.00 mL), few drops of concentrated sulfuric acid was added and the 4.1.4.3. N′-(4-nitrobenzylidene)-2-(4-nitrophenyl)-1H- benzimidazole-5-

9
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

carbohydrazide 4c. Reddish brown powder, 70% yield, m.p. J = 8.00 Hz, Ar-H), 7.89 (d, 2H, J = 8.00 Hz, Ar-H), 8.09 (s, 1H, Ar-H),
293–295 °C, IR (KBr, υmax cm−1): 3390–3247 (NH benzimidazole, NH 8.39 (s, 1H, CH]NH), 9.96 (s, 1H, OH, D2O exchangeable), 11.67 (s,
hydrazone), 3028 (CH aromatic), 1633 (C]O), 1610 (C]C), 1454 (C] 1H, NH, D2O exchangeable), 12.02 (s, 1H, benzimidazole NH, D2O
N), 1382–1509 (NeO); 1HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.70 (d, exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): 114.01, 115.92,
2H, J = 8.00 Hz, Ar-H), 7.68 (d, 1H, J = 8.00 Hz, Ar-H), 7.85 (d, 1H, 116.19, 117.07, 121.81, 125.92, 127.05, 128.48, 128.67, 129.33,
J = 8.00 Hz, Ar-H). 7.95 (d, 2H, J = 8.00 Hz, Ar-H), 8.06–8.08 (m, 2H, 129.47, 147.95, 151.54, 155.09, 159.70, 164.12, 173.40; Elemental
Ar-H), 8.20 (s, 1H, Ar-H), 8.37 (d, 2H, J = 8.00 Hz, Ar-H), 8.63 (s, 1H, analysis calculated for C21H15N5O4 (M.Wt. 401.38): C, 62.84; H, 3.77;
CH]NH), 12.26 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6, N, 17.45; Found C, 63.03; H, 4.00; N, 17.80.
100 MHz, δ ppm): 114.08, 116.44, 116.60, 122.16, 124.56, 124.69,
126.39, 128.22, 128.30, 128.56, 128.78, 141.27, 145.17, 148.07,
151.58, 155.25, 164.69; Elemental analysis calculated for 4.1.4.8. N′-(2,3,4-Trihydroxybenzylidene)-2-(4-nitrophenyl)-1H-
C21H14N6O5 (M.Wt. 430.38): C, 58.61; H, 3.28; N, 19.53; Found C, benzimidazole-5 Carbohydrazide 4 h. Brown powder, 81% yield, m.p.
58.83; H, 3.53; N, 19.81. 261–263 °C, IR (KBr, υmax cm−1): 3448, 3300 (NH benzimidazole, NH
hydrazone), 3156 (OH), 3047 (CH aromatic), 1660 (C]O), 1608
4.1.4.4. N′-(4-(Dimethylamino)benzylidene)-2-(4-nitrophenyl)-1H- (C]C), 1457 (C]N), 1383–1506 (NeO); 1HNMR (DMSO‑d6,
benzimidazole-5-carbohydrazide 4d. Orange powder, 77% yield, m.p. 400 MHz, δ ppm): 6.41 (d, 1H, J = 8.00 Hz, Ar-H), 6.70 (d, 2H,
215–217 °C, IR (KBr, υmax cm−1): 3406, 3393 (NH benzimidazole, NH J = 8.00 Hz, Ar-H), 6.79 (d, 1H, J = 8.00 Hz, Ar-H), 7.60 (d, 1H,
hydrazone), 3046 (CH aromatic), 1610 (C]O), 1598 (C]C), 1452(C] J = 8.00 Hz, Ar-H), 7.77 (d, 1H, J = 8.00 Hz, Ar-H), 7.89 (d, 2H,
N), 1363–1492 (NeO); 1HNMR (DMSO‑d6, 400 MHz, δppm): 2.96 (s, J = 8.00 Hz, Ar-H), 8.12 (s, 1H, Ar-H), 8.50 (s, 1H, CH]NH), 8.54 (s,
6H, 2 CH3), 5.71 (s, 1H, Ar-H), 6.70 (d, 2H, J = 8.00 Hz, Ar-H), 6.76 (d, 1H, OH, D2O exchangeable), 9.49 (s, 1H, OH, D2O exchangeable),
2H, J = 8.00 Hz, Ar-H), 7.57 (d, 3H, J = 8.00 Hz Ar-H), 7.74 (d, 1H, 11.70 (s, 1H, OH, D2O exchangeable), 12.01 (s, 1H, NH, D2O
J = 8.00 Hz, Ar-H), 7.88 (d, 2H, J = 8.00 Hz, Ar-H), 8.33 (s, 1H, CH] exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): 108.05,
NH), 11.60 (s, 1H, NH, D2O exchangeable), 12.82 (s, 1H, NH 111.39, 114.00, 116.74, 121.63, 121.88, 122.21, 125.97, 126.42,
benzimidazole, D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ 128.65, 129.10, 133.17, 147.95, 149.07, 150.03, 151.67, 155.16,
ppm): 54.50, 111.35, 111.92, 112.27, 114.08, 114.15, 116.97, 122.12, 163.53, 172.54; Elemental analysis calculated for C21H15N5O6 (M.Wt.
127.08, 128.56, 128.91, 128.98, 148.60, 149.49, 151.53, 151.81, 433.38): C, 58.20; H, 3.49; N, 16.16; Found C, 58.51; H, 3.78; N, 16.41.
164.07, 172.23; Elemental analysis calculated for C23H20N6O3 (M.Wt.
428.45): C, 64.48; H, 4.71; N, 19.62; Found C, 64.68; H, 4.52; N, 19.85.
4.1.4.9. N′-(4-Methoxybenzylidene)-2-(4-nitrophenyl)-1H-benzimidazole-
4.1.4.5. N′-(4-Chlorobenzylidene)-2-(4-nitrophenyl)-1H-benzimidazole-5- 5-carbohydrazide 4i. Greenish brown powder, 81% yield, m.p.
carbohydrazide 4e. Brown powder, 83% yield, m.p. 210–212 °C, IR 212–214 °C, IR (KBr, υmax cm−1): 3441, 3386 (NH benzimidazole,
(KBr, υmax cm−1): 3432–3327 (NH benzimidazole, NH hydrazone), NH hydrazone), 3032 (CH aromatic), 2927 (CH aliphatic), 1650 (C]
3032 (CH aromatic), 1658 (C]O), 1610 (C]C), 1467 (C]N), O), 1606 (C]C), 1458 (C]N), 1383–1507 (NeO); 1HNMR (DMSO‑d6,
1384–1503 (NeO); 1HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.70 (d, 400 MHz, δ ppm): 3.81 (s, 3H, OCH3), 6.75 (d, 2H, J = 8.40 Hz, Ar-H),
2H, J = 8.00 Hz, Ar-H), 7.52–7.59 (m, 3H, Ar-H), 7.75–7.78 (m, 3H, 7.03 (d, 2H, J = 8.40 Hz, Ar-H), 7.69 (d, 3H, J = 8.00 Hz, Ar-H),
Ar-H), 7.89 (d, 2H, J = 8.00 Hz, Ar-H), 8.12 (s, 1H, Ar-H), 8.49 (s, 1H, 7.87–7.93 (m, 3H, Ar-H), 8.15 (s, 1H, Ar-H), 8.45 (s, 1H, CH]NH),
CH]NH), 11.94 (s, 1H, NH, D2O exchangeable), 12.75 (s, 1H, 11.82 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ
benzimidazole NH, D2O exchangeable); 13C NMR (DMSO‑d6, ppm): 55.77, 112.88, 113.87, 114.12, 114.82, 123.40, 127.42, 128.83,
100 MHz, δ ppm): 113.72, 116.62, 126.52, 128.53, 128.69, 129.08, 129.15, 129.46, 130.45, 138.59, 148.02, 153.06, 153.77, 161.28,
129.42, 129.58, 130.11, 132.80, 133.99, 134.97, 136.17, 146.02, 163.47, 171.02; Elemental analysis calculated for C22H17N5O4 (M.Wt.
151.81, 161.21, 172.78; Elemental analysis calculated for 415.41): C, 63.61; H, 4.13; N, 16.86; Found C, 63.79; H, 4.41; N, 17.10.
C21H14ClN5O3 (M.Wt. 419.83): C, 60.08; H, 3.36; N, 16.68; Found C,
59.92; H, 3.57; N, 16.43.
4.1.4.10. N′-(3,4,5-Trimethoxybenzylidene)-2-(4-nitrophenyl)-1H-
4.1.4.6. N′-(2,4-Dichlorobenzylidene)-2-(4-nitrophenyl)-1H- benzimidazole-5 carbohydrazide 4j. Light green powder, 80% yield, m.p.
benzimidazole-5-carbohydrazide 4f. Greenish brown powder, 73% yield, 295–297 °C, IR (KBr, υmax cm−1): 3441–3422 (NH benzimidazole, NH
m.p. 215–217 °C, IR (KBr, υmax cm−1): 3419–3323 (NH benzimidazole, hydrazone), 3022 (CH aromatic), 2920 (CH aliphatic), 1640 (C]O),
NH hydrazone), 3029 (CH aromatic), 1659 (C]O), 1610 (C]C), 1468 1610 (C]C), 1492 (C]N), 1322–1506 (NeO); 1HNMR (DMSO‑d6,
(C]N), 1385–1468 (NeO); 1HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.70 400 MHz, δ ppm): 3.76 (s, 3H, OCH3), 3.85 (s, 6H, 2 OCH3), 6.70 (d,
(d, 2H, J = 8.40 Hz, Ar-H), 7.53 (d, 1H, J = 8.40 Hz, Ar-H), 7.59 (d, 2H, J = 8.00 Hz, Ar-H), 7.05 (s, 2H, Ar-H), 7.59 (d, 1H, J = 8.00 Hz,
1H, J = 8.40 Hz, Ar-H), 7.71 (s, 1H, Ar-H) 7.78 (d, 1H, J = 8.40 Hz, Ar- Ar-H), 7.76 (d, 1H, J = 8.00 Hz, Ar-H), 7.89 (d, 2H, J = 8.00 Hz, Ar-H),
H),7.89 (d, 2H, J = 8.40 Hz, Ar-H), 8.05 (d, 1H, J = 7.60 Hz, Ar-H), 8.11 (s, 1H, Ar-H), 8.44 (s, 1H, CH]NH), 11.86 (s, 1H, NH, D2O
8.14 (s, 1H, Ar-H), 8.86 (s, 1H, CH]NH), 12.14 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): 56.41, 60.60,
exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): 113.72, 114.02, 104.69, 114.03, 115.20, 116.81, 122.00, 127.00, 127.42, 128.62,
116.88, 122.02, 126.51, 128.48, 128.63, 129.82, 130.11, 131.42, 130.55, 139.56, 147.61, 151.64, 152.16, 153.67, 155.12, 164.25,
134.21, 134.97, 135.34, 142.36, 146.05, 151.63, 155.26, 164.25, 171.98; Elemental analysis calculated for C24H21N5O6 (M.Wt.
172.06; Elemental analysis calculated for C21H13Cl2N5O3 (M.Wt. 475.46): C, 60.63; H, 4.45; N, 14.73; Found C, 60.35; H, 4.66; N, 14.95.
454.27): C, 55.52; H, 2.88; N, 15.42; Found C, 55.37; H, 2.59; N, 15.24.

4.1.4.7. N′-(4-Hydroxybenzylidene)-2-(4-nitrophenyl)-1H-benzimidazole- 4.1.5. General procedure for synthesis of N-substituted-2-(2-(4-

5-carbohydrazide 4 g. Yellowish green powder, 77% yield, m.p. nitrophenyl)-1H-benzimidazole-5-carbonyl)hydrazine-1-carbothioamide
237–239 °C, IR (KBr, υmax cm−1): 3420–3390 (NH benzimidazole, NH 5a-c
hydrazone), 3202 (OH) 3048 (CH aromatic), 1632 (C]O), 1607 Equimolar quantities (2 mmol, 0.50 g) of compound 3 and appro-
(C]C), 1454 (C]N), 1369–1513 (NeO); 1HNMR (DMSO‑d6, priate isothiocyanate in 25 mL of absolute ethanol were heated under
400 MHz, δ ppm): 6.69 (d, 2H, J = 8.00 Hz, Ar-H), 6.85 (d, 2H, reflux for 6 h. The resulting crude solid was filtered off and dried and
J = 8.00 Hz, Ar-H), 7.57 (d, 3H, J = 8.00 Hz, Ar-H),7.74 (d, 1H, used for the following step without further purification.

10
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

4.1.6. General procedure for synthesis of 4-substituted-5-(2-(4- υmax cm−1): 3414, 3303 (NH benzimidazole, NH oxadiazole), 3040 (CH
nitrophenyl)-1H-benzimidazol-5-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione aromatic), 2928 (CH aliphatic), 1532 (CeO), 1502 (C]S), 1196 (CeN);
1
6a-c HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.78 (d, 2H, J = 7.20 Hz, Ar-H),
Appropriate crude thiosemicarbazides 5a-c (10 mmol) were dis- 7.84 (d, 1H, J = 6.80 Hz, Ar-H), 7.92 (d, 1H, J = 6.80 Hz, Ar-H), 7.97
solved in sodium hydroxide solution (20 mL) and the resulting solution (d, 2H, J = 7.20 Hz, Ar-2H), 8.05 (s, 1H, Ar-H), 11.41 (s, 1H, NH
was stirred under reflux for 4 h. The solution was acidified with dilute oxadiazole, D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm):
HCl to pH 3; the formed precipitate was filtered off, washed with dis- 108.18, 111.50, 113.71, 114.76, 118.96, 123.16, 130.32, 132.59,
tilled water, dried, and then recrystallized from ethanol to afford cor- 135.19, 151.78, 154.44, 160.92, 177.84; Elemental analysis calculated
responding 1,2,4-triazoles 6a-c. for C15H9N5O3S (M.Wt. 339.04): C, 53.09; H, 2.67; N, 20.64; S, 9.45;
Found C, 52.79; H, 2.82; N, 20.83; S, 9.19.
4.1.6.1. 4-Ethyl-5-(2-(4-nitrophenyl)-1H-benzimidazol-5-yl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione 6a. Brown powder, 70% yield, m.p. 4.2. Biological evaluation
285–287 °C, IR (KBr, υmax cm−1): 3443, 3321 (NH benzimidazole,
NH triazole), 3039 (CH aromatic), 1501 (C]S), 1142 (CeN); 1HNMR The antimicrobial activity was performed at CO-ADD (The
(DMSO‑d6, 400 MHz, δ ppm): 1.20 (t, 3H, J = 8.00 Hz, CH3), 4.08 (q, Community for Antimicrobial Drug Discovery), funded by the
2H, J = 8.00 Hz, CH2), 6.79 (d, 2H, J = 8.00 Hz, Ar-H), 7.73 (d, 1H, Wellcome Trust (UK) and The University of Queensland (Australia). The
J = 8.00 Hz, Ar-H), 7.88 (d, 1H, J = 8.00 Hz, Ar-H), 7.98 (s, 1H, Ar-H), antimicrobial screenings were performed according to CO-ADD (The
8.14 (d, 2H, J = 8.00 Hz, Ar-H), 14.03 (s, 1H, NH triazole, D2O Community for Antimicrobial Drug Discovery) procedures [58].
exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): 13.93, 19.63,
114.04, 114.16, 114.43, 122.21, 125.39, 130.62, 132.55, 133.82, 4.2.1. Antifungal activity
151.28, 151.48, 154.68, 164.38, 167.31; Elemental analysis Yeast Extract-Peptone Dextrose (YPD) agar was used to culture
calculated for C17H14N6O2S (M.Wt. 366.40): C, 55.73; H, 3.85; N, fungi strains for 3 days at 30 °C. A yeast suspension of 1 × 106 to
22.94; S, 8.75; Found C, 55.92; H, 4.14; N, 23.04; S, 9.05. 5 × 106 CFU/mL (as determined by OD530) was developed in five
colonies. The suspension was thereafter diluted and added to every well
4.1.6.2. 4-Phenyl-5-(2-(4-nitrophenyl)-1H-benzimidazol-5-yl)-2,4- of the compound-containing plates donating a final cell density of fungi
dihydro-3H-1,2,4-triazole -3-thione 6b. Yellowish brown powder, 65% suspension of 2.5 x103 CFU/mL and a total volume of 50 μL. All plates
yield, m.p. 279–281 °C, IR (KBr, υmax cm−1): 3330, 3214 (NH were wrapped and incubated at 35 °C for 24 h without quivering [58].
benzimidazole, NH triazole), 3092 (CH aromatic), 1501 (C]S), 1142
(CeN); 1HNMR (DMSO‑d6, 400 MHz, δ ppm): 6.70 (d, 2H, J = 8.00 Hz, 4.2.2. Antibacterial activity
Ar-H), 7.35 (d, 1H, J = 8.00 Hz, Ar-H), 7.41–7.42 (m, 2H, Ar-H), Cation-adjusted Mueller Hinton broth (CAMHB) was used to culture
749–7.51 (m, 3H, Ar-H),7.53 (s, 1H, Ar-H), 7.61 (d, 1H, J = 8.00 Hz, all bacteria at 37 °C overnight. A specimen of every culture was then
Ar-H), 7.93 (d, 2H, J = 8.00 Hz, Ar-H), 14.20 (s, 1H, NH triazole, D2O diluted 40-fold in fresh broth and incubated at 37 °C for 1.5–3 h. The
exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): 79.20, 79.84, resultant mid-log phase cultures were diluted (CFU/mL measured by
104.46, 108.21, 114.06, 114.15, 121.65, 124.75, 129.26, 129.86, OD600), then added to every well of the compound containing plates,
131.61, 134.41, 135.03, 150.67, 152.23, 152.52, 169.05; Elemental giving a cell density of 5x105 CFU/mL and a total volume of 50 μL. All
analysis calculated for C21H14N6O2S (M.Wt. 414.44): C, 60.86; H, 3.41; the plates were wrapped and incubated at 37 °C for 18 h without qui-
N, 20.28; S, 7.74; Found C, 60.88; H, 3.18; N, 20.00; S, 7.99. vering [58].

4.1.6.3. 4-Allyl-5-(2-(4-nitrophenyl)-1H-benzimidazol-5-yl)-2,4-dihydro- 4.2.3. Cytotoxicity against human embryonic kidney cell line and hemolysis
3H-1,2,4-triazole-3-thione 6c. Yellowish brown powder, 59% yield, m.p. of human red blood cells
268–270 °C, IR (KBr, υmax cm−1): 3449, 3361 (NH benzimidazole, NH Neubauer hemocytometer was used to count manually HEK293 cells
triazole), 3040 (CH aromatic), 1503 (C]S), 1192 (CeN); 1HNMR and then plated in the 384-well plates including the compounds to give
(DMSO‑d6, 400 MHz, δ ppm): 4.74 (d, 2H, J = 4.00 Hz, a density of 5000 cells/well in a final volume of 50 μL. Growth media
NeCH2eCH]CH2), 4.95 (1H, d, Jtrans = 16.00 Hz, was used DMEM supplemented with 10% FBS and the cells were
NeCH2eCH]CH2), 5.18 (d, 1H, Jcis = 8.00 Hz, NeCH2eCH]CH2), brooded together with the compounds for 20 h in 5% CO2 at 37 °C [58].
5.85–5.90 (m, 1H, NeCH2eCH]CH2), 6.78 (d, 2H, J = 8.00 Hz, Ar-H), Three volumes of 0.9% NaCl were used to wash whole Human blood
7.70 (d, 1H, J = 8.00 Hz, Ar-H), 7.83 (d, 1H, J = 8.00 Hz, Ar-H), 7.95 and then pending in same to a concentration of 0.5 × 108 cells/mL, as
(s, 1H, Ar-H), 8.09 (d, 2H, J = 8.00 Hz, Ar-H), 14.12 (s, 1H, NH intended by manual cell count in Neubauer hemocytometer. The 384-
triazole, D2O exchangeable); 13C NMR (DMSO‑d6, 100 MHz, δ ppm): well compound-containing plates were included the rinsed cells for a
46.55, 109.47, 113.89, 114.13, 114.43, 117.91, 122.37, 125.33, final volume of 50 μL. After a 10 min shake on a plate shaker the plates
125.40, 130.41, 132.26, 134.74, 151.61, 152.18, 154.40, 168.02; were then incubated at 37 °C for 1 h. After incubation, the plates were
Elemental analysis calculated for C18H14N6O2S (M.Wt. 378.41): C, centrifuged for 10 min at 1000 g to pellet cells and debris, 25 μL of the
57.13; H, 3.73; N, 22.21; S, 8.47; Found C, 57.34; H, 3.97; N, 21.98; supernatant was then conveyed to a polystyrene 384-well assay plate
S, 8.69. [58].

4.1.7. Synthesis of 5-(2-(4-Nitrophenyl)-1H-benzimidazol-5-yl)-1,3,4- 4.2.4. Screening of antifungal activity

oxadiazole-2(3H)-thione 7 The antifungal activity of compounds 4a, 4e, 4f, Amphotericin B
A mixture of hydrazide 3 (2 mmol, 0.50 g), potassium hydroxide and ketoconazole were determined using the microbroth dilution
(50 mmol, 2.81 g) and carbon disulfide (1.7 mmol, 1.5 mL) in ethanol method using 96-well plates according to Clinical and Laboratory
(70 mL) was stirred under reflux until the evolution of hydrogen sulfide Standards Institute [59] at Microbiology and Immunology department,
ceased (12 h) by tested it with lead acetate. Ethanol was distilled off Faculty of pharmacy, Minia University.
under reduced pressure and the residue was dissolved in water and then
acidified with dilute hydrochloric acid (10%,0.1 N). The resulting 4.2.4.1. Chemicals. Tested compounds (4a, 4e and 4f), Amphotericin B
precipitate was filtered off, washed with water, dried, and recrystallized (positive control), ergosterol and sorbitol were obtained from Sigma-
from absolute ethanol afforded compound 7. Aldrich (S ã o Paulo, SP, Brazil). Dimethylsulfoxide (DMSO) and Tween-
As Greenish brown powder, 70% yield, m.p. 296–298 °C, IR (KBr, 80 were purchased from Labsynth products for Laboratories Ltd.

11
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

(Diadema, SP, Brazil), and Vetec Fine Chemicals Ltd. (Duque de Caxias, 4.2.4.6. The sterol quantitation method (SQM) by spectrophotometric
RJ, Brazil), respectively. method. This test measures the ability of antifungal drugs to inhibit
the synthesis of ergosterol in cell membrane or not by quantitation of
4.2.4.2. Fungal Strains, Media, and growth conditions. Reference the amount of ergosterol after the growth of the fungi before and after
(Candida albicans ATCC 90028) and clinical strains (oral and vaginal exposure of these drugs. The method of Arthington-Skaggs et al. [63]
strains of candida albicans) were used in this study. The isolates were was implemented.
obtained from the collection of the Department of Microbiology and One colony of C. albicans colony from an overnight Sabouraud
Immunology, Faculty of Pharmacy, Minia University. Strains were dextrose agar culture was used were inoculated to 50 mL of Sabouraud
stored as glycerol stock at −70 °C. Strains were subcultured twice on dextrose broth containing 0, 1, 4, 8,…..to 64 μg/ml of Ketoconazole (as
Sabouraud dextrose agar plates (Difco Laboratories, Detroit, MI, USA) a positive control drug which known that it inhibits ergosterol synthesis
to ensure optimum growth, subcultures on Sabouraud dextrose agar in the fungal cell membrane) and the tested compounds (4a, 4e and 4f).
plates were incubated at 35 °C for 24 h. The cultures were incubated for 16 hr with shaking (150 rpm) at 35 °C.
The stationary-phase cells were harvested by centrifugation at
2700 rpm for 5 min and washed once with sterile distilled water. The
4.2.4.3. Determination of the minimum inhibitory concentration
net wet weight of the yeast pellets was determined. Three milliliters of
(MIC). The MIC of the tested compounds (4a, 4e and 4f) were
25% alcoholic potassium hydroxide solution (25 g of KOH and 35 mL of
determined using the microbroth dilution method using 96-well
sterile distilled water, brought to 100 mL with 100% ethanol), was
plates according to Clinical and Laboratory Standards Institute [59]
added to the pellets and vortex mixed for 1 min. Cell suspensions were
against reference and clinical strains of Candida. Tested compounds
transferred to a clean 16 mm borosilicate glass screw-cap tube then,
were dissolved in DMSO to give a stock concentration of 128 µg/mL.
were left in an 85 °C water bath for 1 hr. Tubes were left to cool to room
Microbial suspensions were prepared by transferring cells from the
temperature. Sterols were then extracted by addition of a mixture of
stock cultures to tubes with Trypticase soya broth (Difco Laboratories,
1 mL of sterile distilled water and 3 mL of n-heptane followed by vig-
Detroit, MI, USA) and incubated with agitation for 24 h at 28 °C. The
orous vortex mixing for 3 min. The n-heptane layer was transferred to a
cultures were diluted with broth to reach a final inoculum size of
clean borosilicate glass screw-cap tube and stored at –20 °C for as long
0.5 × 103 to 2.5 × 103 cells/ml 96 µL of Trypticase soya broth and 4 µL
as 24 hr prior to analysis, a 20 µL aliquot of sterol extract was diluted
of desired concentration of the tested compound were added to each
fivefold in 100% ethanol and scanned spectrophotometrically between
well. The candida suspension (100 µL) was added to each well. Control
240 and 300 nm. The presence of ergosterol and the late sterol inter-
of yeasts in the medium without tested compounds was also carried out.
mediate 24(28) Dehydroergosterol [24(28) DHE)] in the extracted
Plates were incubated at 28 °C for 24 h and then yeast growth was
sample resulted in a characteristic four-peaked curve. The absence of
evaluated.
detectable ergosterol in extracts was indicated by a flat line. Any dose-
dependent activity lead to decrease in the height of the absorbance
4.2.4.4. Sorbitol assay (Inhibition of fungal cell wall synthesis). For peaks which corresponded to decreased ergosterol concentration due to
assessment the effect of tested compounds on the cell wall of C. inhibition in both lanosterol 14α-demethylase and squalene epoxidase
albicans, the sorbitol assay was done. The MICs of examined activities. Ergosterol content was calculated as a percentage of the wet
compounds were determined in the presence of sorbitol according to weight of the cell by the following equations:
CLSI method [59]. 96 µL of Sabouraud Dextrose broth (HiMedia
Laboratories, Mumbai, MH, India) and 4 µL of the proper 1: % ergosterol + %24(28) DHE = [(A281.5/290) × F] / pellet
concentration of the tested compounds were added to each well. weight
Next, 100 µL of inoculum (103 CFU/mL) prepared in SDB 2: % 24(28) DHE = [(A 230/518) × F] / pellet weight, % ergos-
supplemented with sorbitol as osmo protective agent to cell wall at a terol = 1 –2
final concentration of 0.8 M was introduced into each well. The plates
were sealed aseptically and incubated at 35 °C. The plates were read at Where F is the factor for dilution in ethanol and 290 and 518 are the
2 and 7 days. Built on the ability of sorbitol to act as a fungal cell wall E values (in percentages per centimeter) determined for crystalline er-
osmotic protective agent, the higher MIC values detected in the plates gosterol and 24(28) DHE, respectively.
with added sorbitol compared to the standard (free from sorbitol)
medium. It means that the cell wall is one of the possible cell targets for 4.2.5. Inhibition of lanosterol 14α-demethylase (CYP51) activity
the compounds examined [60]. Noteworthy compounds developed antifungal activity were
screened for their inhibition activity of Lanosterol 14α-demethylase
4.2.4.5. Ergosterol binding assay. The aim of this test is to assess if the (CYP51) at Confirmatory Diagnostic Unit, VACSERA, Cairo, Egypt.
tested compounds binds to the fungal cell membrane ergosterol or not, Resorufin and 7-ethoxyresorufin are light sensitive. We therefore re-
this experiment was made by Escalante et al. [61] using the previous commend carrying out this procedure under yellow light, in order to
protocols of Leite et al.[62]. Ergosterol was dissolved in DMSO and protect the integrity of the stock solutions. Incubations are set up in a
Tween 80 according to the preferred concentration and volume. The black 96-well plate and consist of substrate (7ER) and CYP51
formed emulsion was then homogenized, heated, and diluted with the Bactosomes in phosphate buffer containing magnesium chloride.
broth culture medium. The MICs of the tested compounds and Reactions are initiated by adding 40 µL of 5x NADPH generating system
amphotericin B (as a positive control drug which known that it binds [this can be omitted from wells containing blanks and standards].
to ergosterol in the fungal cell membrane) against C. albicans were Metabolite (resorufin) formation is measured fluorometrically, using
determined by the micro dilution method, in the presence and absence detection wavelengths chosen to minimize interference from NADPH
of exogenous ergosterol added to the assay medium, in different lines of and 7ER. The substrate, 7-ethoxyresorufin, and metabolite, resorufin,
the same microplate. The concentrations of amphotericin B and tested are both available from Cypex.
compounds ranged from 0.125 to 128 µg/mL in broth medium (100 µL)
supplemented with ergosterol at a concentration of 400 µg/ml. Candida 4.3. Molecular docking
albicans suspension (10 m; 05 McFarland) was added to each well and
plates were incubated at 35 °C for 24 h. MIC was determined. The The crystal structure of the 14α-lanosterol demethylase enzyme co-
vehicle alone run as a negative control and candida cells viability in the crystallized with the posaconazole substrate was acquired from the
presence of ergosterol was also tested. Protein Data Bank (PDB ID: 5FSA). Docking study was carried out using

12
M.M. Morcoss, et al. Bioorganic Chemistry 101 (2020) 103956

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Declaration of Competing Interest novel benzimidazole-1, 3, 4-oxadiazole compounds, Molecules 24 (2019) 191–205,
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