New pyrazolo-triazolo-pyrimidine derivatives as antibacterial agents: Design and synthesis, molecular docking and DFT studies

Article abstract of DOI:10.1016/j.molstruc.2019.127007

A new series of antibacterial pyrazolo-triazolo-pyrimidine derivatives 3a-3i were synthesized in two steps starting from aminopyrazole 1 and characterized by ¹H NMR ¹³C NMR and HRES-MS. Their molecular geometry are also calculated by the Density Functional Theory (DFT) employing B3LYP level with 6-311G (d,p) basis set. All the synthesized compounds were tested for in vitro antibacterial activity against a panel of selected bacterial strains, by application of the Disc-Diffusion and MIC assays, using gentamicin as standard. The interactions of these compounds with the bacteria Pseudomonas aeruginosa (LasR) were performed by molecular docking studies.

Full text of DOI:10.1016/j.molstruc.2019.127007

Journal of Molecular Structure 1199 (2020) 127007
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
New pyrazolo-triazolo-pyrimidine derivatives as antibacterial agents:
Design and synthesis, molecular docking and DFT studies
Mabrouk Horchani a, Amel Hajlaoui a, Abdel Halim Harrath b, Lamjed Mansour b,
,
*
Hichem Ben Jannet a, Anis Romdhane a
a Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity (LR11Es39), Team: Medicinal Chemistry and Natural Products, Faculty of Science of
Monastir, University of Monastir, Avenue of Environment, 5019, Monastir, Tunisia
b King Saud University, Department of Zoology, College of Science, Riyadh, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of antibacterial pyrazolo-triazolo-pyrimidine derivatives 3a-3i were synthesized in two
steps starting from aminopyrazole 1 and characterized by 1H NMR 13C NMR and HRES-MS. Their mo-
lecular geometry are also calculated by the Density Functional Theory (DFT) employing B3LYP level with
6-311G (d,p) basis set. All the synthesized compounds were tested for in vitro antibacterial activity
against a panel of selected bacterial strains, by application of the Disc-Diffusion and MIC assays, using
gentamicin as standard. The interactions of these compounds with the bacteria Pseudomonas aeruginosa
(LasR) were performed by molecular docking studies.
Received 21 June 2019
Received in revised form
27 August 2019
Accepted 29 August 2019
Available online 30 August 2019
Keywords:
Pyrazolo-triazolo-pyrimidine
Antibacterial
© 2019 Published by Elsevier B.V.
Molecular docking
DFT
1. Introduction
structure in a large variety of compounds of great biological and
pharmaceutical value exhibiting anticancer [7], antiviral [8], anti-
Heterocycles have been found a key structural in medical
chemistry and also they are frequently found in large percent in
biomolecules such as enzyme, vitamins, natural products and bio-
logical active compounds. In this context, and as commonly re-
ported that the presence of two or more heterocyclic
pharmacophores linked and/or fused within a same structure
generally could contribute to provide a significant positive effect on
the overall biological efficiency in the resulting poly-heterocycle
[1], we have oriented our research to prepare new classes of het-
erocyclic compounds associating within a same scaffold the pyr-
azole, the pyrimidine and the triazole moieties.
Indeed and as reported in literature, pyrazole and its derivatives
are considered a pharmacologically important active scaffold that
possesses almost all types of pharmacological activities particu-
larly, they are described as anti-HCV [2], antitumor [3], cytotoxic
[4], antioxidant [5] as well as antibacterial agents (Fig. 1A) [6].
On the other hand, pyrimidines, are widely found as the core
inflammatory [9] and antibacterial activities (Fig. 1B) [10].
Finally, triazole and its derivatives have attracted the interest of
medicinal chemists due to their broad spectrum of applications in
medicinal chemistry and biochemical [11]. Thus, there are known
as anti-tumoral [12] anticholinesterase [13], anti-tyrosinase [14],
cytotoxic [15] and antibacterial (Fig. 1C) [16] agents.
On the other hand, and as reported, some works prove that DFT/
B3LYP method has been commonly preferred to study structure,
(QSAR) and many properties of organic molecules, because this
method is efficient and offers an excellent trade-off between
chemical accuracy, biological activity and computational cost
[17e20].
These observations prompted us to synthesize some new poly-
heterocycles bearing in their structures fragments described as
antibacterial agents, as indicated above, such as pyrazole, pyrimi-
dine and triazole derivatives and to investigate their antibacterial
activity against two Gram-positive and four Gram-negative strains.
Further molecular docking studies were carried out to include the
drug-receptor interactions. The new analogues have been theo-
retically investigated by applying density functional theory (DFT) to
understand the structure activity relationship.
* Corresponding author. Tel.: þ 216 73 500 279; Fax: þ 216 73 500 278
E-mail addresses: anis_romdhane@yahoo.fr, romdhaneanis.1@gmail.com
(A. Romdhane).
https://doi.org/10.1016/j.molstruc.2019.127007
0022-2860/© 2019 Published by Elsevier B.V.

2
M. Horchani et al. / Journal of Molecular Structure 1199 (2020) 127007
Fig. 1. Previously reported antibacterial nitrogen heterocycles.
2. Materials and methods
found 290.1167; 1H NMR (300 MHz, DMSO‑d6):
d
(ppm) ¼ 2.73 (s,
3H,H10), 4.55 (s, 2H,H15), 7.43 (t, 1H, J ¼ 7.2 Hz, H14), 7.59 (t, 2H, J¼
2.1. Materials
7.5 Hz, H13þH13’), 8.09 (d, 2H, J ¼ 7.8Hz, H12þ H12’), 9,66 (s, 1H, H5);
13C NMR (75 MHz, DMSO‑d6):
(C9a), 121.7 (C16), 126.9e134.5 (Carom), 143.2 (C11), 145.5 (C5), 147.8
d
(ppm) ¼ 18.6 (C10), 23.1 (C15), 107.9
Melting points were determined on an Electrothermal 9002
melting point apparatus and are uncorrected. IR spectra were
recorded on a FTS-6000 BIO-RAD apparatus. 1H NMR (300 MHz)
and 13C NMR (75 MHz) spectra were recorded in deuterated CDCl3
and DMSO‑d6 on a Bruker AC-300 using non deuterated solvents as
(C9), 151.4 (C9b), 153.8 (C2), 164.7 (C6a).
2.2.2.2. 3b: 2,9-dimethyl-7-phenyl-7H-pyrazolo [4,3-e] [1,2,4] tri-
azolo [1,5-c] pyrimidine. White solid; Yield (%) ¼ 70; m.p (ꢀC) ¼
>300; HRMS [MþH]þ calcd. for (C14H12N6)þ: 265.1202; found
internal reference. All chemical shifts were reported as
d values
265.1211; 1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 2.68 (s, 3H. H10),
(ppm) and coupling constants (J) were expressed in Hz. High Res-
olution Mass Spectra (HRES-MS) were obtained with Micromass
LCT (ESI technique, positive mode) spectrometers. All reactions
were monitored by TLC using aluminum sheets of sds silica gel 60
F254, 0.2 mm. The starting materials 1 were prepared according to
the literature [21].
2.87 (s, 3H, H15), 7.39 (t, 1H, J ¼ 7.2 Hz, H14), 7.56 (dd, 2H, J1¼ 7.5 Hz,
J2¼ 1.8 Hz, H13þH13’), 8.11 (d, 2H, J ¼ 7.5 Hz, H12þ H12’), 9.07 (s, 1H,
H5); 13C NMR (75 MHz, CDCl3):
102.2(C9a), 122.1e129.2 (Carom), 138.1 (C11), 138.4 (C5), 143.3(C9),
d
(ppm) ¼ 13.8 (C10), 14.6 (C15),
146.4 (C9b), 148.9 (C6a), 165.8(C2).
2.2. Methods
2.2.2.3. 3c: 9-methyl-2,7-diphenyl-7H-pyrazolo [4,3-e] [1,2,4] tri-
azolo [1,5-c] pyrimidine. White solid; Yield (%) ¼ 42; m.p (ꢀC) ¼
>300; HRMS [MþH]þ calcd. for (C19H14N6)þ: 327.1367; found
2.2.1. General procedure of synthesis of ethyl (E)-N-(4-cyano-3-
methyl-1-phenyl-1H-pyrazol-5-yl) formimidate
327.1378, 1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 2.93 (s, 3H, H10),
7.28e8.37 (m, 10H, Harom), 9.14 (s, 1H, H5); 13C NMR (75 MHz,
CDCl3):
In a 250 mL flask equipped with a condenser, 0.1 mol of the
precursor 1 and 0.12 mol of the triethyl orthoformate are poured in
150 mL of acetic anhydride; then the mixture is stirred under
reflux. The mixture is allowed to return to room temperature and
the reaction volume is reduced to half by the rotary evaporator. The
mixture is then kept overnight in the refrigerator. The product
obtained by filtration is then recrystallized from hexane.
d
(ppm) ¼ 13.3 (C10); 103 (C9a), 121.5 (C12 þC12’), 126.6 (C14),
127.2 (C16 þC16’), 128.3 (C17 þC17’), 128.7 (C13 þC13’), 129.4 (C18), 130.2
(C15), 137.9 (C11), 138 (C5), 143 (C9), 145.9 (C9b), 148,7 (C6a), 165.4
(C2).
2.2.2.4. 3d: 9-methyl-7-phenyl-7H-pyrazolo [4,3-e] [1,2,4] triazolo
[1,5-c] pyrimidine. White solid; Yield (%)
¼
38; m.p
(ꢀC) ¼ 252e254; HRMS [MþH]þ calcd. for (C13H10N6)þ: 251.1045;
2.2.1.1. Ethyl (E) -N- (4-cyano-3-methyl-1-phenyl-1H-pyrazol-5-yl)
formimidate 2. Yellow solid; Yield (%) ¼ 65; m.p (ꢀC) ¼ 78e80; 1H
found 251.1055; 1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 2.81 (s, 3H,
NMR (CDCl3,300 MHz):
d
(ppm) ¼ 1.36 (t, 3H, J ¼ 7.2 Hz, H14); 4.32
H10), 7.19e8.06 (m, 3H, Harom), 8.05 (d, 2H, J ¼ 7.5 Hz, H12þ12’), 8.33
H
15), 9.10 (s, 1H, H5); 13C NMR (75 MHz, CDCl3):
(q, 2H J ¼ 6.9 Hz, H13); 2,4 (s, 3H, H7); 8,37 (s, 1H, H12); 7.26e7.63
(s, 1H,
(m, 5H, Harom); 13C NMR (CDCl3, 75 MHz):
d
(ppm) ¼ 12.5 (C7); 13.4
d
(ppm) ¼ 13.7 (C10), 122.1 (C9a), 124.1e129.9 (Carom), 138.4 (C5),
(C14); 63.7 (C13þ C4); 114.1 (CN); 123.4e137.6 (Carom); 149.5 (C5);
138.6 (C9), 143.4 (C9b), 148.4 (C6a), 155.3 (C2).
151.1 (C3); 159.7 (C12).
2.2.2.5. 3e: N-(9-methyl-7-phenyl-7H-pyrazolo[4,3-e] [1,2,4]triazolo
[1,5-c]pyrimidin-2-yl)acetamide. White solid; Yield (%) ¼ 41; m.p
(ꢀC) ¼ 151e153; HRMS [MþH]þ calcd. for (C15H13N7)þ: 308.1282;
2.2.2. General procedure of synthesis of pyrazolotriazolopyrimidine
derivatives 3
found 308.1291; 1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 1.89 (s, 3H,
In a 50 mL two-neck flask, 1 mmol of iminoether 2 is dissolved
with 1.1 equivalents of hydrazide and a few drops of acetic acid in
40 mL of anhydrous dioxane. The mixture is stirred under reflux for
24 h, the solid formed by precipitation is filtered and then washed
with petroleum ether.
H
10); 2.61 (s, 3H, H17); 7.26 (t, 1H, J ¼ 7.2 Hz, H14), 7.24e8.22 (m, 5H,
Harom); 8.24 (s, 1H, H5); 11.92 (s, 1H(NH)); 13C NMR (75 MHz,
DMSO‑d6):
d
(ppm) ¼ 14.4 (C10); 20.98 (C17); 100.3 (C9a);
120.2e138.9 (Carom); 142.8 (C5); 154.1 (C9); 156.5 (C9bþC2); 158.6
(C6a); 171.9(C16).
2.2.2.1. 3a: 2-(9-methyl-7-phenyl-7H-pyrazolo[4,3-e] [1,2,4]triazolo
[1,5-c]pyrimidin-2-yl) acetonitrile. White solid; Yield (%) ¼ 52;
m.p(ꢀC) ¼ >300; HRMS [MþH]þ calcd. for (C15H11N7)þ: 290.1154;
2.2.2.6. 3f: 2-ethoxy-9-methyl-7-phenyl-7H-pyrazolo [4,3-e] [1,2,4]
triazolo [1,5-c] pyrimidine. White solid; Yield (%) ¼ 69; m.p

M. Horchani et al. / Journal of Molecular Structure 1199 (2020) 127007
3
(ꢀC) ¼ 182e184; HRMS [MþH]þ calcd. for (C15H14N6O)þ: 295.1307;
solution (in water) at the concentration of 100
m
M and then placed
found 295.1319; 1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 1.27 (t, 3H,
on the surface of the inoculated plates (90 mm diameter). The
plates were kept at 4 ꢀC for 2 h before incubation at 37 ꢀC for 24 h
[22]. Then the diameters of the inhibition zones were measured.
H
16, J ¼ 6,9 Hz), 2.58 (s, 3H, H10), 4.23 (q, 2H, H15, J ¼ 7,2 Hz), 7.28 (t,
1H, J ¼ 7.8 Hz, H14), 7.43 (t, 2H, J1¼ 8.1 Hz, H13þH13’), 8.01 (d, 2H, J ¼
7.5 Hz, H12þ H12’), 8.51 (s, 1H, H5); 13C NMR (75 MHz, CDCl3):
Positive control discs of gentamicin (10 mg/disc, Bio-Rad), were
d
(ppm) ¼ 14.4 (C10), 14 (C16), 62.6 (C15), 100.6 (C9a); 121.6e138.5
included in each assay, and the developing inhibition zones were
compared with those of the reference disc. The 10% DMSO solution
was also tested as negative control. The assays were performed in
triplicate.
(Carom), 141.3 (C5), 154 (C9), 155.9 (C2), 157.3 (C9b), 157.9 (C6a).
2.2.2.7. 3g: 4-methyl-7-((9-methyl-7-phenyl-7H-pyrazolo[4,3-e]
[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)methoxy)-
2H-chromen-2-one.
White solid; Yield (%) ¼ 55; m.p (ꢀC) ¼ >300; HRMS [MþH]þ calcd.
2.2.3.3. Micro-well dilution assay. The minimal inhibition concen-
tration (MIC) values for the antibacterial activity were determined
with the dilution method following the procedure described by
Jabrane et al. [23]. The samples were prepared at a concentration of
for (C24H18N6O3)þ: 439.1519; found 439.1532; 1H NMR (300 MHz,
CDCl3):
d
(ppm) ¼ 2.24 (s, 3H, H10), 2.82 (s, 3H, H9’), 5.42 (s, 2H, H15),
6.08 (s, 1H, H3’), 6.17.(d, 1H, J ¼ 8.7 Hz, H6’), 6.85 (t, 1H, J ¼ 6 Hz, H14),
6.84e8.14 (m, 4H, Harom), 8.12 (d, 2H, J ¼ 7.8 Hz, H12þ H12’), 9.07 (s,
1000
was pipetted into all wells of the microtitre plate before trans-
ferring 100 L of stock solution to the microplate and applying a
series of dilutions. Finally, 50
L of 106 colony forming units (cfu/
mM 10% DMSO solution. Sterile 10% DMSO solution (100 mL)
1H, H5); 13C NMR (75 MHz, CDCl3):
d
(ppm) ¼ 13.9 (C10), 18.6 (C9’),
29.7 (C15), 64.1 (C8’), 67.1 (C9a), 102.2 (C6’), 12.5 (C4a); 112.8 (C3’),
114.3 (C13þC13’), 122.2 (C5’), 125.7 (C7), 127.4 (C14þC14’), 129.2 (C12),
138.3 (C5), 138.4 (C9), 143.5 (C9b), 149.3 (C2), 152.3 (C6'a), 155.1 (C4’),
161.1 (C8'a), 163.6 (C2’).
m
m
mL) (according to Mc-Farland turbidity standards) of standards
microorganism suspensions were inoculated on to microplates and
incubated at 37 ꢀC between 18 and 24 h. At the end of incubation
period, the plates were evaluated for the presence or absence of
growth. All the samples were screened three times against each
microorganism. Gentamicin was used as antibacterial positive
control.
2.2.2.8. 3h: 2-(4-chlorophenyl)-3-((9-methyl-7-phenyl-7H-pyrazolo
[4,3-e] [1,2,4]triazolo[1,5-c]pyrimidin-2-yl) methyl) quinazolin-
4(3H)eone. White solid; Yield (%) ¼ 65; m.p (ꢀC) ¼ 200e202;
HRMS [MþH]þ calcd. for (C28H19ClN8O)þ: 519.1460; found
519.1469; 1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 2.89 (s, 3H, H10),
6.07 (s, 2H, H15), 7.25e8.55 (m,13H, Harom), 9.11 (s,1H, H5); 13C NMR
(75 MHz, CDCl3):
(ppm) ¼ 14.2 (C10), 56.2 (C15), 102.4 (C9a), 112.1
2.2.4. Computational details (DFT studies)
d
The DFT calculations were performed using Gauss View 5 and
GAUSSIAN 09 [24,25]. The molecular structure of the compounds in
the ground state is optimized by using B3LYP/6-311 þ G(d,p) level
of the theory. The most stable and reactive molecule is determined
by using energy gap which is the difference between the HOMO-
LUMO orbitals.
(C12 þC12’), 119.9 (C4'a), 121.9 (C14), 123.2 (C5’), 125 (C9’), 125.2 (C8’),
000
000
125.3 (C6’), 125.4 (C11”þC11 ), 126.3 (C10”þC10 ), 128.5 (C13þ C13’),
00
129.2 (C7’), 131.3 (C12 ), 133.5 (C11), 134.3 (C5), 139.5 (C9), 15.6 (C9b),
155.2 (C2), 156.4 (C8'a), 160 (C6a), 167.7 (C2’), 173.5 (C4’).
2.2.2.9. 3i:
2-(4-methoxyphenyl)-3-((9-methyl-7-phenyl-7H-pyr-
azolo[4,3-e] [1,2,4]triazolo[1,5-c]pyrimidin-2-yl) methyl) quinazolin-
4(3H)eone. White solid; Yield (%) ¼ 43; m.p (ꢀC) ¼ 148e150;
HRMS [MþH]þ calcd. for (C29H22N8O2)þ: 515.1944; found 515.1955;
2.2.5. Molecular docking procedure
Automated docking was used to determine the orientation of
inhibitors bound in the active site of P. aeruginosa (LasR). The three-
dimensional structure of PDB (PDB: 2UV0) were obtained from the
RSCB protein data bank [26]. The molecular docking of the chemical
compounds- LasR binding site was performed using Autodock Vina
software [27].
1H NMR (300 MHz, CDCl3):
d
(ppm) ¼ 2.9 (s, 3H, H10), 3.89 (s, 3H,
00
H13 ), 6.09 (s, 2H, H15), 7.25e8.55 (m, 13H, Harom), 9.12 (s, 1H, H5);
13C NMR (75 MHz, CDCl3):
d
(ppm) ¼ 13.9 (C10), 55.4 (C15), 61.8
00
000
(C13 ), 103.5 (C9a), 113.8 (C11”þC11 ), 114.7 (C12þC12’), 122.1 (C9’),
123.7 (C4'a), 126.2 (C14), 127.3 (C5’), 127.5 (C8’), 129.2 (C6’), 130.3
000
(C13þC13’),133.8 (C10”þC10 ),138.3 (C7’),138.4 (C11),143.4 (C5),146.3
3. Results and discussion
(C9), 149.1 (C2), 151.9 (C9b), 159.5 (C8'a), 161.0 (C6a), 164.3 (C2’), 165.8
00
(C4’), 179.3 (C12 ).
3.1. Chemistry
2.2.3. Antibacterial activity
Justification of much of the chemistry directed to the synthesis
of the compounds comprising nitrogen at the rings fusion, is due to
the application of compounds having interesting antibacterial
properties in the domain of medicinal chemistry. In this respect and
encouraged by some of our previous results [28e30] the aim of this
work was to synthesis some pyrazolo-triazolo-pyrimidine de-
rivatives 3 (Scheme 1) (see Scheme 2).
In order to achieve this aim, it was necessary to first synthesize
the aminopyrazole 1 according to the previously reported method
[21]. Our approach to the target systems 3 was started by the
2.2.3.1. Bacterial srains. The in vitro antibacterial activity of the
synthesized compounds were assayed against six microorganisms,
included four Gram-negative rods: Escherichia coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 27853), Citrobacter freundii (clinical
strain), Proteus mirabilis (clinical strain), and two Gram-positive
cocci: Staphylococcus aureus (ATCC 25923) and Enterococcus faeca-
lis (ATCC 29212) (American Type Culture Collection, Rockville, MD).
The microbial strains were obtained from the culture collection of
the Laboratory of Infectious Diseases and Biological Active Agents,
Faculty of Pharmacy, Monastir, Tunisia.
synthesis of the
a-functionalized iminoether 2. Herein, we are
reporting a simple and scalable methodology for the one-pot syn-
thesis of our key intermediate 2 (65% yield) via condensation re-
action of the precursor 1 with triethyl orthoformate under reflux of
acetic anhydride [31].
2.2.3.2. Disc-Diffusion assay. The antibacterial activity of the pre-
pared compounds was evaluated with the disc diffusion method
using Mueller Hinton Agar (MHA). Inocula were prepared by
diluting overnight (24 h at 37 ꢀC) cultures in Muller Hinton Broth
medium to approximately 106 colony-forming unit per milliliter
(CFU/mL). Absorbent discs (diameter 6 mm, Whatman Paper No. 3)
The structures of the above compounds were established on the
basis of their 1H and 13C NMR spectral data.
Subsequently, the reaction of ethyl (E)-N-(4-cyano-3-methyl-1-
phenyl-1H-pyrazol-5-yl) formimidate 2 with a series of hydrazides
were impregnated with 10 mL of each sample dissolved 10% DMSO

4
M. Horchani et al. / Journal of Molecular Structure 1199 (2020) 127007
Scheme 1. Synthetic route of compounds 3a-3i.
at reflux of dioxane leads to pyrazolo-triazolo-pyrimidine de-
rivatives 3a-3i. The use of a catalytic amount of acetic acid provided
the product in reduced reaction time.Mechanistically, a first
nucleophilic attack of the nitrogen doublet of the eNH2 group on
the iminoether's iminic carbon leads to the departure of an ethanol
molecule. The obtained non isolable intermediate I1 undergoes an
intramolecular cyclization following a second nucleophilic attack
on the carbon of the nitrile function (CN) by the same nitrogen
atom affording another intermediate I2, which gives, after intra-
pyrazolotriazolopyrimidine derivatives 3, except of compound 3e
where the primary amine function was found acetylated with acetic
acid in dioxane.
The structures of the new synthesized pyrazolo-triazolo-
pyrimidine derivatives 3a-3i were evidenced by their spectral
data. The 1H NMR spectra of these compounds showed the disap-
pearance of signals related to methyl and methylene at dH 1.33e1.38
and 4.29e4.36, respectively, of the iminoether 2 and the observa-
tion of signals introduced by hydrazides. Unambiguous proofs for
the obtained products 3a-3i aroused from their 13C NMR data, in
molecular cyclization followed by
a dehydration, the new

M. Horchani et al. / Journal of Molecular Structure 1199 (2020) 127007
5
moiety, which had a very interesting effect towards S. aureus
M) compared to the other com-
(IZ ¼ 23.5 1 mm, MIC ¼ 6.25
m
pounds and Gentamicin (IZ ¼ 8.0 0.8 mm). The data in Table 1
revealed that compounds 3a-3i showed globally significant activ-
ity against the four Gram-negative bacteria used. Indeed, the
compound 3i showed the highest activity against E. coli with IZ and
MIC values of 17.0 0.6 mm and 12.5 mM, respectively. The noted
inhibition zone of the same derivative was found to be comparable
to that of gentamicin (17.5 0.8 mm). Moreover, compounds 3d
(R ¼ H), 3c (R ¼ Ph) and 3h displayed good inhibitory results
against the same strain (IZ ¼ 15.1 1.0, 15.9 0.0 and
15.0 0.0 mm, MIC ¼ 50, 50 and 25
mM, respectively) whereas,
compounds 3f (R ¼ OEt) and 3g (R¼ ((4-methylcoumarin-7-yl)oxy)
methyl) showed the lowest antibacterial potential. Furthermore,
compound 3i ((2-(4-methoxyphenyl)-4-oxoquinazolin-3(4H)-yl)
methyl) was found to be the most active (IZ ¼ 16.8 1.0 mm,
MIC ¼ 12.5
mM) towards P. mirabilis, followed by compounds 3d
(R ¼ H) and 3h (IZ ꢁ 15 mm and MIC ¼ 25
mM). This result shows, in
particular, the importance of the methoxy group in 3i compared to
the chlorine atom in 3h. On the other hand, compounds 3 exhibited
moderate to good activity (IZ ꢁ 12 mm, MIC ¼ 25e200
C. freundii compared to the reference antibiotic (11.8 0.7 mm)
M). Most
mM) against
except compound 3f (IZ ¼ 10.1 0.6 mm, MIC ¼ 400
m
compounds 3a-3i displayed noticeable antibacterial activity against
P. aeruginosa. In this series, compound 3i showed the highest ac-
Scheme 2. Proposed synthetic pathway to compounds 3a-3i.
tivity (IZ ¼ 16.8 1.8 mm, MIC ¼ 12.5
mM) and it was found slightly
more active than the standard gentamicin (IZ ¼ 16.0 0.5 mm)
followed by 3c, 3d and 3h, respectively. It is worth to notice also
that the molecules 3a (R ¼ CH2CN), 3f (R ¼ ethoxy) and 3g were
found to be the less active ones against the same strain. All these
observations clearly indicate that Gram-negative bacteria are more
sensitive to compounds 3 than the gram-positive ones. This finding
can be explained by a preferential interaction between these
compounds and the cell wall of the Gram-negative strains which is
composed of a single layer of peptidoglycan whereas in Gram-
positive bacteria, the cell wall is thicker (15e80 nm) and consist-
ing of several layers of peptidoglycan which could prevent any
interaction with these types of compounds of a particular structure.
fact, the spectra of these compounds showed essentially the
appearance of a new signal at dC 149.1e165.8 due to the quaternary
carbon C2 introduced by the corresponding hydrazide and the
disappearance of the signals relating to the carbons of the ethyl
group (dC 13.4 and 63.7) and the nitrile function (dC 114.1).
In the same context and with regard to the compounds 3a, 3g,
3h and 3i, we noticed the presence of a signal in the zone (dC
23e56) which is reversed on the DEPT 135, attributable to the
methylene group. This data is supported by the appearance of a
singlet between dH 4.55 and 6.09 in the 1H NMR spectrum. The
assignment of several chemical shifts to protons and carbons
constituting the products 3a-i was supported by comparison with
the literature [32,33].
3.3. Molecular docking analysis of compound 3i
Furthermore, the mass spectra (ES-HRMS) of compounds 3
showing the protonated molecular ion peaks [MþH]þ where in
good agreement with the assigned structures.
There are some previous studies that reported new inhibitors
against P. aeruginosa (LasR) using in silico modeling of the ligand-
receptor interaction in order to find a better inhibition efficiency
[34]. Encouraged by this finding and to confirm the antibacterial
effect of the synthesized compounds towards the used Gram-
negative bacteria, especially P. aeruginosa, docking study into the
crystal structure of P. aeruginosa (LasR) was employed. This bacteria
has many potential sites where ligand can be bound. From the
perspective of inhibitors, the most potent site is probably whose
located in the proximity of residues taking key roles in catalytic
functions. Before the docking, co-crystallized ligands and water
molecules of each protein were removed, the molecular docking of
the chemical compounds was performed using Autodock Vina
software [27].
Molecular basis of interactions between target enzyme and the
synthesized ligand 3i (the most potent antibacterial) can be un-
derstood with the help of docking analysis and interactions as
represented in Fig. 2.
In order to provide an explanation and understand the potent
antibacterial activity of compound 3i, it is pertinent to note that the
later showed good docking interactions with the receptor site.
Indeed, the pyrazolotriazolopyrimidine moiety exhibits the
preferred bindings orientation. In this case, the triazole's nitrogen
atom (position 3) showed a hydrogen bonding interaction to
3.2. Biological activity
3.2.1. In vitro antibacterial bioassay
All the newly synthesized compounds were evaluated in vitro
against an assortment of four Gram-negative (Escherichia coli,
Pseudomonas aeruginosa, Proteus mirabilis and Citrobacter freundii)
and two Gram-positive strains (Enterococcus faecalis and Staphylo-
coccus aureus). The activity of all the tested compounds was
compared with that of Gentamicin used as standard reference
antibiotic (10 mg/disc), by measuring the inhibition zone (in mm).
The inhibitory effects of compounds 1, 2, and 3a-3i against these six
strains are given in Table 1. The obtained results revealed that all
synthesized compounds possess significant antibacterial activity
against selected strains. The pyrazoles 1 and 2 were found to be
moderately active only towards P. mirabilis and C. freundii (see
Table 2).
On the other hand, the newly generated compounds 3a-3i have
exerted inhibitory activity against all of tested bacterial strains.
Indeed, these compounds did not show a good activity towards
Gram-positive bacteria (MICꢁ 400
mM) except the derivative 3h
with (2-(4-chlorophenyl)-4-oxoquinazolin-3(4H)-yl)methyl
a

6
M. Horchani et al. / Journal of Molecular Structure 1199 (2020) 127007
Table 1
Antibacterial activities of compounds 1, 2 and 3a-3i expressed in Inhibition Zone (IZ).
Compound Bacteria
Enterococcus faecalis ATCC
29212
Staphylococcus aureus ATCC
25923
Escherichia coli
ATC
Proteus
mirabilis
Citrobacter
freundii
Pseudomonas aeruginosa ATCC
27853
Inhibition Zone (mm)
a
1
2
-
7.5 0.2
8.0 0.8
9.0 0.1
9.5 0.8
8.0 0.4
7.5 0.9
8.7 0.4
7.5 0.1
7.0 0.7
23.5 1.0
7.5 0.2
8.0 0.8
e
8.0 0.8
8.0 1.0
e
e
e
10.5 0.7
11.0 1.0
11.8 1.2
14.0 1.0
15.0 1.0
12.5 1.3
11.0 1.0
11.0 1.1
16.0 1.5
16.8 1.0
17.7 1.2
11.5 0.9
12.0 1.5
12.9 1.0
13.7 0.5
15.1 0.9
13.1 0.7
10.1 0.6
12.3 0.4
15.0 0.3
14.7 0.9
11.8 0.7
e
3a
3b
3c
3d
3e
3f
3g
3h
3i
8.5b 0.8
7.2 0.5
8.5 0.8
10.5 0.1
9.0 0.7
8.5 0.8
9.0 0.7
e
12.5 1.0
13.8 1.0
15.1 1.0
15.9 0.0
13.5 0.8
11.1 0.8
11.5 0.7
15.0 0.0
17.0 0.6
17.5 0.8
12.5 1.0
14.5 1.3
16.4 1.0
15.4 1.2
14.2 1.0
10.0 1.4
13.2 1.0
15.0 0.6
16.8 1.8
16.0 0.5
9.0 0.8
Gentamicin 20.5 0.4
ATCC: American Type Culture Collection.
Gentamicin: reference antibiotic (10
m
g/disc).
a
Inactive.
b
Diameter of the Zone of Inhibition (IZ) expressed in mm including the disk (6 mm). The values are expressed in IZ standard deviation (number of repetitions ¼ 3).
Table 2
Antibacterial activities of compounds 1, 2 and 3a-3i expressed in Minimum inhibitory concentration (MIC).
Compound Bacteria
Enterococcus faecalis ATCC
29212
Staphylococcus aureus ATCC
25923
Escherichia coli ATCC
25922
Proteus
mirabilis
Citrobacter
freundii
Pseudomonas aeruginosa ATCC
27853
MIC (
1
2
mM)
>400
>400
>400
>400
>400
400
>400
>400
400
>400
>400
400
>500
>500
100
100
50
50
50
200
200
25
>500
>500
400
200
50
>500
>500
200
100
50
>500
>500
100
50
25
25
3a
3b
3c
3d
3e
3f
3g
3h
3i
400
>400
>400
>400
>400
>400
6.25
25
25
100
200
200
25
100
400
200
25
50
400
100
25
>400
400
>500
12.5
12.5
50
12.5
residue ASN-H-136 (green color). Moreover, in the pyrazolopyr-
imidine ring Pi eanion interactions were observed with GLU-H-139
(golden color). In addition, the methyl group linked to the pyrazole
ring shows alkyl interactions with LEU-E-40, PHE-H-67 and PRO-E-
41. Furthermore, the ((2-(4-methoxyphenyl)-4-oxoquinazolin-
3(4H)-yl)methyl) introduced by hydrazide showed a Pi-Alkyl
interaction with ALA-H-138 via its phenyl group and carbon-
hydrogen bond with ASN-H-136 and ASP-E-46. Moreover, it is
involved in conventional hydrogen bond interaction with SER-E-40
by its carbonyl focus. This finding clearly reveals that these in-
teractions are the principle factor explaining the inhibition of
P. aeruginosa by 3i compared to the other derivatives.
are indicator of molecular reactivity and properties. The orbital
HOMO is capable to give electrons, while LUMO can take electrons
first [37]. Fig. 3 shows the dispersion and energy levels of the
HOMO and LUMO orbitals calculated at the B3LYP/6-311 þ G(d,p).
As can be seen in Fig. 3a, the highest occupied molecular orbital
(HOMO) is localized on the pyrazole fragment and CN function as
well as NH2 group. While the lowest unoccupied molecular orbital
(LUMO) is localized on almost the entire structure except the
methyl group. Otherwise, the HOMO orbital of the molecule 2 is
confined on the benzene moiety, the pyrazole ring, the methyl
group and partially on the NCHOEt moiety. The same for the LUMO
orbital except for the methyl group as shown in Fig. 3b. On the other
hand, in regards to in Fig. 3c, we note that the HOMO orbital
bounded on the whole structure. In addition, the LUMO is centered
on total structure unless the methyl group.
3.4. DFT studies
The difference between the HOMO-LUMO orbitals is known as
energy gap. This energy range also allows to determine a kinetic
stability, reactivity and chemical hardness-softness of a molecule
reactivity [38,39]. Hard molecules with big HOMO-LUMO gap are
more stable and less reactive. In the opposite case, soft molecules
with small HOMO-LUMO gap are more reactive and less stable [40],
in this context, Fig. 3 shows that aminopyrazole 1 has a large energy
gap (5.06 eV) compared to iminoether 2 (4.59 eV). Moreover, the
gap energy of the latter is greater than that of molecule 3d (4,27eV),
according to these data we can conclude that the
DFT of chemical reactivity, which is titled conceptual DFT, is a
relevant trait of the density functional language for defining and
elucidating important chemical concepts of molecular structure
and reactivity [35]. In this relation, quantum-chemical descriptors
have been widely used in quantitative structureeactivity relation-
ship (QSAR) studies in biochemistry [36].
3.4.1. Frontier molecular orbitals analysis
In accordance with the frontier molecular orbital theory, HOMO
and LUMO are the most substantial factors because these orbitals

M. Horchani et al. / Journal of Molecular Structure 1199 (2020) 127007
7
Fig. 2. Interaction of LasR receptor with compound 3i (PDB: 2UV0).
Fig. 3. Frontier Molecular Orbital of compounds 1 (a), 2 (b) and 3d (c).
pyrazolotriazolopyrimidine derivative 3d is softer and more reac-
tive and less stable by comparison with the iminoether 2 who is
also softer and more reactive and less stable than the amino-
pyrazole 1. The low HOMO-LUMO gap supports the bioactive
molecule [41e44]. In this context, compound 3d giving a gap en-
ergy value lower than that of molecules 1 and 2, has a significant
antibacterial activity against most of the bacterial strains used.
Therefore, heterocyclic molecules according to their softness have
Fig. 4. Molecular Electrostatic Potential of compounds 1 (a), 2 (b) and 3d (c).

8
M. Horchani et al. / Journal of Molecular Structure 1199 (2020) 127007
an influence on the antibacterial activity.
influence in the antibacterial potential.
3.4.2. Molecular electrostatic potential (MEP)
Acknowledgments
Molecular electrostatic potential (MEP) mapping gives infor-
mation about the biological recognition process and hydrogen
bonding interactions and helps to interpret the electrophilic and
nucleophilic reactions [45]. The MEP of compounds 1, 2 and 3d is
presented in Fig. 4. This property is used to predict the behavior and
responsiveness of these molecules. In this case, to fully explain the
mechanism of synthesis of compound 2 starting from the amino-
pyrazole 1, we resort to the electrostatic potential in order to pre-
dict the reactive sites of electrophilic and nucleophilic attack for the
investigated molecule, this theoretical calculation at the B3LYP/6-
311 þ G(d,p) optimized geometry of the synthesized compound 1 is
used. The positive (blue) districts of MEP were related to nucleo-
philic reactivity while the negative (red and yellow) regions explain
the electrophilic reactivity [46,47]. As we can see in Fig. 4a, the MEP
of compound 1 has a single blue zone which is observed around the
nitrogen atom linked to two hydrogen atoms. Therefore, it has a
high electron density, so its free doublet is apt to attack electro-
philic sites preferentially. Indeed, by double nucleophilic attack on
triethyl orthoformate's electrophilic site, we obtain the iminoether
2. On the other hand, the MEP of compound 2 shows that the most
negative regions are located around the iminic carbon (linked to the
ethoxy group) and the carbon of the nitrile function (CN), these
atoms are likely to be attacked by the nitrogen-free doublet of the
hydrazide, to train the pyrazolo-triazolo-pyrimidine derivatives 3a-
3i. In Fig. 4c the MEP of compound 3d shows many negative regions
in the investigated molecule which is explained by its large size.
This electrostatic potential plays an important role to clarify, inter/
intra-molecular interactions, drug-protein interactions and
structure-activity relationship, the negative potential (red-yellow
colored) is localized over the hydrogen atom of the pyrimidine ring
in compound 3d while the positive electrostatic potential (blue
colored) is localized over the tow nitrogen atoms of the triazole
group, these group regions may be well involved in the inhibition of
bacteria.
The authors are grateful to the Ministry of Higher Education and
Scientific Research of Tunisia for financial support. The authors also
extend their appreciation to the Deanship of Scientific Research at
King Saud University for funding this work through the research
group RG-164.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127007.
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