Design and microwave-assisted synthesis of dimers of 1,5-benzodiazepine-1,2,3-triazole hybrids bearing alkyl/aryl spacers and their biological assessment

Article abstract of DOI:10.1016/j.molstruc.2019.06.018

Large series of novel N-bis-1,2,3-triazolo-linked-1,5-benzodiazepin-2-ones (BZD) have been synthesized under microwave irradiation through a Cu(I)-catalyzed double 1,3-dipolar alkyne-azide coupling reaction. This process is of considerable synthetic advantages in terms of time saving and remarkable yields. The chemical structures of the isolated compounds have been elucidated on the basis of extensive spectroscopic methods including 1D & 2D NMR, IR and HRMS. All the synthesized compounds have been evaluated for their antimicrobial and antioxidant activities. In vitro antimicrobial environment, almost all of compounds have shown an interesting activity. Among the dimers of 1,5-benzodiazepine-1,2,3-triazole compounds tested, the results revealed that the potent antibacterial was recorded to compounds 3g,l for Gram-positive (within MIC = 31.25 and 125 μg/mL), and 3h,k for Gram-negative (within MIC = 31.25 and 125 μg/mL). Besides, the results demonstrate that compounds 3h,k have demonstrated the strongest potential against all the tested fungus (within MIC = 62.5 and 125 μg/mL). The antioxidant evaluation indicated that most compounds exhibited a moderate to good biological activity.

Full text of DOI:10.1016/j.molstruc.2019.06.018

Accepted Manuscript
Design and microwave-assisted synthesis of dimers of 1,5-benzodiazepine-1,2,3-
triazole hybrids bearing alkyl/aryl spacers and their biological assessment
Zouhour Jaafar, Sami Chniti, Ahlem Ben Sassi, Hayet Dziri, Sylvain Marque, Marc
Lecouvey, Rafik Gharbi, Moncef Msaddek
PII:
S0022-2860(19)30731-8
DOI:
https://doi.org/10.1016/j.molstruc.2019.06.018
MOLSTR 26660
Reference:
To appear in: Journal of Molecular Structure
Received Date: 2 March 2019
Revised Date: 2 June 2019
Accepted Date: 4 June 2019
Please cite this article as: Z. Jaafar, S. Chniti, A. Ben Sassi, H. Dziri, S. Marque, M. Lecouvey, R.
Gharbi, M. Msaddek, Design and microwave-assisted synthesis of dimers of 1,5-benzodiazepine-1,2,3-
triazole hybrids bearing alkyl/aryl spacers and their biological assessment, Journal of Molecular
Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.06.018.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Design and Microwave-assisted Synthesis of Dimers of 1,5-benzodiazepine-1,2,3-triazole
hybrids bearing alkyl/aryl spacers and Their Biological Assessment
Zouhour Jaafar a, Sami Chniti a, Ahlem Ben Sassi b, Hayet Dziri b, Sylvain Marque c,
Marc Lecouvey d, Rafik Gharbi e, Moncef Msaddek a*
a Laboratory of Heterocyclic Chemistry Natural Products and Reactivity/CHPNR, Faculty of Science of
Monastir, University of Monastir, 5019 Monastir, Tunisia.
b Laboratory of Transmissible Diseases and Biologically Active Substances, Department of Microbiology,
Faculty of Pharmacy, University of Monastir, 5019 Monastir, Tunisia.
c Université de Versailles Saint-Quentin-en-Yvelines, Institut Lavoisier de Versailles (ILV), UMR 8180, 45
avenue des Etats-Unis, 78035 Versailles Cedex, France.
d CSPBAT, CNRS UMR 7244, F-93017, Université Paris 13, Sorbonne Paris City, Bobigny, France.
e Laboratory Applied chemistry and environment, UR13ES63, Faculty of Science of Monastir, University of
Monastir, 5019 Monastir, Tunisia.
E-mail: moncefmsadek@gmail.com
Abstract
Large series of novel N-bis-1,2,3-triazolo-linked-1,5-benzodiazepin-2-ones (BZD) have been
synthesized under microwave irradiation through a Cu(I)-catalyzed double 1,3-dipolar alkyne-
azide coupling reaction. This process is of considerable synthetic advantages in terms of time
saving and remarkable yields. The chemical structures of the isolated compounds have been
elucidated on the basis of extensive spectroscopic methods including 1D & 2D NMR, IR and
HRMS. All the synthesized compounds have been evaluated for their antimicrobial and
antioxidant activities. In vitro antimicrobial environment, almost all of compounds have
shown an interesting activity. Among the dimers of 1,5-benzodiazepine-1,2,3-triazole
compounds tested, the results revealed that the potent antibacterial was recorded to
compounds 3g,l for Gram-positive (within MIC = 31.25 and 125 µg/ml), and 3h,k for Gram-
negative (within MIC = 31.25 and 125 µg/ml). Besides, the results demonstrate that
compounds 3h,k have demonstrated the strongest potential against all the tested fungus
(within MIC = 62.5 and 125 µg/ml). The antioxidant evaluation indicated that most
compounds exhibited a moderate to good biological activity.
Keywords: 1,5-benzodiazepin-2-one, 1,2,3-triazole, microwave irradiation, click chemistry,
antimicrobial activity, antioxidant activity.
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1. Introduction
Among the large varieties of heterocycles, benzodiazepines have found their place in
medicinal chemistry thanks to their clinical success as the most prescribed medicines [1].
Therefore, a considerable interest has been focused on the synthesis of new benzodiazepines.
The use of this class of compounds with therapeutic purposes is not only limited to treatment
of depression [2], epilepsy [3], but also psychomotor agitation [4], seizures and muscle
spasms [5]. Furthermore, many examples of benzodiazepines are described as excellent
samples of anticancer [6], antiviral [7] and antimicrobial agents [8]. Conformational
constraint in their structures plays a key role in the determination of their bioactivities [9].
Interestingly, since N-functionalization of benzodiazepines (BZD) leads to the rigid
analogues, the development of such scaffolds is highly desired [10]. In recent years,
alkylating agents have been extensively studied and leading to the development of many
biologically active compounds, particularly molecules that have been based on a triazole
moiety [11]. Moreover, triazoles are an important class of N-heterocycles [12]. Their
applications have been extended to widespread pathologies such as epilepsy [13],
inflammation [14] and viral diseases [15]. In addition, they are considered to be excellent
antibiotic agents [16]. A recent research in drug discovery has aimed at introducing the 1,2,3-
triazole moiety as a connecting unit to link together two or more pharmacophores. The 1,5-
benzodiazepine scaffold containing triazole moiety has gained popularity thanks to their
pharmacological proprieties [17]. Further, it has been reported that many biologically active
1,2,3-bistriazole derivatives, which have interesting molecular symmetry, have shown a broad
range of clinical applications [18]. In addition, amide-linked 1,4-disubstituted 1,2,3-
bistriazoles present an interesting cytotoxicity [19]. Phenanthroline-2,9-bistriazoles reveal
good anti-selective G-quadruplex intercalators [20] and the deoxystreptamine dimers (sugar-
based bistriazole) serve also as human m-RNA ligands [21]. On the other hand, microbial
infections are the most prevailing and spread infectious diseases worldwide [22].
Furthermore, their serious medical problem is resistance and the rapid rate of their
development has led to increasing levels of resistance to classical antibiotics [23]. Therefore,
the discovery and the development of effective antibacterial and antifungal drugs with novel
mechanisms of action have become priority tasks for infectious-disease-research programs.
On the other hand, it has been established that a free phenolic hydroxyl group connected to a
benzodiazepine ring leads to new hydroxybenzodiazepine systems with a better reducing
ability towards the most recommended AAPH and DPPH peroxyl radicals for measuring
radical-scavenging activity in vitro [24, 25]. Since it looked encouraging, we aimed to
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examine the possibility of our new dimers of 1,5-benzodiazepines-1,2,3-triazoles hybrids
bearing two important phenolic moieties to act as radical scavengers. As part of our ongoing
program directed towards the development of 1,5-benzodiazepine derivatives bearing a
triazole moiety, we have focused our effort on the design of novel symmetric hybrid
conjugates linking the benzodiazepine system to two 1,2,3-triazole units via a copper(I)-
catalyzed 1,3-dipolar cyclisation. The synthesized compounds prompted us to explore the
antimicrobial and the antioxidant effects of these new 1,2,3-triazolo-1,5-benzodiazepin-2-one
derivatives. Their activities have been evaluated and discussed.
2. Experimental section
2.1. Material and instruments
Reagents were commercial products of analytical purity (Merck, Fluka, and Aldrich) and used
1
without prior purification. H, 13C and two-dimensional NMR spectra were recorded on a
Bruker AM-400 spectrometer at room temperature in CDCl3 and DMSO at 300-400 MHz and
at 75-100 MHz with all chemical shifts, reported in ppm, using residual non-deuterated
solvent peaks as internal reference. Coupling constants are given in Hz and the abbreviations
used for signal multiplicity are the following: s (singlet), d (doublet), dd (double doublet),
13
t (triplet), m (multiplet) and (br) broad signal. The C NMR spectra are proton decoupled.
IR spectra (In KBr pellets) were recorded on a Perkine Elmer Spectrum two FT-IR instrument
with the Universal ATR Sampling Accessory, only structurally significant bands are reported.
HRMS spectra were acquired with an electrospray ionisation (ESI) mass spectroscopy data
were recorded on an UPLC Waters device (in positive mode); for the voltages of the mass
spectroscopies, the following abbreviations are used: C Capillary (kV), SC Sampling Cone,
EC Extraction Cone. Calibration was performed with sodium formate (range from 100 to
1000 g.mol-1) and the lockspray (lockmass on the leucine encephaline 556.2771 g.mol-1) was
used without collision energy, the relative intensity of peaks is given in brackets. Microwave
experiments were conducted with a Biotage AB Initiator EXP EU device with a maximum
power of 800 W. Reactions were performed in open-air vessel with a magnetical stirrer.
The temperature was measured by IR detection, after calibration using an optical fiber inside
the reaction mixture. The power was fixed at 400 W and the temperature evolved in restreint-
free conditions. Irradiation was carried out according to the conditions of times and
temperatures indicated in the tables or in the experimental part. All the reactions were
followed by TLC using aluminium sheets of Merck silica gel 60 F254, 0.2 mm. Melting
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points of all the synthesized compounds were determined on BUCHI-510 capillary melting
point apparatus.
2.2. Chemistry
2.2.1. Synthesis of N-(prop-1-yn)-4-(2-hydroxyphenyl)-1,5-benzodiazepin-2-one (2)
In a rounded bottom flask of 25 mL, an excess of propargyl bromide (0.45 mL, 1.5 equiv.,
5.94 mmol) was added to a solution of the compound 1 (1 g, 1 equiv., 3.96 mmol) and NaH
(0.123 g, 1.3 equiv., 5.14 mmol, 60 % dispersion in mineral oil) in N,N-dimethylformamide
(15 mL). The resulting mixture was stirred for 12 h (monitored by TLC) and then organic
materials were extracted with dichloromethane (3 × 30 mL). The combined organic layer was
dried over MgSO4, then filtered. The filtrate was concentrated in vacuum to give a residue,
purified over silica gel by flash column chromatography (Cyclohexane/EtOAc 8:2) to afford
the corresponding compound 2.
Yellow solid; m.p.: 115-117 °C, 0.78 g, yield 78 %. IR (KBr, cm-1): 3376 (O-H), 3320
1
(≡C-H), 1670 (C=O), 1560 (C=N). H NMR (300 MHz, CDCl3): δH 2.25 (t, 1H, H22,
J= 3 Hz), 2.94 (d, 1H, H6a, J= 12 Hz), 4.22 (t, 2H, H20, J= 12 Hz), 4.68 (d, 1H, H6b,
J= 18 Hz), 6.85-6.95 (m, 2H, Ar-H), 7.23-7.36 (m, 4H, Ar-H), 7.66 (d, 1H, Ar-H, J= 6
13
Hz), 7.77 (d, 1H, Ar-H, J= 6 Hz), 13.79 (s, 1H, OH). C NMR (75 MHz, CDCl3):
δC 37.18 (C20), 37.65 (C6), 72.24 (C21), 78.44 (C22), 117.56 (CAr), 117.73 (CAr), 117.98
(CAr), 118.65 (CAr), 121.27 (CAr), 125.55 (CAr), 126.73 (CAr), 128.95 (CAr), 133.65
(CAr), 134.16 (CAr), 137.97 (CAr), 161.65 (C5), 163.78 (C14), 164.59 (C7). ESI(+)-MS
CH3CN [C= 1, SC= 30, EC= 3] m/z (rel. int.): 291 (100, M+H+), 329 (30, M+K+)
HRMS ES+ for C18H15N2O2 m/z: [M+H]+ Calc. 291.1134, found: 291.1124. ESI(-)-MS
CH3CN [C= 1, SC= 30, EC= 2] m/z (rel. int.): 289 (30, [M-H]-), 181 (40), 1116 (100),
HRMS ES- for C18H13N2O2 m/z: [M-H]- Calc. 289.1079, found: 289.1076.
2.2.2. Synthesis of N-bis-1,2,3-triazole linked 1,5-benzodiazepines (3a-l)
In a rounded bottom flask of 25 mL, a mixture of compound 2 (500 mg, 1 equiv., 1.72 mmol)
and DIPEA (0.89 mL, 3 equiv., 5.16 mmol) was stirred in dry DMF (10 mL), followed by
addition of CuI (4.1 mg, 10 mol %, 0.172 mmol) and the appropriate diazide derivative DA1-12
(1 equiv., 1 mmol). The reaction mixture was stirred under microwave irradiation (400 W) for
6-12 min. After completion of the reaction as determined by TLC, the reaction was quenched
with crushed ice (50 g) to avoid the apparition of side products. Organic materials were
extracted with EtOAc (3 × 30 mL), then combined and dried over anhydrous MgSO4. The
crude reaction was filtered and the filtrate was concentrated under reduced pressure. The
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crude material was purified by flash column chromatography on silica gel and concentrated
under vacuum to afford a crude material, purified by column chromatography on silica gel
(cyclohexane/EtOAc from 8:2 to 5:5) as eluent, affording the pure cycloadducts 3a-l in 67-
92% yields.
2.2.2.1. 1,1'-((1,1'-(ethane-1,2-diyl) bis(1H-1,2,3-triazole-4,1-iyl)) bis(methylene)) bis(1,5-
benzodiazepin-2-one) (3a)
Yellow solid; m.p.: 217-219 °C, 460 mg, yield 92%. IR (KBr, cm-1): 3390 (O-H), 1691
(C=O), 1599 (C=N).1H NMR (300 MHz, CDCl3): δH 2.97 (d, 2H, H20a, J= 12 Hz), 4.17 (dd,
2H, H20b, J= 20 Hz, J= 12 Hz), 4.77 (d, 2H, H6a, J= 16 Hz,), 4.86 (s, 4H, H26,26’), 5.13 (d, 2H,
H6b, J= 15 Hz), 6.87-6.94 (m, 2H, Ar-H), 7.02 (d, 2H, Ar-H, J= 6 Hz), 7.28-7.40 (m, 6H,
Ar-H), 7.48 (s, 2H, H25,25’), 7.83 (d, 2H, Ar-H, J= 6 Hz), 8.03 (s, 2H, Htriaz), 13.93 (broad
singlet, 2H, OH). 13C NMR (75 MHz, CDCl3): δC 38.17 (C6,6’), 44.68 (C26,26’), 49.58 (C20,20’),
117.96 (CAr), 118.25 (CAr), 119.09 (CAr), 123.20 (CAr), 125.21 (Ctriaz), 126.05 (CAr), 126.97
(CAr), 127.56 (CAr), 129.28 (CAr), 134.13 (CAr), 135.31 (CAr), 138.29 (CAr), 144.24 (CAr),
162.16 (C5,5’), 164.16 (C14,14’), 164.96 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z
(rel. int.): 731 (20, M+K+), 715 (40, M+Na+), 693 (100, M+H+), 347 (80, 0.5 M+H+), HRMS
ES+ for C38H33N10O4 [M+H]+ calc. 693.2686, found: 693.2696. ESI (-)-MS CH3CN [C= 1,
SC= 30, EC= 2] m/z (rel. int.): 791 (50), 713 (10), 691 (100, [M-H]-), 297 (50), 126 (90),
HRMS ES- for C38H31N10O4 [M-H]- calc. 691.2530, found: 691.2516.
2.2.2.2. 1,1'-((1,1'-(hexane-1,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(methylene))bis(1,5-
benzodiazepin-2-one) (3b)
Yellow solid; m.p.: 231-233 °C, 430 mg, yield 86%. IR (KBr, cm-1): 3338 (O-H), 1691
1
(C=O), 1566 (C=N). H NMR (300 MHz, CDCl3): δH 1.24 (m, 4H, H28,28’), 1.81 (m, 4H,
H27,27’), 2.92 (d, 2H, H6a,6a’, J= 12 Hz,), 4.16-4.23 (m, 6H, H6b,6b’-26,26’), 4.82 (d, 2H, H20a,20a’
,
J= 15 Hz), 5.10 (d, 2H, H20b,20b’, J= 15 Hz), 6.87-6.92 (m, 2H, Ar-H), 6.96 (d, 2H, Ar-H, J=9
Hz), 7.20-7.35 (m, 8H, Ar-H), 7.54 (s, 2H, Htriaz), 7.78 (d, 2H, Ar-H, J= 9 Hz), 8.00 (d, 2H,
13
Ar-H, J= 6 Hz), 13.90 (s, 2H, OH). C NMR (75 MHz, CDCl3): δC 25.77 (C28,28’), 29.82
(C27,27’), 38.24 (C6,6’), 44.73 (C20,20’), 50.09 (C26,26’), 117.74 (CAr), 118.01 (CAr), 118.27 (CAr),
119.12 (CAr), 123.35 (CAr), 124.08 (Ctriaz), 126.05 (CAr), 126.89 (CAr), 127.57 (CAr), 129.34
(CAr), 134.12 (CAr), 135.21 (CAr), 138.36 (CAr), 162.19 (C5,5’), 164.13 (C14,14’), 164.99 (C7,7’).
ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel. int.): 772 (20), 771 (100, M+Na+), 749
(65, M+H+), HRMS ES+ for C42H41N10O4 [M+H]+ calc. 749.3312, found: 749.3309. ESI (-)-
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MS CH3CN [C= 1, SC= 30, EC= 2] m/z (rel. int.): 810 (50), 783 (20), 747 (90, [M-H]-), 213
(100), HRMS ES- for C42H39N10O4 [M-H]- calc. 747.2316, found: 747.2318.
2.2.2.3. 1-((1-(6-azidohexyl)-1H-1,2,3-triazol-4-yl)methyl)-(1,5-benzodiazepin-2-one)
(3b’)
It was obtained as described in the last procedure and was purified as minor product
after purification as yellow solid; m.p.: 117-119 °C, 135 mg, yield 27%. IR (KBr, cm-
1
1): 3386 (O-H), 1690 (C=O), 1598 (C=N). H NMR (300 MHz, CDCl3): δH 1.24-1.42
(m, 4H, H28,29), 1.52-1.60 (m, 2H, H30), 1.84-1.93 (m, 2H, H27), 3.00 (d, 1H, H6b, J= 12
Hz), 3.25 (t, 2H, H31, J= 9 Hz), 4.25 (d, 1H, H6b, J= 12 Hz), 4.28-4.34 (td, 2H, H26, J=
6Hz), 4.90 (d, 1H, H20b, J= 15 Hz), 5.17 (d, 2H, H20a, J= 15 Hz), 6.98 (t, 2H, Ar-H, J=
9 Hz), 7.05 (d, 2H, Ar-H, J=9 Hz), 7.25 (s, 1H, Ar-H), 7.28-7.46 (m, 5H, Ar-H), 7.59
(s, 1H, Ar-H), 7.80 (d, 1H, Ar-H, J= 6 Hz), 8.07 (d, 1H, Ar-H, J= 6 Hz).13C NMR (75
MHz, CDCl3): δC 26.09 (C28), 28.60 (C29), 29.95 (C30), 30.90 (C27), 38.24 (C6), 44.64
(C20), 50.28 (C26), 51.23 (C31), 117.94 (CAr), 118.31 (CAr), 119.18 (CAr), 123.38 (CAr),
124.08 (CAr), 126.11 (CAr), 126.87 (CAr), 127.67 (CAr), 129.37 (CAr), 134.24 (CAr),
135.18 (CAr), 138.22 (CAr), 143.70 (CAr), 162.21 (C5), 164.19 (C14), 164.98 (C7).
ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel. int.): 497 (30, M+K+), 427 (60),
459 (80, M+H+), HRMS ES+ for C24H27N8O2 m/z: [M+H]+ Calc. 459.2348, found:
459.2342. ESI(-)-MS CH3CN [C= 1, SC= 30, EC= 2] m/z (rel. int.): 457 (60, [M-H]-),
416 (20), 252 (40), HRMS ES- for C24H25N8O2 m/z: [M-H]- Calc. 457.2179, found:
457.2173.
2.2.2.4. 1,1'-((1,1'-(1,4-phenylenebis(methylene)) bis(1H-1,2,3-triazole-4,1-diyl))
bis(methylene)) bis(1,5-benzodiazepin-2-one) (3c)
Yellow solid; m.p.: 170-172 °C, 410 mg, yield 82%. IR (KBr, cm-1): 3386 (O-H), 1676
1
(C=O), 1576 (C=N). H NMR (300 MHz, CDCl3): δH 2.99 (d, 2H, H6a, J= 12 Hz), 4.24 (d,
2H, H6b, J= 12 Hz), 4.89 (d, 2H, H20a, J= 15 Hz), 5.17 (d, 2H ,H20b, J= 15 Hz), 5.45 (d, 2H,
H26,26’, J= 15 Hz), 5.54 (d, 2H, H26,26’, J= 15 Hz), 6.95-7.07 (m, 4H, Ar-H), 7.25 (s, 2H,
Ar-H), 7.28 (s, 2H, Ar-H), 7.32-7.47 (m, 8H, Ar-H), 7.59 (s, 2H, Htriaz), 7.84 (d, 2H, Ar-H, J=
9 Hz), 8.07 (d, 2H, Ar-H, J= 9 Hz), 13.96 (s, 2H, OH). 13C NMR (75 MHz, CDCl3): δC 37.15
(C6,6’), 43.68 (C20,20’), 52.67 (C26,26’), 116.93 (CAr), 117.24 (CAr), 118.09 (CAr), 122.29 (CAr),
123.26 (CAr), 125.05 (CAr), 125.89 (CAr), 126.55 (CAr), 127.79 (CAr), 128.28 (Ctriaz), 130.48
(CAr), 133.11 (CAr), 134.11 (CAr), 137.28 (CAr), 143.28 (CAr), 161.12 (C5,5’), 163.05 (C14,14’),
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163.94 (C7,7’). ESI-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel. int.): 807 (10, M+K+), 791
(20, M+Na+), 769 (40, M+H+), 385 (100, 0.5 M+H+), HRMS ES+ for C44H37N10O4 m/z:
[M+H]+ calc. 769.3068, found: 769.2028. ESI-MS CH3CN [C= 1, SC= 30, EC= 2] m/z (rel.
int.): 767 (40), [M-H]-, HRMS ES- for C44H35N10O4 m/z: [M-H]- calc. 767.2843, found:
767.2838.
2.2.2.5. 1,1'-((1,1'-(methylenebis(4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))
bis(methylene))bis(1,5-benzodiazepin-2-one) (3d)
Yellow solid; m.p.: 175-177 °C, 390 mg, yield 78%. IR (KBr, cm-1): 3355 (O-H), 1676
(C=O), 1584 (C=N). 1H NMR (300 MHz, CDCl3): δH 2.90 (d, 2H, H6a, J= 12 Hz), 4.14
(d, 2H, H6b, J=12 Hz), 4.81 (d, 2H, H20a, J= 15 Hz), 5.07 (d, 2H, H20b, J= 15 Hz), 5.39
(s, 2H, H30,30’), 6.88 (t, 2H, Ar-H, J= 9 Hz), 6.96 (d, 2H, Ar-H, J= 9 Hz), 7.15-7.38 (m,
16H, Ar-H), 7.49 (s, 2H, Htriaz), 7.75 (d, 2H, Ar-H, J= 9 Hz), 7.97 (d, 2H, Ar-H, J= 9
Hz), 13.85 (s, 2H, OH). 13C NMR (75 MHz, CDCl3): δC 38.20 (C6,6’), 43.31 (C20,20’),
44.67 (C30), 117.99 (CAr), 118.26 (CAr), 119.09 (CAr), 121.44 (CAr), 123.32 (CAr),
124.24 (CAr), 126.05 (CAr), 126.91 (CAr), 127.54 (Ctriaz), 128.67 (CAr,), 128.69 (CAr),
128.77 (CAr), 129.33 (CAr), 134.11 (CAr), 135.14 (CAr), 138.37 (CAr), 144.27 (CAr),
162.21 (C5,5’), 164.19 (C14,14’), 164.98 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC= 30,
EC= 3] m/z (rel. int.): 853 (40, M+Na+), 831 (70, M+H+), 416 (50, 0.5 M+H+), HRMS
ES+ for C49H39N10O4 m/z: [M+H]+ Calc. 831.3217, found: 831.3213. ESI(-)-MS
CH3CN [C= 1, SC= 30, EC= 2] m/z (rel. int.): 843 (60), 829 (50, [M-H]-), 826 (30),
305 (10), HRMS ES- for C49H37N10O4 m/z: [M-H]- Calc. 829.3023, found: 829.3017.
2.1.2.6. 1,1'-((1,1'-(1,4-phenylene) bis(1H-1,2,3-triazole-4,1-diyl)) bis(methylene)) bis(1,5-
benzodiazepin-2-one) (3e)
Yellow solid; m.p.: 192-194 °C, 420 mg, yield 84%. IR (KBr, cm-1): 3401 (O-H), 1706
1
(C=O), 1591 (C=N). H NMR (300 MHz, CDCl3): δH 3.19 (br, 2H, H6a), 4.33 (br, 2H, H6b),
5.13 (s, 4H, H20,20’) 6.70 (d, 2H, Ar-H, J= 9 Hz), 7.00 (d , 2H, Ar-H, J= 9 Hz), 7.33-7.48 (m,
10H, Ar-H, H27,27’), 7.51 (s, 2H, Htriaz), 7.88-7.96 (m, 4H, Ar-H) , 8.18 (s, 2H, Ar-H), 13.78 (s,
2H, OH). 13C NMR (75 MHz, CDCl3): δC 38.24 (C6,6’), 43.39 (C20,20’), 114.00 (CAr,), 117.62
(CAr), 118.07 (CAr), 118.93 (CAr), 121.45 (C27,27’), 122.99 (C7,7’), 125.64 (Ctriaz), 126.65 (CAr),
127.18 (CAr), 129.85 (CAr) , 133.94 (CAr), 134.70 (CAr), 138.29 (CAr), 143.66 (CAr), 149.19
(CAr), 161.35 (C5,5’), 164.58 (C14,14’), 164.74 (C7,7’) . ESI-MS CH3CN [C= 1, SC= 30, EC= 3]
m/z (rel. int.): 763 (40, M+Na+), 741 (100, M+H+), 385 (20), HRMS ES+ for C42H33N10O4
m/z: [M+H]+ calc. 741.2999, found: 741.3018. ESI-MS CH3CN [C= 1, SC= 30, EC= 2] m/z
7

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(rel. int.): 739 (100, [M-H]-), 334 (50), 217 (20), HRMS ES- for C42H31N10O4 m/z: [M-H]-
calc. 739.2466, found: 739.2467.
2.2.2.7. 1,1'-((1,1'-(1,3-phenylene) bis(1H-1,2,3-triazole-4,1-diyl)) bis(methylene)) bis(1,5-
benzodiazepin-2-one) (3f)
Yellow solid; m.p.: 210-212 °C, 400 mg, yield 80%. IR (KBr, cm-1): 3391 (O-H), 1673
1
(C=O), 1566 (C=N). H NMR (400 MHz, CDCl3): δH 2.94 (d, 2H, H6a, J= 12 Hz),
4.22 (d, 2H, H6b, J= 12 Hz), 4.93 (d, 2H, H20a, J= 16 Hz), 5.22 (d, 2H, H20b, J= 16 Hz),
6.92 (t, 2H, H-Ar, J= 8 Hz), 6.99 (d, 2H, H-Ar, J= 8 Hz), 7.10-7.57 (m, 8H, H-Ar),
7.68 (d, 2H, J= 8 Hz), 7.91 (d, 2H, J= 8 Hz), 8.01 (s, 2H, Htriaz), 8.05 (d, 2H, HAr, J= 8
Hz), 13.93 (s, 2H, OH). 13C NMR (75 MHz, CDCl3): δC 37.41 (C6,6’), 43.70 (C20,20’),
117.24 (CAr), 117.53 (CAr), 118.31 (CAr), 122.14 (CAr), 125.20 (CAr), 126.26 (CAr),
126.63 (CAr), 126.70 (CAr), 126.91 (CAr), 127.26 (Ctriaz), 128.54 (CAr), 129.25 (CAr),
133.41 (CAr), 133.46 (CAr), 134.35 (CAr), 134.65 (CAr), 137.51 (CAr), 161.45 (C5,5’),
163.31 (C14,14’), 164.20 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel.
int.): 779 (70, M+K+), 763 (40, M+Na+), 741 (40, M+H+), 371 (30, 0.5 M+H+), HRMS
ES+ for C42H33N10O4 m/z: [M+H]+ Calc. 741.2778, found: 741.2771. ESI(-)-MS
CH3CN [C= 1, SC= 30, EC= 2] m/z (rel. int.): 845 (60), 836 (20), 823 (80, [M-H]-),
325 (40), 311 (20), HRMS ES- for C42H31N10O4 m/z: [M-H]- Calc. 739.2567, found:
739.2559.
2.2.2.8. 1,1'-((1,1'-(oxybis(4,1-phenylene)) bis(1H-1,2,3-triazole-4,1-diyl)) bis(methylene))
bis(1,5-benzodiazepin-2-one) (3g)
Yellow solid; m.p.: 167-169 °C, 435 mg, yield 87% IR (KBr, cm-1): 3379 (O-H), 1671 (C=O),
1588 (C=N). 1H NMR (400 MHz, CDCl3): δH 2.95 (d, 2H, H6a, J= 16 Hz), 4.19 (d, 2H, H6b,
J= 12 Hz), 4.91 (d, 2H, H20a, J= 16 Hz), 5.18 (d, 2H, H20b, J= 16 Hz), 6.92 (t, 2H, Ar-H, J= 8
Hz), 6.98 (d, 2H, Ar-H, J= 8 Hz), 7.27 (t, 2 H, Ar-H, J= 4 Hz), 7.33-7.37 (m, 8 H, Ar-H), 7.79
(d, 2H, Ar-H, J= 8 Hz), 7.86 (d, 2H, Ar-H, J= 8 Hz), 7.98 (d, Ar-H, J= 8 Hz), 8.04 (s, 2H,
13
Htriaz), 8.07 (d, 2H, Ar-H, J= 4 Hz), 13.88 (s, 2H, OH). C NMR (100 MHz, CDCl3): δC
37.42 (C6,6’), 43.85 (C20,20’), 117.16 (CAr), 117.58 (CAr), 118.46 (CAr), 120.04 (CAr), 121.54
(Ctriaz), 122.40 (CAr), 125.56 (CAr), 126.27 (CAr), 126.89 (CAr), 128.56 (CAr), 128.95 (CAr),
133.53 (CAr), 134.19 (CAr), 137.69 (CAr), 139.60 (CAr), 140.24 (CAr), 144.43 (CAr), 161.43
(C5,5’), 163.38 (C14,14’), 164.43 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel.
int.): 855 (10, M+Na+), 833 (50, M+H+), 417 (100, 0.5 M+H+), HRMS ES+ for C48H37N10O5
m/z: [M+H]+ calc. 833.2948, found: 833.2963. ESI(-)-MS CH3CN [C= 1, SC= 30, EC= 2] m/z
8

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(rel. int.): 831 (100, [M-H]-), 325 (40), 311 (30), 116 (80), HRMS ES- for C48H35N10O5 m/z:
[M-H]- calc. 831.2792, found: 831.2790.
2.2.2.9. 1,1'-((1,1'-(sulfonylbis(4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))
bis(methylene))bis(1,5-benzodiazepin-2-one) (3h)
Yellow solid; m.p.: 185-187 °C, 415 mg, yield 83%. IR (KBr, cm-1): 3402 (O-H), 1668
1
(C=O), 1578 (C=N). H NMR (400 MHz, CDCl3): δH 2.92 (d, 2H, H6a, J= 12 Hz), 4.18 (d,
2H, H6b, J= 12 Hz), 4.86 (d, 2H, H20a, J= 16 Hz), 5.18 (d, 2H, H20b, J= 16 Hz), 6.88 (t, 2H, Ar-
H, J= 8 Hz), 6.95 (d, 2H, Ar-H, J= 8 Hz), 7.01-7.17 (m, 4H, Ar-H), 7.23 (t, 2H, Ar-H, J= 8
Hz), 7.29-7.36 (m, 6H, Ar-H), 7.61 (t, 4H, Ar-H, J= 8 Hz), 7.78 (d, 2H, Ar-H, J= 8 Hz), 7.99
(s, 2H, Htriaz), 8.04 (t, 2H, Ar-H, J= 8 Hz), 13.85 (s, 2H, OH). 13C NMR (100 MHz, CDCl3):
δC 37.22 (C6,6’), 43.77 (C20,20’), 116.98 (CAr), 117.30 (CAr), 118.15 (CAr), 118.93 (CAr), 121.37
(CAr), 122.27 (CAr), 123.96 (CAr), 125.17 (CAr), 125.98 (CAr), 126.62 (CAr), 128.32 (CAr),
131.86 (Ctriaz), 133.17 (CAr), 134.17 (CAr), 137.37 (CAr), 143.56 (CAr), 155.90 (CAr), 161.21
(C5,5’), 163.10 (C14,14’), 164.12 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel.
int.): 903 (10, M+Na+), 881 (100, M+H+), 441 (50, 0.5 M+H+), HRMS ES+ for C48H37N10O6S
m/z: [M+H]+ calc. 881.2618, found: 881.2602. ESI(-)-MS CH3CN [C= 1, SC= 30, EC= 2] m/z
(rel. int.): 879 (100, [M-H]-), 100 (100), 116 (80), 126 (20), HRMS ES- for C48H35N10O6S
m/z: [M-H]- calc. 879.2462, found: 879.2421.
2.2.2.10. 1,1'-((1,1'-(ethane-1,2-diylbis(4,1-phenylene))bis(1H-1,2,3-triazole-4,1-diyl))
bis(methylene))bis(1,5-benzodiazepin-2-one) (3i)
Yellow solid; m.p.: 178-180 °C, 335 mg, yield 67%. IR (KBr, cm-1): 3391 (O-H), 1675
(C=O), 1568 (C=N). 1H NMR (400 MHz, CDCl3): δH 2.88 (d, 2H, H6a, J= 8 Hz), 4.12 (d, 2H,
H6b, J= 8 Hz), 4.78 (d, 2H, H20a, J= 12 Hz), 5.06 (d, 2H, H20b, J= 16 Hz), 5.33-5.45 (2d, 4H,
H29,29’, J= 16 Hz), 6.86 (t, 2H, J= 8 Hz), 6.94 (d, 2H, Ar-H, J= 8 Hz), 7.08-7.23 (m, 8H, Ar-
H), 7.26-7.31 (m, 8H, Ar-H), 7.50 (s, 2H, Htriaz), 7.73 (d, 2H, Ar-H, J= 8 Hz), 7.96 (d, 2H, Ar-
H, J= 4 Hz), 13.83 (s, 2H, OH). 13C NMR (100 MHz, CDCl3): δC 37.23 (C29,29’), 43.79 (C6,6’),
48.73 (C20,20’), 116.99 (CAr), 117.32 (CAr), 118.16 (CAr), 118.95 (CAr), 121.40 (CAr), 121.64
(CAr), 122.28 (CAr), 125.18 (CAr), 125.98 (CAr), 126.64 (CAr), 128.32 (CAr), 131.88 (Ctriaz),
133.19 (CAr), 134.19 (CAr), 137.37 (CAr), 143.57 (CAr), 145.92 (CAr), 161.21 (C5,5’), 163.09
(C14,14’), 164.12 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel. int.): 867 (50,
M+Na+), 845 (100, M+H+), HRMS ES+ for C50H41N10O4 m/z: [M+H]+ calc. 845.2962, found:
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845.2950. ESI(-)-MS CH3CN [C= 1, SC= 30, EC= 2] m/z (rel. int.): 867 (30), 843 (100, [M-
H]-), 827 (10), HRMS ES- for C50H39N10O4 m/z: [M-H]- calc. 843.2252, found: 843.2516.
2.2.2.11. 1,1'-((1,1'-(1,3-phenylenebis(methylene))bis(1H-1,2,3-triazole-4,1-diyl))
bis(methylene))bis(1,5 -benzodiazepin-2-one) (3j)
Yellow solid; m.p.: 143-145 °C, 445 mg, yield 89%. IR (KBr, cm-1): 3402 (O-H), 1660
1
(C=O), 1578 (C=N). H NMR (400 MHz, CDCl3): δH 3.00 (d, 2H, H6a, J= 12 Hz), 4.24 (d,
2H, H6b, J= 12 Hz), 4.87 (d, 2H, H20a, J= 12 Hz), 5.17 (d, 2H, H20b, J= 16 Hz), 5.45 (d, 2H,
H26a, J= 16 Hz), 5.54 (d, 2H, H26b, J= 16 Hz), 6.96 (t, 2H, Ar-H, J= 8 Hz), 7.06 (d, 2H, Ar-H,
J= 4 Hz), 7.20-7.23 (m, 2H, Ar-H), 7.31-7.46 (m, 10H, Ar-H), 7.61 (s, 2H, Htriaz), 7.84 (d, 2H,
Ar-H, J= 4 Hz), 8.09 (d, 2H, Ar-H, J= 8Hz), 13.99 (s, 2H, OH). 13C NMR (75 MHz, CDCl3):
δC 37. 42 (C6,6’), 43.96 (C20,20’), 53.04 (C26,26’), 117.22 (CAr), 117.50 (CAr), 118.33 (CAr),
122.54 (CAr), 125.28 (CAr), 126.15 (CAr), 126.26 (Ctriaz), 126.79 (CAr), 126.91 (CAr), 127.63
(CAr), 128.54 (CAr), 129.25 (CAr), 133.35 (CAr), 133.40 (CAr), 134.41 (CAr), 134.75 (CAr),
137.57 (CAr), 161.40 (C5,5’), 163.35 (C14,14’), 164.21 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC=
30, EC= 3] m/z (rel. int.): 791 (30, M+Na+), 769 (50, M+H+), 385 (100, 0.5 M+H+), HRMS
ES+ for C44H37N10O4 m/z: [M+H]+ calc. 769.2999, found: 769.2990. ESI(-)-MS CH3CN [C=
1, SC= 30, EC= 2] m/z (rel. int.): 769 (30, [M-H]-), 250 (80), 297 (50), 325 (20), 116 (100),
HRMS ES- for C44H35N10O4 m/z: [M-H]- calc. 767.2843, found: 767.2835.
2.2.2.12. 1,1'-((1,1'-(pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(methylene))
bis(1,5-benzodiazepin-2-one) (3k)
Yellow solid; m.p.: 185-187 °C, 355 g, yield 71%. IR (KBr, cm-1): 3391 (O-H), 1675
1
(C=O), 1568 (C=N). H NMR (400 MHz, CDCl3): δH 2.93 (d, 2H, H6a, J= 12 Hz),
4.21 (d, 2H, H6b, J= 12 Hz), 4.92 (d, 2H, H20a, J= 16 Hz), 5.20 (d, 2H, H20b, J= 16 Hz),
6.94 (t, 2H, H-Ar, J= 8 Hz), 6.99 (d, 2H, H-Ar, J= 8 Hz), 7.10-7.52 (m, 9H, H-Ar,),
7.68 (d, 2H, H-Ar, J= 8 Hz), 7.91 (d, 2H, J= 9 Hz), 8.01 (s, 2H, Htriaz), 8.09 (d, 2H,
13
J= 8 Hz), 13.38 (s, 2H, OH). C NMR (75 MHz, CDCl3): δC 37.63 (C6,6’), 43.56
(C20,20’), 117.31 (CAr), 118.31 (CAr), 122.91 (CAr), 125.20 (CAr), 126.26 (CAr), 126.63
(CAr), 126.70 (CAr), 126.83 (Ctriaz), 127.14 (CAr), 128.54 (CAr), 129.25 (CAr), 133.42
(CAr), 133.56 (CAr), 134.61 (CAr), 134.64 (CAr), 137.56 (CAr), 161.71 (C5,5’), 163.40
(C14,14’), 164.45 (C7,7’). ESI(+)-MS CH3CN [C= 1, SC= 30, EC= 3] m/z (rel. int.): 742
(60, M+H+), 423 (60, 0.5 M+H+), 112 (20), HRMS ES+ for C41H32N11O4 m/z: [M+H]+
Calc. 742.2648, found: 742.2656. ESI(-)-MS CH3CN [C= 1, SC= 30, EC= 2] m/z (rel.
10

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int.): 844 (60), 845 (20), 843 (100, [M-H]-), 311 (40), HRMS ES- for C41H30N11O4 m/z:
[M-H]- Calc. 740.2674, found: 740.2679.
2.2.2.13. 1,1'-((1,1'-(3,3'-dimethyl-[1,1'-biphenyl]-4,4'-diyl)bis(1H-1,2,3-triazole-4,1-diyl))
bis(methylene))bis(1,5-benzodiazepin-2-one) (3l)
Yellow solid; m.p.: 167-169 °C, 405 mg, yield 81%. IR (KBr, cm-1): 3391 (O-H), 1660
(C=O), 1582 (C=N). 1H NMR (400 MHz, CDCl3): δH 2.29 (s, 6H, CH3, H32,32’), 3.07 (d, 2H,
H6b, J= 12 Hz), 4.30 (d, 2H, H6a, J= 12 Hz), 5.06 (d, 2H, H20b, J= 16 Hz), 5.31 (d, 2H, H20a,
J= 16 Hz), 7.01 (t, 2H, Ar-H, J=8 Hz), 7.07 (d, 2H, Ar-H, J= 12 Hz), 7.37 (t, 2H, J= 8 Hz),
7.45-7.48 (m, 10H, Ar-H), 7.58 (d, 2H, J= 8 Hz), 7.61 (s, 2H, Htriaz), 7.90 (d, 2H, Ar-H, J= 8
Hz), 8.19 (d, 2H, Ar-H, J= 8 Hz), 14.01 (s, 2H, OH). 13C NMR (100 MHz, CDCl3): δC 17.39
(C32,32’), 37.51 (C6,6’), 43.96 (C20,20’), 117.25 (CAr), 117.54 (CAr), 118.38 (CAr), 122.68 (CAr),
124.88 (CAr), 125.41 (CAr), 125.60 (CAr), 125.67 (CAr), 126.22 (CAr), 126.26 (Ctriaz), 126.87
(CAr), 128.58 (CAr), 129.56 (CAr), 133.23 (CAr), 133.39 (CAr), 134.39 (CAr), 135.30 (CAr),
137.74 (CAr), 140.59 (CAr), 161.45 (C5,5’), 163.43 (C14,14’), 164.34 (C7,7’). ESI(+)-MS CH3CN
[C= 1, SC= 30, EC= 3] m/z (rel. int.): 867 (10, M+Na+), 845 (100, M+H+), 423 (70, 0.5
M+H+), HRMS ES+ for C50H41N10O4 m/z: [M+H]+ calc. 845.3312, found: 845.3312. ESI(-)-
MS CH3CN [C= 1, SC= 30, EC= 2] m/z (rel. int.): 844 (60), 845 (20), 831 (30), 843 (100),
[M-H]-, 325 (70), 311 (40), 100 (100), HRMS ES- for C50H39N10O4 m/z: [M-H]- calc.
843.3156, found: 843.3102.
2.3. Biological evaluation of the synthesized compounds
2.3.1. Antimicrobial activity
2.3.1.1. Microorganisms
The antibacterial activity of the former compounds has been tested against nine
microorganisms, including reference strains consisting of Gram-positive: B. subtilus (ATCC
6633), S. epidermis (CI), S. aureus (ATCC 25923) and S. aureus (ATCC 29213) and Gram-
negative rods: E. coli (ATCC 25922), K. pneumoniae (CI), E. fecalis (ATCC 29212),
S. enterica (CIP 5262) and E. faecium (CI). On the other side, C. albicans (ATCC 90028),
C. glabrata (ATCC 90030) and C. keusei (ATCC 6258) have been used for the determination
of antifungal activity. Antibacterial and antifungal activities were compared with the activities
of the standard drugs Amoxicillin (AMX), Ampiclline (AMP) and Amphotericin (AB).
The microorganism strains used in the biological assays are listed in Figure 3-5. Different
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American-Type Cell Culture (ATCC) reference bacteria and fungi as well as clinical isolates
strains have been used.
2.3.2. Antioxidant activity
All synthesized hydroxybenzodiazepines 3a-l were evaluated for their in vitro antioxidant
activity including three tests DPPH, ABTS and FRAP at several concentrations (0.62-1.25-
2.50-5.0 mg/mL). The antioxidant capacity of the test has been expressed as IC50 values,
which indicates the concentration of sample (mg/mL), required to scavenge 50% of DPPH,
ABTS and EC50 values, that present the effective concentration providing 0.5 of absorbance in
the reducing power assay (FRAP). Trolox has been chosen as the standard. All the tests have
been carried out in triplicate.
3. Results and discussion
3.1. Chemistry
We have initiated our work by the design and the preparation of the dipolarophilic system
starting from 4-(2-hydroxyphenyl)-3H-1,5-benzodiazepin-2-one 2 which has been prepared
according to the method described by Eiden. et al [26] using the 4-hydroxycoumarin and
orthophenylenediamine as starting materials (scheme 1). The propargylation of 1 in dry DMF
using NaH as a catalyst at room temperature has provided the dipolarophile 2 in excellent
1
yield (72%). The H NMR spectrum, recorded at 300 MHz in CDCl3 spectrometer revealed
new signals in the aliphatic region: a doublet at δH= 2.96 ppm and a singlet at δH= 2.25 ppm
1
corresponding to the alkyne protons of the propargyl moiety. In addition, the H NMR
spectrum has shown two doublets at δH= 4.24 ppm and 4.71 ppm, assigned to the methylene
protons H3a and H3b corresponding to the diazepine ring with a coupling constant of 18 Hz for
each other. We notice the disappearance of the NH proton signal and the presence of the
phenolic hydrogen singlet deshielded at 13.90 ppm [27].
The preparation of the diazides (12 diazides) required extensive experiments, following
different methods. i) Starting from the halogenated compounds, SN2 reaction with NaN3
(sodium azide) proceeded for the appropriate time and solvent (in DMSO at room temperature
starting from the dichlorinated derivatives [28], in DMF under reflux from the dibrominated
compounds [29]), leading to desired diazides in good yields (91-98%). ii) For the second
procedure, the diazide compounds have been prepared from the appropriate diamine, through
a two-step process: diazotization with the sodium nitrite (NaNO2) in acidic conditions
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followed by the addition of the sodium azide [30]. The desired diazides (DA1-12) have been
obtained in good yields (74-97%). We have continued our work by the double 1,3-dipolar
cyclization (CuAAC) starting from benzodiazepine 2 as a dipolarophilic system. The 1,6-
diazidohexane (DA2= N3-(CH2)6-N3) was chosen as a model (Scheme 2). We have explored
the suitable conditions carrying out Cu (I)-catalysed Huisgen 1,3-dipolar cyclization with
alkyne 2. The use of 2 equiv. of alkyne was necessary to give access to the N-bis-1,2,3-
triazole linked to the 1,5-benzodiazepine systems. Table 1 summarizes the most relevant
result of this study.
The course of the reaction has been followed by TLC. We observed two products, the
cycloadduct 3b, the major compound and the minor cycloadduct 3b’. The efficiency of the
reaction is due to a weak steric hindrance and the good reactivity of N-propargylated
1
benzodiazepine. The H/13C NMR, 2D experiments data have confirmed the structure of 3b
1
and 3b’. Thus, the H NMR spectrum of 3b’ shows five set of signals for the methylene
protons of the hexylchain and two doublets at 3.00 and 4.25 ppm corresponding to the H6
protons. The benzodiazepine core adopts a unique preferential conformation [31] where its
equatorial proton is deshielded by the C5 aryl ring ᴨ-electron current and the axial proton is
shielded by the benzene ring of the benzodiazepine. While the bis-1,2,3-triazolo-
benzodiazepine 3b displays three signals for the methylene protons of the hexyl chain, one
singlet for the triazolic proton and two doublets at 4.82 and 5.10 ppm (Jgem≈ 15 Hz) represent
the methylene attached to the amido nitrogen.
1
The macrocycle 3b is highly symmetric as shown by its H NMR spectrum which revealed a
second set of two doublets at δH=2.91 and 4.18 ppm (Jgem≈ 12 Hz) indicating the resonance of
the methylene protons of the benzodiazepine cores. The AB system observed at ambient
operating temperature, demonstrates the non-equivalence of the hydrogens of (Ha-Hb) due to
the fact that the 3H-1,5-benzodiazepine ring adopts a unique, non-planar and locked
conformation. Based on these results, we have assumed the importance of the introduction of
triazolyl groups at N1 resulting in perihydrogen interaction between the triazolyl groups and
the other hydrogens of the benzene ring, which could lead to a constrained interesting
conformation of N1-substituted benzodiazepine derivatives. This is further supported by the
diastereotopic nature of the methylene protons of both methylene triazolyl group and the
benzodiazepine core. (Figure 1, 2)
Attempts using 2 equiv. of triethylamine at room temperature, led to the mono and bis
cycloadducts (3b, 3b’) after 24 hours with moderate yields (entry 1). This may be explained
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by the poor solubility of copper iodide in such solvent [32]. In contrast, microwave assisted
cycloaddition in DMF, has given cycloadducts with good conversions (72% for 3b, and 20%
for 3b’) in just few minutes (15 min, entry 4). Optimal conditions have been obtained for this
reaction in dry DMF with 2 equiv., of DIEPA under microwave radiation (400 W, 6 min) in
the presence of a catalytic amount of CuI (entry 5). It is interesting to mention that the
reaction yield has been improved with DIPEA from 72% (entry 4) to 87% (entry 5). These
conditions with a bulkier basis have been found to be the adequate method giving only the
bistriazole in excellent yield with few traces of mono-1,2,3-triazole 3b’. Using these optimal
conditions, the scope of this approach was subsequently explored under microwave irradiation
completed within 6-12 min with various diazides (12 diazides). (Scheme 3)
The consumption of both the azido linker and the alkyne was monitored by a thin layer of
chromatography and allowed the isolation of pure bis-triazoles 3a-l. Structural assignments
for the compounds 3a-l were established by being based on their spectroscopic data mainly
1D, 2D NMR and ESI low and high resolution MS. The structures of 3a-l were confirmed by
13C and DEPT NMR spectra, which present the signals of the benzodiazepine and the
bistriazole systems resonating in the range of 114.0-164.7 ppm, especially with the
remarkable signals in the region of 122.2–124.1 ppm, which can easily be assigned to the C25
of the triazole moiety. It is important to show that, despite the complex topology of the
targets, the 1H and 13C NMR spectra of 3a-l present highly symmetric patterns. For example,
the AB system has been observed in the 1H NMR spectra of all compounds. The reaction took
place regioselectively at the alkynes moiety to produce the corresponding 1,4-disubstituted-
1,2,3-triazole. The reaction was found to be totally regioselective and the bis-1,4-triazole
linked to the benzodiazepine adducts 3a-l were isolated in good yields ranging from 67 to
92% in short reaction times. (Table 2)
3.2. Biological evaluation of the synthesized compounds
3.2.1. Antimicrobial activity
Here, we have investigated the antimicrobial activity of 1,5-benzodiazepines (BZD) and the
series of the N-bis-1,2,3-triazole linked to the 1,5-benzodiazepines conjugates which have
been evaluated through the determination of the minimal inhibitory concentration (MIC) by
the microdilution method. Most of synthesized products have shown, either moderate or
important antibacterial activity against Gram-positive and Gram-negative bacteria with MIC
of 31.25-250 µg/mL, and a remarkable antifungal activity with MIC of 62.5-250 µg/mL.
14

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In vitro, antibacterial and antifungal screening results are summarized in histograms
represented in figure 3, 4 and 5.
In the first screening phase against B. subtilus, two compounds 3g and 3l have exhibited an
excellent activity compared to standard antibiotic amoxicillin (AMX) with MIC= 31.25
µg/mL. Among triazoles derivatives, the compounds 3e, 3g and 3l emerged as potential
antibacterial agents comparable with the standard drugs AMP and AMX against bacterial
pathogens: S. aureus. (Figure 3)
On the other hand, compounds 3b, 3c, 3f, 3g, 3h, 3k and 3l have been significantly the potent
derivatives against most of the used Gram-negative bacteria with a MIC ranging from 31.25
to 125 µg/mL as compared to the benzodiazepines 1 and 2 and to AMX. (Figure 4)
It is also very clear, that most of the tested compounds have displayed an excellent activity
two times higher for 3g and 3i and three times higher for the rest of bis-1,2,3-
triazolobenzodiazepines against E. fecalis compared to the positive control (AMX). It is
worthwhile to notice that compounds 3a, 3b, 3d, 3f, 3g, 3h, 3k and 3l are four times as active
towards E. faecium (CI) than Amoxacilline (AMX). (Figure 4)
The data revealed also that most of bis-1,2,3-triazolobenzodiazepines are very potent
compared to benzodiazepines 1 and 2 without the triazoles moieties. This effect may be
explained by the importance of the triazole unit for the antibacterial activity of the bis-triazolo
benzodiazepine analogues.
Additionally, the results of antifungal screening indicate that all bis triazolobenzodiazepines
showed a very important activity against all used fungal with an MIC = 62.5-250 µg/mL.
Indeed, the compounds 3h and 3k are the most potent antifungal agents showing a MIC of
62.5 µg/mL against C. keusi and C.glabrata. (Figure 5)
Structure-activity relationships (SAR)
Our studies were devoted to the analysis of qualitative and quantitative structure-activity
relationships (SAR) of benzodiazepine derivatives. As described in the literature,
benzodiazepine derivatives revealed to be excellent antimicrobial agents [33]. For the first
screening of the in vivo antimicrobial results, we noticed the presence of activity in our
synthesized compounds. In addition, independently of the nature of the substituent attached to
the benzodiazepine borne by the triazole, compounds 3a-l were found to be more active than
compounds 1 and 2. These results conclude the importance of the introduction of the triazole
moiety via the methylene linker to improve the activity of benzodiazepine 1. Particularly, for
Gram-positive, compounds 3g and 3l can be optimized for the broad spectrum of activity
against bacteria compared to Amoxicillin (AMX). In our case, the compound having ether
15

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group attached to two phenyl rings 3g favors the potency. In fact, the ether group appeared to
be much more important for antibacterial activity [34]. Also, it is interesting to note that the
methyl substitution on the phenyl rings 3l has shown a promising activity. As shown,
compounds possessing electron-donating groups in the links found to enhance the bacterial
inhibition effects. On the other hand, for Gram-negative, compounds with sulfonyl group at
the triazole ring 3h as well as 3k with pyridine group are highly active against E. fecalis, S.
enterica and E. faecium. These strong electron-withdrawing groups induce the potency of
benzodiazepine derivatives. This result is supported by the studies of Konda et al. [35]. For
antifungal activity and according to the minimum inhibitory concentration (MIC) values,
compounds 3h and 3k are highly active against C. keusi and C. glabrata compared to the
standard antifungal agent Amphotericin (AB). In our case, the presence of electron
withdrawing link group increases the antifungal activity of the synthesized benzodiazepine
derivatives.
3.2.2. Antioxidant activity
The DPPH method is known as a convenient method, which is rapid and simple used for the
screening of many samples for radical scavenging activity [36]. These advantages made the
DPPH an interesting method for the evaluation of our compounds. On the other hand, the
assay with the ABTS, as a moderately stable nitrogen center radical known for its high
solubility as compared to the DPPH radical [37], can be used to test the ability of compounds
to donate H· able to neutralize ABTS+ free radicals.
The investigation of antioxidant screening revealed that all the newly of 1,5-benzodiazepines-
1,2,3-triazoles hybrids showed moderate radical scavenging capacity compared to the
standard Trolox, described as a potent antioxidant agent with IC50= 0,126±0,013 mg/ml in
DPPH test and IC50= 0,163±0.021 mg/ml in ABTS test.
As depicted in figure 6, the results showed that compound 1 exhibited an important DPPH
and ABTS scavenging ability with IC50= 3,05±0,05 mg/ml and IC50= 2,43±0,06 mg/ml,
respectively, when compared to compound 2 (IC50= 3,14±0.02 mg/ml and IC50= 2,78±0.12
mg/ml). Such results support the crucial role of the labile hydrogens in the interaction of the
examined compounds with the stable free radicals DPPH and ABTS+.[24]
Furthermore, the lowest capacity of neutralization of DPPH radical was observed for
compound 3b (IC50= 3,01±0.06 mg/mL), while compounds 3h and 3g exhibited the highest
scavenging abilities with IC50= 1,88±0.02 mg/ml and IC50= 2.07±0.03 mg/ml, respectively,
when compared to other derivatives. The compounds 3h and 3g displayed the strongest
16

ACCEPTED MANUSCRIPT
capacity for the neutralization of ABTS+ free radicals with IC50= 1.63±0.04 and 1.91±0.06
mg/mL respectively. These findings may be explained by the presence of sulfonyl group and
oxygen atom in the linker moiety of both structures, which are able to contribute to the
neutralization of free radicals, as well as the presence of phenolic hydroxyl groups connected
to a benzodiazepine ring.
The Ferric Reducing Antioxidant Power (FRAP) is another interesting way to evaluate
antioxidant activity of drugs. It is a technique generally used for experimenting the reducing
ability of antioxidant species by transferring a simple electron [38]. The results by reducing
power assay presented in figure 7 have showed that the activity of all tested compounds
increase compared to Trolox (EC50= 0.072 ± 0.022 mg/mL) as follows:
3h> 3g> 3k> 3l> 3e> 1> 2> 3d> 3j> 3f> 3c> 3i> 3a> 3b.
The highest reducing activity is observed in compounds 3h (EC50= 1.12±0.08 mg/mL), 3g
(EC50= 1.27±0.03 mg/mL), 3k (EC50= 1.50±0.07 mg/mL), 3l (EC50= 1.82±0.13 mg/mL) and
3e (EC50= 2.06±0.05 mg/mL) which is probably due to the presence of different reducing
groups on the linkers of such hybrid dimers.
On the other side, compounds 1 and 2 showed better activity than the rest of bis-1,2,3-triazole
benzodiazepine with Half maximal effective concentration (EC50) of (2.17±0.08),
(2.53±0.06), respectively.
Structure-activity relationships (SAR)
The position and degree of hydroxylation, polarity, solubility and reducing potential are the
main factors influencing the antioxidant activity of phenolic compounds [39]. As shown, the
results of a triplicate assay can only give a suggestion on the potential of the tested
compounds. For 2,2-diphenyl-1-picrylhydrazyl (DPPH) test, the current research revealed the
potential of 4-(2-hydroxyphenyl)-1H-1,5-benzodiazepin-2-one 1 as emerging free radical
scavenger. The study established a structure-activity relationship (SAR) where the hydroxyl
moiety of the 1,5-benzodiazepine was found to play a profound role and influence over
antioxidant activity. The antioxidant potential of our compounds is moderate compared to
Trolox. In fact, the potent antioxidant activity of the new purified compound could be
attributed to the presence of two hydroxyl groups in the benzodiazepine core, which play an
important role in the activation of free radicals. Introduction ether group into 3g increases the
scavenging activity and the presence of a sulfonyl group in 3h has shown a promising
activity. Generally, similar pattern of activity was also observed with 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid (ABTS). On the other hand, Ferric reducing
17

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antioxidant power (FRAP) analysis of the synthesized compounds revealed that they are
moderately to importantly active compared to Trolox.
4. Conclusion
In summary, we have successfully synthesized new benzodiazepine derivatives linked
by 1,4-disubstitued 1,2,3-bistriazoles in a complete regioselective way using different
aliphatic/aromatic moieties. The easy way and the high yields of the obtained products (67-
92%) make this method attractive for the synthesis of potential biologically active molecules.
Their evaluation as antimicrobial agents has revealed that all the screened compounds exhibit
moderate to excellent activities. Only two products (3h,k) have given the highest antifungal
activity. On the basis of current knowledge of SAR, biological evaluation results revealed that
antimicrobial potency is considerably improved by the presence of triazole. Furthermore, all
the synthesized compounds were evaluated for antioxidant activity. Amongst all the tested
compounds, some bis-1,2,3-triazole benzodiazepines have shown an important antioxidant
activity with regard to the starting compounds 1,2 in three tests. Further, it was noticed that 3g
and 3h showed comparatively higher antioxidant activity than other bis-triazole
benzodiazepines. These results illustrate the potential of this new class of molecules for
diverse biologycal applications. Overall results suggested that nature and position of the
substituents attached to the benzodiazepine skeleton had a considerable impact on the
antioxidant effect of the tested molecules. These findings deserve to be studied more
thoroughly in the future.
Acknowledgements
Our acknowledgment is extended to the University of Paris 13, Sorbonne Paris City,
Laboratoiry CSPBAT, Bobigny, France for providing laboratory facilities to bring this work
to light. And thanks are also attributed to Sylvain Marque and Aurélie Damond (Engineering
study) at the University of Versailles Saint-Quentin-en-Yvelines, Versailles, France for
providing mass spectroscopies recorded on an UPLC Waters device.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at xxx
18

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Table 1. Optimization of Cu(I)-catalyzed 1,3-dipolar cyclization for the synthesis of bis-1,2,3-
triazoles 3b and mono-1,2,3-triazole 3b’.a
Yield %c
(ratio 3b/3b’)
25/27
Entry Solvent
Bases
(2 equiv.)
Et3N
Et3N
Et3N
Condition
Time
1
2
3
4
DCM
THF
Toluene
DMF
rt
rt
24 h
24 h
24 h
30/12
45/10
72/20
reflux
Et3N
MWb
15 min
Bold in entry highlights the optimal reaction conditions.
a Alkyne 2 (2 mmol), with diazide DA2 (1 mmol) and 0.2 mmol of CuI.
b The reaction was performed at reflux or under MW irradiation (400W).
c Ratios (3b/3b’) were calculated on the crude product on the basis of integration signal intensities in
1H NMR spectrum.

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Table 2. 1,4-disubstituted bistriazoles 3a-l prepared by the Cu(I)-catalyzed coupling under MW
irradiation
(400W).
Entry
Compd.
3a
linkers
Yield (%)a Time (min)
92
86
6
6
1
2
3b
82
78
8
3
4
3c
10
3d
84
80
8
8
5
6
3e
3f
O
87
83
10
10
7
8
3g
3h
O
S
O
67
89
12
10
9
3i
3j
10
71
81
12
12
11
12
3k
3l
a Isolated yield for the bis-1,2,3-triazole after purification by column chromatography.

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Figure 1. selected 1H NMR chemical shifts of 3b’ (up) and 3b (down) (CDCl3, 300 MHz).

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Figure 2. selected 13C NMR chemical shifts of 3b’ (up) and 3b (down) (CDCl3, 75 MHz).

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Figure 3. Antibacterial activity of synthesized compounds against Gram-positive bacteria.

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Figure 4. Antibacterial activity of synthesized compounds against Gram-negative bacteria.