Synthesis, spectroscopic characterization and DNA/HSA binding studies of (phenyl/naphthyl)ethenyl-substituted 1,3,4-oxadiazolyl-1,2,4-oxadiazoles

Article abstract of DOI:10.1039/d0nj04530f

Two new series of conjugated arylethenyl-1,3,4-oxadiazolyl-1,2,4-oxadiazoles were obtained and spectroscopically characterized in terms of UV-Vis absorption, fluorescence and interaction with CT-DNA and Human Serum Albumin (HSA) biomolecules. Phenyl- and 1-naphthyl-bearing examples were analysed, and the spectroscopic properties of its substitution series were compared, showing extensive conjugation in all compounds and absorption differences due to both the aryl-ethenyl subunit and substituted phenyl/phenylene at the 1,2,4-oxadiazole side. Strong binding interactions of the obtained compounds with CT-DNA and moderate HSA-association capability were observed spectroscopically, and further docking studies were performed. This journal is

Full text of DOI:10.1039/d0nj04530f

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DOI: 10.1039/D0NJ04530F.
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DOI: 10.1039/D0NJ04530F
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Synthesis, spectroscopic characterization and DNA/HSA binding study of
(phenyl/naphthyl)ethenyl-substituted 1,3,4-oxadiazolyl-1,2,4-oxadiazoles
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João C. P. Mayer,a Thiago V. Acunha,b Oscar E. D. Rodrigues,a Davi F. Back,c Otávio
Augusto Chaves,d Luciano Dornellesa* and Bernardo A. Iglesiasb**
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a Departamento de Química, LabSelen-NanoBio, Universidade Federal de Santa Maria, CEP
97105-900, Santa Maria, RS, Brazil.
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Departamento de Química, Laboratório de Bioinorgânica e Materiais Porfirínicos,
Universidade Federal de Santa Maria, CEP 97105-900, Santa Maria, RS, Brazil.
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Laboratório de Materiais Inorgânicos - Departamento de Química, CCNE, UFSM, Santa
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Maria – RS, Brazil, Zip Code 97105-900.
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Instituto SENAI de Inovação em Química Verde, Rua Morais e Silva N° 53, Bloco 09, CEP
20271030, Rio de Janeiro, RJ, Brazil.
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* Corresponding author.
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** Corresponding author.
E-mail adresses: bernardopgq@gmail.com; bernardo.iglesias@ufsm.br (B.A. Iglesias),
ldornel@gmail.com (L. Dornelles).
1

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35 Abstract
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Two new series of conjugated arylethenyl-1,3,4-oxadiazolyl-1,2,4-oxadiazoles
were obtained and spectroscopically characterized in terms of UV-Vis absorption,
fluorescence and interaction with CT-DNA and Human Serum Albumin (HSA)
biomolecules. Phenyl- and 1-naphthyl-bearing examples were analysed, and the
spectroscopic properties of its substitution series were compared, showing extensive
conjugation in all compounds, and absorption differences due to both aryl-ethenyl
subunit and substituted phenyl/phenylene at the 1,2,4-oxadiazole side. Strong binding
interaction of the obtained examples with CT-DNA and moderate HSA-association
capability were observed spectroscopically, and further docking studies were
performed.
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Keywords: 1,2,4-oxadiazoles, 1,3,4-oxadiazoles, photophysical properties, DNA
binding, HSA binding.
50 Introduction
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Heterocyclic compounds, in special azoles, have long been object of much
attention in chemistry.1 Among nitrogen-bearing heterocycles, oxadiazoles represent a
significant portion of target molecules in organic synthesis.2 In addition to the methods
themselves, focus is given to their pharmacological activities and also to their physical-
chemical properties.3 1,2,4- And 1,3,4-oxadiazoles are the most described oxadiazole
regioisomers, and these have been incorporated in a great number of functional
molecules, including antitumor,4 antiviral5 and antidepressant drugs,6 as well as in
polymeric species.7 Moreover, due to its highly conjugated structure, aryl-1,3,4-
oxadiazoles and their vinylogous counterparts have often been incorporated in the
synthesis of fluorescent compounds.8
Molecules containing the di-heterocyclic moiety 1,3,4-oxadiazolyl-1,2,4-
oxadiazole have been studied recently by our group, having electron-withdrawing
properties associated to the oxadiazoles and displaying significant binding affinity for
CT-DNA.9 However, the fluorescence spectroscopic properties of molecules based on
these di-heterocyclic scaffold remained uncharacterized.
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Since there is interest in DNA-binding molecules to be employed as drugs and/ or
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biochemical probes, fluorescent 1,3,4-oxadiazolyl-1,2,4-oxadiazole derivativatives may
deserve attention for the development of such compounds.10 Considering other results
obtained for previous, ferrocene-substituted chemical species,9 it would be plausible to
study any observable fluorescence after replacement of ferrocene by other aryl groups,
making fluorescence spectroscopic characterization of the respective DNA and/or
protein-compound adducts possible.
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In this work, a library of new fluorescent (phenyl/naphthyl)-ethenyl-1,3,4-
oxadiazolyl-1,2,4-oxadiazoles (7aa-be, Fig. 1) were prepared and fully characterized by
analytical techniques. Photophysical properties of these derivatives were evaluated by
UV-Vis and steady-state fluorescence emission spectroscopy. Moreover, DNA and
HSA binding assays were conducted by spectroscopic and theoretical analysis
(molecular docking).
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Fig. 1. Structural representation of the 1,3,4-oxadiazolyl-1,2,4-oxadiazoles 7aa-be.
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84 Experimental
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General analysis
High-resolution mass spectra (HRMS) were obtained for compounds 7aa-be on a
XEVO G2-Q-TOF (Waters) mass spectrometer, operating in ESI(+) mode, at the
Chemistry Department, Federal University of Santa Maria.
Thin layer chromatography (TLC) was performed using Merck Silica Gel GF254,
0.25 mm. For visualization, TLC plates were either placed under ultraviolet light, or
stained with iodine vapor, or acidic vanillin solution followed by heating.
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Proton nuclear magnetic resonance spectra ( H NMR) were obtained at 400 MHz
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in a Bruker Avance III HD NMR spectrometer. Spectra were recorded in either CDCl3
or DMSO-d6 solutions. Chemical shifts are reported in ppm, referenced to the solvent
peak of tetramethyl silane (TMS) as the external reference. Data are reported as follows:
chemical shift (δ) expressed in ppm, multiplicity (br = broad, s = singlet, d = doublet, dd
= doublet of doublets, ddd = doublet of doublet of doublets, dt = doublet of triplets, t =
triplet, m = multiplet, q = quartet), and coupling constant (J) in Hertz and integrated
intensity. Carbon-13 nuclear magnetic resonance (13C NMR) spectra were obtained at
100 MHz in an AVANCE III HD NMR spectrometer. Chemical shifts (δ) are reported
in ppm, referenced to the solvent peak of CDCl3 or DMSO-d6. Deuterated solvents were
acquired from Cambridge Isotope Laboratories®, having TMS added on opening.
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X-Ray crystallography
Crystallographic data were collected on a Bruker D8 Venture Photon 100
diffractometer equipped with an Incoatec IµS high brilliance Mo-Kα X-ray tube with
two-dimensional Montel micro-focusing optics. The structure was solved by direct
methods using SHELXS.11 Subsequent Fourier-difference map analyses yielded the
positions of the non-hydrogen atoms. Refinements were carried out with the SHELXL
package.11 All refinements were made by full-matrix least-squares on F2 with
anisotropic displacement parameters for all non–hydrogen atoms. Hydrogen atoms were
included in the refinement in calculated positions but the atoms (of hydrogens) that are
commenting performing special bond were located in the Fourier map. Drawings were
done using ORTEP for Windows.12 Crystal data and more details of the data collection
and refinements of the ligand in the support information section (Figure S31).
Photophysical analysis
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Electronic UV-Vis absorption spectra were recorded using Shimadzu UV2600
spectrophotometer (data interval, 1.0 nm) using chloroform and DMSO as solvent.
Steady-state fluorescence spectra of samples in CHCl3 and DMSO solutions were
measured with a Varian Cary 50 fluorescence spectrophotometer (emission; slit 2.5
mm) and were corrected according to the manufacturer’s instructions. Fluorescence
quantum yields (f) of the compounds 7aa-ae/7ba-be in solutions were determined by
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comparing the corrected fluorescence spectra with 9,10-diphenylanthracene (DPA) in
DOI: 10.1039/D0NJ04530F
chloroform (f = 0.65, λex = 366 nm) as fluorescence yield standard.13 Collecting the
samples and the standard fluorescence spectra at the same experimental condition, the
fluorescence quantum yield was calculated by Equation 1:
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(1)
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where ΦF, I, A and  are the fluorescence quantum yield, the integral area of
fluorescence emission, the absorbance in exc. and refractive index of selected solvents.
The subscript “std” refers to the standard molecule - in this case, 9,10-
diphenylanthracene (DPA) (ΦF = 0.65 in chloroform solution).
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CT-DNA binding assays
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The interaction between calf-thymus DNA (CT-DNA) and compounds 7aa-
ae/7ba-be were conducted by UV-Vis absorption measurements at room temperature in
PBS buffer at pH 7.2, using DMSO stock solution of derivatives (10-4 M range) at 300
to 700 nm. The DNA pair base concentrations of low molecular weight DNA from CT-
DNA was determined by absorption spectroscopy, using the molar extinction
coefficients 6,600 M−1cm−1 (per base pair) at λmax = 260 nm. Heterocycle compound
solutions in DMSO with PBS buffer were titrated with increasing concentrations of CT-
DNA (ranging from 0-100 μM). The intrinsic binding constants (Kb) of derivatives were
calculated according to the decay of the absorption bands of compounds using Equation
2, through a plot of [DNA]/(εa − εf ) versus [DNA]:
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(2)
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where [DNA] is the concentration of CT-DNA in the base pairs, εa is the extinction
coefficient (Aobs/[compound]), εb and εf are the extinction coefficients of free and fully
bound forms, respectively. In plots of [DNA]/(εa − εf ) versus [DNA], Kb is given by the
ratio of the slope to the interception.
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Also, the standard Gibbs’ free-energy (ΔGº) of CT-DNA:derivative adDdOuI:c1t0.1w03a9s/D0NJ04530F
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calculated from the values of Kb using Equation 3:
(3)
G= −RT lnKb
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where R and T are the gas constant (1.987 kcalK-1mol-1) and temperature (298K),
respectively.
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The competition binding studies were conducted via steady-state fluorescence
emission analysis in the same fluorimeter described in the photophysical analysis. The
derivatives 7ba-be were dissolved in DMSO (stock solution at 10-5 M range) and
competitive studies were performed through the gradual addition of the compounds to a
quartz cuvette (1.0 cm path length) containing ethidium bromide (EB, 2.0 × 10-7 M) and
DNA (2.0 × 10-5 M) in a PBS (pH 7.2). The concentration of derivatives ranged from 0
to 100 μM. Compounds were excited at λexc = 510 nm and steady-state fluorescence
emission spectra were recorded at 550-800 nm range, after 5 min of incubation. The
Stern-Volmer quenching constant (KSV) values were calculated according to the decay
of the emission bands of EB-DNA using Equation 4 through a plot of F0/F versus
[DNA]:
F0
[ ]
= 1+ kq τ0 Q = 1+ K SV
[ ]
Q
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(4)
F
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where F and F0 are the fluorescence intensities in the presence and absence of a
quencher, respectively. KSV, kq, τ0 and [Q] denote Stern–Volmer quenching constant,
bimolecular quenching rate constant, fluorescence lifetime for EB-DNA adducts (23
ns)14 and the concentration of quencher, respectively. According to Equation 4, the KSV
values were calculated from the slope and kq is equal KSV/τ0.
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In order to quantify the displacement ability of the derivatives under study, it was
used minimum ligand concentration that decreases in about 50% of the EB fluorescence
emission (in this case, assumed to be 50% displacement of EB).15 The values for
apparent binding constant with CT-DNA (Kapp) were calculated using Equation 5:
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KEB[EB] = Kapp[compound]
(5)
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where KEB (4.95 × 10 M ) is the DNA-binding constant of EB, [EB] is the EB
DOI: 10.1039/D0NJ04530F
concentration (1.40 × 10-6 M), and [compound] is the concentration of the derivatives
used to obtain 50% reduction in fluorescence emission intensity of EB dye.
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Steady-state fluorescence emission for HSA-binding assays
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The experimental binding ability between human serum albumin (HSA) and
compounds 7aa-ae/7ba-be was performed by steady-state fluorescence emission
measurements at room temperature in PBS buffer (pH 7.2), using DMSO stock solution
of heterocycle derivatives (10-5 M range). As excitation and emission fluorescence
wavelengths were used 290 and 300-500 nm range, respectively. In order to obtain
quantitative values for albumin fluorescence quenching data (KSV and kq values), it was
used the same Stern–Volmer approximation (Equation 4) described in the CT-DNA
binding assays, however for HSA was used τ0 = 5.67 ns as fluorescence lifetime.16
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Generally, double logarithmic approximation (Equation 6) is used to estimate the
Stern-Volmer binding constant (Ka) and number of binding sites (n) for the compound–
HSA adduct:
F − F
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0
log
= log K + nlog
 
Q


a
F
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(6)
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where F0 and F represent fluorescence intensities in the absence and presence of
derivatives, respectively, while [Q] is the quencher concentration. According to
Equation 6, the Ka values were calculated from the interception and n by the slope.
The standard Gibbs’ free-energy (ΔGº) for compound-HSA adduct was calculated
from the Ka values, using Equation 3 previous described in the CT-DNA binding assays.
Molecular docking procedure
The chemical structure for the naphthylethenyl-substituted 1,3,4-oxadiazolyl-
1,2,4-oxadiazoles 7aa-ae and 7ba-be was built and energy-minimized by Density
Functional Theory (DFT) calculations, with B3LYP potential and basis set 6-31G*,
available in the Spartan'18 software (Wavefunction Inc., Irvine, CA, USA).17 The
crystallographic structures of HSA and DNA were obtained from Protein Data Bank
(PDB), with access code 1N5U18 and 1BNA,19 respectively. The molecular docking
studies were performed with GOLD 5.6 software (CCDC, Cambridge Crystallographic
Data Centre, CB2 1EZ, UK).20 The hydrogen atoms were added to the
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biomacromolecules structure according to the data inferred by GOLD 5.6 software on
DOI: 10.1039/D0NJ04530F
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the ionization and tautomeric states. For the albumin, molecular docking calculations
were explored with a 10 Å radius around the three main binding pockets: sites I, II, and
III. On the other hand, for DNA structure, a 5.0 Å radius around the major and minor
grooves of the double helix was defined for the molecular docking calculations. The
number of genetic operations (crossover, migration, mutation) in each docking run used
in the search procedure was set to 100,000. The scoring function used was 'ChemPLP',
which is the default function of the GOLD 5.6 software. The figures were generated by
PyMOL Molecular Graphics System 1.0 level software (Delano Scientific LLC
software San Carlos, CA, USA).21
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Synthetic procedures
All reactants and solvents, unless noted, were commercially available (Sigma-
Aldrich®, Alfa-Aesar®, Merck®, Vetec® and Synth®). Solvents were purified in
accordance to standard procedures, namely by means of distillation and drying over
molecular sieves, as described elsewhere. Intermediates and products were prepared as
described below.
(E)-3-Arylprop-2-enoic acids (2a-b)22
To a round-bottom two-necked balloon, under magnetic stirring, aldehyde 1a-b
(10 mmol) and malonic acid (10 mmol) were dissolved in pyridine (10 mL), and 5 drops
of piperidine was added to the mixture. The system was stirred at reflux temperature for
15 h. Then, the mixture was poured on water-ice (50 mL), and acidified with 6 M
aqueous HCl until pH 2. The suspension was extracted three times with ethyl acetate
(50 ml each time). The combined organic phase was dried over anhydrous magnesium
sulfate, and then filtered. The solvent was removed by rotary evaporation under reduced
pressure, and the product was recrystallized from dichloromethane/hexane.
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(E)-3-Phenylprop-2-enoic acid (cinnamic acid) (2a)
Yield = 0.977 g, 66% (white solid); mp = 126.7 – 130.8 °C. 1H NMR (CDCl3, 400
MHz), δ (ppm): 7.67 – 7.65 (m, 2 H), 7.60 (d, J = 16.0 Hz, 1 H), 7.42 – 7.39 (m, 3 H),
6.52 (d, J = 16.0 Hz, 1 H). 13C NMR (CDCl3, 100 MHz), δ (ppm): 167.5, 143.9, 134.2,
130.1, 128.9, 128.1, 119.2.
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(E)-3-(Naphthalen-1-yl)prop-2-enoic acid (2b)
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Yield = 1.188 g, 60% (yellow solid); mp = 152.7 – 156.8 °C. H NMR (CDCl3,
400 MHz), δ (ppm): 8.40 (d, J = 15.8 Hz, 1 H), 8.18 (d, J = 8.4 Hz, 1 H), 8.01 – 7.92
(m, 3 H), 7.63 – 7.52 (m, 3 H), 6.61 (d, J = 15.7 Hz, 1 H). 13C NMR (CDCl3, 100 MHz),
δ (ppm): 167.6, 140.3, 133.4, 131.1, 130.9, 130.5, 128.8, 127.3, 126.4, 125.8, 125.3,
123.1, 122.0.
Arylamidoximes (4a-e)23
To a round- bottom two-necked balloon, under magnetic stirring, aryl nitrile 3a-e
(15 mmol) was dissolved in ethanol (25 mL) and to the solution were added
hydroxylamine hydrochloride (2.085 g, 30 mmol) and aqueous NaOH (2 equiv, 10 mL).
The mixture was stirred at room temperature for 15 to 24 h, monitoring the reaction by
TLC. After, the solvent was removed under rotary evaporation and to the crude mixture
was added water (20 mL). The mixture was extracted with ethyl acetate (3 x 20 mL) and
the organic layer was dried over anhydrous MgSO4 and filtered. The solvent was
removed by rotary evaporation and the product was recrystallized from chloroform –
hexane.
(Z)-N’-hydroxybenzimidamide (4a)
1
Yield = 2.000 g, 98% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
9.52 (br, 1 H), 7.68 (ddd, J1 = 5.5 Hz; J2 = 3.0 Hz; J3 = 1.5 Hz, 2 H), 7.39 – 7.35 (m, 3
H), 5.69 (br, 2 H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 150.8, 133.3, 128.7, 127.9,
125.3.
(Z)-N’-hydroxy-4-mehylbenzimidamide (4b)
1
Yield = 1.395 g, 62% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
9.46 (br, 1 H), 7.56 (d, J = 8.0 Hz, 2 H), 7,17 (d, J = 7.8 Hz, 2 H), 5.64 (br, 2 H), 2.30
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(s, 3 H). C NMR (DMSO-d6, 100 MHz) δ (ppm): 150.9, 138.3, 130.5, 128.6, 125.3,
DOI: 10.1039/D0NJ04530F
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20.7.
(Z)-N’-hydroxy-4-methoxybenzimidamide (4c)
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Yield = 2.116 g, 85% (grey-white solid). H NMR (DMSO-d6, 400 MHz), δ
(ppm): 9,41 (br, 1 H), 7,61 (d, J = 8.9 Hz, 2 H), 6.92 (d, J = 8.9 Hz, 2 H), 5.66 (br, 2 H),
3.77 (s, 3 H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 159.8, 150.7, 126.7, 125.7,
113.4, 55.1.
(Z)-N’-hydroxy-4-chlorobenzimidamide (4d)
1
Yield = 2.532 g, > 95% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
13
9.68 (s, 1 H), 7.69 (d, J = 7.7 Hz, 2 H), 7.42 (d, J = 8.9 Hz, 2 H), 5.80 (br, 2H). C
NMR (DMSO-d6, 100 MHz) δ (ppm): 150.0, 133.5, 132.2, 128.1, 127.1.
(Z)-N’-hydroxy-4-trifluoromethylbenzimidamide (4e)
1
Yield = 3.029 g, > 95% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
13
9.88 (s, 1 H), 7.90 (d, J = 8.0 Hz, 2 H), 7.72 (d, J = 8.2 Hz, 2 H), 5.92 (br, 2 H). C
NMR (DMSO-d6, 100 MHz) δ (ppm): 149.9, 137.3, 129.1 (q, J = 31.7 Hz); 126.1, 124.9
(q, J = 3.8 Hz); 124.2 (q, J = 272.2 Hz).
Ethyl 3-aryl-1,2,4-oxadiazole-5-carboxylates (5a-e)24
To a round-bottom two-necked balloon mounted with a reflux condenser,
provided with magnetic stirring, a proper amidoxime 4a-e (10 mmol) was dissolved in
tetrahydrofuran (20 mL). Thereafter, N,N’-diisopropylethylamine (2.6 mL, 15 mmol)
was added and the system was cooled to 0 – 5 °C. Then, ethyl oxalyl chloride (1.3 mL,
12 mmol) was added dropwise under stirring; the reaction was warmed to room
temperature and kept under reflux for 2 h. After completion, the reaction mixture was
cooled to room temperature, aqueous 2 N HCl solution (10 mL) and water (100 mL)
were added and the product was extracted with ethyl acetate (3 x 25 mL). The organic
layer was washed with aqueous NaHCO3 and then with water. The organic layer was
dried over anhydrous MgSO4, filtered and the solvent removed by rotary evaporation.
The crude product was purified through flash column chromatography on silica gel,
using hexanes: ethyl acetate (85:15 v/v) and dried under reduced pressure.
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Ethyl 3-phenyl-1,2,4-oxadiazole-5-carboxylate (5a)
1
Yield = 1.809 g, 83% (white solid). H NMR (CDCl3, 400 MHz), δ (ppm): 8.15
(d, 2 H, J = 6.6 Hz), 7.56 – 7.47 (m, 3 H), 4.57 (q, J = 7.1 Hz, 2 H), 1.49 (t, J = 7.1 Hz,
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3 H). C NMR (CDCl3, 100 MHz) δ (ppm): 169.5, 166,7, 154.3, 131.8, 129.0, 127.8,
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125.8, 63.9, 14.1.
Ethyl 3-(4-methylphenyl)-1,2,4-oxadiazole-5-carboxylate (5b)
1
Yield = 1.694 g, 73% (white solid). H NMR (CDCl3, 400 MHz), δ (ppm): 8.03
(d, J = 8.2 Hz, 2 H), 7.29 (d, J = 8.0 Hz, 2 H), 4.56 (q, J = 7.2 Hz, 2 H), 2.41 (s, 3 H),
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1.48 (t, J = 7.2 Hz, 3 H). C NMR (CDCl3, 100 MHz) δ (ppm): 169.5, 166.5, 154.2,
142.3, 129.7, 127.6, 122.9, 63.8, 21.5, 14.0.
Ethyl 3-(4-methoxyphenyl)-1,2,4-oxadiazole-5-carboxylate (5c)
1
Yield = 1.835 g, 74% (white solid). H NMR (CDCl3, 400 MHz), δ (ppm): 8.07
(d, J = 9.0 Hz, 2 H), 6.99 (d, J = 9.0 Hz, 2H), 4.56 (q, J = 7.1 Hz, 2 H), 3.87 (s, 3 H),
13
1.48 (t, J = 7.1 Hz, 3 H). C NMR (CDCl3, 100 MHz) δ (ppm): 169.2, 166.4, 162.5,
154.3, 129.4, 118.1, 114.4, 63.8, 55.4, 14.0.
Ethyl 3-(4-chlorophenyl)-1,2,4-oxadiazole-5-carboxylate (5d)
1
Yield = 2.146 g, 85% (white solid). H NMR (CDCl3, 400 MHz), δ (ppm): 8.08
(d, J = 8.7, 2 H), 7.48 (d, J = 8.7 Hz, 2 H), 4.57 (q, J = 7.2 Hz, 2 H), 1.49 (t, J = 7.2 Hz,
3 H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 168.7, 166.8, 154.0, 138.1, 129.34; 129.0,
124.2, 63.9, 14.0.
Ethyl 3-(4-trifluoromethylphenyl)-1,2,4-oxadiazole-5-carboxylate (5e)
1
Yield = 2.460 g, 86% (white solid). H NMR (CDCl3, 400 MHz), δ (ppm): 8.28
(d, J = 8.0 Hz, 2 H), 7.77 (d, J = 8.1 Hz, 2 H), 4.59 (q, J = 7.1 Hz, 2 H), 1.50 (t, J =
7.1 Hz, 3 H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 168.4, 167.1, 153.9, 133.6 (q, J =
32.8 Hz), 129.1, 128.1, 126.0 (q, J = 3.7 Hz), 123.6 (q, J = 272.8 Hz), 64.0, 13.9.
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In a round-bottom flask, ethyl 3-aryl-1,2,4-oxadiazole-5-carboxylate 5a-e (6
mmol) was dissolved in ethanol (15 mL) under magnetic stirring. Then, hydrazine
hydrate (0.6 mL, 12 mmol) was added and a precipitate of the hydrazide readily formed.
The mixture was kept under stirring at room temperature for 1 h, after which the
precipitated hydrazide was collected by vacuum filtration and washed with cold ethanol
(50 mL). The solid was transferred to another balloon and was dried under reduced
pressure.
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Yield =1.222 g, > 95% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
10.66 (br, 1 H), 8.05 (d, J = 7.9 Hz, 2 H), 7.65 – 7.57 (m, 3 H), 4.99 (br, 2 H). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 169.0, 167.9, 151.7, 131.8, 129.2, 127.0, 125.4.
3-(4-Methylphenyl)-1,2,4-oxadiazole-5-carbohydrazide (6b)
1
Yield = 1.086 g, 83% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
7.92 (d, J = 8.2 Hz, 2 H), 7.38 (d, J = 7.9 Hz, 2 H), 2.38 (s, 3 H). 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 168.9, 167.9, 151.8, 141.9, 129.8, 127.1, 122.7, 21.0.
3-(4-Methoxyphenyl)-1,2,4-oxadiazole-5-carbohydrazide (6c)
1
Yield = 1.264 g, 90% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
7.98 (d, J = 9.0 Hz, 2 H), 7.13 (d, J = 9.0 Hz, 2 H), 3.84 (s, 3 H). 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 168.8, 167.7, 162.0, 157.8, 151.8, 128.9, 117.8, 114.8, 55.4.
3-(4-Chlorophenyl)-1,2,4-oxadiazole-5-carbohydrazide (6d)
1
Yield =1.428 g, > 95% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
10.78 (br, 1 H), 8.04 (d, J = 8.7 Hz, 2 H); 7.66 (d, J = 8.7 Hz, 2 H), 4.95 (br, 2 H). 13C
NMR (DMSO-d6, 100 MHz) δ (ppm): 169.2, 167.2, 151.6, 136.7, 129.5, 128.9, 124.4.
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3-(4-trifluoromethylphenyl)-1,2,4-oxadiazole-5-carbohydrazide (6e)
1
Yield = 1.306 g, 80% (white solid). H NMR (DMSO-d6, 400 MHz), δ (ppm):
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NMR (DMSO-d6, 100 MHz) δ (ppm): 169.5, 167.0, 151.6, 131.7 (q, J = 32.1 Hz),
DOI: 10.1039/D0NJ04530F
129.4, 128.0, 126.3 (q, J = 3.6 Hz), 123.7 (q, J = 272.7 Hz).
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(E)-3-Aryl-5-(5-(2-arylethen-1-yl)-1,3,4-oxadiazol-2-yl)-1,2,4-oxadiazoles
be)9,26
(7aa-
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To a round-bottom, two-necked balloon, carboxylic acid 2a-b (0.5 mmol) and a
proper hydrazide 6a-e (0.5 mmol) were suspended in dichloromethane (7.0 mL), and
triethylamine (5 equiv.) was added. Then, TBTU (1.1 equiv.) was added, and the
mixture was stirred at room temperature for 2 h. After completion, as verified by TLC,
4-toluenesulfonyl chloride (3 equiv.) was added to the reaction vessel and the mixture
was stirred at room temperature for 2 h. The reaction was quenched using 5.0 mL of
aqueous 35% ammonia solution, and stirring was continued for additional 15 min.
Dichloromethane (20 mL) was added and the organic phase was transferred to an
Erlenmeyer flask, and then it was dried over anhydrous magnesium sulphate. The
resulting phase was filtered and solvent was removed by rotary evaporation under
reduced pressure. The crude product was purified through flash chromatography
employing hexanes/ ethyl acetate/ dichloromethane (7:2:1, v/v) mixture, and the solvent
was removed under reduced pressure.
(E)-3-phenyl-5-(5-(2-phenylethen-1-yl)-1,3,4-oxadiazol-2-yl)-1,2,4-oxadiazole (7aa)
1
Yield = 0.114 g, 72% (white solid). mp = 151.8 – 153.8 °C. H NMR (CDCl3,
400 MHz), δ (ppm): 8.20 (d, J = 8.1 Hz, 2 H), 7.85 (d, J = 16.5 Hz, 1 H), 7.63 – 7.60
13
(m, 2 H), 7.57 – 7.51 (m, 3 H), 7.47 – 7.44 (m, 3 H), 7.13 (d, J = 16.5 Hz, 1 H). C
NMR (CDCl3, 100 MHz) δ (ppm): 169.5, 166.2, 162.8, 152.6, 142.5, 134.1, 132.0,
130.8, 129.1, 129.0, 127.9, 127.7, 125.4, 108.3. HRMS-ESI(+) m/z, calcd. for
C18H13N4O2 [M + H]+: 317.1039; found: 317.1026.
(E)-3-(4-methylphenyl)-5-(5-(2-phenylethen-1-yl)-1,3,4-oxadiazol-2-yl)-1,2,4-
oxadiazole (7ab)
1
Yield = 0.134 g, 81% (white solid); mp = 169.9 – 171.4 °C. H NMR (CDCl3,
400 MHz), δ (ppm): 8.08 (d, J = 8.2 Hz, 2 H), 7.84 (d, J = 16.5 Hz, 1 H), 7.62 – 7.59
(m, 2 H), 7.47 – 7.42 (m, 3 H), 7.32 (d, J = 7.9 Hz, 2 H), 7.12 (d, J = 16.4 Hz, 1 H),
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2.43 (s, 3H). C NMR (CDCl3, 100 MHz) δ (ppm): 169.6, 166.2, 162.6, 152.7, 142.5,
DOI: 10.1039/D0NJ04530F
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142.5, 134.2, 130.8, 129.7, 129.1, 127.9, 127.7, 122.7, 108.4, 21.6. HRMS-ESI(+) m/z,
calcd. for C19H15N4O2 [M + H]+: 331.1195; found: 331.1197.
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(E)-3-(4-methoxyphenyl)-5-(5-(2-phenylethen-1-yl)-1,3,4-oxadiazol-2-yl)-1,2,4-
oxadiazole (7ac)
1
Yield = 0.151 g, 87% (white solid); mp = 163.7 – 166.0 °C. H NMR (CDCl3,
400 MHz), δ (ppm): 8.12 (d, J = 8.9 Hz, 2 H), 7.83 (d, J = 16.5 Hz, 1 H), 7.62 – 7.59
(m, 2 H), 7.47 – 7.42 (m, 3 H), 7.12 (d, J = 16.5 Hz, 1 H), 7.01 (d, J = 8.9 Hz, 2 H),
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3.87 (s, 3 H). C NMR (CDCl3, 100 MHz) δ (ppm): 169.2, 166.2, 162.6, 162.5, 152.7,
142.4, 134.1, 130.8, 129.4, 129.1, 127.9, 117.8, 114.5, 108.4, 55.4. HRMS-ESI(+) m/z,
calcd. for C19H15N4O3 [M + H]+: 347.1144; found: 347.1142.
(E)-3-(4-chlorophenyl)-5-(5-(2-phenylethen-1-yl)-1,3,4-oxadiazol-2-yl)-1,2,4-
oxadiazole (7ad)
1
Yield = 0.130 g, 74% (white solid), mp = 176.7 – 179.2 °C. H NMR (CDCl3,
400 MHz), δ (ppm): 8.14 (d, J = 8.8 Hz, 2 H), 7.85 (d, J = 16.5 Hz, 1 H), 7.62 – 7.59
(m, 2 H), 7.51 (d, J = 8.8 Hz, 2 H), 7.48 – 7.43 (m, 3 H), 7.13 (d, J = 16.5 Hz, 1 H). 13C
NMR (CDCl3, 100 MHz) δ (ppm): 168.8, 166.3, 163.0, 152.5, 142.7, 138.4, 134.1,
130.9, 129.4, 129.2, 129.1, 127.9, 124.0, 108.3. HRMS-ESI(+) m/z, calcd. for
C18H12ClN4O2 [M + H]+: 351.0649; found: 351.0645.
(E)-3-(4-(trifluoromethyl)phenyl)-5-(5-(2-phenylethen-1-yl)-1,3,4-oxadiazol-2-yl)-1,2,4-
oxadiazole (7ae)
1
Yield = 0.086 g, 45% (white solid), mp = 192.0 – 195.3 °C. H NMR (CDCl3,
400 MHz), δ (ppm): 8.33 (d, J = 8.1 Hz, 2 H), 7.86 (d, J = 16.5 Hz, 1 H), 7.80 (d, J =
8.2 Hz, 2 H), 7.65 – 7.57 (m, 2 H), 7.49 – 7.41 (m, 3H), 7.14 (d, J = 16.5 Hz, 1 H). 13C
NMR (CDCl3, 100 MHz) δ (ppm): 168.6, 166.4, 163.3, 152.5, 142.8, 134.2, 133.8 (q,
J = 32.9 Hz), 130.9, 129.2, 128.9, 128.2, 128.0, 126.1 (q, J = 3.7 Hz), 123.6 (q, J =
272.5 Hz), 108.3. HRMS-ESI(+) m/z, calcd. for C19H12F3N4O2 [M + H]+: 385.0912;
found: 385.0908.
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oxadiazole (7ba)
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Yield = 0.101 g, 55% (yellow solid); mp = 155.8 – 159.8 °C. H NMR (CDCl3,
400 MHz), δ (ppm): 8.64 (d, J = 16.2 Hz, 1 H), 8.25 – 8.20 (m, 3 H), 7.93 (d, J =
8.2 Hz, 1 H), 7.89 (d, J = 8.2 Hz, 1 H), 7.85 (d, J = 7.2 Hz, 1 H), 7.63 (t, J = 7.7 Hz,
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1 H), 7.57 – 7.51 (m, 5 H), 7.21 (d, J = 16.2 Hz, 1 H). C NMR (CDCl3, 100 MHz) δ
(ppm): 169.6, 166.2, 162.8, 152.6, 139.4, 133.8, 132.0, 131.3, 131.2, 129.7, 129.0,
128.9, 127.8, 127.8, 127.2, 126.4, 125.5, 125.0, 123.0, 110.7. HRMS-ESI(+) m/z, calcd.
for C22H15N4O2 [M + H]+: 367.1195; found: 367.1195.
(E)-3-(4-methylphenyl)-5-(5-(2-(naphthalen-1-yl)ethen-1-yl)-1,3,4-oxadiazol-2-yl)-
1,2,4-oxadiazole (7bb)
Yield = 0.114 g, 60% (yellow solid); m. p. = 135.4 – 139.1 °C. 1H NMR (CDCl3,
400 MHz), δ (ppm): 8.64 (d, J = 16.2 Hz, 1 H), 8.24 (d, J = 8.4 Hz, 1 H), 8.09 (d, J =
8.2 Hz, 2 H), 7.93 (d, J = 8.2 Hz, 1 H), 7.89 (d, J = 8.4 Hz, 1 H), 7.85 (d, J = 7.3 Hz,
1 H), 7.62 (t, J = 7.6 Hz, 1 H), 7.57 – 7.51 (m, 2 H), 7.33 (d, J = 8.0 Hz, 2 H), 7.20 (d,
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J = 16.2 Hz, 1 H), 2.43 (s, 3 H). C NMR (CDCl3, 100 MHz) δ (ppm): 169.6, 166.2,
162.6, 152.7, 142.5, 139.3, 133.8, 131.4, 131.3, 131.2, 129.8, 128.9, 127.7, 127.2,
126.5, 125.5, 125.0, 123.1, 122.7, 110.8, 21.6. HRMS-ESI(+) m/z, calcd. for
C23H17N4O2 [M + H]+: 381.1352; found: 381.1364.
(E)-3-(4-methoxyphenyl)-5-(5-(2-(naphthalen-1-yl)ethen-1-yl)-1,3,4-oxadiazol-2-yl)-
1,2,4-oxadiazole (7bc)
Yield = 0.139 g, 70% (yellow solid); m. p. = 162.7 – 166.0 °C. 1H NMR (CDCl3,
400 MHz), δ (ppm): 8.65 (d, J = 16.2 Hz, 1 H), 8.25 (d, J = 8.5 Hz, 1 H), ), 8.15 (d, J =
9.0 Hz, 2 H), 7.94 (d, J = 8.1 Hz, 1 H), 7.90 (d, J = 7.7 Hz, 1 H), 7.86 (d, J = 7.3 Hz,
1 H), 7.63 (t, J = 7.6 Hz, 1 H), 7.58 – 7.52 (m, 2 H), 7.22 (d, J = 16.2 Hz, 1 H), 7.03 (d,
13
J = 9.0 Hz, 2 H), 3.88 (s, 3 H). C NMR (CDCl3, 100 MHz) δ (ppm): 169.3, 166.2,
162.6, 162.5, 152.8, 139.4, 133.8, 131.5, 131.3, 131.2, 129.5, 128.9, 127.2, 126.5,
125.5, 125.0, 123.1, 117.9, 114.5, 110.8, 55.4. HRMS-ESI(+) m/z, calcd. for
C23H17N4O3 [M + H]+: 397.1301; found: 397.1305.
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(E)-3-(4-chlorophenyl)-5-(5-(2-(naphthalen-1-yl)ethen-1-yl)-1,3,4-oxadiazol-2-yl)-
4
5
6
7
8
483
484
485
486
487
488
489
490
491
1,2,4-oxadiazole (7bd)
Yield = 0.118 g, 59% (yellow solid); m. p. = 179.4 – 181.6 °C. 1H NMR (CDCl3,
400 MHz), δ (ppm): 8.66 (d, J = 16.2 Hz, 1 H), 8.25 (d, J = 8.5 Hz, 1 H), 8.16 (d, J =
8.8 Hz, 2 H), 7.96 (d, J = 8.2 Hz, 1 H), 7.91 (d, J = 8.1 Hz, 1 H), 7.87 (d, J = 7.2 Hz,
1 H), 7.64 (t, J = 8.4 Hz, 1 H), 7.59 – 7.55 (m, 2 H), 7.52 (d, J = 8.8 Hz, 2 H), 7.23 (d,
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13
J = 16.2 Hz, 1 H). C NMR (CDCl3, 100 MHz) δ (ppm): 168.8, 166.3, 162.9, 152.5,
139.5, 138.3, 133.8, 131.3, 131.2, 131.2, 129.4, 129.0, 128.9, 127.2, 126.5, 125.5,
125.0, 123.8, 123.0, 110.5. HRMS-ESI(+) m/z, calcd. for C22H14ClN4O2 [M + H]+:
401.0805; found: 401.0790.
492
493
494
(E)-3-(4-(trifluoromethyl)phenyl)-5-(5-(2-(naphthalen-1-yl)ethen-1-yl)-1,3,4-oxadiazol-
2-yl)-1,2,4-oxadiazole (7be)
495
496
497
498
499
500
501
502
Yield = 0.085 g, 39% (yellow solid); m. p. = 203.2 – 206.4 °C. 1H NMR (CDCl3,
400 MHz), δ (ppm): 8.72 (d, J = 16.2 Hz, 1 H), 8.38 (d, J = 8.1 Hz, 2 H), 8.27 (d, J =
8.5 Hz, 1 H), 7.98 (d, J = 8.2 Hz, 1 H), 7.93 (d, J = 7.7 Hz, 1 H), 7.90 (d, J = 7.1 Hz,
1 H), 7.83 (d, J = 8.2 Hz, 2 H), 7.66 (t, J = 7.6 Hz, 1 H), 7.61 – 7.56 (m, 2 H), 7.27 (d,
13
J = 16.20 Hz, 1 H). C NMR (CDCl3, 100 MHz) δ (ppm): 168.6, 166.4, 163.2, 152.5,
139.7, 133.8, 133.6, 131.4, 131.3, 131.2, 129.0, 128.8, 128.2, 127.3, 126.5, 126.1 (q, J =
3.3 Hz), 125.6, 125.1, 123.6 (q, J = 273.4 Hz), 123.0, 110.6. HRMS-ESI(+) m/z, calcd.
for C23H14F3N4O2 [M + H]+: 435.1069; found: 435.1053.
503
504 Results and Discussions
505
506
Synthesis of the target compounds
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508
509
510
511
512
513
The obtention of the target compounds 7aa-be followed the method
previously adapted by our group,9 from Stabile et al (2010),26 which involves
condensation of a carboxylic acid and a carbohydrazide, followed by
cyclodehydration. Firstly, (E)-3-arylacrylic acids (2a-b) were prepared by
Knoevenagel-Doebner condensation of aldehydes (1a-b) and malonic acid in
pyridine.22 1,2,4-Oxadiazole-5-carbohydrazides (6a-e) were obtained by literature
procedures, in three steps. In the first, nitriles (3a-e) were treated with hydroxylamine
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518
hydrochloride and aqueous sodium hydroxide in ethanol, giving the rDeOspI: e10c.1t0i3v9e/D0NJ04530F
amidoximes (4a-e).23 These were reacted with ethyl oxalyl chloride under basic
conditions (DIPEA) in THF, forming ethyl 1,2,4-oxadiazole-5-carboxylates (5a-e).24
The esters were then converted to hydrazides (6a-e), employing hydrazine hydrate in
ethanol at room temperature.25 (Scheme 1).
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Scheme 1. Synthetic route to the acid 2 and to hydrazides 6a-e. Conditions: i) 1a-b, malonic acid,
piperidine, pyridine, 100 °C, 15 h. ii) NH2OH.HCl, EtOH, r. t., 15 h. iii) EtO2CC(O)Cl, DIPEA, THF,
reflux, 2h. iv) NH2NH2.H2O, EtOH, r. t., 1 h.
523
524
525
526
527
528
529
530
531
532
533
534
535
Finally, the 1,3,4-oxadiazolyl-1,2,4-oxadiazoles 7aa-be were prepared through
TBTU-promoted condensation of 3-arylacrylic acids 2a-b with the 1,2,4-oxadiazole-5-
carbohydrazides 6a-e in the presence of triethylamine, followed by cyclodehydration of
the diacylhydrazine intermediates employing 4-toluenesulfonyl chloride in a one-pot
procedure. A substituent series of 10 compounds was prepared for comparison. The
yields of examples bearing electron-withdrawing groups were lower than those analogs
bearing electron-donating groups. The results are shown in the Table 1.
1
13
The identity of compounds 7aa-be was confirmed by means of H and C NMR
spectroscopy, and High-Resolution Mass Spectroscopy (HRMS), as shown in Synthetic
Procedures section. 1H and 13C spectra of compounds 7aa-be are listed in
Supplementary Information (Figures S1 – S20), as well as HRMS spectra (Figures S21
– S30). For X-Ray data of compound 7aa, see Supplementary Information (Figure S31).
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539
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540
Table 1. Substituent series of the 1,3,4-oxadiazolyl-1,2,4-oxadiazoles 7aa-be.a
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Entry
R1
R2
Yield (%)b
7aa
7ab
7ac
7ad
7ae
-
7ba
7bb
7bc
7bd
7be
Ph
Ph
Ph
Ph
Ph
H
CH3
OCH3
Cl
CF3
-
72
81
87
74
45
-
55
60
70
59
39
-
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
H
CH3
OCH3
Cl
CF3
542
543
544
545
23
24
25
26
aReaction conditions: i) 2a-b (0.5 mmol), 6a-e (0.5 mmol), TBTU (1.1 equiv), Et3N (5.0 equiv), DCM (7 mL), for 2 h
b
at room temperature; ii) TsCl (3.0 equiv), 2 h at room temperature. Isolated yield after column chromatography on
flash silica, with hexanes/ethyl acetate/dichloromethane (7:2:1, v/v) mixture as eluent.
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58
59
60
546
547
548
549
550
551
552
553
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555
556
557
558
559
560
561
562
563
564
UV-Vis absorption and fluorescence emission properties for derivatives 7aa-ae and
7ba-be
The absorption UV-Vis and steady-state fluorescence emission spectra for
derivatives 7aa-ae in CHCl3 are shown in Fig. 2 and the photophysical data are listed in
Table 2. All derivatives showed transition bands located at ultraviolet (UV) range,
around 250-400 nm. The electronic transitions which can be related to π→π* type-
transitions are observed and no significative shift were observed when change the
solvent polarity (see Table 2). The presence of a naphthyl moiety in the compounds
7ba-be caused a significant change in the maximum absorption of the less energetic
transition. This fact can be attributed to the greater contribution of aromaticity from
naphthyl group. The UV-Vis spectra for the compounds 7aa-ae in DMSO and 7ba-be
in CHCl3 and DMSO are listed in the Supplementary Information (see Figures S32-
S33).
As example, steady-state fluorescence emission spectra for the compounds 7ba-
be in argon saturated CHCl3 solution are shown in Fig. 3. In general, all heterocycles
contain one emission peak in the blue to cyan range (400 to 500 nm). As example,
emission band shifts (~50-60 nm) can be seen when comparing the derivatives 7aa
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566
567
568
569
570
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572
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576
577
578
(phenyl) and 7ba (naphthyl) in the same solvent. All the compoundsDOsI:h1o0.w103e9d/D0NJ04530F
fluorescence emission with moderate quantum yields (Φf) in both CHCl3 or DMSO
solutions. The Φf values for these compounds also show differences; for example,
compound 7ba (naphthyl and Ph groups), compound 7bc (naphthyl and 4-OCH3Ph
groups) and compound 7be (naphthyl and 4-CF3Ph groups) in CHCl3 solution have
distinct Φf values, which explains the influence of the donor/acceptor electronic
properties, attributed to the resonance stabilization structures in the excited state (Table
2).
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Large Stokes Shifts (SS) were observed in all derivative compounds, mainly in
DMSO solution, and it can be assigned to the ICT state that exists in these structures
(Table 2). Steady-state fluorescence emission spectra for compounds 7aa-ae in CHCl3
and DMSO and 7ba-be in DMSO are listed in the Supplementary Information (see
Figures S34-S35). The visible appearance of representative examples 7aa and 7ba in
solution (DMSO) is shown in the Supplementary Information (see Figure S36).
1.0
0.8
0.6
0.4
7aa
7ab
7ac
7ad
0.2
7ae
0.0
250
300
350
400
450
500
Wavelenght (nm)
579
580
Fig. 2. UV-Vis absorption spectra of derivatives 7aa-ae in CHCl3 solution ([ ] = 2.00 x 10-5 M).
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8
100
80
60
40
20
0

= 358 nm
exc
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7ba
7bb
7bc
7bd
7be
400
450
500
550
600
650
700
Wavelenght (nm)
582
28
583
584
585
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600
601
602
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604
Fig. 3. Steady-state emission spectra of derivatives 7ba-be (λexc = 358 nm) in saturated argon chloroform
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solution ([ ] = 1.00 x 10-6 M).
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605
Table 2. Photophysical data of compounds 7aa-ae and 7ba-be.
in CHCl3
Compound
7aa
λ, nm (ε; M-1cm-1)a
323 (38,250)
Emission, nm (Φf)b
426 (0.103)
432 (0.158)
429 (0.212)
425 (0.147)
429 (0.170)
473 (0.274)
491 (0.335)
480 (0.015)
482 (0.357)
481 (0.482)
in DMSO
SS (nm)c
103.0
107.0
107.0
104.0
104.0
114.0
131.0
124.0
126.0
122.0
E0-0 (eV)d
3.48
7ab
325 (42,700)
3.44
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7ac
252 (29,350), 322 (40,850)
262 (28,850), 321 (36,500)
253 (30,450), 325 (37,650)
261 (sh), 359 (38,300)
360 (43,100)
3.48
7ad
3.55
7ae
3.46
7ba
3.04
7bb
3.03
7bc
356 (40,250)
3.15
7bd
260 (52,950), 356 (36,300)
359 (40,400)
3.06
7be
3.06
Compound
7aa
λ, nm (ε; M-1cm-1)a
324 (39,050)
Emission, nm (Φf)b
426 (0.097)
433 (0.154)
428 (0.199)
425 (0.134)
429 (0.168)
473 (0.300)
490 (0.348)
480 (0.142)
481 (0.377)
481 (0.501)
SS (nm)c
102.0
107.0
103.0
102.0
104.0
116.0
133.0
131.0
126.0
125.0
E0-0 (eV)d
3.50
7ab
326 (40,350)
3.42
7ac
325 (39,600)
3.43
7ad
259 (31,100), 323 (37,550)
325 (39,750)
3.52
7ae
3.40
7ba
259 (32,150), 357 (31,800)
357 (27,700)
3.00
7bb
2.94
7bc
258 (39,400), 349 (19,250)
256 (47,250), 355 (23,950)
256 (46,200), 356 (25,450)
3.16
7bd
2.97
7be
3.00
606
607
608
a[ ] = 2.00 x 10-5 M; b[ ] = 10-6 M range at 298K, using 9,10-Diphenylanthracene (DPA) in CHCl3 as standard (Φf =
0.65); cStokes Shift equation: Δλ = λemisson - λabsorption; dE0-0 = 1240 / λ (in eV); sh = shoulder.
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DNA-binding assays by absorption UV-Vis analysis
The interaction of compounds 7aa-ae and 7ba-be with CT-DNA model was
studied by UV–Vis spectroscopy in DMSO(2%)/Tris-HCl buffer pH 7.2 mixture
solution, at 250-500 nm range. All compounds interact with CT-DNA and gives an
absorbance change in ultraviolet region. As example, the effect of different
concentrations of DNA titration on the absorption spectra using compounds 7aa and
7ba are shown in Fig. 4.
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In this experiment, upon increase concentration of CT-DNA (0-100 μM range)
DOI: 10.1039/D0NJ04530F
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into solution of derivatives 7aa and 7ba reveals hypochromicity profile in the
transitions around 300-360 nm. For all derivatives, was not observed any hypso or
bathochromic shifts, indicating non-electrostatic interaction of the molecules and CT-
DNA (Fig. 4). The decrease intensity changes of the related transition bands could be
accounted by the interaction of the aromatic portion of the compounds, probably via
hydrophobic forces (van der Waals, H-bonding or π-stacking) with the DNA
biomolecule or the presence of an aromatic moiety in the structure by covalent
interactions, as previously reported in the literature.27
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The hypochromicity parameter (H%) and intrinsic binding constant (Kb) values
for the compounds were calculated and summarized in Table 3. In the present study, the
derivatives demonstrated strong binding forces to CT-DNA (Kb ~105 to 106 M-1). These
binding constant values are associated to the compound-DNA complex stability in
solution, while the Gibb´s free energy indicates the spontaneity of derivative-DNA
binding process (Table 3). The UV-Vis CT-DNA spectra of derivatives 7ab-ae and
7bb-be can be found in the Supplementary Information and presented a similar behavior
(Figures S37-S44). Moreover, molecular docking calculations between compounds and
DNA was performed, reinforcing the experimental results.
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Fig. 4. UV-Vis titration absorption spectra of derivative (a) 7aa and (b) 7ba, in a DMSO (2%)/Tris-HCl
buffer (pH 7.2) solution. The concentration of CT-DNA ranged from 0 to 100 μM. Insert graph shows the
plot of [DNA]/(εa – εf) versus [DNA].
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EB-DNA competitive assays by steady-state fluorescence emission analysis
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In order to further confirm the binding affinity of the studied compounds with CT-
DNA, competitive binding assays via fluorescence quenching analysis were conducted
in the presence of the commercial intercalator (EB) into CT-DNA solution. Steady-state
fluorescence emission analysis of fixed concentration of CT-DNA:EB adduct in the
presence of derivatives 7aa and 7ba are shown in Figures S45-S54 in the
Supplementary Information depict the CT-DNA:EB without and in the presence of 7ab-
ae and 7bb-be.
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The CT-DNA:EB adduct formation shows a strong fluorescence emission at λ =
645 nm when excited at λexc = 510 nm and the corresponding fluorescence intensity
emission decreased upon successive addition of the compounds under study, being in
accordance for some heterocyclic compounds reported in the literature.9,27 This fact
could be assigned to the competition of derivatives with the intercalator EB into CT-
DNA strands. The Stern-Volmer quenching constant (KSV) and bimolecular quenching
rate constant (kq) values are presented in the Table 3. In this context, the KSV values
suggest weak competition mode of EB-binding (KSV ~ 102 M-1) in the case of phenyl
derivatives 7aa-ae and good competition mode of EB-binding (KSV ~ 103-104 M-1) in
the case of naphthyl derivatives 7ba-be. Moreover, the kq values for all derivatives
indicated a probably static interaction (kq ~ 1010-1011 M-1s-1) between derivatives and
CT-DNA:EB adduct (see Table 3). Again, molecular docking calculation were
performed, corroborating with the experimental results.
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672
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Table 3. Hypochromicity (H%), bathochromic shift (), Intrinsic binding constant (Kb), Stern-Volmer
DOI: 10.1039/D0NJ04530F
quenching constant (KSV), quenching rate constant (kq) and apparent binding constant (Kapp) values for the
interactions of compounds with calf thymus DNA (CT-DNA).
Absorption-DNA
Emission EB-DNA
9
ΔGº
(kcal
Kapp
Compound
H
Kb
Q
KSV
kq

(nm)b
0.0
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(M-1)h
(%)a
(M-1)c
(%)e
(M-1)f
(M-1s-1)g
mol-1)d
7aa
7ab
7ac
7ad
7ae
7ba
7bb
7bc
7bd
7be
8.00
7.70
1.07 x 106
4.07 x 106
1.16 x 106
6.76 x 105
2.00 x 106
4.94 x 105
2.15 x 106
1.00 x 105
1.87 x 106
3.65 x 106
−8.22
−9.01
−8.27
−7.95
−8.59
−7.76
−8.63
−6.82
−8.550
−8.945
10.00
7.40
7.48 x 102
5.24 x 102
1.26 x 103
4.41 x 102
9.28 x 102
1.24 x 103
1.69 x 104
1.37 x 103
1.49 x 104
2.50 x 104
3.25 x 1010
2.28 x 1010
5.48 x 1010
1.92 x 1010
4.03 x 1010
5.39 x 1010
7.34 x 1011
5.95 x 1010
6.48 x 1011
1.08 x 1012
6.44 x 105
6.58 x 105
6.51 x 105
6.77 x 105
6.70 x 105
6.47 x 105
4.38 x 106
6.48 x 105
4.48 x 106
3.85 x 106
0.0
8.65
0.0
12.10
5.30
8.50
0.0
11.00
10.80
7.10
0.0
8.20
0.0
9.65
0.0
61.40
13.20
57.20
69.00
11.30
9.00
0.0
0.0
9.05
0.0
b
c
685
686
687
688
689
aH(%) = (Absinitial - Absfinal)/(Absinitial) × 100;  (nm) = final - initial; Intrinsic binding constant by UV-Vis CT-
DNA analysis; Gibb´s free energy (R = 1.987 cal mol-1 K-1 and T = 298K); Q(%) = (Eminitial - Emfinal)/(Eminitial) ×
d
e
f
g
100; Stern-Volmer quenching EB-DNA constant (KSV) by steady-state fluorescence emission spectra; Bimolecular
Stern-Volmer quenching rate EB-DNA constant (kq) by steady-state fluorescence emission spectra (EB-DNA – τ0 =
23 ns); hDNA-binding apparent constant (Kapp);
690
691
692
693
694
695
696
697
698
699
700
701
702
26

Page 27 of 38
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DOI: 10.1039/D0NJ04530F
703
HSA-binding by fluorescence quenching emission assays
4
5
6
7
8
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
HSA-binding properties can be easily determined by steady-state fluorescence
emission assays through the fluorescence quenching of tryptophan (Trp) and/or tyrosine
(Tyr) amino acid residues. The HSA pocket structure presents one and seventeen Trp
and Tyr amino acid residues, respectively.28 Therefore, the interaction between human
serum albumin and derivatives was determined through the fluorescence quenching
assays.
9
10
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14
15
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19
20
21
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53
54
55
56
57
58
59
60
As example, the steady-state fluorescence emission for HSA without and in the
presence of successive additions of derivatives 7aa (ranging from 0 to 100 µM) at room
temperature (298K) is shown in Fig. 5, while Figures S55-S63 in the Supplementary
information depicts the spectra results for the other derivatives. The HSA exhibited a
strong fluorescence emission peak at λem = 329 nm when excited at 290 nm.
Fluorescence intensities of HSA gradually reduced upon successive additions of each
compound under study, and a slightly blue shift in all cases was also observed, which
suggests that 7aa-ae/7ba-be interact with albumin structure, mainly with the main
fluorophores present in the biomacromolecule and the shift may be explained by
conformational changes and/or perturbation on the microenvironment around the
albumin’s fluorophores upon ligand binding.
The fluorescence quenching in a biomolecule can be induced by different
mechanisms, which are in general classified into dynamic or static process. In order to
evaluate the main fluorescence quenching mechanism behavior induced by the
compounds 7aa-ae and 7ba-be, the well-known Stern–Volmer approximation was
applied in this study (see Experimental section).
The Stern-Volmer quenching (KSV) and bimolecular quenching rate (kq) constant
values for the interaction between HSA and the derivatives are shown in Table 4. In all
cases, moderate KSV values (102 – 104 M-1) and high kq values in about one/two orders of
magnitude larger (e.g. 1011 – 1012 M-1s-1) than the diffusional collision quenching rate
constant (kdiff ≈ 7.40 × 109 M−1s−1, according to the Smoluchowski-Stokes-Einstein
theory at 298K),29 indicate that the main fluorescence quenching mechanism is via static
process. Therefore, there is a ground-state association between HSA:7aa-ae and
HSA:7ba-be.
27

New Journal of Chemistry
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736
737
738
739
740
741
742
743
Since the Ka values for each compound are in the order of 10 – 10 M (Table 4)
DOI: 10.1039/D0NJ04530F
indicate moderate interaction ability. In addition, the number of binding sites (n) for all
compounds are in the range of 0.70-1.90 (Table 4), suggesting the presence of one or
two possible binding sites in the HSA subunits (depending on the ligand structure).
Overall, the presence of different units (phenyl or naphthyl) in the organic ligands under
study, cause variation in the binding constant values and there is an indication that both
derivatives 7aa-ae and 7ba-be can be possible transported and biodistributed by serum
albumin in the human bloodstream.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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57
58
59
60
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
Fig. 5. HSA-binding emission spectra with derivative 7aa in a DMSO (2%)/Tris-HCl buffer (pH 7.2)
solution. The concentration of compound ranged from 0 to 100 μM. Insert graph shows the plot of F0/F
versus [compound].
28

Page 29 of 38
New Journal of Chemistry
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DOI: 10.1039/D0NJ04530F
759
Table 4. HSA-binding data of derivatives 7aa-ae and 7ba-be.
Compound
Q (%)a
17.80
21.70
19.40
8.55
KSV (M-1)b
1.55 x 103
2.32 x 103
1.40 x 103
8.96 x 102
8.20 x 102
2.79 x 103
9.88 x 103
1.02 x 104
3.86 x 103
3.27 x 103
kq (M-1 s-1)c
2.73 x 1011
4.09 x 1011
2.47 x 1011
1.58 x 1011
1.44 x 1011
4.92 x 1011
1.74 x 1012
1.80 x 1012
6.80 x 1011
5.76 x 1011
Ka (M-1)d
1.10 x 103
1.12 x 103
1.24 x 103
1.68 x 102
2.07 x 102
3.87 x 103
5.20 x 103
7.50 x 103
3.33 x 103
3.96 x 103
ΔG0 (kcal/mol)e
−4.14
nf
7aa
1.90
1.88
1.29
0.70
0.78
1.07
1.32
1.86
0.95
1.12
7ab
−4.16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7ac
−4.22
7ad
−3.03
7ae
13.75
40.80
51.25
53.95
29.50
28.45
−3.16
7ba
−4.90
7bb
−5.06
7bc
−5.28
7bd
−4.80
7be
−4.90
760
761
762
763
764
aQuenching = (Intinitial – Intfinal) / Intinitial × 100;
bStern-Volmer quenching HSA constant by steady-state emission spectra;
cStern-Volmer rate quenching HSA constant by steady-state emission spectra (τ0 = 5.67 x 10-9 s);
dModified Stern-Volmer binding HSA constant by steady-state emission spectra;
eGibb´s free-energy for HSA-molecule interaction;
765
766
fNumber of binding sites;
767
768
769
770
771
772
773
774
775
776
777
778
Molecular docking calculations for the interaction between biomacromolecules and
7aa-ae / 7ba-be
Molecular docking technique is an attractive tool in drug design since it can
evaluate biomacromolecule-drug interactions from an atomic point of view, gaining
insights into the experimental results.30 Thus, in order to complement the experimental
data at predict the best-fit orientation of naphthylethenyl-substituted 1,3,4-oxadiazolyl-
1,2,4-oxadiazoles within DNA strands and HSA, as well as identify the main
intermolecular forces involved in the interaction process, molecular docking
calculations were carried out.
29