The relationship between solvatochromic properties and in silico ADME parameters of new chloroethylnitrosourea derivatives with potential anticancer activity and their β-Cyclodextrin complexes

Article abstract of DOI:10.1016/j.saa.2021.119579

In view of the anticancer effect of nitrosoureas a set of four new N-(2-chloroethyl)-N-nitrosourea (CENU) derivatives was synthesized. An in silico absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) prediction study revealed that the

Full text of DOI:10.1016/j.saa.2021.119579

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
The relationship between solvatochromic properties and in silico ADME
parameters of new chloroethylnitrosourea derivatives with potential
anticancer activity and their b-Cyclodextrin complexes
b
c
a
Hassina Fisli a, , Andreas Hennig , Mohamed Lyamine Chelaghmia , Mohamed Abdaoui
⇑
a Laboratory of Applied Chemistry, Université 8 Mai 1945 Guelma, Algeria
b Institute of Chemistry of New Materials, Universität Osnabrück, Germany
c Laboratory of Industrial Analysis and Materials Engineering, Université 8 Mai 1945 Guelma, Algeria
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
A new series of N-(2-chloroethyl)-N-
nitrosourea (CENU) derivatives was
synthesized.
In silico ADME/Tox prediction study
suggested CENUs as potential
anticancer drugs.
Solvatochromism of CENUs evaluated
by the Kamlet-Taft, Catalán and
Laurence models.
ꢀ
ꢀ
Solvatochromic comparison method
revealed excellent correlations.
Host-guest complexes of the CENUs
with b-CD were explored.
a r t i c l e i n f o
a b s t r a c t
Article history:
In view of the anticancer effect of nitrosoureas a set of four new N-(2-chloroethyl)-N-nitrosourea (CENU)
derivatives was synthesized. An in silico absorption, distribution, metabolism, excretion and toxicity
(ADME/Tox) prediction study revealed that the CENU derivatives satisfied all the required criteria for oral
administration and introduced them as remarkable anticancer candidates in the central nervous system
(CNS). A comparative solvatochromic study including the Kamlet-Taft, Catalán and Laurence models indi-
cated that the solvatochromic behavior of the CENUs depended on both, unspecific and specific solvent–
solute interactions. In detail, the solvatochromic effect of the solvent polarity on the absorption and emis-
sion maxima was significant for all CENUs, whereas the solvatochromic effect of the solvent’s ability to
donate or accept hydrogen bonds on the absorption and emission maxima was critically dependent on
the electron density of the N’-aryl group. From the solvatochromic comparison method, excellent corre-
lations (r ꢁ 0.890) were obtained between the ADME parameters and the solvatochromic regression coef-
ficients obtained by the Catalán model. As potential stabilizers, inclusion complexes of the investigated
CENU derivatives with b-cyclodextrin (b-CD) were also explored. The spectrofluorimetric host–guest
experiments included double-reciprocal Benesi-Hildebrand plots as well as the molar ratio and continu-
ous variation plots (Job’s plots), which established a 1:1 b-CD to CENU binding stoichiometry and rela-
tively high affinities of b-CD for CENU derivatives.
Received 9 September 2020
Received in revised form 28 January 2021
Accepted 30 January 2021
Available online 13 February 2021
Keywords:
Nitrosourea
ADME/Tox
Solvatochromism
LSER
Spectrofluorimetry
b-cyclodextrin
Ó 2021 Elsevier B.V. All rights reserved.
⇑
Corresponding author at: Laboratory of Applied Chemistry, Université 8 Mai 1945 Guelma, P.O. Box 401, 24000 Guelma, Algeria.
E-mail address: fisli.hassina@univ-guelma.dz (H. Fisli).
https://doi.org/10.1016/j.saa.2021.119579
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
1. Introduction
for solvatochromic behavior in twelve different solvents and we
found, in fact, significant correlations between the regression coef-
ficients from the solvatochromic studies and the ADME parameters
from the in silico studies of the CENU derivatives. In particular,
excellent correlations (r ꢁ 0.890) were revealed between the solva-
tochromic analysis of selected solvent sets according to the Catalán
model and the HIA, Caco-2, MDCK, BBB, and PPB ADME parameters.
Since the currently used CENUs are known to be quite unstable,
too hydrophobic and of limited aqueous solubility, and often show
major unfavorable side effects, the interaction of the new CENU
derivatives with b-cyclodextrin (b-CD) was also investigated,
which appears as an excellent candidate to optimize CENUs actions
[2,11–13]. b-CD is widely used due to its low cost and its capacity
to interact with a wide variety of molecules, including pesticides
and drugs [5,6,14,15], and we determined herein the stoichiometry
and binding constants (K) of the CENU/b-CD inclusion complexes
by fluorescence spectroscopy [16–25]. The contribution of our
research leads to provide a theoretical basis for developing new
drugs, drug carriers, and new forms of drugs, and can be exploited
in the future to obtain more detailed information about the CD
inclusion process of CENUs.
Nitrosoureas
and,
in
particular,
N-(2-chloroethyl)-N-
nitrosoureas (CENUs) are potential drug candidates for antitumor
chemotherapy since they are active on a large number of tumors
[1]. Given their potential, the chemistry of CENUs is very diverse
and has been the object of intensive studies, which revealed that
their primary mode of action involves alkylation, whereas other,
yet to be confirmed mechanisms have been also invoked [1,2]. Pre-
vious structure–activity relationships found that small substituent
changes can largely influence the chemical as well as the biological
properties of CENUs, e.g., their cytotoxicity, mutagenicity, tumori-
genicity, and control for histological variation of tumors [3]. For
example, the alkylating activity of various N’-aryl-N-nitrosoureas
in experimental medicinal studies has been related to the induc-
tive effect and the hydrogen bond-forming ability of the sub-
stituents at the urea nitrogen atoms [4].
As a continuation of our previous work [5,6], we report herein a
new series of CENUs derived from primary arylamines (aniline and
anilines substituted at the ortho position: 2-fluoroaniline, 2-
methylaniline and 2-methoxyaniline). The chemical structures of
the synthesized CENU derivatives are summarized in Fig. 1 and
include 1-(2-chloroethyl)-3-phenyl-1-nitrosourea (PNU), 1-(2-chl
oroethyl)-3-(2-fluorophenyl)-1-nitrosourea (2FPNU), 1-(2-chloroe
thyl)-3-(2-methylphenyl)-1-nitrosourea (2MPNU) and 1-(2-chlor
oethyl)-3-(2-methoxyphenyl)-1-nitrosourea (2MOPNU). The selec-
tion of CENUs examined in this study expands the common struc-
tural motifs that have been used in experimental medicinal studies
[2,4,7] and introduces structural variations at the ortho-position of
the N’-aryl group. This allows to dissect electronic factors from
steric factors for controlling in vitro and in vivo actions of these
compounds. The latter can be neglected in our series of CENUs
[8], because the ortho-position of the N’-aryl group is thought to
play no role as a reaction center in the generation of the active
antitumor species [4].
The new CENU derivatives were successfully synthesized and
characterized and their potential anticancer activity was assessed
by in silico investigations of their absorption, distribution, metabo-
lism, and excretion (ADME) properties. With the goal to study
structure–activity relationships and to potentially relate the bio-
logical activity of CENUs to their physicochemical properties, a
detailed solvatochromic study was performed to examine the
effects of the various ortho-substituents on non-specific and speci-
fic interactions of the CENU derivatives with the surrounding sol-
vent [9,10]. A better understanding of solvent–solute interactions
has the potential to enhance the pharmacologically pertinent prop-
erties of CENUs and to improve their application potential as can-
cer chemotherapeutic agents. Therefore, the absorption and
emission maxima and the Stokes shifts of the CENUs were evalu-
ated with the Kamlet-Taft, Catalán, and Laurence et al. models
2. Experimental
2.1. Synthesis
The synthesis of CENU derivatives was performed according to a
general procedure reported in Refs. [2,26] and the details of the
synthesis are provided as Supplementary Material.
2.2. In silico ADME study
The ADMET parameters and the pharmacokinetic profile along
with the druglikeness of the investigated CENU derivatives were
evaluated by the freely offered web servers SwissADME-(http://
www.swissadme.ch/) and PreADMET (https://preadmet.bmdrc.kr/
).
2.3. Spectroscopic measurements
Fluorescence excitation (for mabs) and emission (for mem) spectra
were recorded in 1-cm quartz glass cuvettes at room temperature
on a Shimadzu RF-5301PC type spectrofluophotometer equipped
with a Xenon lamp as excitation source. All the solutions were
freshly prepared and mixed well by an UP50H ultrasonic processor
(Hielscher Ultrasonics GmbH, Germany). All measurements were
made at least in duplicate.
2.3.1. Solvatochromic studies
Stock solutions of the CENU derivatives were freshly prepared
for each measurement in the respective solvent (methanol, etha-
nol, 2-propanol, dimethyl sulfoxide (DMSO), acetonitrile,
dimethylformamide (DMF), acetone, dichloromethane (DCM),
tetrahydrofuran (THF), ethyl acetate (EA) and cyclohexane) to
obtain working solutions of 1.00 ꢂ 10ꢃ5 M. For measurements in
water, CENU stock solutions in acetonitrile were diluted with
water to afford solutions containing 2 vol% acetonitrile in water.
Then, fluorescence excitation and emission spectra of the working
solutions were recorded and the wavelengths of the excitation and
emission maxima were converted into wavenumbers. Plots of the
linear solvation energy relationship (LSER) were obtained by mul-
tiple linear regression analysis using Excel Solver. Therefore, the
experimental absorption and emission maxima, mabs and
mem, as
Fig. 1. Chemical structures of the CENU derivative molecules used in this study.
well as the Stokes shift, em, were used as the
D
m
=
mabs
ꢃ
m
2

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
solvent-dependent property in the Kamlet-Taft (mx in Eq. 1), Cat-
alán (A in Eq. 2), and Laurence (ma in Eq. 3) models, which gave with
the empirical solvatochromic model parameters (Table S1) the fit-
ted lines including the respective regression and correlation
coefficients.
N-(2-chloroethyl)-N-nitrosourea derivatives as the major product.
In the cases of PNU, 2MPNU and 2MOPNU, double nitrosation
has not been observed, which can be ascribed to steric constraints
for 2MPNU and 2MOPNU as well as to an intramolecular hydrogen
bond (Fig. S3) between the oxygen atom of the nitroso group and
the hydrogen atom of the neighboring amide group [29].
2.3.2. Correlation analysis between solvatochromic behavior and
ADME parameters
3.2. In silico ADME predictions
In order to reveal correlations of the solvatochromic regression
coefficients and the ADME parameter, the respective values were
plotted against each other and analyzed by linear regression anal-
ysis using Microsoft Office Excel Solver to obtain the correlation
coefficients.
Nowadays, modern drug discovery involves searching drug can-
didates with suitable absorption, distribution, metabolism, and
excretion (ADME) properties to improve pharmacological activity.
Therefore, in silico ADME screens became increasingly important
to select the most promising compounds, because they minimize
the risk of drug candidates failing in late-stage development and
allow to reject compounds with inappropriate ADME properties
before substantial time and money are invested in synthesis and
testing [30]. The physicochemical properties and pharmacokinetic
parameters of our synthesized CENU derivatives were determined
in silico by the SwissADME and Pre-ADMET softwares to explore
their drug-like characteristics and the principal descriptors are
given in Tables 1 and 2.
With respects to the physicochemical properties displayed in
Table 1, the investigated derivatives present zero violation for Lip-
inski’s Rule of Five for oral drug availability: (1) The molecular
weight (MW) of the CENU derivatives is between 227.65 and
257.67 g/mol and, thus, below 500 g/mol, which ensures easy
transportation, absorption and diffusion of compounds; (2) the
estimated octanol/water partition coefficients (log P values) were
between 1.91 and 2.27 and, thus, lower than 4.15 suggesting a
good permeability across the cell membrane; (3) the number of
hydrogen bonds to be donated by the solute to the water molecules
in an aqueous solution (HBD) is less than 5, one HBD for all the
CENU derivatives; and (4) the number of hydrogen bonds to be
accepted by the solute from the water molecules (HBA) is less than
10, between 3 and 4 HBA. Moreover, the number of rotatable bonds
in the investigated derivatives were between 6 and 7 and, thus, less
than 10, and the topological polar surface area (TPSA) was lower
than 140 Å2, namely 61.77 and 71.00 Å2, with consequent percent-
ages oral absorption (%ABS) of 87.68 and 84.50% suggesting good
permeability, absorption and transport via biological membranes.
Consequently, the examined CENU derivatives should theoretically
manifest good oral availabilities as drugs. This was confirmed by
high bioavailability scores of 0.55 for all the examined CENU
derivatives and pan assay interference compounds (PAINS) pre-
sented zero alerts to all the hits. Finally, in accordance with our
straight-forward synthesis, the synthetic accessibility scores of all
the derivatives were between 2.22 and 2.41.
Additionally, in silico studies were performed using the Pre-
ADMET software to obtain important pharmacokinetic parameters,
namely Caco-2 (human colon adenocarcinoma) and MDCK (Madin-
Darby canine kidney cells) permeability coefficients, the in vitro
skin permeability, the percentage of human intestinal absorption
(HIA), blood brain barrier partition coefficients (BBB), and percent-
age of human plasma protein binding (PPB). This allows a first
assessment of the epithelial and endothelial permeability for oral
and transdermal drug delivery. Moreover, the Pre-ADMET calcula-
tions include the Ames test for checking toxicity and a prediction
for rodent carcinogenicity assay results. The results generated at
this regard are shown in Table 2.
The efficacy of drugs, including anticancer drugs, is mainly
dependent on the ability of the compounds to permeate across cell
membranes [31]. When evaluating the absorption properties for
the investigated CENU derivatives, HIA values showed that all the
derivatives can be classified as well-absorbing compounds (70 to
100%) with high HIA values from 95.04 to 95.61%. They exhibited
2.3.3. Host-guest studies with b-CD
Host-guest experiments of the CENUs with b-CD were per-
formed at room temperature in acetonitrile. For the investigation
of the effect of b-CD on the fluorescence properties of CENUs and
for the determination of the binding constants, the concentration
of each CENU derivative was kept constant at 1.00 ꢂ 10ꢃ5 M, while
the b-CD concentration was varied from 0.00 to 10.00 ꢂ 10ꢃ5 M.
Fluorescence data for the continuous variation plots (Job’s plots)
and molar ratio plots were obtained at 1.00 ꢂ 10ꢃ5 M total concen-
tration of b-CD and CENU. All fluorescence spectra were measured
using identical experimental conditions (excitation wavelength,
monochromator slit widths, etc.).
3. Results and discussion
3.1. Synthesis of CENU derivatives
First, various arylamines (aniline, 2-fluoroaniline, 2-
methylaniline and 2-methoxyaniline) were reacted with 2-
chloroethyl isocyanate to afford the respective 2-chloroethylurea
(CEU) derivatives (Fig. S1). The structure of the CEUs was con-
firmed by 1H NMR spectroscopy, which indicated the presence of
aromatic protons in the region from 6.95 to 9.50 ppm, by triplets
attributed to the chloromethylene group in the range of 3.68 to
3.95 ppm and a quadruplet of the CH2N group between 3.40 and
3.78 ppm, and by electrospray ionization mass spectrometry
(ESI-MS), which gave the expected molecular ion peaks.
The subsequent nitrosation reaction was carried out with
sodium nitrite, which is not regiospecific and gives the N’-aryl-N-
nitrosoureas as major and the N’-aryl-N’-nitrosoureas as a minor
product (Fig. S2) as revealed by thin layer chromatography. Sepa-
ration of the two isomers by column chromatography on silica
gel was, however, straightforward and the structure of the CENUs
was confirmed by 1H NMR spectroscopy and ESI-MS. In ESI-MS, all
CENUs gave [MꢃNO + H]+ ion peaks corresponding to the loss of
NO due to the instability of the N-nitroso group under ESI-MS con-
ditions [27].
In 1H NMR, successful nitrosation was confirmed by disappear-
ance of the exchangeable N-H protons and by appearance of an
A2X2 system with a triplet between 3.10 and 3.75 ppm correspond-
ing to the chloromethylene group and a triplet between 3.65 and
4.32 ppm corresponding to the CH2NNO group. As expected, the
nitroso group has a strong deshielding effect and the peak corre-
sponding to the CH2N group was significantly shifted downfield
upon nitrosation. A challenge in the synthesis of CENUs is the
regioselectivity of the nitrosation reaction [26,28], which may lead
to the formation of two regioisomers (Fig. S3), from which only the
N-(2-chloroethyl)-N-nitrosourea derivatives are of therapeutic
interest, whereas the N-(2-chloroethyl)-N’-nitrosoureas cannot
form a vinyl carbonium ion and, thus, lack oncostatic properties
[1,2]. Under kinetic control, we were able to obtain the desirable
3

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
Table 1
Swiss ADME physicochemical and ADME prediction of analyzed CENU derivatives.
Compd
Lipinski’s parameters
nROTBe
TPSAf
%ABSg
BIO Scoreh
PAINS #alertsi
SAccessj
MWa
Log Pb
HBAc
HBDd
Violations
PNU
227.65
245.64
241.67
257.67
1.91
2.27
2.25
1.97
3
4
3
4
1
1
1
1
0
0
0
0
6
6
6
7
61.77
61.77
61.77
71.00
87.68
87.68
87.68
84.50
0.55
0.55
0.55
0.55
0
0
0
0
2.22
2.41
2.27
2.39
2FPNU
2MPNU
2MOPNU
a
b
c
d
e
f
Molecular weight (g/mol).
Logarithm of partition coefficient between n-octanol and water.
Number of H-bond acceptors (O and N atoms).
Number H-bond donors (OH and NH groups).
Number of rotatable bonds.
Topological polar surface area (Å2).
Percentage of absorption.
Bioavailability Score.
Number of Pan Assay Interference Structures alerts.
Synthetic Accessibility.
g
h
i
j
Table 2
pre-ADMET absorption, distribution, metabolism, excretion and toxicity prediction of analyzed CENU derivatives.
c
Compd
HIA (%)a
Caco-2 (nm/s)b
MDCK (nm/s)
Skin_Permeability
BBBd
PPB (%)e
Ames_test
Carcino_Mouse
Carcino_Rat
PNU
95.14
95.61
95.37
95.04
15.20
20.37
10.93
20.53
39.61
129.45
56.19
35.71
ꢃ2.60
ꢃ3.26
ꢃ2.53
ꢃ3.26
0.70
0.73
0.71
0.83
82.92
88.03
91.67
74.59
mutagen
mutagen
mutagen
mutagen
negative
positive
positive
positive
negative
negative
negative
negative
2FPNU
2MPNU
2MOPNU
a
b
c
Percentage of human intestinal absorption.
Adenocarcenoma cell permeability.
Maden Darby Canine Kidney cell permeability.
Blood-Brain Barrier penetration.
d
e
PlasmaProtein Binding.
medium cell permeability in Caco-2 (4 to 70 nm/s) and MDCK (25
to 500 nm/s) models with permeability coefficients ranging from
10.93 to 20.53 nm/s and 35.71 to 129.45 nm/s, respectively. In con-
trast, the skin permeability values were negative suggesting that
transdermal delivery would not be a viable administration route
for CENU derivatives.
Since N-nitrosourea-type anticancer drugs are especially
applied in the central nervous system (CNS), their permeability
through the BBB was the most interesting parameter. Generally,
BBB values are a direct measure of penetration of a drug into the
CNS. Compounds which exhibit BBB penetration values higher than
0.40 are considered as active in the CNS and compounds that have
values below 0.40 are inactive in the CNS [32]. All the CENU deriva-
tives analyzed showed penetration values higher than 0.40 ranging
from 0.70 to 0.83. Thus, it is highly likely that they can cross the
BBB and are active in the CNS. Moreover, the investigated deriva-
tives showed PPB values lower than 90%, from 74.59 to 88.03,
except 2MPNU (91.67%), which indicates that the compounds will
not be trapped by too strong binding to proteins in the blood
plasma.
The Ames test predicted that all the CENU derivatives are muta-
gens and the carcinogenicity prediction suggested that all the
examined CENU derivatives except PNU are not carcinogenic in
mice, but that they may be carcinogenic in rats.
In summary, the results of the evaluation of the ADME/Tox
properties suggested that the investigated CENU derivatives satisfy
all the required criteria for oral administration, which renders
them suitable drug lead structures for further optimization and
evaluation.
Stokes shift are influenced by the nature and the position of func-
tional groups in the molecule, by the geometry of the molecule in
the ground and excited states, and by the nature of the surround-
ing solvent environment [33]. Molecules with solvent-sensitive
spectroscopic properties are very common, but it is often challeng-
ing to provide an unambiguous explanation of the origin of this
solvent dependence. Therefore, considerable efforts have been
deployed to correlate spectral properties with macroscopic and
empirical solvent parameters [34].
The solvent effect has been estimated with Kamlet-Taft [35],
Catalán [36] and Laurence et al. models [37]. The solvation effects
treatment supposes attractive solute–solvent interactions and
allows evaluating the ability of examined compounds to interact
with the surrounding media. These methods treat specific and
non-specific types of solute–solvent interactions separately, which
gives their importance and significance. This knowledge can be
used to extrapolate the dependence of these spectral properties
in several solvents and to establish the nature of environments in
macromolecules.
The photophysical properties of the CENU derivatives were
investigated to determine the effect of substituent, to examine
the solvatochromic properties, and to find out the effect of non-
specific and specific interactions with the surrounding solvents
for the investigated CENU derivatives. Therefore, absorption and
emission spectra were measured in solvents of different types with
varying polarity, polarizability, hydrogen bond donating (HBD)
ability (protic solvents) and hydrogen bond accepting (HBA) ability
(aprotic solvents), namely the polar HBD solvents water, methanol,
ethanol, and 2-propanol, the polar non-HBD solvents dimethyl sul-
foxide (DMSO), dimethyl formamide (DMF), acetonitrile, and ace-
tone, and the apolar, non-HBD solvents DCM, THF, EA, and
cyclohexane. The resulting absorption and emission frequencies
and Stokes shifts of the CENU derivatives investigated in selected
solvents are presented in Table 3.
3.3. Solvatochromism of the CENU derivatives
The photophysical properties of molecules, such as the absorp-
tion and emission maxima, the shape of the spectral band, and the
4

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
Table 3
Absorption and emission frequencies and Stokes shifts (cmꢃ1) of the CENU derivatives investigated in different solvents.
CENU derivative
PNU
2FPNU
2MPNU
2MOPNU
mx (cmꢃ1
)
mabs
mem
D
m
mabs
mem
D
m
mabs
mem
D
m
mabs
mem
Dm
Water
37,174
37,313
37,174
37,593
37,593
37,313
37,593
37,453
37,174
37,593
37,593
37,878
27,855
28,248
28,248
28,169
28,089
27,932
27,932
27,932
27,173
27,247
27,472
28,409
9319
9065
8926
9424
9504
9381
9661
9521
10,001
10,346
10,121
9469
37,174
37,313
37,313
37,313
37,453
37,174
37,313
37,735
37,313
37,593
37,735
38,022
27,932
28,409
28,571
28,571
27,548
27,247
27,322
27,700
27,855
27,855
28,089
27,548
9242
8904
8742
8742
9905
9927
9991
10,035
9458
9738
9646
10,474
37,174
37,593
37,735
38,167
37,313
37,453
37,313
37,878
37,593
37,735
38,314
37,453
27,624
27,932
28 169
28,089
27,322
27,173
27,322
27,472
27,173
27,322
27,100
27,397
9550
9661
9566
10,078
9991
37,037
37,593
37,735
37,878
37,313
37,735
37,313
37,453
37,453
36,900
37,174
37,593
28,089
27,027
28,248
28,011
27,548
27,855
26,809
27,624
27,472
27,322
27,173
27,472
8948
10,566
9487
9867
9765
9880
10,504
9829
9981
9578
Methanol
Ethanol
2-propanol
DMSO
DMF
10,280
9991
Acetonitrile
Acetone
DCM
EA
THF
10,406
10,420
10,413
10,066
10,056
10,001
10,121
Cyclohexane
Table S1 displays the solvent parameters [36–38] used in the
multiple regression analysis fit. In order to dissect various
solvent-specific effects, the solvatochromic analysis of the CENU
derivatives was separately performed with all solvents, polar sol-
vents only (water, methanol, ethanol, 2-propanol, DMSO, DMF,
acetonitrile and acetone), and aprotic non-HBD solvents only
(DMSO, DMF, acetonitrile, acetone, DCM, EA; THF and
cyclohexane).
The key result of the solvatochromic analysis includes the sus-
ceptibility constants p, a, and b, which present a measure for the
influence of each solvent parameter on the spectral property. To
facilitate the evaluation of the different contributions of the sol-
vent parameters on the spectroscopic properties, a ‘‘percentage
contribution” is also established on the basis of the absolute values
of p, a, and b (for example:
% + b % = 100%).
Quantification of solute–solvent interactions, from the absorp-
p* % = |p|/(|p| + |a| + |b|) such that p* % +
a
tion and emission bands and Stokes shifts, by the Kamlet-Taft sol-
vatochromic comparison method shows that not only the polarity
of the medium but also the hydrogen-bonding properties of the
solvent affect the solvatochromic behavior of the investigated
CENU derivatives.
3.3.1. Multiparametric approach of Kamlet-Taft
Non-specific and specific solvent–solute interactions effects on
absorption and emission spectra and Stokes shift of the investi-
gated CENU derivatives were interpreted by a LSER using the fol-
lowing Kamlet-Taft equation [39]:
Specifically, the solvatochromic contribution of the solvent
dipolarity/polarizability (
p*) to the position of the absorption max-
0
mx
=
mx + p
p
*+ a
a
+ bb
ð1Þ
imum,
m
abs, was ꢁ 40% for all CENUs except for 2MOPNU in aprotic
solvents (25%), and the sensitivity of electron-rich 2MPNU and
where
m
x and
m
0x are the solvent-dependent property and the regres-
2MOPNU towards the HBA ability (b) was higher than to the
sion value of this solvent-dependent property in a reference solvent,
respectively. The index of solvent dipolarity/polarizability, *,
quantifies the ability of the solvent to stabilize a charge or a dipole
by virtue of its dielectric effect. The measure of the solvent
HBD ability (
unsubstituted PNU appeared to be more sensitive to
The mostly negative sign of the p and a coefficients indicate a bath-
ochromic shift of the absorption maximum with an increase in
and for all CENUs, whereas PNU, 2MPNU and 2MOPNU show a
a
) of the solvent, whereas electron-poor 2FPNU and
p
a
than to b.
p*
hydrogen-bond donor (HBD) acidity,
a, illustrates the ability of a
a
solvent to donate a proton in a solvent-to-solute hydrogen bond.
The measure of the solvent hydrogen-bond acceptor (HBA) basicity,
b, illustrates the solvent’s ability to accept a proton (or, vice versa,
to donate an electron pair) in a solute-to-solvent hydrogen bond.
The regression coefficients p, a, and b, in Eq. 1, estimate the relative
susceptibilities of the solvent-dependent solute property mx to the
indicated solvent parameters [38]. This LSER has been extensively
used in the modelling of solvation effects [6,40].
hypsochromic shift with increasing b. This suggests a stabilization
of the electronic excited state relative to the ground state except
for these three CENUs in solvents with strong HBA ability.
The position of the emission maximum,
also largely dependent on the solvent dipolarity/polarizability
*) exceeding 30% contribution in most cases and a negative sign
mem, of all CENUs was
(p
of the p coefficient. The exception was 2MOPNU, which had a con-
tribution of ꢄ25%, and a positive p coefficient indicating a hyp-
The results collected in Table S2 are obtained from the fit of the
multiple regression analysis of the absorption and emission ener-
gies and Stokes shift employing Eq. 1 and data in Tables 3 and S1
sochromic shift of the emission maximum with an increase in p*.
Interestingly, the dependence on the HBA and HBD ability of the
solvent appeared to be reversed in comparison to the absorption
maximum. The sensitivity of electron-rich 2MPNU and 2MOPNU
towards the HBA ability (b) appeared equal to or lower than to
(a). Plots of obtained calculated data according to Eq. 1, mx calc., ver-
sus the corresponding experimental data, mx exp., presented on
Fig. 2 are used to check the success of the quantification and inter-
pretation of solvent effects on the shifts of the absorption and
emission maxima and Stokes shift of examined molecules.
Overall, the Kamlet-Taft model revealed mostly moderate linear
correlations when all solvents (see Fig. 2a to c and r = 0.448–0.945
in Table S2) and when only aprotic solvents (see Fig. 2a’’ to c’’ and
r = 0.161–0.859 in Table S2) were considered, whereas better cor-
relations were obtained for polar solvents only (see Fig. 2a’ to c’
and r = 0.582–0.998 in Table S2). A possible reason for the partly
significant deviations from linearity is that the Kamlet-Taft model
does not consider potential steric interactions of substituents at
the ortho position and/or the presence of intramolecular hydrogen
bonds [6].
the HBD ability (
and unsubstituted PNU appeared to be less sensitive to
a
) of the solvent, whereas electron-poor 2FPNU
than to
a
b or similarly sensitive. The mostly positive sign of the a and b coef-
ficients for all CENU derivatives except in aprotic solvents demon-
strates a hypsochromic shift with increases in both, solvent HBD
and HBA ability.
In view of the contrasting results for the solvatochromic effects
on the absorption and emission maxima in different solvents, the
dependence of the Stokes shift,
Dm, on the solvent parameters
was less clear. Overall, it appears that the Stokes shift decreases
for the three most electron-poor 2FMPNU, PNU, and 2MPNU with
increasing HBD and HBA ability (negative sign for a and b).
5

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
Fig. 2. Kamlet-Taft LSER plots of the calculated values of (a)
mabs, (b)
mem, and (c)
D
m
plotted against the corresponding experimental data in all solvents (no prime), polar
solvents (’), and aprotic solvents (’’).
3.3.2. Multiparametric approach of Catalán
Catalán [36] proposed a refinement of the Kamlet-Taft model by
splitting the solvent dipolarity/polarizability parameter, *, into
solvent polarizability (SP) and solvent dipolarity (SdP) parameters
according to Eq. 2:
both, the Catalán and the Kamlet-Taft equations, do not take into
account [6]. Nonetheless, the satisfactory correlations of absorp-
tion and emission maxima as well as the Stokes shift indicate that
the Catalán model gives also a correct interpretation of LSER of the
investigated CENU derivatives in the solvents used.
p
Gratifyingly, the main results from the LSER according to the
Catalán equation agreed with the results from the Kamlet-Taft
analysis. The solvatochromic contribution of the solvent polarity
parameters, solvent polarizability (SP) and solvent dipolarity
(SdP), to the position of the absorption and emission maxima
was significant for all CENUs (combined ꢁ 45%) except for aprotic
solvents only. The Catalán analysis additionally revealed larger
contributions of the polarizability for electron-poor 2FPNU and
unsubstituted PNU and larger contributions of the dipolarity for
electron-rich 2MPNU and 2MOPNU to the overall solvent polarity
effect. Also, the higher sensitivity of the absorption maxima of
electron-rich 2MPNU and 2MOPNU towards the HBA ability (SB)
than to the HBD ability (SA) of the solvent, the reversed behavior
for electron-poor 2FPNU and unsubstituted PNU, as well as the
reversal of this trend in the emission maxima was clearly con-
firmed by the Catalán model when polar solvents were not
excluded from the analysis. Moreover, the general trend for the
regression coefficients s, d, a and b obtained by the Catalán model
was in accordance with the respective coefficients p, a, and a from
the Kamlet-Taft model.
A = A0 + sSP + dSdP + aSA + bSB
ð2Þ
where SP is the solvent polarizability, SdP is the solvent dipolarity,
SA is the solvent’s hydrogen bond donor strength and SB is the sol-
vent’s hydrogen bond acceptor strength. s, d, a and b are the regres-
sion coefficients. Thus, the contribution of Catalán’s parameters to
the absorption and emission frequencies and Stokes shift of the
CENU derivatives could be estimated.
The results of the Catalán model (Fig. 3 and Table S3) were gen-
erated from the fit of the multiple regression analysis of the
absorption and emission energies and Stokes shift using Eq. 2
and the data in Tables 3 and S1 (b). As it can be observed from
Fig. 3 and from the r values in Tables S2 and S3, the multilinear
analysis of mabs, mem and Dm data according to the Catalán equation
produced the same quality of correlation between the predicted
and experimental spectroscopic properties as the Kamlet-Taft
model for all CENU derivatives investigated in all solvents (see
Fig. 3d to f and r = 0.421–0.878 in Table S3), in aprotic solvents
(see Fig. 3d’’ to f’’ and r = 0.365–0.929 in Table S3), and in polar sol-
vents only (see Fig. 3d’ to f’ and r = 0.565–0.980 in Table S3). As in
the Kamlet-Taft model, the deviations from linearity might be
ascribed to steric interactions of substituents at ortho position
and/or the presence of intramolecular hydrogen bonding, which
3.3.3. Multiparametric approach of Laurence
Laurence et al. have recently modified the LSER by introducing
new parameters [33,37]:
6

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
Fig. 3. Catalán LSER plots of the calculated values of (d)
m
abs, (e)
m
em, and (f)
D
m
versus their corresponding experimental data in all solvents (no prime), polar solvents (’), and
aprotic solvents (’’).
ma
=
m0 + diDI + eES + aa1 + bb1
ð3Þ
solvatochromic shifts of the absorption and emission maxima,
which is also confirmed with the Laurence model. However, the
where DI are dispersion and induction interactions, ES the elec-
dependence of the absorption and emission maxima of the
electron-rich 2MPNU and 2MOPNU compared to the electron-
poor 2FPNU and unsubstituted PNU became not apparent in the
regression coefficients obtained with the Laurence model, not even
in the best correlated data. This indicates that the Laurence model
may not provide a correct interpretation of LSER of CENU deriva-
tives in the solvents used, although similar limitations as for the
Kamlet-Taft and Catalán model have been noted [6].
trostatic interactions between permanent dipoles of the solute and
the solvent, and a1 and b1 the HBD and HBA ability of the solvent,
respectively, where a1 and b1 have the same meaning as the
respective solvent parameter values in the Kamlet-Taft and Catalán
models. The symbols di, e, a, and b represent the corresponding
regression coefficients.
The multiple linear regression analysis of the photophysical
properties in Table 3 with the solvent parameters in Table S1 (c)
with the Laurence equation Eq. 3, yields the results in Fig. 4 and
the calculated values displayed in Table S4. The correlation coeffi-
cients r obtained with the Laurence model (Table S4) displayed the
same quality of correlation between the predicted and experimen-
tal spectroscopic properties as the Kamlet-Taft (Table S2) and Cat-
alán model (Table S3) for all CENU derivatives investigated in all
solvents (see Fig. 4g to i and r = 0.186–0.852 in Table S4) and in
aprotic solvents (see Fig. 4g’’ to i’’ and r = 0.304–0.912 in
Table S4), whereas the correlation in polar solvents only was
clearly better with the Laurence model and consistently ꢁ 0.849
for the emission maximum and Stokes shift (see Fig. 4h’ and i’
and Table S4).
3.3.4. Discussion of the solvatochromism of the CENU derivatives
Overall, it was found that the solvent dependence of the absorp-
tion and emission maxima of the CENU derivatives was satisfacto-
rily correlated with the solvent parameters of all three
solvatochromic models. The dissection of the single parameter
p*
for non-specific solvent effects in the Kamlet-Taft equation into
dipolarity, SdP, and polarizability, SP, in the Catalán model or into
dispersion and induction interactions, DI, and electrostatic interac-
tions, ES, in the Laurence model did not significantly improve the
quality of the correlations.
The multiparametric analysis of the data collected in Tables S2
to S4 indicated that most of the solvatochromic effects on the
absorption and emission maxima are attributed to both, non-
specific solute–solvent interactions (to generally > 30%) and speci-
fic solute–solvent interactions, i.e. the solvent’s HBD and HBA abil-
ity (to generally < 70%). This is in accordance with the structure of
all investigated CENU derivatives, which indicate the possibility for
hydrogen bonding with the solvent due to the presence of hydro-
In general, the trends from the Kamlet-Taft and Catalán models
seem to be reflected in the regression coefficients obtained with
the Laurence model. For example, the Kamlet-Taft and Catalán
analysis consistently indicated that the contributions from the
HBA and HBD ability of the solvent do not exceed 70% and that
the solvent polarity parameters contribute more than 30% to the
7

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
Fig. 4. Laurence LSER plots of the calculated values of (g)
mabs, (h)
m
em, and (i)
D
m
versus their corresponding experimental data in all solvents (no prime), polar solvents (’), and
aprotic solvents (’’).
gen bond-accepting (carbonyl C=O, nitroso N=O) and hydrogen
bond-donating groups (amide N-H).
Interestingly, we found in the multiparametric analysis using
the Kamlet-Taft and Catalán models that the contribution of the
parameter reflecting the HBA ability (b and SB) of the solvent to
amino group, which is influenced by the nature of the substituent
in the aromatic ring (substituents with an electron-donating effect
weaken the intramolecular hydrogen bonding while substituents
with an electron-withdrawing effect strengthen the intramolecular
hydrogen bonding). Furthermore, where the strength of the
the solvatochromic shift of the absorption maximum,
m
abs, of the
intramolecular bonding is small, it is expected that the influence
of intermolecular hydrogen bonding is large [41]. This interpreta-
tion is in line with a steric hindrance and/or the electron-
donating effect caused by the ortho-methoxy and ortho-methyl
groups. Noteworthy, a steric effect influences the interaction with
all the solvents to the same extent, while intramolecular hydrogen-
bonding influences the interaction with the protic solvents more
than with the aprotic solvents [42]. Hence, the absorption and
emission spectra and Stokes shift of the examined CENU deriva-
tives are mainly dependent on the polarity of the solvents, whereas
the detailed solvatochromic response is dependent on the exact
chemical structures of the chromophores and the solvents.
Besides, the presence of an aromatic group is expected to confer
all CENU derivatives a pronounced solvent polarity. This is nicely
confirmed by the data in Tables S2 to S4, which shows that the
general solvent effects display a predominant influence on the sol-
vatochromic shifts of the investigated CENU derivatives.
electron-poor CENUs, PNU and 2FPNU, is unexpectedly small. The
low sensitivity of the two CENU derivatives to the hydrogen bond
basicity of the medium may be explained by the possible existence
of an intramolecular hydrogen bond N=O. . .HN in PNU and 2FPNU
(Fig. S3), which would effectively prevent the formation of hydro-
gen bonds of the urea N-H with a hydrogen bond-accepting solvent
[5,6]. In contrast, the sensitivity of the electron-rich CENUs,
2MPNU and 2MOPNU, to the HBA ability of the medium is more
pronounced, in particular when aprotic solvents are excluded from
the Catalán model, indicating that the intramolecular hydrogen
bond is significantly weakened or even absent in 2MPNU and
2MOPNU.
It is also interesting to note that the position of the emission
maximum, mem, of all CENUs are more dependent on the HBA abil-
ity of the solvent than the absorption maxima. This indicates that
the intramolecular hydrogen bond may be significantly loosened
in the excited state. This interpretation is in accordance with the
observation that the emission maxima of the electron-poor PNU
and 2FPNU are more dependent on the HBA ability of the solvent
than on the HBD ability, whereas the electron-rich 2MPNU and
2MOPNU are equally dependent on HBA and HBA ability.
3.4. Solvent effect on the structure-activity relationship
It is well known that the molecular structure and the physico-
chemical properties governs the interactions of drug molecules
with their environment and, thus, have a major influence on their
behavior in biological processes of the human body. Since the sol-
The degree of intramolecular hydrogen bonding is, apparently,
influenced by the electron density on the nitrogen atom of the
8

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
vatochromism of the investigated CENU derivatives similarly
reflects their interactions with the surrounding solvent molecules,
we speculated whether relationships between polarity and hydro-
gen bonding interactions as revealed by the solvatochromic analy-
sis and selected structural properties related to the ADME
parameters could be established.
Therefore, the regression coefficients obtained from LSER with
the Catalán model from absorption frequencies (from all solvents,
polar, and aprotic solvents tested, respectively) were used and
the estimated ADME parameters (HIA, Caco-2, MDCK, BBB penetra-
tion and PPB property) were plotted against (|s|+|d|), i.e., the sum of
the absolute values of the regression coefficients of the solvent
polarizability (SP) and the solvent dipolarity (SdP), respectively.
The latter were thought to serve as a measure of the importance
of non-specific interactions.
This analysis revealed acceptable linear dependencies (Table 4,
ra values) between the solvatochromic regression coefficients and
some ADME parameter suggesting correlations between the differ-
ent ADME parameters and non-specific solute–solvent interac-
tions. In particular, a very good correlation was found between
the BBB permeation (r = 0.997 for polar solvents) and non-
specific solvent–solute interactions as expressed by (|s|+|d|). Since
the ability of a molecule to permeate across a cell membrane is
mostly influenced by the hydrophilicity/lipophilicity balance it is
reasonable that the non-specific interactions to overall solvation
effects are correlated with lipophilicity and thus increase the per-
meation [31]. Similarly, an increased lipophilicity should also show
a good correlation with unspecific protein binding expressed as
PPB, which was, in fact, observed (r = 0.999 for aprotic solvents).
In view of this, it is intriguing to see that the correlation with
HIA, Caco-2 and MDCK absorption parameters was, at best, moder-
ate (with r < 0.80), whereas the correlation with BBB permeation
was very high (Table 4, ra values).
It was further attempted to include (|a|+|b|) from the absorp-
tion frequencies of the Catalán model in the correlation with the
ADME parameters, which is the sum of the absolute values of
the regression coefficients of the solvent’s hydrogen bond donor
strength, SA, and the solvent’s hydrogen bond acceptor strength,
SB. This significantly improved all correlations (Table 4, rb val-
ues). In particular the correlation coefficients between the HIA,
Caco-2 and MDCK permeabilities and the regression coefficients
in polar solvents increased dramatically to r = 0.890, 0.897,
and 0.974, which clearly suggests that the hydrogen bond inter-
actions are the main contributors in governing HIA, Caco-2 and
MDCK absorption properties.
3.5. Effect of b-CD complexation
Since the CENU derivatives investigated were found to be sensi-
tive to the surrounding environment (polarity, dipolarity, polariz-
ability, HBD and HBA strengths), changes of the spectroscopic
properties can also be used to follow encapsulation into CD cavity.
Therefore, the complexation was followed by spectrofluorometry
in acetonitrile [6] and the fluorescence spectra of the four investi-
gated CENU derivatives with increasing concentrations of b-CD are
shown in Fig. 5.
Inclusion of a fluorescent guest into a supramolecular host
changes the spectral properties of the guest and the most common
outcome for CDs is fluorescence enhancement. This can be attribu-
ted to protection against collisional quenching, changes in the
microenvironment, or a decreased flexibility of the guest due to
confinement in the host cavity [43]. Most prominent is the reloca-
tion from a more hydrophilic environment (most often water) into
the hydrophobic environment of the CD cavity.
We can deduce, from the fluorescence spectra of the investi-
gated CENU derivatives in Fig. 5, that the addition of increasing
concentrations of b-CD causes significant increase in their fluores-
cence emission intensities with significant changes in the position
of the emission maxima, where red shifts of 4, 4, 4 and 10 nm for
PNU, 2FPNU, 2MPNU and 2MOPNU, respectively, of the original
peak positions were observed. Since we have established above
that the shift of the maximum emission wavelength is sensitive
towards a change in the environment surrounding the fluorophore,
the red shift represents a clear indication for binding with the CD
cavity. We propose complex formation between b-CD and each
CENU derivative, in which each of the investigated CENU deriva-
tives is included, or at least partially included, into the b-CD cavity
in acetonitrile solutions.
The stoichiometry and the corresponding binding constants of
the inclusion complexes of the four CENU derivatives with b-CD
were assessed using Benesi-Hildebrand relations as expressed by
Eqs. (4) and (5) [44]. In the case that only the 1:1 complexes are
formed, the binding constants’ values for the formation of 1:1 com-
plexes, can be calculated based on the following Benesi-Hildebrand
relation [44]:
1
1
1
¼
þ
ð4Þ
I ꢃ I0 ðI1 ꢃ I0Þ ðI1 ꢃ I0ÞK1½CDꢅ
where I0, I and I1 are the fluorescence intensities of the fluorophores
without b-CD, with a particular concentration of b-CD, and at the
maximum concentration of b-CD used, respectively, [CD] is the total
concentration of b-CD molecule added during the titration, and K1
represents the binding constant for 1:1 complex formation.
For 2:1 (host:guest) inclusion complexes, the following Benesi-
Hildebrand relation [44] can be used:
In summary, these correlations confirm the importance of the
relative contributions from the non-specific and specific solute–
solvent interactions on ADME parameters of CENU derivatives.
They may even pave the way for generating new equations that
demonstrate exact relationships between solute–solvent interac-
tions and structure–activity parameters.
1
1
1
¼
þ
ð5Þ
2
I ꢃ I0 ðI1 ꢃ I0Þ
ðI1 ꢃ I0ÞK1K2½CDꢅ
Table 4
Correlation between the ADME parameters and the sum of the regression coefficients from Catalán model, for the investigated CENU derivatives.
ADME parameter
All solvents tested
HIA
Caco-2
MDCK
BBB
PPB
a
r
0.122
0.789
0.520
0.890
0.795
0.808
0.243
0.626
0.569
0.897
0.599
0.664
0.140
0.956
0.326
0.974
0.536
0.610
0.675
0.697
0.997
0.999
0.805
0.931
0.100
0.275
0.792
0.802
0.999
0.999
rb
a
Polar solvents
r
r
r
r
b
a
b
Aprotic solvents
r
a : Correlation coefficient, r, between the ADME parameters and the sum of the regression coefficients (|s|+|d|) obtained from the solvatochromic analysis of the absorption
maxima with the Catalán model.
b
r
: Correlation coefficients, r, between the ADME parameters and the sum of the regression coefficients (|s|+|d|) and (|a|+|b|) from the absorption maxima of the solva-
tochromic analysis with the Catalán model.
9

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
Fig. 5. Emission enhancement of the investigated CENU derivatives : (a) PNU, (b) 2FPNU, (c) 2MPNU and (d) 2MOPNU in presence of increasing concentrations of b-CD.
[CENU] = 1.00 ꢂ 10ꢃ5 M; [b-CD] = 0–10.00 ꢂ 10ꢃ5 M.
where K2 represents the binding constant for the second step of the
2:1 complex formation.
Benesi-Hildebrand plots yielded straight lines with excellent
linear regressions (r = 0.974, 0.982, 0.987 and 0.981, respectively)
for 1:1 (host:guest) inclusion complexes (Fig. 6), whereas no linear
relationship was obtained for the 1/(I - I0) versus 1/[CD]2 plots
(Fig. 7). This confirms the supposed 1:1 b-CD:CENU inclusion com-
plexes and rules out the possibility of forming 2:1 inclusion com-
plexes between b-CD and each CENU derivative. The
experimental binding affinities, K1, were 1316, 600, 2200 and
5071 Mꢃ1 for the CD-PNU, CD-2FPNU, CD-2MPNU and CD-
2MOPNU complexes, respectively.
Fig. 7. Benesi-Hildebrand plots for the 2:1 inclusion complexes.
The binding stoichiometry of b-CD-CENU inclusion complexes
was further confirmed by the continuous variation method [45]
and the molar ratios method [46]. According to continuous varia-
tion method, equimolar (1.00 ꢂ 10ꢃ5 M) solutions of each CENU
derivative and b-CD were prepared and mixed to afford varying
molar fractions of b-CD (between 0.20 and 0.80), while the total
concentration of the species ([b-CD] + [CENU]) was kept constant.
After stirring, the emission was measured for all solutions and the
fluorescence intensity (FI) was plotted as a function of the [b-CD]/
([b-CD] + [CENU]) ratio. In Fig. 8 are presented Job’s plots, showing
maxima at a molar fraction of 0.5, indicating that the stoichiometry
of the complexes is 1:1.
Fig. 6. Benesi-Hildebrand plots for the 1:1 inclusion complexes.
10

H. Fisli, A. Hennig, Mohamed Lyamine Chelaghmia et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 253 (2021) 119579
Commonly, the major driving force for b-CD complexation in
water is the hydrophobic effect, which enables hydrophobic drug
molecules to bind to the hydrophobic CD cavity. Since our studies
have been performed in acetonitrile as a solvent, a major contribu-
tion from the hydrophobic effect is thus less likely. Potential other
contributions to the observed affinity and selectivity originate
from hydrogen bonding of the guest with primary or secondary
hydroxyl groups of b-CD or from van-der-Waals interactions. Very
reasonable is also a contribution from C-H-p interactions between
the inner cavity hydrogen atoms with the highly polarizable aro-
matic rings of the CENU derivatives.
The binding selectivity clearly correlates with the electron den-
sity of the aniline ring, such that the CENU derivative with the
highest electron density (2MOPNU carrying an ortho-methoxy
group) binds strongest and the CENU derivative with the lowest
electron density (2FPNU with an ortho-fluorine group) binds weak-
est. This is well in line with increased van-der-Waals and C-H-
p
interactions of the electron-rich CENU derivatives. A contribution
from steric crowding, which disfavors the complexation, is not
observed since the most bulky ortho-methoxy-substituted CENU
derivative binds strongest.
Fig. 8. Continuous variation plots.
In the molar ratios method, the FI is expressed as a function of
[b-CD]/[CENU] ratio. Therefore, equimolar stock solutions of CENU
and b-CD were prepared (1.00 ꢂ 10ꢃ5 M) and varying volumes of
the b-CD stock solution (from 0.4 to 6 mL) were mixed with a fixed
volume (2 mL) of the CENU solution. This gave solutions with iden-
tical total concentration of the two species, b-CD and each CENU
4. Conclusion
A new series of four potential anticancer agents derived from
CENU was synthesized. The ADME/Tox prediction study revealed
that the investigated CENU derivatives can be good candidates as
anticancer agents for CNS-related cancers with high probability
of crossing the BBB. The contribution of specific and non-specific
solvent interactions to the observed solvatochromic behavior was
quantitatively evaluated with the multiparametric Kamlet-Taft
solute–solvent interaction model, which revealed the dependence
of the solvatochromic behavior of the investigated CENU deriva-
tives on both, the polarity of the medium and the hydrogen-
bonding properties of the solvent. Furthermore, the application of
Catalàn and Laurence models has shown that the non-specific
interactions are the main factors contributing to the solva-
tochromism exhibited by the investigated CENU derivatives. It
was also shown that the solvatochromic properties of these substi-
tuted CENU derivatives are influenced by the type of substituent.
The evidence for solvation effects on the structure–activity rela-
tionship of the investigated molecules was obtained by correlating
HIA, Caco2, MDCK, BBB and PPB parameters with the contributions
of non-specific and specific solute–solvent interactions, demon-
strating satisfactory relationships between solute–solvent interac-
derivative (1.00
ꢂ
10ꢃ5 M), while the molar ratio [b-CD]/
[nitrosourea] was varied from 0.2 to 3. From the molar ratios
method, the abrupt variations of the slopes in plots FI against
molar ratios [b-CD]/[CENU] at about 1:1 mol ratio, as shown in
Fig. 9, indicates the formation of 1:1 inclusion complexes and con-
firmed the formation of an association between b-CD and CENU
with a 1:1 M ratio of b-CD to CENU.
The binding constant, K1, reflects the strength of the binding
interactions of b-CD with guest molecules and, in the case of CENU
derivatives, relatively high affinities and a certain selectivity were
obtained. The large binding constant values of some b-CD-CENU
complexes demonstrate efficient complexation of the drug mole-
cules with b-CD, whereas the inclusion interactions of b-CD and
the investigated CENU derivatives can be affected by many factors.
It is generally believed that the driving forces involved in the for-
mation of CD inclusion complexes include electrostatic interac-
tions, van-der-Waals forces, hydrophobic interactions, and
hydrogen bonding, which cooperatively govern the stability of
the inclusion complex. The relative importance of each of these
contributions depends on the properties of the specific guest being
included [47]. For the CENU derivatives, the binding affinity to b-
CD is in the order 2MOPNU > 2MPNU > PNU > 2FPNU.
tions
and
structure–property
parameters.
Based
on
spectrofluorimetric analysis, binding constant values and stoi-
chiometries for the inclusion complexes between b-CD and each
CENU derivative were evaluated. Both methods, the continuous
variation and the molar ratios method, support the formation of
1:1 inclusion complexes. We have found that many factors highly
influence the inclusion complexation of guest CENU derivatives
with b-CD, including simultaneously, the hydrophobicity and sub-
stituent effect of the guest, which determine the stability of host–
guest complexes through the hydrophobic and hydrogen-bonding
interactions.
In view of these points, it is reasonable to propose that, studying
CENU derivatives’ spectral properties under different conditions
will lead to better explaining their biological activities and better
development of anticancer CENU drugs.
CRediT authorship contribution statement
Hassina Fisli: Investigation, Formal analysis, Writing - original
draft. Andreas Hennig: Conceptualization, Writing - review & edit-
Fig. 9. Molar ratios plots.
11

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Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
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