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