Synthesis, structure, computational and molecular docking studies of asymmetrically di-substituted ureas containing carboxyl and phosphoryl hydrogen bond acceptor functional groups

Article abstract of DOI:10.1016/j.molstruc.2019.127360

Two asymmetrically substituted ureas N,N ?-(2-carboxylphenyl)phenyl urea (L₁) and diethyl 4-(3-phenylureido)benzylphosphonate (L₂) were synthesized and characterized by spectroscopic and X-ray crystallographic analysis. The two compo

Full text of DOI:10.1016/j.molstruc.2019.127360

Journal Pre-proof
Synthesis, structure, computational and molecular docking studies of asymmetrically
di-substituted ureas containing carboxyl and phosphoryl hydrogen bond acceptor
functional groups
Obinna C. Okpareke, William Henderson, Joseph R. Lane, Sunday N. Okafor
PII:
S0022-2860(19)31469-3
DOI:
https://doi.org/10.1016/j.molstruc.2019.127360
MOLSTR 127360
Reference:
To appear in: Journal of Molecular Structure
Received Date: 15 May 2019
Revised Date: 21 September 2019
Accepted Date: 2 November 2019
Please cite this article as: O.C. Okpareke, W. Henderson, J.R. Lane, S.N. Okafor, Synthesis, structure,
computational and molecular docking studies of asymmetrically di-substituted ureas containing carboxyl
and phosphoryl hydrogen bond acceptor functional groups, Journal of Molecular Structure (2019), doi:
https://doi.org/10.1016/j.molstruc.2019.127360.
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Synthesis, structure, computational and molecular docking studies of asymmetrically di-
substituted ureas containing carboxyl and phosphoryl hydrogen bond acceptor
functional groups
Obinna C. Okpareke1,2*, William Henderson1 Joseph R. Lane1 and, Sunday N. Okafor3
,
1School of Science, University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand
2Department of Pure and Industrial Chemistry, University of Nigeria, 410011 Nsukka, Enugu
State, Nigeria
3Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria, 410011
Nsukka, Enugu State, Nigeria
Corresponding author email:obinna.okpareke@unn.edu.ng
1

Abstract
Two asymmetrically substituted ureas N,N -
́
(2-carboxyl phenyl)phenyl urea (L1) and diethyl 4-
(3-phenylureido)benzylphosphonate (L2) were synthesized and characterized by spectroscopic
and X-ray crystallographic analysis. The two compounds crystallized in the centrosymmetric
monoclinic crystal system and P21/n space group. The carboxyl substituted urea L1 crystallized
with one molecule in the asymmetric unit. A hydrogen-bonded dimer is formed between the
carboxyl group of the urea and a second molecule of the compound. The urea functional group is
involved in both intramolecular and intermolecular hydrogen bonding with the carboxyl and
carbonyl oxygens, respectively. The interplay of intermolecular and intramolecular hydrogen
bonding in the compound results in a 2-D hydrogen-bonded structure. The phosphoryl-
substituted urea L2, on the other hand, crystallized with two molecules in the asymmetric unit,
resulting in a hydrogen-bonded tetramer in the crystal lattice. Non-covalent interactions (NCI)
analysis of the two compounds revealed the presence of competitive interactions between the
urea functional group and the carboxyl and phosphoryl substituents in L1 and L2, respectively.
Molecular docking calculations predicted favorable binding interactions between the ureas and
the two anticancer protein targets EGFR kinase (2J5F) and anaplastic lymphoma kinase (5J7H).
Keywords: Synthesis, structure, non-covalent interaction, molecular docking
2

Graphical Abstract
3

1. Introduction
A wide range of biological and chemical interactions, including interactions between
proteins and drugs, catalysts and their substrates, self-assembly of nanomaterials and even simple
chemical reactions are dominated by non-covalent interactions. Among these interactions are
hydrogen bonding, dipole-dipole interactions, steric repulsion, and London dispersions [1-3].
Hydrogen bonding, due to its molecular robustness, pronounced directionality and relatively high
strength have been the subject of a significant number of publications [4-10]. Hydrogen bonds
play a very important role in chemical reactivity, solvation, and most importantly the
advancement of supramolecular chemistry, with the aim of designing and controlling crystal
structures with interesting architectures. This goal has however remained elusive. The ability to
understand and predict the formation of a hydrogen bond is of great importance to the chemical
scientist [11-14].
Different functional groups containing some donor and acceptor groups including FH, OH, NH,
SH, F, O, N, and S, as well as
different hydrogen-bonded structures [15-17]. Among these groups, the N,N
ureas have proven to be very reliable supramolecular building blocks due to their ability to form
persistent one dimensional (1-D) -tape hydrogen-bonded chains (Figure 1) in a variety of
environments including solutions [18, 19], gels and fibers [20, 21], as well as in the solid-state
[22, 23]. The N,N disubstituted ureas can act as both hydrogen bond donors through their two
π-electrons, have been used as structure-directing motifs in
՛
-disubstituted
α
՛
-
NH protons and acceptors through the lone pairs of the C=O group. The 1-D motifs in
disubstituted ureas have been consistently exploited for the design of some fascinating hydrogen-
bonded architectures [24-27].
Despite the exceptional ability of N,N՛ -disubstituted ureas to form highly robust crystalline
solids of targeted architecture, there is still a limited level of reliability as the vast majority of the
functional groups may assume alternative motifs in the solid-state depending on the presence of
other competing functional groups or sterically bulky substituents [28]. The number of reports on
the use of hydrogen bonding as a structure control element in crystal engineering and self-
assembly of molecular structures is on the increase and scientists have continued to examine the
connection between molecular perturbation and crystal packing in the solid-state [10]. However,
understanding of how a functional group or alkyl chain modification will alter the
4

supramolecular architecture of a hydrogen-bonded structure is still evolving. Our interest in
asymmetrically substituted urea compounds is as a result of the possibility of incorporating
different functionalities to form designer compounds with interesting physical, chemical and
biological properties. Here we illustrate the formation of unpredictable hydrogen-bonded motifs
in some N,N’-disubstituted ureas containing carboxyl (COOH) and phosphoryl (P=O)
functionalities and their structural, chemical and biological properties.
Figure 1: One-dimensional α-tape urea chain.
2. Experimental
2.1. General Experimental Methods
All chemicals were obtained from Sigma Aldrich and were used without further purification.
Micro-analytical data were collected by the Campbell Microanalytical Laboratory, Department
of Chemistry, University of Otago, New Zealand. ESI-mass spectra were recorded in positive ion
mode on a Bruker MicrOTOF instrument in methanol. Infrared spectra were recorded as KBr
5

discs on a Perkin-Elmer Spectrum 100 Fourier Transform Infrared Spectrometer with a
31
1
wavenumber range of 4000-400 cm-1. P{1H} and H NMR spectra were recorded on a Bruker
Avance(III) 400 MHz NMR instrument in DMSO-d6 or CDCl3 at 300 K. Chemical shifts were
quoted to external H3PO4 (31P) and SiMe4 (1H). Melting points were recorded on a Reichert–Jung
thermovar instrument as solid samples on glass slides.
2.2. X-ray crystallography
Diffraction data were collected at 100 K on an Agilent (Supernova, single source at offset Atlas)
diffractometer equipped with an EOS CCD area detector and a 4-axis KAPPA goniometer.
Graphite monochromated Cu-Kα radiation (λ=1.54184 Å) was used. Data integration, scaling,
and empirical absorption correction was carried out using the CrysAlis-Pro program package
[29]. The structures were solved with intrinsic phasing method in ShelXT and refined by Matrix-
least-square against F2. The non-hydrogen atoms were refined anisotropically, and hydrogen
atoms were placed at idealized positions and refined using the riding model. All calculations
were implemented in OLEX2 program package [30]. Important crystallographic and refinement
parameters are presented in Table 1.
2.3. Quantum chemical methods
Quantum chemical calculations were carried out using the Gaussian 2009 program suite [31] on
the University of Waikato’s high-performance computing facility. Geometry optimizations and
harmonic frequency calculations in the gas phase were completed with the B3LYP functional
and repeated with the B3LYP-D3 by adding the keyword ‘‘empirical dispersion=gd3’’ and using
the 6-311G++(d,p) basis set. The absence of imaginary frequencies in the calculated vibrational
frequencies of the compounds showed that the optimized geometries were the true minima.
Single point energy calculations at the B3LYP-D3 level of theory using 6-311G++(d,p) basis set
were completed using the experimental crystal structures. Noncovalent interaction (NCI)
analysis was undertaken on these single point electron densities using a locally developed
program, Bonder, and visualized with graphics software; Visual Molecular Dynamics (VMD).
6

2.4. Synthesis and characterization of urea ligands
2.4.1. N,N -(2-carboxyl phenyl)phenyl urea (L1)
́
This compound was synthesized using a modified literature method [32]. A mixture of
anthranilic acid (6.85 g, 0.05 mmol) and phenyl isocyanate (5.6 mL, 4.96 g, 0.05 mmol) were
reacted in dry THF (50 mL) under reflux for 3 h. After cooling to room temperature, aqueous
NH4Cl (10 %, 200 mL) was added and the resulting precipitate was filtered off and washed with
water (70 mL). The product was recrystallized from a water-ethanol mixture 1:3, 150 mL and
o
dried overnight under vacuum. Yield (8.46 g, 73 %). Melting range: 190-193 C. Elemental
analysis: Calculated %; C, 65.40; H, 4.76; N, 11.17, Found%; C, 65.48; H, 4.71; N, 11.12. ESI-
1
MS: m/z 255.08(69 %) [M–H]+, 511.17(100 %) [2M–H]+. H NMR (400 MHz) DMSO-d6,
δ
ppm: 10.35 (s, 1H; CO2H), 9.75 (s, 1H; NH), 8.34 (d, 1H; NH;
J = 5.6 Hz), 7.9-8.2 (m; 9H; Ar).
13C NMR (100 MHz): 169.87 (1C, COOH), 152.77 (1C; C=O), 142.68 (1C; C–N; Ar), 140.17
(1C; C–N; Ar), 134.15 (1C; Ar), 131.43 (1C; Ar), 129.15 (2C; Ar), 122.58 (1C; Ar), 121.3 (1C;
Ar), 120.32 (1C; Ar), 119.26 (2C; Ar), 115.99 (1C; Ar). IR (cm-1): 3301(br)
H, 1664(s) C=O, 1599(s) ѵsNH, 1584(m) NH, 1415(s) ѵsC=C, 1277(m) ѵasNCN, 1173(s)
ѵsC–N, 1044(m) C=N.
ѵO-H, 3134(m) ѵN-
ѵ
ѵ
ѵ
2.4.2. Diethyl 4-(3-phenylureido)benzyl phosphonate (L2)
To a mixture of diethyl 4-aminobenzyl-1-phosphonate (0.535 g, 0.0022 mol, 1 equiv) in diethyl
ether (20 mL) was added phenyl isocyanate (0.30 mL, 0.0025 mol, 1 equiv) dropwise and
refluxed with gentle heat until a white precipitate appeared. The filtered product was washed
with diethyl ether (15 mL) and recrystallized from ethanol to give a colorless solid. This was
o
dried overnight in a vacuum. Yield (0.62 g 83 %). Melting range: 150-154 C. Elemental
analysis: Calculated %; C 59.66, H 6.59, N 7.73. Found %; C 59.61, H 6.31, N 7.68. ESI-MS,
m/z 385.12(100%) [M+Na]+, 747.25(14.31%) [2M+Na]+, 1109.30(5.25%), [3M+Na]+. 1H NMR
(400 MHz) CDCl3,
δ
ppm: 8.54 (s, 1H; NH), 8.12 (s, 1H; NH), 7.4-7.0 (m, 9H; Ar), 4.1 (m, 4H;
= 8.6 Hz), 1.34 (t, 6H; CH3,
= 8.2 Hz). 13C NMR (100 MHz)
CH2, ester), 3.17 (d, 2H; CH2,
J
J
CDCl3: 153.58 (s, C=O), 139.66 (s, C–N; Ar), 138.4 (s, C–N; Ar), 129.91 (d, 2C; Ar), 128.84 (s,
2C; Ar), 123.81 (s, 1C; Ar), 122.26 (s, 1C; Ar), 120.03 (d, 2C; Ar,), 118.84 (s, 2C; Ar), 62.81 (d,
7

2C; CH2), 32.38 (d, 1C; CH2), 16.45 (d, 2C; CH3). 31P{1H} NMR: 27.80 (s). IR (cm-1); 3346(br)
O-H, 3200(s) N-H, 1708(s) C=O, 1233(s) P=O, 1029(s) C=N, 976(s) -OR.
ѵ
ѵ
ѵ
ѵ
ѵ
ѵP
Table 1: Crystal data and structure refinement parameters for urea ligands L1 and L2
L1
L2
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
C14H12N2O3
256.26
100.00(10)
monoclinic
P21/n
11.3928(3)
4.8487(10)
22.1770(5)
96.467(2)
1217.27(5)
4
C18H21N2O4P
360.34
99.98(11)
monoclinic
P21/n
10.91600(10)
11.35370(10)
29.6292(2)
98.9670(10)
3627.27(5)
8
b/Å
c/Å
β/°
Volume/Å3
Z
ρcalcg/cm3
1.398
0.830
1.320
1.560
ꢀ
/mm-1
F(000)
536.0
1520.0
Crystal size/mm3
Radiation
0.1072 × 0.0873 × 0.0603 0.1173 × 0.0955 × 0.0336
CuK = 1.54184 Å) CuK = 1.54184 Å)
6.04 to 147.986
5, - -13 12, -13
36
34555
α
(
λ
α (λ
2Θ range for data collection/° 8.02 to 147.48
-14
≤
h
≤
≤
19
13, -5
≤
k
≤
≤
h
≤
≤ k ≤ 14, -36 ≤ l
Index ranges
27
≤
l
≤
Reflections collected
Independent reflections
4687
2336 [Rint = 0.0138, Rsigma 7266 [Rint = 0.0292, Rsigma
=
= 0.0196]
2336/0/173
1.073
0.0242]
7266/0/454
1.058
Data/restraints/parameters
Goodness-of-fit on F2
R1 = 0.0349, wR2 = 0.0836 R1 = 0.0583, wR2 = 0.1492
R1 = 0.0404, wR2 = 0.0882 R1 = 0.0726, wR2 = 0.1569
Final R indexes [I≥2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3 0.22/-0.21
1.52/-0.88
3. RESULTS AND DISCUSSION
3.1. Synthesis and characterization
The ureas L1 and L2 (Scheme 1 and 2) were synthesized by the reaction of the corresponding
amines and phenyl isocyanate in refluxing tetrahydrofuran and diethyl ether respectively. The
insoluble product precipitated out of the solution on cooling. The products were recrystallized
8

from a 1:3 water-ethanol mixture for L1 and ethanol for L2. The compounds L1 and L2 have been
synthesized previously [32, 33]; however, there are no reports on the chemical or structural
characterization of both compounds in the literature.
L1
Scheme 1: Reaction scheme for the synthesis of N,N ́-(2-carboxyl phenyl)phenyl urea (L1)
L2
Scheme 2: Reaction scheme for the synthesis of diethyl 4-(3-phenylureido)benzyl phosphonate
(L2)
ESI-mass spectra of the carboxyphenyl-substituted urea L1 (Figure S1 of the
supplementary information) in methanol solution and negative ion mode showed pseudo
molecular ion peaks at m/z 255.08 and 511.17 for [M-H]+ and [2M-H]+ respectively. The
spectrum for the phosphoryl-substituted urea L2 showed an ion at m/z 385.12 for the sodium
adduct of the compound. Addition of two drops of NaCl solution to the analyte solution resulted
in a very intense peak for the sodium adduct. Two other molecular ion peaks were observed in
the spectra at m/z 747.25 and 1109.30 corresponding to [2M+Na]+ and [3M+Na]+.
1
In the H NMR spectra of the compounds, L1 (Figure S2) showed a singlet at 10.35 ppm
resulting from the carboxyl proton. Singlet peaks appeared at 9.75 and 8.35 ppm corresponding
to the two NH protons of the urea. The phenyl ring protons were observed as multiplets at 7.9–
8.5 ppm. The more downfield NH proton of L1 is probably as a result of intramolecular hydrogen
bonding as evident in the crystal structure of the compound. The phosphoryl substituted urea L2
(Figure S3) showed singlets 8.54 and 8.12 ppm for the two NH protons and a multiplet at 7.4
ppm corresponding to the phenyl protons in the compound. Another multiplet observed at 4.1
ppm corresponds to the POCH2 protons of the phosphoryl ester. The spectra also showed a
9

doublet at 3.17 ppm resulting from the protons of the PCH2 group. The triplet at 1.34 ppm is
from the methyl groups of the phosphoryl ester.
The 13C NMR of the urea L1 (Figure S4) recorded in DMSO-d6 showed a carboxyl carbon peak
at 169.87 ppm and carbonyl carbon peak appearing at 152.77 ppm. Similar chemical shifts were
reported for urylene dicarboxylic acids [34]. The peaks at 142.68 and 140.17 ppm correspond to
the phenyl carbons bonded to the urea nitrogen. The peaks are further downfield than the other
phenyl carbon peaks due to the deshielding effect of the urea nitrogen bonded to it. The peaks
around 134.55–115.99 ppm are from the remaining phenyl carbons of the urea. The 13C{1H}
NMR spectrum of L2 was similar to L1 and also presented in the supplementary information
(Figure S5). The peak at 153.58 ppm corresponds to the urea carbonyl carbon while the peaks at
139.66 and 138.4 ppm are as a result of the urea nitrogen bonded carbons. The peaks appearing
around 129.91–118.84 ppm correspond to the other phenyl carbons in the compound. The
doublet at 62.8 ppm is for the two CH2 carbons of the phosphoryl ester. The doublet is
presumably due to P-C coupling. These peaks are further downfield than the peaks for the
bridging PCH2 carbon (32.38 ppm) due to the electron-withdrawing effect of the phosphoryl
oxygens. The doublet at 16.45 ppm is a result of the two methyl protons of the phosphoryl ester.
The 31P{1H} NMR spectrum of L2 (Figure S6) shows a singlet at 27.8 ppm corresponding to the
PO3Et2 phosphorus.
The infrared spectra of L1 and L2 (Figures S7 and S8) show stretching frequencies at
3301 cm-1 for the carboxyl OH in L1. The carbonyl stretching frequencies appear at 1664 cm-1
and 1708 cm-1 for L1 and L2 respectively. The N-H stretching frequencies appear around 3200 -
3100 cm-1 for both L1 and L2 and the P=O stretching frequency was observed as an intense peak
at 1232 cm-1 for L2.
3.2. Structural Analysis
3.2.1. N,N ́-(2-carboxyl phenyl)phenyl urea (L1)
The compound L1 crystallized in a centrosymmetric monoclinic crystal system and P21/n
space group. The molecular structure of the compound is shown in Figure 2, and selected bond
lengths and angles are presented in Table 2. The core of the urea structure is essentially planar
with the COOH functional group twisted out of the plane of the adjacent urea by a torsion angle
of 41.26o defined by C1-N2-C2-C3. The urea carbonyl oxygen is also slightly out of the plane of
10

the urea functional group with a torsion angle of 9.68o defined by the torsion angle involving
atoms O1-C1-N1-C9. The hydrogen-bonded structure of the compound (Figure 3) shows a
complex interplay of intra and intermolecular hydrogen bonding. There is strong intramolecular
hydrogen bonding between the carboxylic acid group and the adjacent urea N-H. Ureas are
known to form 1-D bifurcated hydrogen bonds between the two N-H donors of the urea and the
lone pairs on the carbonyl oxygen of a second molecule of the urea [35]. These urea N-H…..O
bonds are reported to be so strong that they can persist even in the presence of other strong
hydrogen bond donor groups[36]. However, the presence of carboxylic acid groups on the
adjacent phenyl of the N͵N՛ -disubstituted urea L1 probably initiated some competition between
the different functional groups in the crystal lattice. This competition must have resulted in the
rearrangement of the hydrogen bonding architecture of the urea ligand from the usual bifurcated
one-dimensional α-network of bonds to a two-dimensional hydrogen-bonded chain (Figure 4).
Figure 2: Molecular structure of the N,Nʹ-(2-carboxyl phenyl)phenyl urea L1. Ellipsoids are
drawn at 50% probability.
The 2-D hydrogen-bonded structure consists of three distinct hydrogen bonding
interactions which include: i. Intramolecular hydrogen bond interaction between the carboxylic
acid acceptor functional group and one of the urea nitrogen donors, ii. a linear α-tape inter-
11

molecular hydrogen bond interaction between the second N-H donor and the urea carbonyl
acceptor group and iii. a hydrogen-bonded dimer between the carboxylic acid functional group
and another molecule containing the carboxylic acid moiety. The hydrogen bond distances and
angles are presented in Table S1 of the supplementary information, shows that the
intramolecular bond distances between the urea nitrogen and the oxygen of the carboxylate (N2-
O2 = 2.683Å) are shorter than the intermolecular NH…..O bond length of 2.876Å which is
comparable to the average N-H…..O hydrogen bond distance of 2.85Å [27]. The proximity of the
competing carboxylic acid group to the urea N-H-donor group is probably the reason for the
strong intramolecular hydrogen bond between the urea N-H functional group and one of the
carboxyl oxygens (O2) orthogonal to the N-H-donor group. These intra-molecular hydrogen
bonds tend to have disrupted the usual bifurcated α-tape hydrogen bond chain usually observed
in N͵N՛ -substituted ureas of this kind. The carboxyl group also forms a hydrogen-bonded dimer
with a second molecule through the carboxylic acid functional group. The relatively strong inter-
molecular CH…..O hydrogen bond has a rather short bond distance (DHA 2.581Å) and an almost
linear hydrogen bond angle (ے
DHA =173o).
The second urea N-H-donor group forms a continuous linear chain of hydrogen bonds with the
carbonyl oxygen of a second molecule of the urea, resulting in an array of unexpected 2-D
hydrogen-bond motifs (Figure S9) held together by intramolecular hydrogen bond and stabilized
by intramolecular and inter-molecular C-H……O and NH……O interactions in the crystal lattice
(Figure 4). Related compounds exhibiting self-complementary hydrogen bonds between urea
and carboxyl groups were reported by Zhao et al. [34].
12

Table 2 : Selected bond lengths and angles for ureas L1 and L2
Bond Angles (o)
Bond Lengths (Å)
L1
O2 – C8
1.2366(17)
1.3141(16)
1.2295(17)
1.3580(18)
1.4215(17)
1.3834(17)
1.3994(17)
1.382(2)
C9 – N2 – C1
C2 – N1 – C1
O3 – C8 – O2
C7 – C8 – O2
C7 – C8 – C3
N2 – C1 – O1
N1 – C1 – N2
C3 – C2 – N1
125.17(12)
124.34(12)
122.61(12)
123.24(12)
114.15(11)
124.13(12)
113.21(12)
120.63(12)
O3 – C8
O1 – C1
N2 – C1
N2 – C9
N1 – C1
N1 – C2
C3 – C4
L2
P1A – O3A
1.5785(18)
O3A – P1A – C14A 103.88(11)
O4A – P1A – O3A 106.96(10
O4A – P1A – C14A 108.17(10)
P1A – O4A 1.5710(17)
P1A – O2A 1.4741(18)
P1A – C14A 1.789(2)
P1B – O3B
P1B – O2B
P1B – O4B
P1B – C14B 1.792(3)
O3A – C17A 1.462(3)
O4A – C15A 1.456(3)
O1B – C1B 1.220(3)
O1A – C1A 1.219(3)
N1B – C8B 1.404(3)
N1B – C1B 1.376(3)
N1A – C2A 1.408(3)
N1A – C1A 1.372(3)
N2B – C1B 1.375(3)
N2B – C2B 1.399(3)
N2A – C1A 1.367(3)
N2A – C8A 1.409(3)
O2A – P1A – O3A
O2A – P1A – O4A
113.92(10)
108.63(10)
1.565(3)
1.456(2)
1.601(3)
O2A – P1A – C14A 114.86(11)
O3A – P1B – O4B
O2B – P1B – C14B
O2B – P1B – O3B
O2B – P1A – O4B
O2B – P1B – C14B
O4B – P1B – C14B
102.43(15)
104.02(13)
115.60(17)
114.43(14)
116.26(15)
102.23(15)
C17A – O3A – P1A 122.64(17)
C15A – O4A – P1A 123.73(15)
C1B – N1B – C8B
C1A – N1A – C2A
C1B – N2B – C2B
C1A – N2A – C8A
C15B – O3B – P1B
C17 – O4B – P1B
128.5(2)
128.1(2)
128.8(2)
128.1(2)
124.2(2)
126.0(3)
Note: A and B refer to two independent molecules
13

Figure 3: Interplay of inter and intra-molecular hydrogen bonding in the structure of L1. The
intramolecular bonds are shown in red while the intermolecular bonds are green in color. The
relative proximity of the carboxyl oxygen to the urea NH contributes to the distortion of the
usual α-tape synthon prevalent in di-substituted ureas.
Figure 4: Intermolecular CH----OH and NH----O=C stabilizing interactions in the hydrogen-
bonded structure of L1.
14

3.2.2. Diethyl 4-(3-phenylureido)benzylphosphonate (L2)
The phosphoryl ester functionalized urea (L2) crystallized in a centrosymmetric P21/n crystal
system. The crystal structure of the compound showed the presence of two similar but
distinctively different molecules in the asymmetric unit. The molecular structures of the two
molecules are labeled (A) and (B) (Figure 5) and selected bond lengths and angles are presented
in Table 2. The crystal structure of molecule B showed a disorder on one of the ethyl groups of
the phosphoryl ester, which was modeled in two positions and a constraint introduced to stabilize
the molecule. Only one position is shown in Figure 5 for clarity. The structures of the
compounds show significant differences in the orientations of the substituted phosphoryl ester.
The two POEt groups in the molecule (A) are pointed in opposite directions with one of the
groups oriented towards the adjacent phenyl ring. In the second molecule (B), the POEt groups
are pointed in the same direction and away from the adjacent phenyl ring. Also, in the molecule
(A), P=O points in the opposite direction to the C=O, but in the same direction in the molecule
(B). These differences in geometric orientation of the structures also result in slight differences
in the bond distances and angles of the two structures. For example, the P1-O3 bond has a bond
length of 1.5785(18) Å in (A) and 1.5665(3) Å in (B), while the O4-P1-C14 angle in (A) is
108.17(10)o and that in (B) is 102.23(15)o. Even though there was a slight disorder in one of the
ethyl carbons of the phosphoryl ester group of the structure (B), the overall structure of the urea
was preserved. The two molecules are linked by the bifurcated hydrogen bonds between the
phosphoryl oxygen of one molecule and the two N-H-donor groups of the second molecule. The
two molecules then form one bifurcated hydrogen bond with each of the two molecules of the
urea, resulting in a hydrogen-bonded tetramer (Figure 6). The urea tetramer shows two different
bifurcated hydrogen bonds at each end of the molecule. The following hydrogen bond distances
and angles,[2.924 Å; 156o; N(1A)-H(1A)-O(2B), 2.837 Å; 162o; N(2A)-H(2A)-O(2B)] and
[2.870 Å; 160o; N(1B)-H(1B)-O(2A), 2.843 Å; 158o; N(2B)-H(2B)-O(2A)] were recorded for the
two different hydrogen bonding interactions observed in the tetramer. The tendency for urea
compounds to form 1-D hydrogen-bonded chains using the two NH proton donors and the
carbonyl proton acceptor in a bifurcated hydrogen bond motif according to Etter et al. [37]
comes from the linear mode of approach of the N–H donors to the lone pairs on the C=O
hydrogen bond acceptor. This process is however disrupted by the presence of a competing
phosphonate hydrogen bond acceptor, resulting in the rearrangement of the predicted hydrogen
15

bonding architecture of the urea in the crystal lattice. This rearrangement is predicted to be the
reason for the formation of a chain of bifurcated hydrogen-bonded tetramers shown in Figure 6.
Figure 5: Molecular structure of L2 showing two independent molecules in the asymmetric unit.
16

Figure 6: A side view of the hydrogen-bonded tetramer of L2 showing different hydrogen bond
distances at each end of the tetramer.
3.4. Non-covalent interactions
Due to the rich and challenging bonding patterns in crystalline solids, it can be difficult to
experimentally rationalize the different bonding distributions in crystalline compounds,
especially those exhibiting various degrees of non-covalent interactions. Consequently,
researchers have increasingly turned to various theoretical approaches to investigate the nature
and strength of non-covalent interactions. A number of these are based on topological analysis of
the electron density [38-41], including the Electron Localization Function (ELF) [42], the
Quantum Theory of Atoms in Molecules (QTAIM) [43] and most recently the Non-Covalent
Interaction index (NCI) [44]. The NCI theory is based on the reduced density gradient(s),
1
|∇ꢅ|
1
ꢀ =
2(3ꢁꢂ)ꢃ/ꢄ ꢅ(ꢆ)ꢇ/ꢄ
A combination of the reduced density
s and the density ρ allows a spatial representation of
bonding regions in real space. Regions of high reduced density and low density correspond to
non-interacting density tails, while regions of low reduced density and low density correspond to
17

noncovalent interactions. The different types of interactions can now be distinguished by plotting
the sign of the second eigenvalue sign λ2 as the ordinate against the reduced density gradient
s(ρ). Analysis of the sign of λ2 thus helps to ascertain the different types of weak interactions and
whether these are attractive or repulsive, whereas the density itself provides information about
the strength of the interactions. Attractive non-covalent interactions are associated with regions
where the electron density is domiciled with respect to the plane perpendicular to the bond path
and have values of (λ2 < 0) while repulsive non-covalent interactions appear at values of (λ2
>
0)[45, 46].
The non-covalent interactions of disubstituted ureas L1 and L2 were investigated with a locally-
developed program, Bonder, which provides numerically equivalent results to existing NCI
codes such as NCIplot, NCImilano or Multifwn. However, Bonder offers some advantages to
these existing codes, particularly for larger molecules, in that it analyzes each discrete non-
covalent interaction separately, rather than constructing a single sparse matrix, including the
many regions where there are not non-covalent interactions. Input geometries were taken from
the crystal structures of the ligands, and the wave functions files were generated at the DFT level
of theory using the B3LYP-D3 functional and the 6-311++G(d,p)** basis set.
In order to unambiguously characterize interactions from the two main competing groups in the
hydrogen-bonded structure of the carboxyl-substituted urea L1, the NCI analysis of this
compound was performed in two parts. The 2-D electron density plots and 3-D isosurface
troughs for the carboxylic acid dimer end of the structure are presented in Figure 7. The 2D-
plots and their corresponding 3D isosurface representation indicate the presence of the three
main categories of interactions. The first is the very strong intermolecular hydrogen bonding
interactions in the carboxylic acid dimer shown as a deep blue peaks at the far end of the signλ2
axis with a density value of
ρ = 0.47 and λ2 < 0 (Figure 7a). The high ρ value of this interaction
is indicative of very strong attractive hydrogen bonding interaction and corresponds to two deep
blue compact pill-shaped isosurface volumes between the two carboxylic acid groups in the 3-D
isosurface plot, Figure 7b. There are also the green isosurface volumes between the two blue
isosurfaces corresponding to weak non-bonding van der Waal interactions between the adjacent
carboxylic acid hydrogens. This weak interaction corresponds to the peaks at critical density
value ρ = 0.009 a.u. and λ2 > 0 in the 2-D plot.
18

The second set of interactions in the urea dimer are the benzene ring closure interactions
which are at the positive end of the 2-D plot with a reddish-green trough at a density value of
ρ
=
0.023 a.u. and λ2 > 0 corresponding to red cigar-shaped isosurfaces at the center of the benzene
rings. These red-colored isosurfaces are as a result of steric interactions within the benzene ring.
The third and final set of interactions that observed in the dimer are the intra-molecular
interactions, including the NH---OC intramolecular hydrogen bonding interactions and the weak
CH---OC intramolecular interactions. The NH---OC intramolecular hydrogen bonding
interaction between the urea NH functional group and one of the carboxylic oxygens appears as a
low density, low gradient deep blue peak (
ρ
= 0.38 a.u. a.u. and λ2 < 0) on 2-D plot and
= 0.5)
characteristic of strong intramolecular hydrogen bonding [46]. The 3-D isosurface (
s
indicated by a round blue pill-shaped trough and a reddish-green almond-shaped trough. The
blue isosurface area represents the directional attractive intramolecular hydrogen bonding
interaction between the carboxyl O acceptor and the N-H donor, corresponding to the low
low = 0.038 a.u. and λ2 < 0 on the 2-D electron density plot. The reddish-green almond-shaped
isosurface results from some steric crowding within the five-membered ring formed by the
intramolecular hydrogen bond interaction [47]. This steric interaction corresponds low and low
peak of the 2-D electron density plot with = - 0.020 a.u. and λ2 > 0. The weaker
intramolecular CH--- OC interactions are represented by the symmetric peaks at = 0.020 a.u.
s and
ρ
s
ρ
ρ
ρ
and -0.015 a.u. on both sides of the signλ2 axis of the 2-D electron density plot respectively.
These interactions correspond to the bi-centric stabilizing blue end of the flat isosurface and the
multi-centric non-stabilizing end of the isosurface resulting from the strain inside the five-
membered ring formed by the interaction. Similar symmetric interactions has been reported for
intra-molecular CH----OC interactions in
diethyl- ’-palmitoyl thiourea [48].
N
-acetyl-phenylalanyl-amide (NAPA) [47], and N,N-
N
19

a
b
Figure 7: (a) The plot of the reduced density gradient (s) against signλ(ρ) for the COOH dimer
in the crystal structure of urea ligand L1. (b) Gradient isosurface representation for carboxylic
acid dimer (s=0.5). The surfaces are scaled in blue, green and red coloration according to the
sign (λ2)ρ, from -0.2 to 0.2 a.u. Blue indicates strong, attractive interactions, and red indicates
strong repulsive interactions or steric clashes.
20

The primary interaction in the second part of the hydrogen-bonded urea L1 is the α–urea
tape N-H----O-C intermolecular hydrogen bonding. A separate 2-D electron density plot shows a
bluish-green peak with ρ = 0.018 a.u. from a strong stabilizing intermolecular hydrogen bond
interaction and corresponding to the single blue pill-like isosurface volume between the N-H
donor and the carbonyl acceptor functional group of another molecule of the ligand (Figure
S10). The value of ρ for this interaction is slightly less negative than the density value for the
intramolecular NH---OC interaction found in the first part of the structure (Figure 7), which
indicates a higher stabilizing effect for the intramolecular hydrogen bonding interaction relative
the intermolecular hydrogen bonding interaction. This explains the distortion of the usual α-tape
hydrogen bonding architecture of the urea to form a 2-D supramolecular structure.
The 2-D plots and the corresponding isosurface troughs for the urea ligand L2 are
presented in Figure 8. The first interaction in order of strength is the strong intermolecular
hydrogen bonding interaction between the lone pairs on the phosphoryl oxygen and the NH
protons of the urea functional group. The peaks at ρ = 0.022 a.u.; λ2 < 0 in the 2-D electron
density plot corresponding to dark blue pill-like isosurface volumes on the right-hand side of the
3-D isosurface representation. Another peak directly to the right of the first one with ρ = 0.018
a.u. and λ2 < 0 is for the second intermolecular hydrogen bonding interaction in the bifurcated
arrangement and corresponds to the second pill-like isosurface on the right-hand side of the 3D
plot and attached to the little bluish-green isosurface volume. The bluish-green isosurface trough
between the two pill-like volumes is a result of some weak van der Waals interaction between
the two adjacent hydrogens of the urea with ρ = 0.01 a.u. and the red tint on the green isosurface
must have resulted from multicentric repulsive interactions within the four-membered ring
formed by the urea nitrogens and carbonyl carbon. At the other end of the 2D electron density
plots with ρ = 0.02 a.u. ; λ2> 0 is a reddish-green peak indicative of strong repulsive or steric
interaction and corresponds to the red-isosurface volumes at the center of the benzene rings in
the dimer.
21

Figure 8: The 2-D electron density plots for the phosphoryl urea dimer (top) and the
corresponding 3-D isosurface representations (bottom) (s = 0.5 a.u.) ( -0.05 < ρ < 0.05 a.u.).
These red isosurface volumes at the center of the benzene rings are a result of strong
multicentric steric repulsion within the benzene ring. Apart from the strong, attractive hydrogen
bonding interactions between the phosphoryl oxygen acceptor and the urea N-H donors, there is
also the presence of strong, attractive non-bonding CH-----OC interactions. The two almond-
shaped bicolored (blue/red) isosurfaces depict some fairly strong attractive interactions with
critical density values of ρ = 0.017 a.u. representing some bicentric directional stabilizing
interaction between the phenyl carbons on each side of the carbonyl group and the carbonyl
oxygen. The red lower end of the isosurface is a result of a strain in the five-membered ring
22

formed by the interaction due to the multicentric nature of the density around the ring backbone.
This interaction corresponds to the peaks on the 2D Bonder plot with ρ = 0.015 a.u. and λ2> 0;
characteristic of ring closure interactions [46]. Similar interactions were reported for 2-{[2-
(phenylsulfonyl)hydrazinylidene]methyl}benzoic acid [49].
3.6. Molecular Docking
The synthesized compounds L1 and L2 were studied for their binding affinities through
molecular docking against specific proteins implicated in cancer. The two proteins used are
epidermal growth factor receptor kinase - EGFR kinase (PDB code: 2J5F) and anaplastic
lymphoma kinase - ALK (PDB code: 5J7H). EGFRs are a large family of receptor tyrosine
kinases (TK), which are one of the main tumor makers in many types of cancer [50]. They are
expressed in different types of cancer, including breast, lung, oesophageal, head, and neck [51].
They play critical roles in the complex, signaling cascade that modulates growth, differentiation,
adhesion, migration, and survival of cancer cells. Owing to the multidimensional roles they play
in the progression of cancer, EGFR, and it's family members have emerged as attractive
candidates for anti-cancer therapy [52]. Specifically, the aberrant activity of EGFR has been
associated with cellular proliferation and apoptosis, resulting in the development and growth of
tumor cells[53]. The increasing knowledge of the structure of EGFR has provided the impetus
for focused-oriented development of new anticancer chemotherapies.
ALK regulates the development and maintenance of the nervous system. Detailed studies
by Griffin et al., 1999 and Gascoyne et al., 2003 revealed that ALK fusion genes were drivers of
inflammatory myofibroblastic tumors and diffuse large B-cell lymphoma [54, 55]. Also, the
amplification or activating point mutations of the ALK gene have been reported in
neuroblastoma [56], anaplastic thyroid cancer [57], and ovarian cancer [58]. The use of ALK as
oncogenic drug target was further amplified when a novel fusion gene involving ALK and the
echinoderm microtubule-associated protein-like 4 (EML4) gene was identified in approximately
5% of non-small-cell lung cancer (NSCLC) [59], independent of other oncogenic driver
mutations like (EGFR) or Kirsten rat sarcoma virus (KRAS) mutations. In the US, new cases of
ALK+ lung cancer are estimated to exceed 8000 per year. This finding has made ALK a valid
molecular target, especially for NSCLC patients.
23

The solubility and binding modes of substituted urea compounds containing carboxyl and
phosphoryl acceptor functionalities, L1 and L2 were calculated and compared with the results for
N,N´-diphenylurea without carboxyl and phosphoryl functional groups L3, (Figure S11). The
docking protocols were validated, and the results are presented in Figure S12.
Table 3: Binding energy of the synthesized compounds in the drug targets, 2J5F and 5J7H
Drug target: 2J5F
Drug target: 5J7H
GBV/WSA dG London dG GBV/WSA dG
G (kJ/mol) G (kJ/mol) ꢀG (kJ/mol)
London dG
Comp
L1
ꢀG (kJ/mol)
ꢀ
ꢀ
-44
-46
-35
-25
-27
-19
-28
-29
-44
-51
-34
-56
-75
-22
-23
-16
-33
-31
L2
L3
Native ligand -46
Doxorubicin -65
The presence of the carboxylate and the phosphonate ester groups did not significantly affect the
aqueous solubility of the compounds (Table S1). Likewise, the partition coefficient was within
the range as prescribed by Lipinski's rule of five (logP ≤ 5) [60]. The implication of this is that
the compounds L1-L3 will not be soluble should they be considered as drugs because they will
not be bioavailable in the systemic circulation if orally administered. The compounds showed
reasonable binding energy with the targets (Figure 9). With the drug target 2J5F, phosphoryl-
substituted urea L2 showed comparable binding affinities with both the co-crystallized ligand and
the standard drug doxorubicin (Table 3). The carboxylate and phosphonate ester groups
significantly increased the binding affinities of the synthesized compounds (L1 and L2) when
compared to L3 without such groups (Table 3). There appears to be no significant difference in
the binding affinities of carboxylate urea L1 and the phosphonate urea L2 against the two tested
drug targets. The phosphoryl urea L2 showed higher binding affinity with the drug target 2J5F,
with the two scoring functions (London dG and 2J5F). Compound L2 against 2J5F showed no
significant difference in its binding affinity when compared to co-crystallized ligand but showed
slight differences with the standard drug (doxorubicin).
24

A
B
Figure 9: The binding mode of drug target 2J5F with (A) compound L1 (B) compound L2. Note;
blue dotted line = hydrogen bond; red dotted line = Pi bond
3.6.1. Chemical interaction of epidermal growth factor receptor kinase - EGFR kinase (2J5F)
and anaplastic lymphoma kinase (5J7H) with target drug doxorubicin and urea ligands L1 and
L2
A number of amino acid residues from the epidermal growth factor receptor kinase – EGFR
Kinase (25JF) interacted with the standard drug doxorubicin, and a complex involving the
following amino acids were formed; ASP 855, ARG 841, ASN 842, GLU 762, LEU 844, ALA
743, VAL 726, ASP 800, LEU 718, GLY 719, CYS 797 (Figure S13). The urea L1 interacted
favourably with 8 amino acid residues including ASP 855, GLU 762, LEU 844, ALA 743, VAL
726, LEU 718, THR 854 and LYS 745 (Figure S14), while ASP 855, PHE 723, LEU 844, ALA
743, LEU 718, THR 854 and CYS 797 and LYS 745 were involved in complex formation with
urea L2 (Figure S15). Similar binding interactions were observed for the anaplastic lymphoma
kinase (5J7H) receptor where hydrogen bonding interactions were observed between ASP 1203
and ASP 1270 amino acid proteins of 5J7H and N12 and C30 of the doxorubicin with bond
distances of 2.98 and 3.65Å respectively. There was also H-acceptor interaction between the O8
and LYS 1150 at a distance of 3.17Å. The study also showed ionic bonding interaction of N12
with ASP 1203 through its OD1 and OD2 at a distance of 3.32 and 2.98 Å respectively. 5LEU
1122 and GLY 1202 and 6-membered rings of doxorubicin interacted through pi-H bonding.
25

Essentially ASP 1203, ASP 1270, LYS 1150, LEU 1122 and GLY 1202 of anaplastic lymphoma
kinase were responsible for doxorubicin binding to the receptor. In the urea L1, the phenyl rings
interacted with ASP 1203 through pi-H bonding at a distance of 4.44 Å. N1 and O1 of the urea
bonded with MET 1199at distances of 3.10 and 2.90 Å respectively. Other pi-H bonding
interactions were observed between L1 and L2 with other amino acid residues LEU 1122VAL
1130, GLY 1202 and ASP 1203.
4. Conclusion
Asymmetrically substituted ureas containing COOH and P=O hydrogen bond acceptor
functional groups were synthesized and structurally characterized. The crystal structure of the
carboxyl substituted urea L1 indicated structural rearrangement of the predicted 1-D α-tape motif
of the urea to a 2-D hydrogen bond chain of carboxylic acid dimer and a linear α-urea chain. The
presence of the P=O in the urea L2 resulted in the distortion of the predictable hydrogen bonding
architecture of urea to a phosphoryl directing hydrogen-bonded tetramer. The carbonyl groups of
the ureas were not involved in hydrogen bonding for the two compounds. NCI analysis revealed
strong inter/intramolecular hydrogen bonding interactions in the urea compounds, indicating that
the carboxyl and phosphoryl directing structures are probably the thermodynamically preferred
structures in the crystal lattice. The protein and DNA docking results show increased binding
affinities for the ureas containing carboxylate L1 and phosphoryl L2 functional groups relative to
the urea without these functional groups L3. Comparable ionic, hydrogen bonding and π-binding
interactions were observed for the studied ureas and the standard drug doxorubicin.
Acknowledgment
The authors acknowledge Dr. Judith Burrows for the X-ray intensity data and Assoc Professor
Graham Saunders for help with X-ray structure solution, University of Waikato, New Zealand,
for funding and Tertiary Education Trust Fund Nigeria, for a scholarship to O.C.O.
Competing Interests.
We declare that none of the authors have any competing interests in the manuscript
26

Appendix A: Supplementary material
CCDC 1915786-1915787 contain the supplementary crystallographic data for compounds L1
and
L2.
These
data
can
be
obtained
free
of
charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033).
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