Synthesis, crystal structure and biological evaluation of new phosphoramide derivatives as urease inhibitors using docking, QSAR and kinetic studies

Article abstract of DOI:10.1016/j.bioorg.2019.01.064

In an attempt to achieve a new class of phosphoramide inhibitors with high potency and resistance to the hydrolysis process against urease enzyme, we synthesized a series of bisphosphoramide derivatives (01–43) and characterized them by various spectroscopic techniques. The crystal structures of compounds 22 and 26 were investigated using X-ray crystallography. The inhibitory activities of the compounds were evaluated against the jack bean urease and were compared to monophosphoramide derivatives and other known standard inhibitors. The compounds containing aromatic amines and their substituted derivatives exhibited very high inhibitory activity in the range of IC₅₀ = 3.4–1.91 × 10^?10 nM compared with monophosphoramides, thiourea, and acetohydroxamic acid. It was also found that derivatives with P[dbnd]O functional groups have higher anti-urease activity than those with P[dbnd]S functional groups. Kinetics and docking studies were carried out to explore the binding mechanism that showed these compounds follow a mixed-type mechanism and, due to their extended structures, can cover the entire binding pocket of the enzyme, reducing the formation of the enzyme-substrate complex. The quantitative structure-activity relationship (QSAR) analysis also revealed that the interaction between the enzyme and inhibitor is significantly influenced by aromatic rings and P[dbnd]O functional groups. Collectively, the data obtained from experimental and theoretical studies indicated that these compounds can be developed as appropriate candidates for urease inhibitors in this field.

Full text of DOI:10.1016/j.bioorg.2019.01.064

Accepted Manuscript
Synthesis, Crystal Structure and Biological Evaluation of New Phosphoramide
Derivatives as Urease Inhibitors Using Docking, QSAR and Kinetic Studies
Khodayar Gholivand, Mahsa Pooyan, Fahimeh Mohamadpanah, Foroogh
Pirastefar, Peter C. Junk, Jun Wang, Ali Asghar Ebrahimi Valmoozi, Ahmad
Mani-Varnosfaderani
PII:
S0045-2068(18)31251-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.064
YBIOO 2768
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
1 November 2018
20 January 2019
27 January 2019
Please cite this article as: K. Gholivand, M. Pooyan, F. Mohamadpanah, F. Pirastefar, P.C. Junk, J. Wang, A. Asghar
Ebrahimi Valmoozi, A. Mani-Varnosfaderani, Synthesis, Crystal Structure and Biological Evaluation of New
Phosphoramide Derivatives as Urease Inhibitors Using Docking, QSAR and Kinetic Studies, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.064
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Synthesis, Crystal Structure and Biological Evaluation of New
Phosphoramide Derivatives as Urease Inhibitors Using Docking, QSAR
and Kinetic Studies
Khodayar Gholivand,*a Mahsa Pooyan,a Fahimeh Mohamadpanah,a Foroogh Pirastefar,a
Peter C. Junk,b Jun Wang,b Ali Asghar Ebrahimi Valmoozi,a Ahmad Mani-
Varnosfaderania
a Department of Chemistry, Faculty of Science, Tarbiat Modares University, Tehran, Iran
b College of Science & Engineering, James Cook University, Townsville, Queensland
4811, Australia
* Corresponding author’s E-mail address: gholi_kh@modares.ac.ir
1

Abstract:
In an attempt to achieve a new class of phosphoramide inhibitors with high potency and
resistance to the hydrolysis process against urease enzyme, we synthesized a series of
bisphosphoramide derivatives (01−43) and characterized them by various spectroscopic
techniques. The crystal structures of compounds 22 and 26 were investigated using X-ray
crystallography. The inhibitory activities of the compounds were evaluated against the
jack bean urease and were compared to monophosphoramide derivatives and other
known standard inhibitors. The compounds containing aromatic amines and their
substituted derivatives exhibited very high inhibitory activity in the range of IC50 = 3.4–
1.91 × 10−10 nM compared with monophosphoramides, thiourea, and acetohydroxamic
acid. It was also found that derivatives with P=O functional groups have higher anti-
urease activity than those with P=S functional groups. Kinetics and docking studies were
carried out to explore the binding mechanism that showed these compounds follow a
mixed-type mechanism and, due to their extended structures, can cover the entire binding
pocket of the enzyme, reducing the formation of the enzyme-substrate complex. The
quantitative structure-activity relationship (QSAR) analysis also revealed that the
interaction between the enzyme and inhibitor is significantly influenced by aromatic rings
and P=O functional groups. Collectively, the data obtained from experimental and
theoretical studies indicated that these compounds can be developed as appropriate
candidates for urease inhibitors in this field.
Keywords: bisphosphoramide derivatives; urease enzyme; inhibitory activity; docking;
Kinetics; QSAR.
2

1. Introduction
Control and inhibition of urease enzyme is the most important step in agricultural
productivity and the treatment of diseases caused by enzyme disorders. The urease
enzyme is present in a variety of microorganisms, such as some eukaryotes and
prokaryotes, and as a catalyst converts urea into ammonia and carbon dioxide or
carbamate during the reaction of hydrolysis [1-3]. The high and uncontrolled activity of
urease results in excessive ammonia release and increased pH of the environment,
bringing about damaging consequences in medicine and agriculture, such as
gastrointestinal infections and destruction of plant roots [2, 4-12]. Inhibition of urease
enzyme seems to be the only way to deal with these negative consequences. Among
various compounds identified as urease inhibitors, monophosphoramide derivatives are
broadly considered as the most effective ones [1, 13-16]. Despite their high inhibitory
ability, these compounds have attracted less attention due to their instability in aqueous
media [17]. In order to overcome this issue, the compounds containing a P−C linkage
(phosphonates and phosphinates) have been reported to be good substitutes for the
monophosphoramide compounds, which have exhibited relatively less inhibitory activity
than the monophosphoramides [9, 18, 19]. Regarding the mentioned problem, in this
work, we introduced a new framework for phosphoramides with high hydrolytic stability
as urease inhibitors. Several studies have demonstrated that the functional groups of P=O
and P−N in monophosphoramide derivatives have the largest effect on enzyme inhibition
[5, 9, 15, 19-21]. Inspired by these reports, we considered the various categories of
bisphosphoramide derivatives containing functional groups of P=O and P−N as urease
inhibitors. Accordingly, 43 bisphosphoramides were offered with the general formula of
3

(R1)(R2)P(Y)X(Y)P(R1)(R2) (Y = O and S; R1 and R2 = C6H5, C6H5O, C6H5NH, C2H5O;
X = various aliphatic and aromatic diamines). Out of the compounds, 26 of them were
reported in our previous publications [22-27] and others were newly synthesized in the
present study, see Figure 1.
In continuation of the work, the inhibitory activity of these compounds was evaluated
against the jack bean urease and compared to the previously reported
monophosphoramides [1, 2, 5, 6, 15, 20, 21, 28]. Kinetic and docking studies were
conducted to explore the mode of interaction and also to gain an insight into the reason
behind the relatively high difference in inhibitory activity of these compounds with
monophosphoramides. Also, structural parameters affecting the inhibitory activity of the
compounds were obtained through QSAR studies using Genetic Algorithm-Artificial
Neural Networks (GA-ANN).
In general, with the aim of developing and improving the hydrolytic stability of urease
inhibitors, a new framework for phosphoramide inhibitors of urease enzyme with high
potency and resistance to the hydrolysis process was introduced. Furthermore, by using
computational and experimental methods the mechanism of interaction between these
compounds with the urease enzyme, as well as significant factors affecting this
interaction were explored.
4

Figure 1 Schematic of newly synthesized compounds in the present work.
2. Results and discussion
2.1. Chemistry
bisphosphoramide derivatives (01−43) were synthesized from the reaction of 2 mmol of
R1R2P(O)Cl with 1 mmol of the corresponding diamine in the presence of triethylamine
in acetonitrile or dichloromethane solution at 0 °C according to our previously reported
5

procedures [22-27]. The pathway for the synthesis of target compounds is described in
Scheme 1. The structures of synthesized derivatives were characterized by FT-IR,
13
31
1H-NMR, C-NMR, P-NMR spectroscopy techniques, and elemental analysis. Also,
the structures of compounds 22 and 26 were furthermore identified by X-ray single-
crystal structure analysis. Bisphosphoramides 09, 11, 12, 19, 20, 22, 25-28, 34-36 and 40-
43 have been synthesized for the first time and their structure and purity were also
confirmed by spectral data. A summary of these data is presented in Table 1. In addition,
six monophosphoramide derivatives were synthesized [6, 29] to investigate their
inhibitory properties and to compare them with bisphosphoramide derivatives under
identical conditions (Figure 3B).
Scheme 1. Synthetic route of bisphosphoramide derivatives, each color represents a category of bisphosphoramide
derivatives with their corresponding diamines.
In the IR spectra of the newly synthesized compounds, two bonds appeared in the range
of 1185 to 1295 cm−1 and 811 to 815 cm−1, which are assigned to the P=O and P=S
groups, respectively (Table 1). The absorption bands of the P-N stretching vibrations for
all compounds were found in the range of 754−982 cm−1. The 31P-NMR chemical shift of
these compounds was observed to be in two different ranges of −6.4 to 16.23 ppm and
also 64.35 to 69.5 ppm, attributed to derivatives containing, respectively, P=O and P=S
6

groups. Comparison of the 31P-NMR spectra of the newly synthesized compounds shows
that the phosphorus chemical shifts of the compounds 19 and 20, resulted from the strong
inductive effect of the phenoxy groups, shift to lower field and appear in the range of
1
−6.41 to −6.48 ppm. The H-NMR spectra of the compounds 34-36 and 40-43 display
triplet and multiplet signals at around 1.08 and 3.68 ppm, which are related to the methyl
1
and the methylene protons in the CH3-CH2 and OCH2 groups, respectively. In the H-
NMR spectra of compounds 11, 12, 19 and 20, the methylene protons have appeared as a
1
multiplet signal in the range of 2.99−3.57 ppm. The H-NMR and 31P-NMR results
presented here are in good agreement and consistent with the previously reported spectral
results for the corresponding compounds [22-24, 26]. See Supplementary Information for
full details the spectral data.
7

Table 1. A summary of the spectral data of newly synthesized compounds
com FT-IR data (KBr pellet, cm−1):
selected bands:
1H NMR (500.13 MHz, d6–DMSO,
31P NMR (202.45 MHz, d6-
DMSO, 25°C, H3PO4 external);
δ= ppm
25°C, TMS); δ=
1195s (P=O), 960s (P–N).
6.76 (s, 4H, C6H4), 6.61 (d, 2H, NH),
7.28–8.15 (m 10H C6H5).
16.1 (d)
09
11
1185s, (P=O), 1119s, 931m,
725m (P–N).
3.32-3.57 (m, 2H, CH2), 8.09 (d, 2 H,
2JPH= 12.1 Hz, NH).
16.20 (d)
16.23 (d)
−6.48 (d)
1193s (P=O), 929m (P–N).
3.16-3.43 (m, 4H, CH2), 8.10 (d, 2H,
12
19
2JPH 11.45 Hz, NH).
1196s (P=O), 1000m, 946m
(P–O), 764m, (P–N).
2.99-3.01 (m, 2H, CH2), 8.77 (d, 2H,
2JPH=10.20 Hz, NH).
1232s, 1185s (P=O), 991s,
943s (P–O), 762m (P–N).
3.12-3.41 (m, 4H, CH2), 8.69 (d, 2H,
2JPH=10.75 Hz, NH).
−6.41 (d)
20
22
1208s (P=O), 941 (P–O), 758
(P–N).
1.44–1.49 (m, 2H, CH2–CH2), 2.79–
2.85 (m, 4H, CH2–CH2), 5.19 (m, 2 H,
NH).
5.46 (m)
1209s (P=O), 937 (P–O), 756
(P–N).
1H NMR (300.13 MHz): 0.61-0.81 (m,
6H, CH3), 3.34-3.70 (m, 4H, C-CH2),
5.26 (m, 2H, NH).
6.04
25
1220 (P=O), 922 (P–O), 756
(P–N).
2.49–2.57 (m, 6H, N–CH3), 3.07–3.15
(m, 4H, CH2–N).
6.79 (m)
3.31 (d)
26
27
1205s (P=O), 1072w, 991m
(P–O), 933s, 754m (P–N).
1H NMR (300.13 MHz): 5.31 (d, 2H,
2JPH = 9.69 Hz, NH).
1209s (P=O), 1064s (P–O),
922m, 755m (P–N).
6.37 (m, 2H, NH).
3.6 (d)
28
34
35
1028s (P–O), 956s (P–N),
811s (P=S).
1.08 (t, 12H, 4CH3), 3.68–4.45 (m, 8H,
4CH2).
69.5 (d)
1028 (P–O), 965m (P–N),
815m (P=S).
1H NMR (300.13 MHz): 1.09-1.44 (t,
12H, CH3), 3.68-4.42 (m, 8H, OCH2),
8.64 (d, 1H, 2JPH= 8.30 Hz, NH).
31P NMR (121.49 MHz): 64.35
(d)
1024s (P–O), 959s (P–N),
814m (P=S).
1.17-1.19 (t, 12H, CH3), 3.71 (s, 2H,
CH2), 3.93-4.03 (m, 8H, OCH2), 8.25
(d, 1H, 2JPH= 8.09 Hz, NH).
65.63 (d)
36
1212s (P=O), 1038s (P–O),
982s (P–N).
1.19 (t, 12H, 4CH3), 3.95–4.50 (m, 8H,
4CH2).
2.9 (d)
40
41
1224s (P=O), 1027s (P–O),
976m (P–N).
1H NMR (300.13 MHz): 1.19-1.24 (t,
12H, CH3), 3.92-4.06 (m, 8H, OCH2),
8.03 (d, 1H, 2JPH= 9.30 Hz, NH).
31P NMR (121.49 MHz): 2.52 (d)
1224s (P=O), 1027s (P–O),
976m (P–N).
1H NMR (300.13 MHz): 1.16-1.21 (t,
12H, CH3), 3.65 (s, 2H, CH2), 3.86-
3.92 (m, 8H, OCH2), 7.85 (d, 1 H,
2JPH= 9.26 Hz, NH).
31P NMR (121.49 MHz): 2.72 (d)
31P NMR (121.49 MHz 2.81 (d)
42
43
1235s (P=O), 1040s
(P–O), 972m (P–N).
1H NMR (300.13 MHz): 1.16-1.21 (t,
12H, CH3), 2.48 (m, 4H, CH2), 4.00-
3.92 (m, 8H, OCH2), 7.82 (d, 1 H,
2JPH= 9.00 Hz, NH).
8

2.2. Crystal structure analysis of compounds 22 and 26
For X-ray analysis, suitable single crystals of compounds 22 and 26 were obtained by a
slow evaporation method using methanol solvent. X-ray crystallographic data and
ORTEP diagrams of both compounds 22 and 26 are shown in Table 2 and Figure 2A,
respectively. Selected bond distances and angles are listed in Table S1 (see the
Supporting Information). The X-ray diffraction data analysis reveals that 22 crystallizes
in the monoclinic space group P21/c and its asymmetric unit contains one molecule. In
this molecule, two phosphoryl groups are found in the syn direction toward each other.
Due to the different orientations of the phenoxy and phenylamino rings attached to the
phosphorus atoms and the different torsion angles of phosphorus atoms (N3-P2-N4-C16=
62.0(3) ), (N2-P1-N1-C1= –55.4(4) ), the molecule doesn’t have an inversion center
(Table S1). In the crystal structure, each molecule is connected to six neighboring
molecules via N3-H3⋯O1, N2-H2⋯O1 and N4-H4⋯O3 (dH3⋯O1= 2.005, dH2⋯O1 =
2.027 and dH4⋯O3 = 2.030 Å) hydrogen bonds (Table S2), leading to the formation of
two-dimensional layers consisting of (8), (18) and (10) rings along the ab-plane,
as shown in Figure 2B. Single-crystal X-ray structural analysis of 26 indicates that it
crystallizes in a monoclinic system, space group P21/c and contains half of the molecule
in the asymmetric unit and the other half is created by a center of inversion.
9

Table 1. Crystal data and structural refinement parameters for compounds 22 and 26
Compound
Empirical formula
Formula weight
22
C27H30N4O4P2
536.49
26
C28H32N4O4P2
550.52
T(K)
100(2)
100(2)
Crystal system, space group
monoclinic, P21/c
10.735(2)
monoclinic, P21/c
12.1461(4)
13.4965(4)
8.9082(3)
(Å)
(Å)
(Å)
9.851(2)
24.672(5)
90
90
92.99(3)
102.250(10)
90
90
V
Z
2605.5(9)
1427.07(8)
2
1.281
0.192
580
4
Dcalc (Mg m-3)
Absorption coefficient (mm-1)
1.368
0.208
1128
Crystal size (mm)
rang for data collection
Limiting indices
0.9 × 0.3 × 0.01
1.9-24.99
−12≤ h ≤ 12
−11 ≤ k ≤ 11
−27 ≤ l ≤ 28
19407
0.25 × 0.20 × 0.20
1.72-30.57
−17≤ h ≤ 17
−19 ≤ k ≤ 19
−12 ≤ l ≤ 12
31888
Total reflections
Unique reflections (Rint)
Completeness to
Data/restraints/parameters
Refinement method
4353 [R(int) = 0.0614]
94.8% ( = 25.00)
4353/0/335
Full-matrix least squares on
F2
4384[R(int) = 0.0186]
99.9% ( = 30.57)
4384/0/173
Full-matrix least squares
Goodness-of-fit (GOF) on F2
Final R indices [I ˃ 2σ(I)]
1.163
1.002
R1=0.0578, wR2=0.1546
R1=0.0644, wR2=0.1579
0.458 and −0.503
R1=00340, wR2=0.1030
R1=0.0369, wR2=0.1071
0.413 and −0.311
R indices (all data)
Largest difference in peak and hole (e Å-3)
10

Figure 2. (A) ORTEP diagrams for the molecular structure of 22 and 26 (the thermal ellipsoids are drawn at the 50%
probability level) with the atom-numbering scheme and schematic representation of intramolecular hydrogen bonds.
Symmetry code: i: -x, -y, 1-z. (B) 2D representation of hydrogen bond interactions in ab-plane, which shows each
molecule is connected to six neighboring molecules through hydrogen bond interactions. (C) Representation of the
hydrogen bonding interactions in 26
Compared to 22, in this molecule, the phosphoryl groups are in the opposite direction
from each other, and due to the replacement of hydrogen substitutes attached to nitrogen
atoms with methyl groups, each molecule is connected to only four neighboring
molecules through four hydrogen bonds (N1-H1N⋯O1 (dH1N⋯O1=1.958 (8))) (Table
S2). These connections lead to the creation of two-dimensional layers consisting of
(26) rings along the bc-plane, (Figure 2C). Therefore, it can be expected that when the
functional groups attached to nitrogen atoms change from methyl to hydrogen, different
positions can be provided for binding to active sites of urease enzyme. Further
information about the crystal packing is given in the “Support Information”.
11

Figure 3. (A) Five categories of synthesized bisphosphoramide compounds. (B) Inhibitory amounts of
monophosphoramide compounds. PPDa (PPD was used as reference).
2.3. Urease inhibitory activity
Five categories of bisphosphoramide compounds, including bis(P,P-diphenyl
phosphinicamide), bis(diethyl phosphoramidate), bis(diphenyl phosphoramidate),
bis((phenylamino)(phenoxy)phosphinicamide), bis(diethyl phosphoramidothioate) (see
Figure 3A), with different aliphatic and aromatic diamines, were synthesized and their
inhibitory activity was determined in vitro. The results of the inhibition assays against
jack bean urease are presented in Table 3. In this analysis, the inhibitory activity of some
monophosphoramides was also evaluated as shown in
Figure 3B.
Phenylphosphorodiamidate (PPD) was used as the reference compound for the assay, and
its value is also given in Figure 3B. The results of inhibition analysis indicated that
derivatives containing aromatic diamines with long spacers between the two amino
groups and aromatic substituted derivatives such as phenyl and phenoxy groups have a
higher inhibitory effect than others. These derivatives with a range of
IC50 = 3.4–1.91 × 10–10 nM have greater inhibitory potency than the positive control
12

(with IC50 of 21 nM) and other known standard inhibitors such as thiourea and
acetohydroxamic acid [30, 31]. Compared to the monophosphoramides activities reported
in this work (IC50= 21–495 nM) and previously reported monophosphoramides [1, 2, 5, 6,
15, 20, 21, 28], these group of compounds show potent inhibitory activities against jack
bean urease. Among the derivatives of bisphosphoramide containing aromatic diamines,
Compound 12 with IC50 of 1.91 0.03 × 10−10 nM is considered as one of the most
potent inhibitors. The change in the substitutions attached to the phosphorus atoms from
phenyl to ethoxy caused a significant reduction in inhibition activity. On the other hand,
the displacement of the P=O moiety with the P=S moiety led to a sharp decrease in
inhibition activity. In general, the category of compounds of bis diethyl
phosphoramidothioate containing aliphatic diamines (with IC50= 11200−23000 µM)
showed the least inhibitory activity and the category of bis P,P-diphenyl phosphinicamide
compounds with aromatic diamines (with IC50= 1.91 × 10−13–0.0028 µM) showed the
highest inhibitory activity in this assay. According to these interpretations, aromatic
diamines and the P=O moiety have a significant effect on the inhibitory activity of these
derivatives. These results were supported by a molecular docking study, so that the
investigation of the binding energy values clearly indicated that the lowest and highest
binding energy belongs to compounds 12 (–9.05 kcal/mol) and 30 (–3.45 kcal/mol),
respectively, which indicates the high affinity of 12 for interaction with urease enzyme.
Data related to the binding energy of the compounds are listed in Table 3.
13

Table 2. Inhibitory activity (IC50), binding energy and experimental and predicted pIC50 values of the synthesized
bisphosphoramide derivatives against jack bean urease.
pIC50
∆Gbinding
(kcal/mol)
Comp
X
R2
R1
Y
IC50(µM) ± SDa
ref
Exp
1.56
Pred
1.31
1.18
1.18
0.15
ethane-1,2-diamino
ethane-1,2-diamino
butane-1,4-diamino
piperazine-1,4-diyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
O
O
O
O
2.7 ± 0.21 × 10-2
2.9 ± 1.0 × 10-2
3.23 ± 1.8 × 10-2
1.92 ± 0.05
[23]
[23]
[23]
[23]
01
02
03
04
7.36
7.45
7.26
8.23
1.53
1.49
0.28
2,2-dimethylpropane-1,3-
diamino
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
O
O
O
1.55
1.53
1.30
1.35
8.28
8.09
8.1
2.8 ± 1.6 × 10-2
2.9 ± 1.2 × 10-2
2.32 ± 1.5
[23]
[23]
[23]
05
06
07
propane-1,2-diamino
N,N'-dimethylethane-1,2-
diamino
0.36
0.09
cyclohexane-1,2-diamino
benzene-1,4-diamino
4,4'-Biphenyldiamino
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
O
O
O
2.45
2.55
5.01
2.40
2.23
5.25
3.5 ± 1.87 × 10-3
2.8 ± 0.35 × 10-3
9.7 ± 0.7 × 10-6
[23]
-
08
09
10
9.02
8.42
8.53
[26]
(4,4' methylene
bis(phenyldiamino))
phenyl
phenyl
phenyl
phenyl
O
O
12.00
12.71
11.93
12.63
9.78 ± 0.05 × 10-13
1.91 ± 0.03 × 10-13
-
-
11
12
9.24
9.27
(4,4' ethylene
bis(phenyldiamino))
ethane-1,2 diamino
propane-1,3 diamino
butane-1,4-diamino
piperazine-1,4-diyl
benzene-1,4-diamino
4,4'-biphenyldiamino
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
Phenoxy
phenoxy
O
O
O
O
O
O
1.51
1.58
1.42
1.59
2.11
2.46
1.37
1.40
1.37
1.41
1.52
1.93
7.02
7.58
6.3
3.08 ± 0.17 × 10-2
2.60 ± 0.39 × 10-2
3.72 ± 0.55 × 10-2
2.56 ± 0.43 × 10-2
7.64 ± 0.071 × 10-3
3.41 ± 0.03 × 10-3
[23]
[23]
[25]
[25]
[24]
[26]
13
14
15
16
17
18
7.71
8.26
8.57
(4,4' methylene
bis(phenyldiamino))
phenoxy
phenoxy
phenoxy
phenoxy
O
O
2.53
2.75
2.56
2.40
8.63
7.64
2.90 ± 0.55 × 10-3
1.75 ± 0.07 × 10-3
-
-
19
20
(4,4' ethylene
bis(phenyldiamino))
a Values are the mean ± SD.
14

Table 3 (continued)
X
pIC50
∆Gbinding
(kcal/mol)
Comp
R2
R1
Y
IC50(µM) ± SDa
ref
Exp
1.44
1.52
1.38
1.62
Pred
1.56
1.65
1.57
1.47
ethane-1,2-diamino
propane-1,3-diamino
butane-1,4-diamino
piperazine-1,4-diyl
phenoxy
phenoxy
phenoxy
phenoxy
phenyl amino
phenyl amino
phenyl amino
phenyl amino
O
O
O
O
6.68
7.71
6.71
7.98
3.58 ± 0.25 × 10-2 [27]
21
22
23
24
3.02 ± 0.05 × 10-2
4.16 ± 1.3 × 10-2
2.37± 0.10 × 10-2
-
[27]
[25]
2,2-dimethylpropane-
1,3-diamino
phenoxy
phenoxy
phenyl amino
phenyl amino
O
O
1.45
1.65
1.43
1.26
3.48 ± 0.3 × 10-2
2.20 ± 0.29 × 10-2
-
-
25
26
6.93
7.05
N,N'-
dimethylethylenediamin
o
benzene-1,4-diamino
4,4'-Biphenyldiamino
ethane-1,2 diamino
propane-1,3 diamin0
butane-1,4-diamin0
(piperazine-1,4-diyl)
phenoxy
phenoxy
ethoxy
ethoxy
ethoxy
ethoxy
phenyl amino
phenyl amino
ethoxy
O
O
S
S
S
S
1.59
1.83
1.92
2.81
2.56 ± 1.6 × 10-2
1.46 ± 0.11 × 10-2
-
-
27
28
29
30
31
32
7.85
8.36
3.74
3.45
3.86
4.55
4.04
4.08
4.30
4.36
4.04
3.93
4.05
4.11
1.12 ± 0.06 × 10+4 [22]
1.22 ± 0.02 × 10+4 [22]
2.00 ± 0.03 × 10+4 [22]
2.30 ± 0.01 × 10+4 [22]
ethoxy
ethoxy
ethoxy
2,2-dimethylpropane-
1,3-diamino
ethoxy
ethoxy
S
1.80 ± 0.03 × 10+4 [22]
33
4.25
4.08
4.06
benzene-1,4-diamino
4,4'-Biphenyldiamino
ethoxy
ethoxy
ethoxy
ethoxy
S
S
9.00 ± 0.09 × 10+3
8.00 ± 0.29 × 10+3
-
-
34
35
3.95
3.90
4.09
4.11
4.82
4.77
(4,4' methylene
bis(phenyldiamino))
ethoxy
ethoxy
S
6.00 ± 0.03 × 10+3
-
36
3.77
4.09
5.69
ethane-1,2 diamino
propane-1,3 diamin0
(piperazine-1,4-diyl)
benzene-1,4-diamino
4,4'-biphenyldiamino
ethoxy
ethoxy
ethoxy
ethoxy
ethoxy
ethoxy
ethoxy
ethoxy
ethoxy
ethoxy
O
O
O
O
O
2.28 ± 0.02
1.17 ± 0.09
[22]
[22]
37
38
39
40
41
0.35
0.06
0.04
0.04
0.28
0.26
0.86
3.46
3.67
4.54
5.11
5.08
9.10 ± 0.01 × 10-1 [22]
0.55
2.80 ± 0.07 × 10-1
2.40 ± 0.13 × 10-1
-
-
0.61
0.96
(4,4' methylene
bis(phenyldiamino))
ethoxy
ethoxy
ethoxy
ethoxy
O
O
0.72
1.44
0.96
1.56
1.90 ± 0.27 × 10-1
1.70 ± 0.22 × 10-1
-
-
42
43
5.99
6.68
(4,4' ethylene
bis(phenyldiamino))
a Values are the mean ± SD.
15

2.4. Kinetics of urease inhibition by compound 12
G.W. McCarty and co-workers determined the inhibitory mechanism of the
monophosphoramide compounds [21]. These derivatives have a slow-binding
competitive inhibitory mechanism that competes with urea due to their small structure
and similarity to urea substrates for binding to the same location. The inhibitory
mechanism of bisphosphoramide derivatives against the urease enzyme has not been
investigated so far. We expected these compounds to have a different mechanism due to
their extended structure and high inhibitory activity compared to monophosphoramide
derivatives. To conduct kinetic studies, 12 was selected as the most active compound for
determining the mechanism of urease inhibition. The data obtained from Lineweaver–
Burk plots revealed a mixed–type mechanism for 12, in which, and , in the
presence of composition 12 increased and decreased respectively (see Figure 4A). The
values of Ki and KI were calculated by plotting separate graphs of slopes and y-intercepts
of the Lineweaver–Burk plot versus the concentration of inhibitor respectively [32],
(Figures 4(B) and (C)). Obtained values of Ki and KI were 5.7 × 10-5 and 0.014 µM
respectively, revealing the strong tendency of the compound 12 to the free jack bean
urease. It has been argued that this compound, due to its extended structure, can
encompass all binding sites of the enzyme, thus reducing the possible formation of the
enzyme-substrate complex.
16

Figure 4. (A) Lineweaver-Burk plot of the inhibition of the jack bean urease activity in the absence and in presence of
compound 12. (B) and (C) secondary replots of the Lineweaver−Burk plot, slope and 1/Vmax app versus various
concentrations of compound 12, respectively.
2.5. Structural analysis of docking
To explain and understand the mechanism of enzyme inhibition and binding mode of
bisphosphoramide derivatives inside the binding pocket of enzyme, molecular docking of
these compounds on the crystal structure of jack bean urease was investigated. According
to Figure 5A, the results obtained from molecular docking showed that π-π stacking,
π-sulphur interaction, hydrogen bonds and hydrophobic interactions are the main
interactions between bisphosphoramide derivatives and urease enzyme and that these
compounds completely covered the binding pocket due to their extended structures.
Docking results including the free energy of binding
and inhibition constant
(Ki) are presented in Table 3 and Table S5, respectively. One of the strategies to inhibit
enzymes is to restrict flap mobility. The Cys592 residue (also known as CME592) is one
of the most important residues of jack bean urease and is located on the mobile flap at the
active site entrance. The interaction of the compounds with Cys592 significantly reduces
17

the activity of the enzyme. In the analysis of compounds binding model, it was observed
that π-sulphur interactions of phenyl substituents of bisphosphoramides with flap residues
Cys592 and hydrogen bonding or –alkyl interactions of compounds with flap residue
Arg439 are constant in compounds containing aromatic rings. The compound 12 with
9.05 kcal/mol binding free energy and inhibition constant 231.92 nM has the best
interaction with the enzyme compared to other compounds. As shown in Figure 5A, the
binding mode of 12 is such that it covers the entire catalytic site. There are four
interactions involving hydrogen bonding interactions between 12 and the active site
residues. The first interaction is between the P=O oxygen atom of compound 12 and the
amino group of His593 (d=1.78 Å), and the second and third interactions are observed
separately between each of the two amine groups of Arg609 and the P=O oxygen atom
(the length of each hydrogen bond is 2.16 and 2.28 Å, respectively), the last interaction
with length of 2.56 Å is found between the carbonyl oxygen of Asp 494 and the hydrogen
of the amino group of 12, (Figure 5B). Moreover, two π–sulphur and π–alkyl interactions
were found between two flap residues of the active site entrance (Cys592 and Arg439)
and the phenyl rings of the amine chain, which resulted in a significant decrease in the
enzyme activity in comparison with compounds containing aliphatic amines, as shown by
Figure 5A.
18

Figure 5. Plausible binding mode of the most active compound (12) within the binding pocket of jack bean urease: (A)
left: The enzyme and inhibitor are shown as surface and cpk in green color, respectively, which indicates the entire
coverage of the binding pocket by the inhibitor; (A) middle: The enzyme and 12 are demonstrated as ribbon and stick
forms, respectively; (A) right: The dotted lines illustrate various interactions between 12 and the active site residues,
which including hydrogen bond (green), π–sulphur (yellow) and π–alkyl (pink) interactions. For clarity only interacting
residues are labeled; the two nickel ions are represented as blue spheres. (B) 2D interaction diagram of 12 with the
target enzyme. Hydrogen bond interactions are indicated with green dashed lines.
To further investigate the effect of π-interactions on the inhibitory potency of compounds
due to the presence of phenyl rings in the structure of synthesized compounds, we
performed a comparison (the k-mean clustering) between the aromatic index and the
binding energy. The results showed that there is a reasonable relationship between them.
As shown in Figure 6, the compounds were classified into four categories. The
compounds containing aromatic diamines are in the same category having less binding
19

energy than other groups and thus have higher inhibitory power. According to the
diagrams of the aromatic index relative to the free energy of binding, the structures
containing the fewer number of phenyl rings have more binding energy. Information
extracted from docking simulation reveals that compounds containing P=S substitutes
have lower inhibitory activity than the P=O compounds, which is consistent with
experimental results, (Figure 6). In addition, further analysis of the results shows that the
monophosphoramides have more binding energy than those of some bisphosphoramide
derivatives, which can be attributed to the difference in their mode of interaction with the
enzyme (Table S5). As mentioned above, bisphosphoramides are not able to interact with
nickel ions due to their extended structure but cover the entire binding pocket. While
monophosphoramides, thanks to their small structures, can easily enter the active site
pocket of the urease enzyme and connect to the nickel ions inside of its active site [1, 5,
6, 15]. Following these studies, the QSAR study was conducted to achieve a logical
relationship between the activity and structure of the compounds.
Figure 6. Clustering of compounds (assayed bis and mono phosphoramides) based on binding energy and aromatic
index.
20

2.6. QSAR analysis
QSAR calculations were performed to determine the most important descriptors affecting
the inhibitory power using Genetic Algorithm and Artificial Neural Network (GA–ANN)
method. Of the 101 structural and electronic descriptors, six descriptors were selected as
the most important descriptors using the GA method, which are: rotatable bond fraction
(RBF), 31P chemical shift (δ), total energy of the molecule (Etotal), total charge (Qtotal), the
number of aromatic and aliphatic rings (nCIC), and the number of aromatic bonds (nAB).
These six selected descriptors and –logIC50 were used as input and output of the ANN
model, respectively. The statistical parameters obtained from the model such as
correlation coefficient (R2) and root mean square error (RMSE) are 0.988 and 0.331,
respectively, which indicates a strong correlation between independent and dependent
variables and appropriate accuracy of the model shown in Table 4. The predicted
–logIC50 values are presented in Table 3, and the relationship between experimental and
predicted values of biological activity is illustrated in Figure 7.
Figure 7. Plot of experimental pIC50 versus predicted pIC50 of assayed bis and mono phosphoramides by GA–ANN
model.
21

To further study the validity of the model used, Leave-One-Out (LOO) and Leave-
Multiple-Out (LMO) cross-validation methods were used. In the first cross-validation
model, a molecule is considered as a prediction set and the model is developed using
other molecules. However, in the second cross-validation model, the numbers of
molecules (M) are set aside as a prediction set. In the LMO method, we set the M equal
to six, and the L6O model was repeated 200 times. The data from LOO and L6O cross-
validations are shown in Table 4. The high values of Q2LOO and R2L6O are indicative of the
stability and the predictive ability of the generated model.
Table 3. Statistical parameters obtained from the QSAR model.
Validation
Training
Q2
RMSELOO R2
RMSEL6O
Rt2
RMSEt
GA-ANN
LOO
L6O
0.827
1.355
0.804
1.805
0.988
0.331
R2 is a correlation coefficient of the training set; RMSEt is a root mean square error of the training set;
t
Q2LOO is a correlation coefficient of leave-one-out cross validation; RMSELOO is a root mean square error LOO–CV
According to the diagram of the selected descriptors importance (shown in Figure 8),
molecule total energy descriptor (Etotal) has the most significance among chosen
descriptors. The study of the total energy of the synthesized compounds indicated that the
bisphosphoramide compounds have lower total energy and are more stable than the
monophosphoramide compounds synthesized in this work. Another important factor
affecting inhibitory activity is the nCIC descriptor which represents the number of rings
in the structure of the compounds and is associated with the number of aromatic and
aliphatic rings, whose presence in the construction of molecules provides information on
the hydrophobicity and rigidity of the compounds.
22

Figure 8. Relative importance of the selected descriptors in GA-ANN model.
.
Investigating the effect of the number of rings on inhibitory activity showed that
increasing the number of rings in the structure has a positive impact on the value of
pIC50, which can be due to an increase in the number of hydrophobic and π interactions
between the enzyme and the inhibitor as can be seen in Figure 9. The RBF descriptor
represents the flexibility and rigidity of compounds and is influenced by the type of bond
and the number of carbon atoms and the nature of substituents (such as aromatic and
aliphatic substituents) in the compounds. The phosphorus chemical shift is the third most
important descriptor that is altered by changing the functional groups attached to the
phosphorus atom. The study of the relationship between pIC50 and chemical shift of
phosphorus showed that the oxygen and sulfur atoms connected to the phosphorus atoms
have the largest effect on the chemical shift of phosphorus atoms. Thus, in terms of the
chemical shift of phosphorus, the compounds were classified into two groups: P=O with
chemical shift in the range of –7.3 to 31.56 ppm and P=S with chemical shift in the range
of 63.73 to 76.59 ppm. It has also been observed in the inhibitory behavior of these
compounds that the ones with P=S moiety had less inhibitory activity than those with a
P=O moiety, as shown in Figure 9. The total charge descriptor (Qtotal) is influenced by
23

heteroatoms and hydrogen bond donor and acceptor groups in the structure of compounds
that significantly affect the inhibitory potency.
Figure 9. Relationship and changes of pIC50 values of assayed all compounds relative to Etotal and nCIC.
2.7. Analysis of molecular electrostatic potential (MESP)
In order to rationalize the results of theoretical and empirical studies on the inhibition of
most of the derivatives containing the P=O functional group in comparison to the
compounds containing the P=S functional group and determining the reactive sites in the
structure of the molecules, their molecular electrostatic potential (MESP) surfaces were
calculated using the B3LYP/6–311+G** method. The color spaces in the MESP maps
represent the electrostatic potential. The red area is an electron-rich region and a
nucleophilic center in the structure, the blue part is an electron deficient region known as
the electrophilic center, and the yellow areas are less electron rich regions. As shown in
Figure 10, from the comparison of electrostatic potential maps of compounds 11 and 36,
it can be concluded that the oxygen atoms attached to the phosphorus atoms, were located
24

in the red areas and have the most negative potential than sulfur atoms, which represents
their strong tendency to participate in nucleophilic reactions. Blue regions that describe
electropositive regions or electron deficiency regions are located on the hydrogen atoms
attached to nitrogen atoms and contribute as electron receptors to the interactions
between the inhibitor and the enzyme. The yellow color of the phenyl rings is due to the
delocalized electrons of the phenyl rings, which have an influence on the formation of π-
interactions and increasing the inhibitory activity of the compounds.
Figure 10. Comparison of the electrostatic potential surfaces of compounds 11 and 36. In this diagram, the red and blue
areas indicate the richest density electron region and the poorest one.
3. Conclusions
Investigating the results of the bisphosphoramide derivatives inhibitory test showed that
these derivatives have a different behavior as compared to monophosphoramide
compounds regarding the type of inhibitory mechanism (they have a mixed type
inhibitory mechanism) and the ability to inhibit urease enzymes (with the inhibitory range
of 23000–1.91 × 10−13 µM). The docking analysis revealed that these compounds are
connected to the key residues of the entrance and inside of the binding pocket via
25

hydrogen bonds, hydrophobic and π–interactions. It has also been found that these
compounds do not interact directly with nickel because of their extended structures, but
react with other active site residues and occupy the entire binding pocket, which was
supported by information derived from kinetic studies. The results obtained from docking
analysis and experimental studies similarly indicated that the derivatives containing
aromatic amines in their structure have more inhibitory activity than derivatives
containing aliphatic amines. Also, in a closer examination, it was determined that
inhibitors containing the P=O functional group are more potent than the inhibitors
containing P=S. Following these studies, QSAR calculations were performed to
determine the most significant descriptors affecting the inhibitory activity of the
synthesized bisphosphoramides using the Genetic Algorithm and the Artificial Neural
Network (GA–ANN) method. The total energy of the molecule (Etotal), the number of
rings (nCIC), a rotatable bond fraction (RBF), 31P chemical shift (δ), the number of
aromatic bonds (nAB) and total charge (Qtotal) descriptors were selected as the most
critical parameters influencing the inhibitory activity. The chemical shift of phosphorus
atoms is one of the parameters affecting the inhibitory power of these compounds. The
sulfur and oxygen atoms connected to the phosphorus atom are the main factors in
chemical shift and differences in the ability of the inhibitors. These differences were
justified by analysis of molecular electrostatic potential surfaces of the compounds. In
general, the logical relations between theoretical and empirical results shows that an
increase in effective functional groups (P=O, P–N, and aromatic substitutes) in the
compounds, despite extending the structure, has a positive effect on the inhibitory activity
by changing the type of mechanism and the interaction model of the inhibitor with the
26

enzyme. This information can be used as an introduction to the synthesis of targeted
inhibitors.
4. Materials and methods
4.1. Instruments
All chemicals and solvent were purchased from commercial suppliers (Sigma–Aldrich,
Merck) and all of them were of reagent grade and were used without further purification.
Nuclear magnetic resonance spectra (NMR) were recorded on a Bruker Avance DRX 500
1
MHz spectrometer. TMS (tetramethylsilane) in H and 13C NMR spectroscopy and
31
H3PO4 85% in P NMR spectroscopy were used as internal standards. Infrared spectra
(IR) were recorded on a Nicolet 510P spectrophotometer using KBr disks. Melting points
were determined by an electrothermal instrument. UV–Vis spectra were recorded on a
Perkin–Elmer Lambda 25 spectrophotometer. Instrumentation details for the single
crystals X-ray analysis of 22 and 26 are provided in the supporting information.
Crystallographic data of 22 and 26 have been deposited with the CCDC and have been
given the deposition numbers 1581447 and 1584194 for 22 and 26, respectively.
Molecular docking simulations were performed to obtain the ligand-protein interaction
information using the ADT software (AutoDockTools package, version 1.5.6) along with
AutoDock 4.2 and hybrid Lamarckian Genetic Algorithm (LGA) [33]. The three-
dimensional crystal structure of jack bean urease was obtained with 1.49 Å resolutions
from the Protein Data Bank (PDB code: 4GY7).
27

4.2. Synthesis
Compounds (01–08), 10, (13–18), 21, 23, 24, (29–33) and (37–39) were synthesized and
characterized according to the procedure reported in our previous work [22-27]. The
synthesis procedures and information relating to the identification of compounds 09, (11-
12), (19, 20, 22), (25–28), (34–36) and (40–43) are provided in the Supporting
Information. Figure 1 shows the newly synthesized compounds in this work.
4.3. Urease inhibition assay
Urease inhibitory activity was determined by measuring the absorption of ammonia
released using the indophenol method which has been explained by Weather Burn [34].
At first, 1 mL of buffer solution (NaH2PO4/Na2HPO4, 70 mM, pH 7.4), 1 mL of inhibitor
solution with different concentrations (in order to dissolve some of the compounds, a
mixture of water and dimethyl sulfoxide solvents was used; the highest concentration of
dimethyl sulfoxide used in the assays was 4 %.), and 10 µL of jack bean urease solution
(2.5 mg/mL, 12.5 U/mL) were mixed together in test tubes and incubated for 30 min at
37 °C. Then, 1 mL of urea solution (6 mg/mL) was added to the assay mixture and again
was incubated for 30 min at 37 °C. Afterward, 1mL of phenol reagent (1.0 g phenol, 5
mg sodium nitroprusside in 100 mL water) was added to the mixture. After mixing, 1 mL
of alkali reagent (0.5 g NaOH, 870 µL sodium hypochlorite in 100 mL water) was added
and the final mixture was incubated for 30 min at 37 °C. The absorbance of the resulting
colored mixture was measured using UV–Vis spectrophotometer at 625 nm. The control
solution contained the same mix without inhibitor. The inhibitory assays for all
compounds were performed in triplicate, and phenylphosphorodiamidate (PPD) was used
28

as reference inhibitor in the inhibitory assay. The inhibition percentage [INH (%)] was
calculated using the following formula:
INH (%) = 100 – ((AINH /AB) × 100)
Eq. 1
Where and
are the absorbances of control solution and inhibitor solution,
respectively. The IC50 values for urease inhibition were determined using the non-linear
curve fitting program PRISM 6.07 (GraphPad Prism).
4.4. Kinetics study
Lineweaver-Burk plots of the velocities inverse value (1/v) versus the substrate
concentration inverse value (1/[Urea]) were applied to determine the mechanism of
enzyme inhibitory in synthesized bisphosphoramide compounds. The values of kinetic
parameters (Km, Vmax) were determined using these plots in the absence and presence of
inhibitor and at different concentrations of urea. and
values were determined
for at least 4 concentrations of inhibitor. The values of Ki and KI inhibitory constants
were determined using two plots. The first of which is plot of 1/Vmax vs. different
concentrations of inhibitor, and other plot is the plot of the slope vs. inhibitor
concentration. The intersection of each of them on the -axis will be equivalent to −KI
and −Ki, respectively. In this study, the slopes were obtained from the Lineweaver–Burk
lines.
29