Phosphorhydrazides as urease and acetylcholinesterase inhibitors: biological evaluation and QSAR study

Article abstract of DOI:10.1007/s13738-016-0836-8

New phosphorhydrazide compounds (1–7, 13, 15–16) and thiophosphorhydrazide (14 and 17) were synthesized and characterized by ³¹P, ¹³C, ¹H NMR and IR spectroscopy. Furthermore, the crystal structure of compound (C₆

Full text of DOI:10.1007/s13738-016-0836-8

J IRAN CHEM SOC
DOI 10.1007/s13738-016-0836-8
ORIGINAL PAPER
Phosphorhydrazides as urease and acetylcholinesterase
inhibitors: biological evaluation and QSAR study
Lida Asadi2 · Khodayar Gholivand1 · Karim Zare2
Received: 3 November 2015 / Accepted: 26 February 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract New phosphorhydrazide compounds (1–7, 13,
15–16) and thiophosphorhydrazide (14 and 17) were syn-
step of organic nitrogen mineralization to produce
ammonia and carbamate. The carbamate produced dur-
ing this reaction spontaneously decomposes, at physi-
ological pH, to give a second molecule of ammonia
and bicarbonate [3–6]. The hydrolysis of the reaction
products causes an abrupt overall pH increase, the
major cause for the negative side effects of the action
of urease both for human and animal health, and for
agriculture. Urease inhibitors have also been proposed
to control urea hydrolysis in soil [6–16]. In particular,
compound with the P(X)–NH2 segment (X = O, S)
have received considerable attention as urease inhibi-
tors. The most effective inhibitors are substituted both
thiophosphinicamide and phosphinicamide families.
Many phosphoramide compounds are the most effective
compounds currently available to retard the hydrolysis
of urea fertilizer in soil and to decrease ammonia vola-
tilization and nitrite accumulation in soils treated with
urea. (Thio)phosphoramide inhibited urease by form-
ing a chelated complex with the nickel ion in the active
site of enzyme. Phosphoramide acts as urease inhibi-
tors by coordinating itself to the nickel ions of active
site [15]. The oxygen or sulfur atoms and amide group
of the inhibitor molecule are engaged in the formation
of a bridge between the Ni(1) and Ni(2) ions. In this
work, we have compared the effectiveness of new (thio)
phosphorhydrazide compounds (1–17) to inhibit jack
bean urease toward (thio)phosphoramides. In addition,
we carried out quantitative structure–activity relation-
ship (QSAR) studies on the aforementioned derivatives
to gain an understanding of the activity shown by such
compounds, to propose its union mode to the urease
active site, and to try to create the correlation between
the electronic and structural parameters in contrast to
the inhibition potency.
1
thesized and characterized by 31P, 13C, H NMR and IR
spectroscopy. Furthermore, the crystal structure of com-
pound (C6H5NH)(C6H5O)P(O)(NH–NH2) (2) was investi-
gated. The activities of derivatives on acetylcholinesterase
(AChE) and urease were determined. Quantitative struc-
ture–activity relationship (QSAR) was used to understand
the relationship between molecular structural features and
inhibitory. DFT–QSAR models for enzymes demonstrated
the importance of ELUMO parameter in describing the anti-
AChE and anti-urease activities of the synthesized com-
pounds. The correlation matrix of QSAR models and dock-
ing analysis confirmed that electrophilicity descriptor can
control the influence of the polarizability properties of N–H
functional group of PAH derivatives in the inhibition of
enzymes.
Keywords Phosphorhydrazide · Crystal structure ·
Urease inhibitor · Anti-AChE · QSAR
Introduction
Urease catalyzes the hydrolysis of urea in plants, algae,
fungi, and several microorganisms [1, 2], in the final
* Khodayar Gholivand
gholi_kh@modares.ac.ir
1
Department of Chemistry, Tarbiat Modares University,
P.O. Box: 14115-175, Tehran, Iran
2
Department of Chemistry, Islamic Azad University, Science
and Research Branch, Tehran, Iran
1 3

J IRAN CHEM SOC
N‑Hydrazino (O‑phenyl)(N‑phenyl) amidophosphate (2)
Materials and methods
Instrument
This compound was synthesized similar to the preparation
method of compound 1 using of (C6H5O)P(O)(NHC6H5)
Cl [18] instead of (C6H5O)2P(O)Cl. Powder sample; m.p.
229 °C, 1H NMR (500.13 MHz, d6–DMSO, 25 °C, TMS);
δ = 6.46 (d, 2 H, 2JPNH = 32.6 Hz, NH–NH), 6.84 (t, 1 H,
OC6H5), 7.14 (t, 1 H, HNC6H5), 7.20 (m, 2 H, HNC6H5),
7.22 (d, 2 H, OC6H5), 7.33 (t, 2 H, OC6H5), 7.85 (d, 2 H,
3JPNH = 8.5 Hz, NH) ppm. 13C NMR (125.75 MHz, d6–
The enzyme AChE (human erythrocyte; Sigma, Cat.
No. C0663) and BChE (bovine erythrocyte, Sigma, Cat.
No. B4186), Triton X-100, bovine serum albumin, Ace-
tylcholinesterase (AChE, EC 3.1.1.7), alpha-naphthyl
acetate, beta-naphthyl acetate, fast blue RR, DMSO,
Sodium dodecyl sulfate (SDS) were all from Sigma-
Aldrich. Acetylthiocholine iodide (ATCh, 99 %, Fluka),
5, 5′-dithiobis (2-nitrobenzoic acid)) (DTNB, 98 %,
Merck), Na2HPO4, NaH2PO4 (99 %), (thio)hydrazide,
triethylamine (99.5 %, Merck), CDCl3 (99 %, Sigma
Aldrich), (C6H5O)P(O)Cl2 (99 %, Merck), (CH3O)2P(S)
Cl, (CH3CH2O)2P(S)Cl and (CH3CH2O)2P(O)Cl (97 %,
3
DMSO, 25 °C, TMS); δ = 117.37 (d, JPC = 7.4 Hz, 2
3
C, ortho-HNC6H5), 120.71 (d, JPC = 4.5 Hz, 2 C, ortho-
OC6H5), 124.12 (s, 1 C, para-OC6H5), 128.03 (s, 1 C,
para-HNC6H5), 129.39 (s, 2 C, meta-OC6H5), 135.94 (s, 2
C, meta-HNC6H5), 143.80 (s, 1 C, ipso-HNC6H5), 150.90
2
(d, JPC = 6.6 Hz, 1 C, ipso-OC6H5) ppm. 31P{1H} and
31P NMR (202.45 MHz, d6–DMSO, 25 °C, H3PO4 exter-
Sigma Aldrich) were used as supplied. H, 13C, and 31P
1
2
nal); δ = −6.45 (s) (d, JPNH = 32.9 Hz) ppm. IR (KBr):
spectra were recorded on a Bruker Advance DRX 500
spectrometer. 1H and 13C chemical shifts were deter-
mined relative to internal TMS, and 31P chemical shifts
were determined relative to 85 % H3PO4 as the exter-
nal standard. Infrared (IR) spectra were recorded on a
Shimadzu model IR-60 spectrometer using KBr pellets.
Melting points of the compounds were obtained with an
electrothermal instrument. UV spectrophotometer was
operated using a PERKIN-ELMER Lambda 25. The cor-
relation analysis was performed by the Statistical Pack-
age for Social Scientists (SPSS), version 16.0 for Win-
dows [17].
υ˜ = 3225 (N–H), 3066 (N–H), 1605 (s), 1499 (s), 1219
(P=O), 950 (P–O), 751 (P–N).
N,N′‑Di hydrazino phosphoramidic acid 4‑chloro phenyl
ester (3)
A solution of hydrazine hydrate (4 mmol) in THF was
added at 0 °C to a solution of (p-ClC6H5O)P(O)Cl2
(1 mmol) in THF. After 4 h stirring, the solvent was
removed in vacuum and the resulting white powder was
washed with distilled water. Powder sample; m.p. 171 °C,
1H NMR (300.13 MHz, d6–DMSO, 25 °C, TMS), δ = 7.32
(d, 3JHH = 8.6 Hz, 2H, Ar–H), 7.15 (d, 3J HH = 9.0 Hz, 2H,
Synthesis
2
Ar–H), 7.07 (d, JPNH = 31.0 Hz, 2H, NH), 5.66 (br, 4H,
2NH2) ppm. 13C NMR (75.47 MHz, d6–DMSO, 25 °C,
The synthesis pathway of compounds 1–17 was repre-
sented in Scheme 1.
2
TMS), δ = 151.21 (d, JPC = 6.2 Hz, Cipso), 128.84 (s),
3
127.06 (s), 121.80 (d, JPC = 4.9 Hz, Cortho(ppm. 31P
NMR (121.49 MHz, d6–DMSO, 25 °C, H3PO4 external),
N‑Hydrazino diphenyl amidophosphate (1)
δppm = −5.44(m) ppm. IR (KBr) υ˜ = 3355–3207 (N–H),
1210 (P=O), 979 (P–N), 924 (N–N) cm−1
.
A solution of hydrazine hydrate (2 mmol) in THF was
added at 0 °C to a solution of (C6H5O)2P(O)Cl (1 mmol)
in THF. After 4 h stirring, the solvent was removed in
vacuum and the resulting white powder was washed with
distilled water. Powder sample; m.p. 92 °C, 1H NMR
(300.13 MHz, d6–DMSO, 25 °C, TMS); δ = 7.38 (t, 4H),
7.25 (d, 4H), 7.19 (t,2H), 6.87 (d,2JPNH = 38.4 Hz,1H,
N–Hamid), 3.99 (2H, NH2) ppm. 13C NMR (75.46 MHz,
N,N′‑Di hydrazino phosphoramidic acid 4‑methyl phenyl
ester (4)
This compound was synthesized similar to the prepara-
tion method of compound 3 using of (p-CH3C6H5O)P(O)
Cl2 instead of (p-ClC6H5O)P(O)Cl2. Powder sample; m.p.
1
2
153 °C, H NMR (300.13 MHz, d6–CDCl3, 25 °C, TMS);
TMS); δ = 150.57 (d, JPC = 6.3 Hz, Cipso), 129.72 (s,
2
Cmetha), 124.77 (s, Cpara), 120.50 (d,3JPC = 4.6 Hz,
Cortho) ppm.31P NMR (121.49 MHz, d6–DMSO, 25 °C,
H3PO4 external), δ = 1.05(m) ppm. IR (KBr) υ˜ = 3327–
3211 (N–H), 1217 (P=O), 1071 (P–O), 934 (P–N), 905
δ = 6.98–7.06 (q, 4H, Ar–H), 6.01 (d, JPNH = 31.9 Hz,
2H, 2NH), 5.84 (br, 4H, 2NH2), 2.26 (s, 3H, p-CH3)
ppm. 13C NMR (75.46 MHz, DMSO, 25 °C, TMS);
2
δ = 150.40 (d, JPC = 6.3 Hz, Cipso), 131.62 (s), 129.44
3
(s), 119.80 (d, JPC = 4.8 Hz, Cortho), 20.26 (s) ppm. 31P
(N–N) cm−1
.
1 3

J IRAN CHEM SOC
O
C
S
P
R1
R3
NH NH
e
d
i
S
P
z
a
b
r
S
P
a
c
i
R2
m
e
s
S
P
+
Hydrazine
R1 & R2 = OC2H5; R3 = NH2 (10)
R1 & R2 = OCH3; R3 = NH2 (11)
R1
R1
R2
NH NH
R1
R2
Cl
R2
+
R1 & R2 = OC2H5 (14)
T
h
i
o
s
e
m
i
c
a
S
r
S
b
a
z
i
d
e
C
P
OR
OR'
X
NH NH
X=O, S
R1 & R2 = OC2H5; R3 = NH2 (12)
P
Cl
Cl
O
C
O
P
Cl
+ Semicarbazide
R1
R3
NH NH
O
P
R2
R1 = OC6H5; R2 = Cl; R3 = NH2 (5)
R1
R1 = Cl-OC6H4; R2 = Cl; R3 = NH2 (6)
R1 = C6H5; R2 = Cl; R3 = NH2 (7)
Cl
R2
R1 = OC6H5; R2 =OC6H5; R3 = NH2 (8)
R1 = OC2H5; R2 =OC2H5; R3 = NH2 (9)
R1 = CH3-OC6H4; R2 = NH2-C(O)-NH-NH; R3 = NH2 (13)
+
H
X
y
d
r
a
z
i
n
e
P
R1
R2
R3 NH NH
R1 & R2= OC6H5; R3 = H; X = O (1)
R1 = NH-C6H5; R2= OC6H5; R3 = H; X = O (2)
R1 = Cl-OC6H4; R2 = NH-NH2; R3 = H; X = O (3)
R1 = CH3-OC6H4; R2 = NH-NH2; R3 = H; X = O(4)
R1 = Cl-OC6H4; R2 = NH-NH-C6H5; R3 = C6H5; X = O (15)
R1 = CH3-OC6H4; R2 = NH-NH-C6H5; R3 = C6H5; X = O (16)
R1 & R2 = OC2H5; R3 = C6H5; X = S (17)
Scheme 1 Preparation of PHA derivatives (1–17)
NMR (121.49 MHz, d6–DMSO, 25 °C, H3PO4 external),
(s, C=O), 151.20 (d, Cipso, 2JPC = 6.0 Hz); 130.00 (s, Cpara),
3
δ = −4.69(m) ppm. IR (KBr) υ˜ = 3300–3250 (N–H), 1214
125.05 (s, Cmetha), 120.82 (d, Cortho, JPC = 5.0 Hz) ppm
(P=O), 1034 (P–O), 979 (P–N), 922 (N–N) cm−1
.
31P NMR (161.81 MHz, DMSO,25 °C, H3PO4external),
δppm = 1.98 (d, 2JPNH = 34.8 Hz). IR (KBr) υ˜ = 3423–3234
(N–H), 1665 (C=O), 1212 (P=O), 969 (P–N) cm−1.
Chloro N‑(semicarbazido) phosphoramidic acid phenyl
ester (5)
Chloro N‑(semicarbazido) phosphoramidic acid 4‑chloro
A solution of semicarbazide (1 mmol) and triethylamine
(1 mmol) in THF was added at 0 °C to a solution of (C6H5O)
P(O)Cl2 (1 mmol) in THF. After 4 h stirring, the solvent was
removed in vacuum and the resulting white powder was
washed with distilled water. Powder sample; m.p. 152 °C.
1H NMR (400 MHz, d6–DMSO, 25 °C, TMS); δ = 7.63
phenyl ester (6)
This compound was synthesized similar to the preparation
method of compound 5 using of (p-CH3C6H5O)P(O)Cl2
instead of (C6H5O)P(O)Cl2. Powder sample; m.p. 174 °C,
1H NMR (300.13 MHz, d6–DMSO, 25 °C, TMS); δ = 7.64
(s, 1 H, N–Hamid), 7.48 (d, 2JPNH = 35.6 Hz, 1 H, N–HPNH),
7.43–7.24 (4H, m, Ar–H), 5.84 (s, 2 H, NH2) ppm. 13C
NMR (75.47 MHz, d6–DMSO, 25 °C, TMS); δ = 159.51
2
(s,1H, N–Hamid), 7.45 (d, JPNH = 35.6 Hz,1 H, N–HPNH),
7.39–7.15 (5H, m, Ar–H), 5.85 (s,2H, N–Hamid) ppm. 13C
NMR (100.51 MHz, d6–DMSO, 25 °C, TMS); δ = 159.60
1 3

J IRAN CHEM SOC
(s, 1 C, C=O), 149.5 7 (d, Cipso,2JPC = 6.4 Hz).), 129.48 (s,
3JPC = 4.0 Hz, Cortho amine) ppm. 31P NMR (101.25 MHz,
d6–DMSO, 25 °C, H3PO4 external), δ = 11.46(dt) ppm.
IR (KBr) υ˜ = 3318–3267 (N–H), 1211 (P=O), 936 (P–N)
Cpara), 128.63 (s, Cmetha), 122.23 (d,3JPC = 4.7 Hz, Cortho
)
ppm. 31P NMR (161.83 MHz, d6–DMSO, 25 °C, H3PO4
external), δ = 2.08 (d,2JPNH = 35.6 Hz) ppm. IR (KBr)
υ˜ = 3420–3231 (N–H), 1664 (C=O), 1229 (P=O), 965
cm−1
.
(P–N) cm−1
.
N,N′‑bis(phenyl hydrazino) phosphoramidic acid 4‑methyl
phenyl ester (16)
Chloro phenyl N‑(semicarbazido) phosphoramidic acid (7)
This compound was synthesized similar to the prepara-
tion method of compound 15 using of (p-CH3C6H5O)P(O)
Cl2 instead of (p-ClC6H5O)P(O)Cl2. Powder sample; m.p.
196 °C, 1H NMR (250.13 MHz, d6–DMSO, 25 °C, TMS);
δ = 7.10 (m, 2JPNH = 34.0 Hz, 2 H, N–HPNH), 7.13–7.07 (8
H, Ar–H), 7.09 (d, 3JPNH = 31.3 Hz, 2 H, N–H), 6.89–6.85
(4H, Ar–H), 6.99–6.64 (2H, Ar–H), 2.28 (s, 3 H, p-CH3)
ppm. 13C NMR (75.47 MHz, d6–DMSO, 25 °C, TMS);
This compound was synthesized similar to the prepa-
ration method of compound 5 using of (C6H5)P(O)
Cl2 instead of (C6H5O)P(O)Cl2. Powder sample; m.p.
199 °C, 1H NMR (300.13 MHz, d6–DMSO, 25 °C, TMS);
δ = 7.78–7.40 (5H, m, Ar–H), 7.32 (s, 1 H, N–Hamid), 7.18
2
(d, JPNH = 27.0 Hz, 1 H, N–HPNH), 5.79 (s, 2 H, NH2)
ppm. 13C NMR (75.47 MHz, d6–DMSO, 25 °C, TMS);
1
2
δ = 159.85 (s, C=O), 131.72 (d, Cipso, JPC = 16.6 Hz),
δ = 150.31 (d, JPC = 4. 5 Hz, Cipso phenyl), 149.11 (d,
131.50 (s, Cmetha), 129.13 (s, Cpara), 128.21 (d, Cortho,
2JPC = 13.9 Hz) ppm. 31P NMR (121.49 MHz, d6–DMSO,
25 °C, H3PO4 external); δ = 20.93 (d,2JPNH = 27.0 Hz)
ppm.IR (KBr) υ˜ = 3412–3213 (N–H), 1662 (C=O), 1204
3JPC = 6.4 Hz, Cipso amine), 131.26 (s, Cmeta), 128.43 (s, Cme-
tha amine), 120.46 (s, Cpara amine), 118.128 (s, Cortho), 112.558
4
(d, JPC = 4.2 Hz, Cortho amine), 20.289 (s, p-CH3) ppm. 31P
NMR (101.25 MHz, d6–DMSO, 25 °C, H3PO4 external);
2
(P=O), 973 (P–N)cm−1
.
δ = 11.28 (d, JPNH = 34.0 Hz). IR (KBr) υ˜ = 3302–3202
(N–H), 1219 (P=O), 937 (P–N) cm−1
.
N,N′‑Bis(O,O‑diethyl phosphorothioyl)hydrazide (14)
Diethyl N‑(phenyl hydrazino) amidothiophosphate (17)
A solution of hydrazine hydrate (3 mmol) in THF was
added at 0 °C to a solution of (C2H5O)2P(O)Cl (2 mmol) in
THF. After 4 h stirring, the solvent was removed in vacuum
and the resulting white powder was washed with distilled
water. Powder sample; m.p. 84 °C, 1H NMR (300.13 MHz,
A solution of phenyl hydrazin (2 mmol) in THF was added
at at the room temperature to a solution of (C2H5O)P(S)
Cl2 (1 mmol) in THF. After 4 h refluxing, the solvent was
removed in vacuum and the resulting white powder was
washed with distilled water. Powder sample; m.p. 73 °C,
1H NMR (300.13 MHz, d6–DMSO, 25 °C, TMS), δ = 7.57
(s, 1 H, N–H), 7.002 (d, 3JHH = 6.0 Hz, 1 H, N–H), 7.004–
2
d6–DMSO, 25 °C, TMS), δ = 7.20 (d, JPNH = 38.1 Hz,
2H, N–H), 4.0 (m, 8H, CH2), 1.20–1.25 (t, 3JHH = 7.0 Hz,
12H, CH3) ppm. 13C NMR (75.46 MHz, d6–DMSO, 25 °C,
TMS), δ = 62.29 (s, CH2), 15.72 (s, CH3) ppm. 31P NMR
(121.49 MHz, d6–DMSO, 25 °C, H3PO4 external) ppm.
δ = 70.90 (m). IR (KBr) υ˜ = 3390–3251 (N–H), 1024
6.69 (m, 5 H, Ar–H), 4.08–3.98 (m, 10 H, 2C2H5) ppm. 13
C
NMR (75.46 MHz, d6–DMSO, 25 °C, TMS), δ = 149.73
(m, Cipso amine), 128.47 (s, Cpara), 118.25 (d, 4JPC = 5.1 Hz,
(P–O), 965 (P–N), 897 (N–N), 795 (P=S) cm−1
.
Cortho), 112.32 (d, JPC = 4.2 Hz, Cmeta), 62.45 (d,
5
3JPC = 4.9 Hz, CCH3), ppm. 31P NMR (101.25 MHz,
d6–DMSO, 25 °C, H3PO4 external), δ = 71.94 (m,
2JPNH = 43.2 Hz) ppm. IR (KBr) υ˜ = 3307–2978 (N–H),
N,N′‑bis(phenyl hydrazino) phosphoramidic acid 4‑chloro
phenyl ester (15)
802 (P=S), 959 (P–N) cm−1
.
A solution of phenyl hydrazin (4 mmol) in THF was added
at 0 °C to a solution of (p-ClC6H5O)P(O)Cl2 (1 mmol)
in THF. After 4 h stirring, the solvent was removed in
vacuum and the resulting white powder was washed
with distilled water. Powder sample; m.p. 167 °C, 1H
NMR (250.13 MHz, d6–D2O, 25 °C, TMS), δ = 7.36
Crystal structure determination
X–Ray data of compound 2 were collected on a Bruker
SMART 1000 CCD area detector with graphite mono-
chromated Mo-Kα radiation (λ = 0.71073 Å) and refined
by full-matrix least-squares methods against F2 with
SHELXL97 [19]. CCDC 1027079 contain the supplemen-
tary crystallographic data for compound 2. These data can
be obtained free of charge via http://www.ccdc.cam.ac.uk/
conts/retrieving.html, or from the Cambridge Crystal-
lographic Data Centre, 12 Union Road, Cambridge CB2
2
(m, 2 H, N–HPNH, JPNH = 35.3 Hz), 7.17 (d,2H, N–H,
3JPNH = 31.8 Hz), 7.13–7.07 (m, 10 H, Ar–H), 6.87–6.84
(m, 4 H, Ar–H), ppm. 13C NMR (75.47 MHz, d6–DMSO,
25 °C, TMS), δ = 150.60 (s, Cipsophenyl), 149.99 (s, Cipso
amine), 129.20 (s, Cmetha), 128.45 (s, Cmetha amine), 127.94 (s,
Cpara), 122.47 (d, Cpara amine), 118.31 (s, Cortho), 112.56 (d,
1 3

J IRAN CHEM SOC
1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@
>1500 U/L, α-ketoglutarate 4.0 mM, buffer pH 8.2 0.1
and phosphoramidate inhibitors. The reconstituted reagent
has a reagent blank absorbance less than 1.0 at 340 nm.
ccdc.cam.ac.uk.
Human ChE assay
Statistical analysis for QSAR model
Human AChE activity measurements were performed
essentially according to the method of Ellman [20]. The
reaction was carried out at 37 °C in 70 mM phosphate
buffer (Na2HPO4/NaH2PO4, pH 7.4) containing the AChE
enzyme (10 µl volume, diluted 100 times in phosphate
buffer, pH 7.4), DTNB (5,5′-dithiobis (2-nitrobenzoic
In order to identify the effect of physicochemical parameters
on the AChE inhibition activity, QSAR studies were under-
taken using the approach described by Hansch and Fujita.
The stepwise multiple linear regression procedure is a com-
mon method in QSAR studies for selection descriptors. The
MLR method performed by the software package SPSS 16.0
was used for selection of the descriptors. The electronic and
structural descriptors are obtained by either the quantum
chemical calculations, theoretical and experimental stud-
ies. The electronic descriptors include the energy of frontier
orbital (EHOMO and ELUMO), electrophilicity (ω), polarizabil-
ity (PL, the charge difference between the atoms in func-
tional groups) and the net atomic charges (Q). Also hydro-
phobic coefficient (logP), dipole moment (μ) and molecular
acid)) (10−4 M concentration) and ATCh (1.35 × 10−4
M
concentration). Each compound was dissolved in dime-
thyl sulfoxide (DMSO), and then added to buffer for
in vitro cholinesterase assays. The highest concentra-
tion of DMSO used in the assays was 5 %. In independ-
ent experiments without the inhibitor, 5 % DMSO had no
effect on the inducing activity of enzyme. The absorbance
change at 37 °C was monitored with the spectrophotom-
eter at 412 nm for 3 min and three replicates were run in
each experiment. In the absence of inhibitor, the absorb-
ance change was directly proportional to the enzyme level.
The reaction mixtures for determination of IC50 values, the
median inhibitory concentration, consisted DTNB solution,
100 μl; inhibitor, x μl; acetylthiocholin idide (ATCh) solu-
tion, 40 μl; phosphate buffer (850-×) μl and hAChE solu-
tion, 10 μl.
volume (Mv) are the structural descriptors. EHOMO, ELUMO
,
ω, P, Q, μ and Mv values are obtained from the DFT results.
The logarithm of partition coefficient (logP) is measured
by the shake–flask and theoretical methods. The toxici-
ties of phosphorhydrazide analogues are expressed in terms
of p(IC50) or −log(IC50) as an anti-cholinesterase activity.
The descriptor values were related with toxicity using MLR
analysis. MLR of descriptors, selected for biological activity,
gives rise to the problem of multicollinearity. This problem
can be solved using the principal component analysis (PCA).
These linear combinations form a new set of variables,
namely principal components (PCs), which are mutually
orthogonal. The first PC contains the largest variance and
the second new variable contains the second largest variance,
and so on. The variable selection in this PCA study was
performed using the Fisher’s weights. The descriptors with
higher correlation coefficient and lower correlation (|r| < 0.5)
to p(IC50) were selected to carry out stepwise MLR analysis
and to optimize the QSAR equation [22]. The stable geom-
etry structures of compounds were further fully optimized
using the Density functional theory (DFT) at the B3LYP/6–
311+G** level of theory. Natural population analysis (NPA)
was performed at the same level using the Reed and Wein-
hold scheme [23]. All quantum chemical calculations were
carried out using the Gaussian 03 program package [24].
Urease inhibitory activity
Antiurease inhibition activity was tested by reported pro-
tocol [21]. Urea has been determined by the direct method
where urea condenses with diacetyl to form a chromagen
and an indirect method where ammonia is measured as a
product of urease action on urea. The liberated ammo-
nia has been measured using Nessler’s reagent and by the
Berthelot reaction. Talke and Schubert introduced a totally
enzymatic procedure in 1965 utilizing urease and Gluta-
mate Dehydrogenase. The present procedure is based on a
modification of their method.
Urea + H2O → 2NH3 + CO2
NH3 + α-Ketoglutarate + NADH + H+
→ L-glutamate + NAD+ + H2O
Urea is hydrolyzed by urease to produce ammonia
and carbon dioxide. The liberated ammonia reacts with
α-ketoglutarate in the presence of NADH to yield gluta-
mate. An equimolar quantity of NADH undergoes oxidation
during the reaction resulting in a decrease in absorbance
that is directly proportional to the urea nitrogen concentra-
tion in the sample. Concentrations of reagents are NADH
0.3 mM, urease 1500 U/L, glutamate dehydrogenase
Results and discussion
Spectral study
Spectroscopy data and phosphorus–hydrogen (2JPNH
)
coupling constants and δ(31P) of compounds 1–17 are
1 3

J IRAN CHEM SOC
Table 1 Selected spectroscopic
NMR and IR experimental data
of the products
No.
δ(31P)P=X(O,S)
δ(1H)N–H
2JPNH(hydrazide)
νP=X(O,S)
νN–H
References
a
1
−5.44
−6.45
−4.69
1.05
7.07
7.85
6.01
6.87
7.45
7.48
7.41
7.82
7.02
7.39
7.48
7.89
7.27
7.20
7.17
7.09
7.57
38.4
32.9
31.0
31.9
35.6
35.6
27.0
38.0
32.2
39.9
41.2
35.1
31.0
38.1
35.3
34.0
43.2
1209
1218
1214
1217
1212
1228
1203
1215
1216
790
3354/3207
3224
a
a
a
a
a
a
2
3
3300/3250
3327/3210
3423/3234
3419/3231
3412/3212
3300
4
5
1.98
6
2.08
7
2.10
8
2.61
[27]
[27]
[27]
[27]
9
6.21
3423
10
11
12
13
14
15
16
17
69.89
74.03
68.66
8.48
3445
814
3440
792
3168/3415
3432
[27]
a
1226
795
a
a
a
a
70.90
11.46
11.28
71.94
3390/3251
3318
1210
1218
802
3302
3307
a
This work
summarized in Table 1. Phosphorus chemical shifts δ(31P)
were observed in the range of −6.45 ppm (2) to 74.03 ppm
(11). As 31P NMR spectra reveal the effect of X on δ(31P),
in P=X moiety this comparison can be made that 10, 11,
12, 14, 17 compounds consisting P=S moeity show a high
upfield shift rather than the compounds with P=O moi-
ety. That is to say, the presence of a sulfur atom leads to
the deshielding of phosphorus atom in these derivatives.
31P NMR spectra designate different patterns, compounds
1, 3, 4, 14 indicate singlet pattern, although the rest are
appeared as doublets. This splitting pattern arises from
the spin couplings between the phosphorus nucleus and
NH protons, while a doublet of triplet appeared in com-
pound 15 is achieved from the spin couplings between the
phosphorus nucleus and NH and one equivalent hydrogen
Crystal structures
Colorless single crystal of compound 2 which is suitable
for X-ray diffraction analysis was grown from a THF/Chl-
roform mixture after slow evaporation at room tempera-
ture. The crystal data of X-ray analysis is given in Table 2.
Molecular structure is shown in Figs. 1 for compound 2.
Compound 2 crystallize in the triclinic crystal system with
space group P−1. The phosphorus atom has a slightly dis-
torted tetrahedral configuration in compound 2, that is,
the surrounding angles around the P atom are in the range
of 95.75(9)°–116.22(9)°. The P=O bond distance is in 2
1.4812(15) Å. The P–Nα bond distance in 2 1.6423(18) Å
is shorter than the single bond P–N distance of 1.77 Å.
The N(3)–H(3NA)…O(1A), N(3)–H(3NA)…N(1), N(1)–
H(1N)…N(3A), N(2)–H(2N)…O(1), N(1A)–H(1NA)…
N(3), N(2A)–H(2NA)…O(1A) and N(3A)–H(3NC)…O(1)
hydrogen bonds produce along a linear direction (Table 3;
Fig. 2). The polymeric chains are formed by hydrogen
bonds in the crystal lattice with cyclic R22(8) motifs [25] via
N(2A)–H(2NA)…O(1A) [d = 2.864(2) Å] (Fig. 2). Three
molecules linked in the crystal lattice with cyclic R23(9)
motifs which the molecules are connected to each other
via two N(2A)–H(2NA)…O(1A) [d = 2.864(2) Å], N(3)–
H(3NA)…O(1A) [d = 2.995(3) Å] and N(3)–H(3NA)…
O(2) (d = 3.438 Å) hydrogen bonds (Fig. 2). The asymmet-
ric unit is composed of two independent molecules (Fig. 3).
2
atoms in amine (NH) group. The JPNHα was observed for
2
all compounds. The comparison of the JPNHα values indi-
cate that the compounds with the P=S group (10, 11, 12,
14, 17) have a larger coupling than molecules with P=O
2
group. The greatest and lowest JPNHα values are observed
for 17 (2JPNHα = 43.24 Hz) with the (S)P–NHα–βHN–P(S)
skeleton and 1 (2JPNHα = 30.99 Hz) with the (O)P–NHα–
βHN skeleton. The analysis of the IR spectra indicates that
the vibrational bands of compounds 1–17 appear in the
range of 3300–3445 cm−1 for NH group also the funda-
mental υ(P=X) stretching modes observed in the range of
790 cm−1 (P=S) to 1209 cm−1 (P=O).
1 3

J IRAN CHEM SOC
Table 2 Crystallographic data of compound 2
Biological activity
2
Anti‑AChE activity
Empirical formula
C12H14N3O2P
Formula weight
263.23
To test the experimental anti-acetylcholinesteras (anti-
ChE) [22] activity of the synthesized titled compounds,
we evaluated the inhibitory potential of titled compounds
against AChE enzyme by Ellman assay [20]. The inhibitory
ability of selected compounds against AChE were in 2.34
(11) to 35.52 (12) mM (Table 4). The inhibitory of com-
pound 4 is higher than compound 3 with the (R–C6H4O)
(O)P–(NH–NH2)2 (R=CH3, Cl). Comparison of 9 with the
(C2H5O)2(O)P–NH–HN–C(O)NH2 structure and 10 with
the (C2H5O)2(S)P–NH–HN–C(O)NH2 structure revealed
the inhibitory of P=O (9) > P=S (10) including NH–HN–
C(O)NH2 moiety.
Temperature (K)
100(2)
Wavelength (Å)
0.71073
Triclinic, P-1
Crystal system, space group
Unit cell dimensions
a (Å)
9.6500(8)
10.2212(9)
13.6586(12)
97.589(2)
105.344(3)
90.617(2)
1286.33(19)
4, 1.359
b (Å)
c (Å)
α (°)
β (°)
γ (°)
V (Å3)
Z, calculated density (Mg m−3
)
Absorption coefficient (mm−1
)
0.212
Anti‑urease activity
F(000)
552
Thirteen PAH 1 and 3–14 were evaluated for their inhibi-
tory activities against jack bean urease. Percent inhibition at
a 1 mM concentration of compounds 1 and 3–14 was ini-
tially determined, and the results were reported in Table 4.
Most of the phosphorhydrazides showed good inhibi-
tory activities, especially PAH 9 (IC50 = 1.208 mM) and
11 (IC50 = 1.611 mM) exhibited potent in vitro inhibitory
activities. In general, compounds 5–11 with one NH–HN–
C(O)NH2 group exhibited better inhibitory activities than
compound 12. According to Table 4 data, compound 3
exhibited better inhibitory activity than 4. Compounds 1, 3,
and 4, the order of inhibitory activities showed the potency
of 3 > 1 > 4. It was possibly due to the difference between
the electro affinities of chlorine, methylen, and proton.
Compound 3 with halogen atom in the benzene ring showed
better inhibitory activity than those with methyl substitutes.
Crystal size (mm)
0.35 × 0.35 × 0.05
2.01–29.00
θ range for data collection (°)
Limiting indices
−13 ≤ h ≤ 13,
−13 ≤ k ≤ 13,
−18 ≤ l ≤ 18
Reflections collected/unique
15,665/6783
[R(int) = 0.0281]
Completeness to theta
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices
99.3 %
Semi-empirical from equivalents
Full-matrix least-squares on F2
6783/0/326
1.008
R1 = 0.0520, wR2 = 0.1508
R1 = 0.0612, wR2 = 0.1583
0.547 and −0.440
R indices (all data)
Largest diff. peak and hole (e Å−3
)
Fig. 1 ORTEP representation of compound 2
1 3

J IRAN CHEM SOC
Table 3 Hydrogen-bond distances (Å) and angles (°) for 2
D–H…A
d(D-H) d(H····A) < DHA d(D····A)
N(3)–H(3NA)…O(1A)1
N(3)–H(3NA)…N(1)1
N(1)–H(1N)…N(3A)2
N(2)–H(2N)…O(1)3
N(1A)–H(1NA)…N(3)4
N(2A)–H(2NA)…O(1A)2 0.91
N(3A)–H(3NC)…O(1)5
0.85
0.93
0.93
0.92
0.92
0.80
2.11
2.61
2.05
1.99
2.15
2.03
2.24
158
115
166
169
172
153
168
2.995(3)
3.115(3)
2.956(3)
2.903(2)
2.943(3)
2.864(2)
3.074(3)
1
Symmetry transformations used to generate equivalent atoms:
x,
2
3
4
y, z; −x + 1, −y + 1, −z + 1; −x + 2, −y, −z + 1; −x + 2,
−y + 1, −z + 1; 5 x, y + 1, z
Fig. 3 The asymmetric unit is composed of two independent mol-
ecules
Therefore, PCA method was used to reduce the independ-
ent variables. The principal components (PCs) as a new set
of variables (mutually orthogonal) were obtained by this
method. The first PC contains the largest variance and the
second PC contains the second largest variance [26]. The
variable selection in PCA was performed using the Fisher’s
weights approach and the results are summarized as the fol-
lowing Eqs. (1a, 1b):
PC1 = −0.340QN(α) − 0.307QN(β) + 0.308PN–H(α)
+ 0.378PN–H(β) + 0.230EHOMO + 0.431ELUMO
Fig. 2 Graph sets and a model to describe the hydrogen-bonded clus-
ter in compound 2
− 0.419ω + 0.306µ + 0.091 log P
(1a)
QSAR analysis
PC1 = −0.319QN(α) − 0.028QN(β) + 0.254PN–H(α)
+ 0.157PN–H(β) − 0.332EHOMO − 0.133ELUMO
AChE‑QSAR
The docking data provides important basic information
including the interaction model, the effective functional
groups as well as the electronic and structural properties on
the inhibition mechanism. These data can be guide to create
and to develop of the QSAR models. So far, no model in
literatures has been suggested to explain of the inhibition
mechanism PHAs against urease and AChE enzymes. Here,
we explain the inhibition mechanism of human AChE and
urease by theoretical QSAR models. The electronic and
structural descriptors were obtained by quantum chemical
calculations (Table 5). The number of the independent vari-
ables is more than the tested compounds shown in Table 5.
+ 0.193ω − 0.270µ − 0.475 log P
(1b)
The results showed the total variance of the first, sec-
ond and third factor PC as 39.2 and 24.8 %, respectively.
Also, from the above equations, it was deduced that the
electronic parameters (Q, P, EMO and ω) are predominated
from those related to structural parameters (logP and μ).
Consequently, these eleven descriptors with higher corre-
lation coefficient were selected to carry out the stepwise
multiple linear regression (MLR) analysis, which led to an
optimal QSAR equation based on the anti-AChE potency
(Eq. 2):
1 3

J IRAN CHEM SOC
Table 4 experimental
anti-AChE and urease activitis
of the PAH analogous for the
below moiety
No.
PAH analogous
R1
Biological activity References
IC50 (mM)
R2
R3
X
AChE
Urease
a
a
a
a
a
a
a
1
(C6H5O)
(C6H5O)
(C6H5–NH)
NH–NH2
NH–NH2
Cl
H
O
O
O
O
O
O
O
O
O
S
7.006
–
3.087
–
X
2
(C6H5O)
H
R3
3
(Cl–C6H4O)
(CH3–C6H4O)
(C6H5O)
H
8.690
5.157
9.886
2.831
5.370
4.020
7.130
7.140
2.340
35.52
–
2.213
50.037
7.251
4.745
2.364
4.137
1.208
2.210
1.611
94.843
2.544
2.623
–
P
β
α
R1
N
N
4
H
H
H
R2
5
C(O)NH2
C(O)NH2
C(O)NH2
C(O)NH2
C(O)NH2
C(O)NH2
C(O)NH2
C(S)NH2
C(O)NH2
P(S)(C2H5O)2
NH–NH–C6H5
NH–NH–C6H5
NH–NH–C6H5
6
(Cl–C6H4O)
(C6H5)
Cl
7
Cl
8
(C6H5O)
(C6H5O)
(C2H5O)
(C2H5O)
(CH3O)
[27]
[27]
[27]
[27]
9
(C2H5O)
10
11
12
13
14
15
16
17
(C2H5O)
(CH3O)
S
(C2H5O)
(C2H5O)
NH–NH–C(O)NH2
(C2H5O)
NH–NH–C6H5
NH–NH–C6H5
(C2H5O)
S
[27]
a
(CH3–C6H4O)
(C2H5O)
O
S
a
a
a
a
–
(Cl–C6H4O)
(CH3–C6H4O)
(C2H5O)
O
O
S
–
–
–
–
–
a
This work
Table 5 Quantum-chemical and theoretical descriptors for compounds computed at B3LYP/6–311+G** level
No.
Electronic
Hydroph.
Steric
QP
QN(α)
QN(β)
PP=X
PN–H(α)
PN–H(β)
EHOMO
ELUMO
ω
μ
logP
Mv
1
2.359
2.456
2.452
2.165
2.162
1.985
2.440
2.447
1.963
1.953
1.974
2.054
−0.829
−0.887
−0.837
−0.812
−0.812
−0.823
−0.800
−0.808
−0.797
−0.797
−0.798
−0.855
−0.672
−0.745
−0.656
−0.495
0.408
−3.455
−3.570
−3.535
−3.215
−3.210
−3.050
−3.483
−3.512
−2.562
−2.546
−2.616
−2.726
1.228
1.314
1.246
1.268
1.238
1.246
1.214
1.221
1.208
1.209
1.222
1.310
1.239
1.152
1.020
0.903
0.904
0.899
0.864
0.864
0.868
0.868
0.862
1.305
−0.244
−0.235
−0.247
−0.268
−0.263
−0.280
−0.246
−0.270
−0.231
−0.234
−0.238
−0.248
−0.019
−0.007
−0.014
−0.039
−0.044
−0.059
−0.028
−0.022
−0.022
−0.022
−0.031
0.029
0.077
0.064
0.073
0.103
0.108
0.131
0.086
0.086
0.076
0.077
0.087
0.043
6.886
7.521
8.989
8.146
6.926
3.093
7.067
7.169
7.246
7.007
9.156
9.703
0.04
126.643
182.634
189.315
150.991
179.817
171.836
245.822
163.656
161.607
127.581
182.117
194.763
3
−0.16
1.71
0.19
1.03
−0.26
1.37
0.89
1.25
1.35
2.15
1.84
4
5
6
7
−0.496
−0.485
−0.485
−0.486
−0.486
−0.450
−0.861
8
9
10
11
12
14
Fisher’s statistic [27]. The high determination coefficient
(R2 = 0.828) and low residuals (Sreg = 11.320) are signifi-
cant. High variance inflation factor (VIF >10) (Table 6) are
associated with the multicollinearity problem. Therefore,
the variables with a high VIF are candidates for exclusion
from the model. The improvement in Eq. (2) was carried
out by omitting compound 4 as outland data from the tested
compounds and replacing ω with ELUMO as well as PN–H(α)
with QN(α). Multiple regression performed using the remain-
ing seven parameters yielded the following model with
log(1/IC50) = +125.038QN(α) + 0.893QN(β) + 96.777PN–H(α)
+ 10.301PN–H(β) − 29.318EHOMO + 115.504ELUMO
+ 12.042ω − 1.345µ + 2.122 log P − 23.942
n = 11; R2 = 0.828; Sreg = 0.291;
r = 0.530; Fstatistic = 1.203
(2)
where, n is the number of compounds, r is the correlation
coefficient, R2 is the determination coefficient of regression,
Sreg is the standard deviation of regression and Fstatistic is the
1 3

J IRAN CHEM SOC
Table 6 VIFa values of theoretical QSAR equations
EHOMO (r = +0.692), EHOMO and PN–H(α) (r = −0.670),
with EHOMO and logP (r = +0.627), respectively.
The effect of hydrophobicity and polarizability in mod-
ulating inhibition activity against AChE enzyme may be
due to the presence of phosphoryl (Pδ+–Oδ−), carbonyl
(Cδ+–Oδ−) and amine groups (Hδ+–Nδ−); where permanent
polarization was seen due to electronegativity difference
between the atoms.
Independent AChE
variable
Urease
Equation (2) Equation (3) Equation (4) Equation (5)
QN(α)
QN(β)
PN–H(α)
PN–H(β)
EHOMO
ELUMO
ω
372.737
1.820
134.265
1.996
3.438
1.891
1.827
2.823
4.655
7.474
7.836
318.146
67.465
9.092
269.233
71.560
217.570
4.045
4.668
1.507
7.370
Urease‑QSAR
69.705
1.010
4.856
Also the interaction of urease as a secondary target was inves-
tigated against the tested compounds (1–17) and the same
procedures were carried out. The QSAR modification model
based on anti-urease potency produced the following equation:
μ
121.108
103.360
5.432
6.286
152.001
109.226
3.042
4.045
logP
VIF = 1/(1 − R2i ); where, Ri is the multiple correlation coefficient
of the ith independent variable on all of the other independent vari-
ables
a
log(1/IC50) = +12.765QN(α) + 0.678QN(β) + 7.064PN–H(α)
− 1.040PN–H(β) − 28.525EHOMO − 22.165ELUMO
− 77.456ω − 0.418µ − 0.283 log P + 4.668
n = 12; R2 = 0.960; Sreg = 0.237;
increasing of R2 = 0.822 and decreasing of Sreg = 0.292
(Eq. 3).
log(1/IC ) = +1.002Q
N(β)
50
+ 20.819P
− 1.520P
N–H(α)
N–H(β)
− 0.383µ + 0.036 log P − 17.384
= 1.324
r = 0.168; Fstatistic = 5.312
(4)
+ 12.071E
+ 47.292E
LUMO
HOMO
As a result, as shown in Table 6, the regression equa-
tion is not favorable with the VIF >10. The improvement
in Eq. (4) was performed by excluding PN–H(α) and ω. Con-
sequently, a new multiple regression was resulted without
colinearity (Eq. 5).
2
n = 10; R = 0.822; S = 0.292; r = 0.495; F
reg
statistic
(3)
The correlating parameters have VIF <10, thus there
is no colinearity problem (Table 6). In this equation, the
inhibitory potency of AChE is influenced mainly by the
electronic parameters (as frontier molecular orbital ener-
gies). ELUMO with the coefficient value of +47.292 has the
highest contribution to log(1/IC50) rather than the structural
parameters. The positive sings of ELUMO in log(1/IC50)
disclose that the compound with lower molecular orbital
(ELUMO) is indicative of higher toxicity against AChE
enzyme. The correlation matrix was used to determine
the interrelationship between the independent variables
(Table 7). Table 7 shows that the majority of regression
coefficients among; were higher than 0.70, showing that
they were closely correlated. Therefore, orthogonalization
of the molecular descriptors was conducted [28]. Orthogo-
nalization of molecular descriptors is undertaken to avoid
collinearity among variables and model overfitting. The
high interrelationships were observed between ELUMO and
log(1/IC ) = +7.213Q
50
+ 0.694Q
− 2.186P
N(β) N–H(β)
N(α)
− 3.165E
+ 50.169E
− 0.263µ
HOMO
LUMO
− 0.576 log P + 10.652
2
n = 12; R = 0.947; S = 0.193;
reg
(5)
r = 0.020; F
= 10.179
statistic
The model described by Eq. (5), similar to Eq. (3),
depicts the share of molecular orbital energy variables in
the inhibition of urease. The most effective variable in the
interaction of urease and PAH derivatives was ELUMO with
the coefficient value of +50.169. The high interrelation-
ship in Table 8 (r = +0.727) between ELUMO descriptors
and PN–H(β) showed that the properties of derivatives affect
in the inhibition of urease. It means that polarizability and
Table 7 Correlation matrix for
AChE-QSAR in Eq. (2)
Selected variables
QN(β)
1.000
PN–H(α)
PN–H(β)
EHOMO
ELUMO
μ
logP
QN(β)
PN–H(α)
PN–H(β)
EHOMO
ELUMO
μ
0.076
−0.300
−0.242
−0.451
−0.081
0.077
1.000
0.192
1.000
0.107
−0.670
−0.472
−0.045
−0.482
1.000
0.692
0.528
0.647
0.325
1.000
0.661
0.033
1.000
−0.045
logP
−0.372
−0.670
1.000
1 3

J IRAN CHEM SOC
Table 8 Correlation matrix for
urease-QSAR in Eq. (5)
Selected variables
QN(α)
1.000
QN(β)
PN–H(β)
EHOMO
ELUMO
μ
logP
QN(α)
QN(β)
PN–H(β)
EHOMO
ELUMO
μ
0.455
−0.785
−0.089
−0.535
−0.157
0.352
1.000
−0.526
−0.309
−0.599
−0.242
0.046
1.000
0.217
1.000
0.506
0.490
0.417
0.727
1.000
0.687
0.384
0.318
1.000
0.662
logP
−0.150
1.000
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Conclusion
New (thio)phosphorhydrazide compounds (1–17) were
synthesized and characterized by spectroscopy methods.
The cyclic motifs of compound 2 were further determined
by X-ray crystallography. The inhibitory potencies of the
selected compounds against human AChE and urease were
studied. PCA–QSAR was deduced that the electronic param-
eters are predominated from those related to structural
parameters. DFT–QSAR models for AChE (R2 = 0.822
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enzymes. The correlation matrix of QSAR models and dock-
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Acknowledgments The financial support of Tarbiat Modares Uni-
versity’s Research Council is gratefully acknowledged.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea-
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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