Synthesis of α-oxycarbanilinophosphonates and their anticholinesterase activities: the most potent derivative is bound to the peripheral site of acetylcholinesterase

Article abstract of DOI:10.3109/14756366.2012.663362

A novel method has been developed for the synthesis of α- oxycarbanilino phosphonates through a reaction of α-hydroxyphosphonates with isocyanate under microwave irradiation. The synthesized compounds were evaluated for their acetylcholinesterase (AChE) inhibition potency through IC50determination. Molecular modelling studies suggest that the most potent inhibitor (compound 4h, IC50 = 6.36 M) is bound to the peripheral site of AChE, which suggests that it decreases the catalytic activity not through binding to the active site but through blocking the entrance of the active site gorge. This puts forward the potential of compound 4h and its derivatives to be used in the design of dual inhibitors: inhibition of the catalytic activity of AChE and of amyloid β aggregation.

Full text of DOI:10.3109/14756366.2012.663362

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 576–582
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.663362
RESEARCH ARTICLE
Synthesis of α-oxycarbanilinophosphonates and their
anticholinesterase activities: the most potent derivative is
bound to the peripheral site of acetylcholinesterase
Babak Kaboudin1, Saeed Emadi2, Mohammad Reza Faghihi1, Maryam Fallahi1, and
Vahid Sheikh-Hasani2
1Department of Chemistry and 2Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences
(IASBS), Gava Zang, Zanjan, Iran
Abstract
A novel method has been developed for the synthesis of α-oxycarbanilino phosphonates through a reaction of
α-hydroxyphosphonates with isocyanate under microwave irradiation. The synthesized compounds were evaluated
for their acetylcholinesterase (AChE) inhibition potency through IC50determination. Molecular modelling studies
suggest that the most potent inhibitor (compound 4h, IC50 = 6.36 µM) is bound to the peripheral site of AChE, which
suggests that it decreases the catalytic activity not through binding to the active site but through blocking the
entrance of the active site gorge. This puts forward the potential of compound 4h and its derivatives to be used in
the design of dual inhibitors: inhibition of the catalytic activity of AChE and of amyloid β aggregation.
Keywords: Organophosphates, α-oxycarbanilinophosphonates, α-hydroxyophosphonates, AChE inhibitors,
peripheral site of AChE, molecular modelling
Introduction
AChE by anti-AD drugs such as tacrine10, donepezil11,
and huperzine12 increases the ACh levels and improves
neurotransmission in cholinergic synapses13. AChE is
also used widely as a target in the studies of compounds
with insecticidal activities, as all commercially available
organophosphate compounds are believed to exert their
effects through potent reversible or irreversible inhibi-
tion of this enzyme14. ere are two major binding sites
in AChE, a narrow active site gorge, at the base of which
is located the catalytic triad or acylation site (A-site), con-
sisting of H447, E334, and S203 (Electrophorus electricus
AChE numbering). e other binding site is peripheral
anionic site (PAS) which spans from residue W286, at the
mouth of the gorge, to D74, at the boundary between PAS
and A-Site15,16. Inhibitors of AChE bind to A-site (organo-
phosphates14, rivastigmine17, tacrine10) or PAS (prop-
idium18) or to both of them (donepezil11) in AChE.
Phosphonic acids are of growing importance in under-
standingandmodulatingbiologicalprocesses1–3.Inrecent
years, the synthesis of α-substituted phosphoryl deriva-
tives (phosphonic and phosphinic acids) have attracted
significant attentions, due to their biological activities
with broad application as enzyme inhibitors, antime-
tabolites and antibiotics2. Among α-functionalized phos-
phonic acids, α-hydroxyphosphonic derivatives have
potential biological activities such as anti-bacterial4,
herbicidal5 and fungicidal6.
Acetylcholinesterase (AChE) hydrolyzes the neu-
rotransmitter acetylcholine (ACh) and is the most widely
used target enzyme in studies with the purpose to synthe-
size new and more effective therapeutic agents to treat
patients with diseases such as Alzheimer’s disease (AD)
or myasthenia gravis7–9. Reversible inhibition of brain
Address for Correspondence: Prof. Babak Kaboudin, Department of Chemistry, Institute for Advanced Studies in Basic Sciences (IASBS),
Gava Zang, Zanjan 45137–66731, Iran. Tel: +98 241 4153220. Fax: +98 241 4153232. E-mail: kaboudin@iasbs.ac.ir; Dr. Saeed Emadi,
Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Gava Zang, Zanjan 45137–66731, Iran.
Tel. +98 241 4153005. Fax: +98 241 4153004. E-mail: emadi@iasbs.ac.ir
(Received 15 October 2011; revised 15 January 2012; accepted 17 January 2012)
576

Synthesis of α-oxycarbanilinophosphonates 577
e carbamates have attracted lots of attention due
to applications such as elastase and cholinesterase inhi-
bition19. ey are derived from carbamic acid and are
insecticidal in a similar fashion as organophosphates and
are widely used in homes, gardens and agriculture. ey
inhibit cholinesterase enzymes (acetyl- and butyrylcholin-
esterases), and terminate nerve impulse transmission. Due
to relatively low mammalian oral and dermal toxicity, they
have found wide application in lawn and garden settings.
Besides the above applications, recent studies have
shown that a number of organophosphates could also
be useful as potential chemotherapeutic agents20,21 and
inhibitors of different enzymes22. Although, because
of these applications and potential usages, extensive
studies on the synthesis of 1-hydroxyphosphonates and
carbamate derivatives have been undertaken, relatively
few papers have been published on the synthesis of the
compounds containing two functionalities in the same
molecule, i.e. organophosphates and carbamates23. e
conjunction of two functionalities would presumably
afford more versatile biological activity and specifically,
less powerful inhibition of AChE, which would make
them beneficial in designing new drugs to be used in the
treatment of diseases such as AD or myasthenia gravis
(Scheme 1).
Despite wide range of biological activities of
1-hydroxyphosphonates and carbamate derivatives, the
synthesis and study of anticholinesterase activities of
novel α-oxycarbanilinophosphonates have received lit-
tle attention. To the best of our knowledge, only one syn-
thetic route to α-oxycarbanilinophosphonates has been
reported. e method involves prolonged heating (3
days) of α-hydroxyphosphonates with phenylisocyanate
at 50°C in the presence of tin octanoate as a catalyst23.
However, the method has problems including harsh
reaction conditions, low yields, long reaction times,
using of Lewis acid and side reactions. On the other
hand without using tin octanoate as catalyst, N-phenyl
isocyanurates has been obtained as the major product.
Recently we have reported a novel method using ultra-
sound energy for the one-pot synthesis of α-oxycarbanil
inophosphonates24.
microwave-assisted methodology for the synthesis of
organic compounds26–28, hereby we report a new method
for the synthesis of α-oxycarbanilinophosphonates from
the reaction of 1-hydroxyphosphonate with isocyanate
under microwave irradiation, producing good to high
yields of α-oxycarbanilinophosphonates.
In the continuation of our studies on the synthesis
and biological activities of novel organophosphates29,30
,
we decided to investigate to what extent the inhibitory
effect of the synthesized α-oxycarbanilinophosphonates
toward electric eel AChE would be affected upon the
inclusion of different substitutions in the R and R’ groups.
We have shown, through molecular modelling, the most
potent synthesized α-oxycarbanilinophosphonates
inhibits AChE through binding to the peripheral site and
not to the catalytic site of the enzyme.
Materials and methods
General
Chemicals were either prepared in our laboratories or
purchased from Merck, Fluka, and Aldrich Chemical
Companies. All yields refer to isolated products. NMR
spectra were taken by a 250 Brucker with the chemical
shifts being reported as δ ppm and couplings expressed
in Hertz. Silica gel column chromatography was carried
out with Silica gel 100 (Merck No. 10184). UV spectra
were determined on a Varian Cary 100 instrument. A
microwave reactor (Milestone, Micro SYNTH Microwave
Labstation for Synthesis) was used for all experiments
with a continuous power source (1000 W max).
General procedure for the synthesis of diethyl
(2-4-dichlorophenyl) hydroxymethylphosphonate (2h)
is compound was obtained according to the method
of our previously published article31. Magnesium oxide
(2 g) was added to a stirred mixture of diethyl phosphite
(20 mol) and 2,4-dichlhorobenzaldehyde (20 mol) at
room temperature. After 2 h, the mixture was washed
with dichloromethane (4 × 50 mL), dried with CaCl2, and
solvent evaporated to give crude product. Product was
crystallized from EtOAc/n-hexane.
e application of microwave energy to accelerate
organic reactions is of increasing interest and offers
several advantages over conventional techniques25.
Syntheses that normally require lengthy periods
can be achieved conveniently and very rapidly in a
microwave oven. As part of our efforts to explore the
mp 92–94°C (n-hexane-EtOAc) (Lit. report: 90–91°C)31.
1H NMR (CDCl3, 250 MHz): δ = 1.16 (3H, t, J = 7.0 Hz), 1.36
(3H, t, J = 7.0 Hz), 3.90–4.28 (5H, m), 5.83 (1H, d, JHP
=
19.5 Hz), 7.15-7.34 (3H, m); 13C NMR (CDCl3, 62.9 MHz):
δ = 16.2, 16.4, 63.1 (d, JPC = 6.9 Hz), 63.2 (d, JPC = 6.9 Hz),
68.8 (d, JPC = 158.1), 128.5, 129.6, 129.7, 132.32; 31P NMR
(CDCl3/H3PO4, 101 MHz): δ = 20.80.
General procedure for the synthesis of diethyl[(3-
chloro-4-methylanilinocarbonyl)oxy](2,4-dichloro
phenyl) methyl phosphonate (4h)
Diethyl (2-4-dichlorophenyl) hydroxymethylphospho-
nate 2h (2 mmol) was added to dry toluene (8 mL) under
argon. 3-Chloro-4-methylisocyanate (3 mmol) was
added stepwise for 45 min to the reaction mixture at 50°C
at ambient pressure in a microwave reactor. e reaction
Scheme 1. Structure of 1-hydroxyphosphonates and α-oxycarba
nilinophosphonate.
© 2013 Informa UK, Ltd.

578 B. Kaboudin et al.
mixture was stirred for 45 min under microwave irradia-
tion. e solvent was evaporated and the crude product
was chromatographed on silica gel (n-hexane:EtOAc =
40/60) to give the pure product in 80% isolated yield.
mp 146–148°C (n-hexane-EtOAc). 1H NMR (CD3SOCD3,
250 MHz): δ = 1.09–1.25 (6H, m), 2.21 (3H, S), 3.96-4.15
(4H, m), 6.67 (1H, d, JHP = 18.8 Hz), 7.20–7.56 (6H, m), 10.17
(1H, br, NH); 13C NMR (CD3SOCD3, 62.9 MHz): δ = 16.4,
16.6, 19.2, 63.7 (d, JPC = 6.9 Hz), 63.9 (d, JPC = 6.9 Hz), 68.5 (d,
Scheme 2. Synthesis of 1-hydroxyphosphonates.
afford diethyl 1-hydroxyphenylmethylphosphonate 2a
in 85% isolated yield after 2 h. When the phenylisocya-
nate was added to diethyl 1-hydroxyphenylmethylphos-
phonate 2a in toluene, it afforded the corresponding
α-oxycarbanilinophosphonate 4a in 88% isolated yield
under microwave irradiation for 45 min. ese results
prompted us to extend this process to other 1-hydroxy-
phosphonates and isocyanates. Interestingly, 1-hydroxy-
phosphonates reacted smoothly with isocyanates under
microwave irradiation to produce the corresponding
α-oxycarbanilinophosphonates in good yields (Table 1
and Scheme 3). As shown in Table 1, treatment of diethyl
1-hydroxyphenylmethylphosphonate (2a) with phenyl-
isocyanate afforded diethyl[(anilinocarbonyl)oxy](phe-
nyl)methylphosphonate (4a) in 88 % isolated yield. Other
different substituted 1-hydroxyarylmethylphosphonates
also reacted with phenylisocyanate to give the desired
compounds (4b–4e) in good yields. Treatment of diethyl
1-hydroxy-3-(2-nitrophenyl)allyl phosphonate (2f), as
an α,β-unsaturated 1-hydroxyphosphonate, also reacted
with phenylisocyanate under ultrasonic irradiation to
give the desired compound 4f in 71% yield. e reaction
of diethyl (2-thiophenyl) hydroxymethylphosphonate
(2g), as a heterocyclic 1-hydroxyphosphonate, with phe-
nyl isocyanate gave 78% desired product 4g. e reaction
of substituted isocyanates with 1-hydroxyphosphonate
gave corresponding α-oxycarbanilinophosphonate 4h
and 4i in good yields. e reaction of cyclohexylisocya-
nate, as an aliphatic isocyanate with diethyl 1-hydroxy-
phenylmethylphosphonate 2a gave the desired product
4j in 80% isolated yield.Diethyl 1-hydroxy-1-naphthyl-
methyl phosphonate (2k), as polynuclear 1-hydroxyaryl-
methylphosphonate, also reacted with benzylisocyanate
under microwave irradiation, gave the desired com-
pound 4l in 86 % isolated yield.
J
PC = 173.6 Hz), 117.6, 118.7, 129.5, 130.4, 131.5, 131.7, 133.6,
137.6, 152.3; 31P NMR (CDCl3/H3PO4, 101 MHz): δ = 16.47.
Anal. Calcd for C19H21NCl3O5P. C, 47.60; H, 4.42; N, 2.92%.
Found: C, 47.48; H, 4.28; N, 2.81%.
Biochemical methods: AChE inhibition assay
Inhibitory activities of synthesized α-oxycarbanilinophos-
phonates were determined at 25°C by the colorimetric
method of Ellman et al.32. e assay solution contained
0.1 M phosphate buffer, pH 8, 0.3 mM 5,5’-dithiobis(2-
nitrobenzoic acid) (DTNB), 0.125 units of AChE (Sigma
Chemical Co. from electric eel (Electrophorous electricus)),
and 0.2 mM acetylthiocholine iodide as the substrate in a
total volume of 2.5 mL. In order to provide sufficient time
for interaction with either PAS and/or active site of the
enzyme, particularly in competition with the substrate, the
synthesized compounds were added to the assay solution
and preincubated with the enzyme for 10 min at 25°C. In
order to solubilize the synthesized compounds, the reac-
tion mixture contained 2% ethanol (50 µL in 2.5 mL reac-
tion mixture volume). Control experiments were run and
it was seen that no inhibitory effect was exerted on the
enzyme by ethanol. e changes in absorbance at 412 nm
were recorded for 5 min with a Camspec 501 Single Beam
Scanning UV/Vis Spectrophotometer. Percent inhibi-
tions were calculated and the concentration of the com-
pounds that produced 50% inhibition of AChE (IC50) was
determined through the use of Excel-Solver program. e
IC50’s are the result of three independent experiments and
reported as the average value SEM. e dose response
curves were fit by nonlinear regression.
Computer-aided molecular modelling
Molecular Docking was carried out using AutoDock 4.2
and AutoDockTools version 1.5.4 with standard param-
eters33. Crystal structure of EeAChE was obtained from
protein data bank (pdb code 1C2O) and ligand structure
was constructed and energy minimized using PRODRG
online server34. All calculations were performed with the
use of a PC with Intel Core 2 Quad 2.66 GHz Processor.
Images were generated with PMV33.
AChE inhibitory activity
Newly synthesized α-oxycarbanilinophosphonates were
assayed for AChE (Electrophorus electricus) inhibition
potency by the Ellman method32. e results are summa-
rized in Table 1.
As it can be seen in Table 1, oxycarbanilinophospho-
nates have generally shown three kinds of effects on the
target enzyme AChE: (1) no inhibition toward AChE
(compounds 4a–4c, 4e, 4g, and 4j–4k), (2) moderate
inhibitory effects (compounds 4d and 4l with IC50’s of
161.42 and 418.29 µM, respectively) and (3) the most
potent inhibitory effects (compounds 4f, 4h, and 4i with
IC50’s of 10.35, 6.36, and 7.47 µM, respectively).
In compound 4f there is no chemical group in the
para position of the aromatic ring and instead there is
Results and discussion
We have reported synthesis of α-hydroxyphosphonate
by the reaction of aldehyde with diethylphosphite in the
presence of magnesia (Scheme 2)31.
Initially, we carried out the reaction of benzaldehyde
1a with diethylphosphite in the presence of magnesia to
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of α-oxycarbanilinophosphonates 579
Table 1. Synthesis of novel α-oxycarbanilinophosphonates (4) and their anticholinesterase activities.
Entry
a
Product
Yielda %
Mp (°C)
117–119
31P NMR δ (ppm)
AChE IC50(µM)
No inhibition
88
18.82
b
c
82
85
131–133
152–154
18.54
18.95
No inhibition
No inhibition
d
e
f
81
79
71
78
80
135–137
128–130
137–139
138–140
146–148
18.65
19.08
16.90
17.22
16.90
161.42 14.25
No inhibition
10.35 1.16
g
h
No inhibition
6.363 0.31
i
79
165–167
16.90
7.47 0.12
j
80
77
86
97–99
19.36
17.90
18.74
No inhibition
No inhibition
418.29 29.56
k
l
108–110
112–114
aYields refer to the isolated pure products after column chromatography.
a nitro group in the ortho position. e IC50 of the com-
pound is 10.35 µM which is much lower than compound
4d and shows considerably higher inhibitory activity.
Nitro group has caused the aromatic ring to become
highly electron-deficient which makes it prepared to
interact through π–π interaction with another aromatic
ring system in the enzyme. Also the nitro group itself can
make H-bond with potential side chains in the enzyme.
© 2013 Informa UK, Ltd.

580 B. Kaboudin et al.
e fulfillment of these two potentials has presumably
made the IC50 of compound 4f that was much lower than
compound 4d.
oxygen of the carbamate group is also ready for a strong
H-bond because of the presence of a chlorine atom on
the proximal aromatic ring. ese two changes are pre-
sumably the reasons for its very low IC50 of 7.47 µM.
e highest inhibitory activity was obtained with com-
pound 4h with an IC50 of 6.36 µM. Although we have not
tried out to determine the inhibition mechanism, con-
sidering the fact that it can be deduced from molecular
modelling results that the mode of inhibition is of mixed
type inhibition, in the case of pure noncompetitive inhi-
bition, Ki would be equal to IC50, that is 6.36 µM35. is
deduced Ki of compound 4h is comparable to and in a
number of cases more potent than reported Ki’s for syn-
thetic anticholinesterases in the literature36–38. e pres-
ence of two chlorine atoms at ortho and para positions
of the aromatic ring has made the ring very electron-
deficient and consequently well prepared for potential
π–π interactions with aromatic side chains in the PAS.
Also the acyl oxygen of the carbamate group can poten-
tially make strong H-bond with an acceptor group in the
enzyme because of its orientation and also because of the
presence of a chlorine atom in the meta position of the
aromatic ring close to carbamate group (in the Molecular
modelling section we will explain more about the specific
interactions of this compound with the enzyme).
Molecular modelling studies
In order to understand better the mode of interaction
between the synthesized α-oxycarbanilinophosphonates
and AChE, we studied the interactions between com-
pound 4h and AChE through molecular modelling.
Compound 4h was the most potent inhibitor, and could
be considered as a representative member.
Our molecular docking calculations showed that
compound 4h could only interact with peripheral site of
AChE. e binding site was defined as a sphere around
the Trp 286 with an arbitrary distance that included Ser
203 in the active site, together with the fact that a num-
ber of interacting residues around the drug and the drug
itself were fully flexible. After 100 runs we observed that
Trp 286 and Tyr 341 could interact through π–π interac-
tions with both of the phenyl rings attached to phospho-
nate and carbamate moieties (Figure 1). As it is clear from
Figure 1, there are also other residues whose interactions
with the drug would be important in its proper orienta-
tion in the PAS. Asp 74 can interact electrostatically with
the partially positive charge of the phosphorous atom
and Tyr 72 may participate in another π–π interaction.
We can also observe from Figure 1 that the compound 4h
prefers to enter the gorge structure from its phenyl ring
proximal to the carbamate group. And the other phenyl
group proximal to the phosphonate group has been
remained in the outside of the gorge structure. Figure 2
is a surface polarizability map of AChE that shows tri-
dimensionally the drug and enzyme during binding
interactions, together with the degree of the polariz-
ability of the enzyme’s surface. e binding energies
showed that the Asp 74 could have a determining role in
making it favourable energetically the orientation of the
drug shown in Figure 2, where the Asp 74 is visible at the
Almost the same explanation as that provided for
compound 4h can also be provided for compound 2i.
Here, there is a nitro group at para position of the aro-
matic ring instead of two chlorine atoms at para and
ortho positions in compound 4h. e nitro group has also
caused the ring to become electron-deficient. e acylic
Scheme 3. Synthesis of α-oxycarbanilinophosphonates.
Figure 1. Stereo image presentation of peripheral site residues of the enzyme and Ser 203. e interacting residues are Trp 286 and Tyr
341. As it can be seen both of the phenyl rings of the compound 4h are interacting with the above-mentioned amino acid residues of the
PAS through π–π interactions. e interactions of other residues such as Asp 74 and Tyr 72 are important in the appropriate orientation of
the drug.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of α-oxycarbanilinophosphonates 581
Acknowledgments
e authors gratefully acknowledge support by the
Institute for Advanced Studies in Basic Sciences (IASBS)
Research Council under grant No. G20109IASBS120.
Declaration of interest
e authors report no conflicts of interest.
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Conclusions
We have synthesized a family of α-oxycarbanilinophos-
phonates through a new synthetic methodology and
have examined their inhibitory activities against electric
eel AChE. ese compounds have been synthesized by
a reaction of α-hydroxyophosphonates with isocyanate
under microwave irradiation. Variations in the aromatic
rings proximal to phosphonate and carbamate moieties
together with altering the distance between the aromatic
ring and phosphonate group led us to identify that pres-
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as nitro and chlorine, respectively, in the aromatic rings
enhanced the AChE inhibitory potency. Molecular mod-
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