A convenient method for the synthesis of phosphorothioates and their anticholinesterase activites

Article abstract of DOI:10.1080/10426500490422245

A simple, efficient, and general method has been developed for the synthesis of phosphorothioates through a one-pot reaction of alkyl halides with the mixture of diethyl phosphite in the presence of NH₄OAc/S/CaO under solvent-free conditions. T

Full text of DOI:10.1080/10426500490422245

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Phosphorus, Sulfur, and Silicon and the Related
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A CONVENIENT METHOD FOR THE SYNTHESIS
OF PHOSPHOROTHIOATES AND THEIR
ANTICHOLINESTERASE ACTIVITIES
a
a
a
Babak Kaboudin , Saeed Emadi & Hamid Norouzi
a
Institute for Advanced Studies in Basic Sciences , Zanjan, Iran
Published online: 12 Aug 2010.
To cite this article: Babak Kaboudin , Saeed Emadi & Hamid Norouzi (2004) A CONVENIENT METHOD FOR THE SYNTHESIS OF
PHOSPHOROTHIOATES AND THEIR ANTICHOLINESTERASE ACTIVITIES, Phosphorus, Sulfur, and Silicon and the Related Elements,
179:3, 585-590, DOI: 10.1080/10426500490422245
To link to this article: http://dx.doi.org/10.1080/10426500490422245
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Phosphorus, Sulfur, and Silicon, 179:585–590, 2004
Copyright ꢀC Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500490422245
A CONVENIENT METHOD FOR THE SYNTHESIS
OF PHOSPHOROTHIOATES AND THEIR
ANTICHOLINESTERASE ACTIVITIES
Babak Kaboudin, Saeed Emadi, and Hamid Norouzi
Institute for Advanced Studies in Basic Sciences, Zanjan, Iran
(Received September 9, 2003)
A simple, efficient, and general method has been developed for the
synthesis of phosphorothioates through a one-pot reaction of alkyl
halides with the mixture of diethyl phosphite in the presence of
NH OAc/S/CaO under solvent–free conditions. The anticholinesterase
4
activities of eight different phosphorothioates were investigated on
acetylcholinesterase from electric eel.
Keywords: Ammonium acetate; anticholinesterase activity; calcium
oxide; phosphorothioate; sulfur
Phosphorothioates have found a wide range of application in the ar-
eas of industrial, agricultural, and medicinal chemistry owing to their
biological and physical properties as well as their utility as synthetic
1
intermediates. One of the useful properties of phosphorothioate com-
pounds is their relatively low stability and rapid metabolic breakdown
in plants, in animal organism, in soil, and in other components of
the environment with the formation of products that are safe for hu-
man beings and domestic animals. Phosphorothioates have been pre-
pared as pesticides and thio-analogues of biologically active phosphoric
2
mono- and diesters. In recent years a number of phosphorothioates
3
,4
have been introduced as potential chemotherapeutic agent and in-
5
hibitors of different enzymes. Despite their wide range of pharmaco-
logical activity, industrial and synthetic applications, the synthesis of
phosphorothioates has received little attention. The following methods,
We thank the Institute for Advanced Studies in Basic Sciences for supporting this
work. We are also grateful to Dr. E. Shams for helpful discussion and Mr. K. Navaee for
technical assistance.
Address correspondence to Babak Kaboudin, Department of Chemistry, Institute
for Advanced Studies in Basic Sciences (IASBS), Gava Zang, Zanjan 45195-159, Iran.
E-mail: kaboudin@iasbs.ac.ir
5
85

5
86
B. Kaboudin et al.
not generally applicable, have been reported in the literature: (i) reac-
6
7
tion of dialkyl phosphites with sulfenyl chlorides, sulfenyl cyanides,
8
9
10
thiosulfonates, disulfides, and sulfur, (ii) condensation of phospho-
rchloridate with thiols,11 and (iii) treatment of phosphites with thiols
in the presence of tellurium chloride in a redox-type reaction. How-
ever, all of these methods have problems, including drastic reaction
conditions and also some severe side reactions. In recent years the use
of reagents and catalysts immobilised on solid supports has received
considerable attention. Such reagents not only simplify purification
processes but also help prevent release of reaction residues into the
1
2
environment. We recently reported a novel synthetic preparation of
phosphorothioates by reaction of diethyl phosphite with alkyl halides in
the presence of a mixture of ammonium acetate/sulfur/alumina under
1
3
solvent-free conditions using microwave irradiation. This methodol-
ogy prompted us to try the use of other metal oxides in place of the acidic
alumina in the above reaction for the preparation of phosphorothioates.
We found that replacing of alumina with calcium oxide is a convenient
solid media that does not need microwave irradiation in first step of
above reaction. We here report that diethyl phosphite in the presence
of ammonium acetate/sulfur/calcium oxide under solvent–free condi-
tions is a convenient reagent for the preparation of phosphorothioates
(
Scheme 1). The anticholinesterase activities of eight different phos-
phorothioates were investigated on acetylcholinesterase from electric
eel.
SCHEME 1
As shown in Table I, a wide range of alkyl halides in the presence of
a mixture of ammonium acetate/sulfur/calcium oxide and diethyl phos-
phite, gives the required products 2 in excellent yields. The reactions
were clean with no tar formation. Ammonium formate (NH4O2CH) is
not as effective as ammonium acetate and gave low yields of the re-
quired product. Other ammonium salts (NH4Cl, NH4Br, NH4PF6) are
not effective and did not give any product.
In summary, a simple work-up, low consumption of solvent, fast re-
action rates, mild reaction condition, good yields, relatively clean with
no tar formation in the course of the reaction make this method an at-
tractive and a useful contribution to present methodologies. Indeed, a

Phosphorothioates
587
TABLE I Reaction of Alkyl Halides in the Presence of
a Mixture of Ammonium Acetate/Sulfur/Calcium Oxide
and diethyl Phosphite, under Solvent-Free Conditions
Using Microwave Irradiation
Reaction time
2
R
X
(min)
Yield %a
a
a
b
c
d
e
f
PhCH2
PhCH2
Ph2CH
Br
Cl
Br
Br
Br
Cl
Cl
Cl
Cl
2
2
3
2
2
2
5
3
2
80
83
73
65
80
78
76
60
73
PhCH2CH2
p-NO2C6H4CH2
p-MeC6H4CH2
o-MeC6H4CH2
m-MeC6H4CH2
1-C10H7CH2
g
h
aIsolated yields.
wide range of alkyl halides was converted to the corresponding phos-
phorothioates using this method.
ANTICHOLINESTEREASE PROPERTIES
Acetylcholinesterase is the most widely used target enzyme in stud-
ies with the purpose to synthesize new and more effective therapeutic
agents to treat patients with diseases such as Alzheimer’s disease or
myasthenia gravis.1
4−16
Also, acetylcholinesterease is the most widely
used target in the studies of compounds with insecticidal activities,
as all commercial organophosphate compounds are believed to exert
2
b
their effects through inhibition of the enzyme. Assays showed that all
compounds inhibited the enzyme to different degrees and these results
enhanced the importance of the introduced protocol for the synthesis of
new phosphorothioates.
Results of the anticholinesterease assay of eight phosphorothioate
derivatives are summarized in Table II. The range of inhibitory activ-
ities varies from 30.2–77.5% for 2a–2h. The results show that com-
pounds containing two aromatic rings in their structure (2b and 2h)
have greatest inhibition in comparison with other compounds listed in
Table I. This occurs through favorable binding of these compounds with
1
7
the active site residues of the enzyme through π-π interactions. Other
derivatives of phosphorothioates displayed different levels of inhibition
of acetylcholinesterease at the same concentrations. The minimum in-
hibitory property was recorded with compound 2f having a nitro group

5
88
B. Kaboudin et al.
TABLE II Anticholinesterease Activity of Some
Phosphorothioates 2a–h
2
R
Activity remained (%)a
Inhibition (%)b
a
b
c
d
e
f
PhCH2
Ph2CH
PhCH2CH2
p-NO2C6H4CH2
p-MeC6H4CH2
o-MeC6H4CH2
m-MeC6H4CH2
1-C10H7CH2
62.6
22.5
65.6
69.8
52.3
55.5
58.5
41.4
37.4
77.5
34.4
30.2
47.7
44.5
41.5
58.6
g
h
aThe activities were compared to the activity of a control and expressed
as percent inhibition.
bResults shown are averages of triplicate experiments.
in the para position of the aromatic ring (30.2%). Other compounds
inhibit the enzyme in ranges between compounds having two aromatic
ring structures and compound 2d having one aromatic ring with a para
nitro group on it.
DATA CONCLUSIONS
With the use of the proposed method, we have synthesized phos-
phorothioate compounds shown to have inhibitory effects on acetyl-
cholinesterease. It seems that hydrophobic π-π interactions play the
central role in showing the highest inhibitory activities of the com-
pounds synthesized in this study.
EXPERMENTAL
All chemicals were commercial products and distilled or recrystallized
before use. Acetylcholinesterease from electric eel (EC 3.1.1.7), actetyl-
choline chloride and gelatin were from Sigma and were used with-
out further treatment. A commercially available pulse microwave at
2
450 MHz (900 W) was used in all experiments. The infrared (IR)
spectra were determined using a FT-IR Brucker-Vector 22. NMR spec-
tra were taken with a DMX-500 Bruker Advance instrument 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). Merck Silica-gel 60 F254 plates (No. 5744)
were used for the preparative TLC. Acetylcholinesterease activity was

Phosphorothioates
589
measured titrimetrically18 using an automatic dispenser (765 Dosimat,
Metrohm), a pH meter (713 Metrohm) and at controlled temperature
◦
(
25 C), by the use of a high-precision temperature-controlled water
pump (Multi Temp III Pharmacia).
General Procedure for the Synthesis
of Phosphorothioates
This solvent-free reaction method is operationally simple. 10 mmol
of the reagent were prepared by the combination of ammonium ac-
etate (10 mmol, finely ground), sulfur (10 mmol) and calcium oxide
(
CaO, 2.5 g) in a mortar and pestle by grinding them together until
a fine, homogeneous, powder was obtained (5–10 min). Diethyl phos-
phite (10 mmol) was added to this mixture and the mixture was shaken
for 10 min. The alkyl halide (9 mmol) was added to this reagent (solid
alkyl halides need to be grinded) and was irradiated by microwave for
2–5 min using 600 W (A kitchen-type microwave was used in all ex-
periments). Chromatography through a plug of silica gel with EtOAc/n-
hexane (1:9 to 5:5) and evaporation of the solvent under reduced pres-
sure gave the pure products as oils in 60–83% yields. All products
gave satisfactory spectral data in accord with the assigned structures.
1
For 2a (S-Benzyl O, O-Diethyl Phosphorothioate) as an example H-
NMR (CDCl3/TMS-500 MHz): 1.31 (6H, t, J = 7.1 Hz), 4.01–4.25 (6H,
m), 7.27–7.29 (5H, m); 31P-NMR (CDCl3/H3PO4-85%): 27.05 ppm; C-
NMR: 16.39, 35.4, 63.9, 128.0, 129.1, 137.9; IR (neat): 1260 (P O),
13
−
1
1
162(P O Et) cm .
ANTICHOLINESTEREASE ACTIVITIES
Acetylcholinesterease activity was measured titrimetrically.18 The as-
say was carried out in potassium phosphate buffer (pH 7.0). A solution
of 7.5 mL of acetylcholine chloride (0.005 M), 7.5 mL of salt-gelatin
mixture [containing sodium chloride (0.2 M) and magnesium chloride
◦
(
0.04 M) in aqueous 0.01% gelatin] was added to a dual wall cell at 25 C.
This solution was stirred continuously to ensure complete mixing and
−
9
thermal exchange. After this step, the enzyme (100 µL, 7.7 × 10 M)
was added to the mixture. In order to titrate the acetic acid produced by
enzymatic reaction, NaOH (0.15 M) was added in micro liter amounts
with Dosimat to maintain the pH of the mixture at 7.0 for 5 min. Ac-
tivities were then measured according to the following formula:
Units/mg = [mL base added/min × base molarity × 1000]/[mg en-
zyme in reaction mixture]

5
90
B. Kaboudin et al.
Phosphorothioates were dissolved in methanol and then added as
the final ingredients before addition of the enzyme. The maximum
concentration of methanol in the reaction mixture was 1%.
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