Environmentally benign synthesis and antimicrobial study of novel chalcogenophosphates

Article abstract of DOI:10.1016/j.bmcl.2014.03.008

We report in this work an environmentally benign zinc mediated synthesis of aryl and benzyl phosphorochalcogenoates in ethanol within a short reaction time. In vitro antimicrobial study along with statistical analysis and seed germination assay were perfo

Full text of DOI:10.1016/j.bmcl.2014.03.008

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2198–2201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Environmentally benign synthesis and antimicrobial study of novel
chalcogenophosphates
Shubhanjan Mitra a, Sayani Mukherjee b, Sukanta K. Sen b, Alakananda Hajra a,
⇑
a Department of Chemistry, Visva-Bharati (A Central University), Santiniketan 731235, India
b Department of Botany, Visva-Bharati (A Central University), Santiniketan 731235, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report in this work an environmentally benign zinc mediated synthesis of aryl and benzyl phospho-
rochalcogenoates in ethanol within a short reaction time. In vitro antimicrobial study along with statis-
tical analysis and seed germination assay were performed. These chalcogenophosphates possess strong
antimicrobial activity against the reference strains. The antibacterial activity was determined against four
standard strains (Bacilus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa). The
antifungal activity was evaluated against one fungal strain Candida albicans.
Received 31 December 2013
Revised 27 February 2014
Accepted 4 March 2014
Available online 12 March 2014
Keywords:
Chalcogenophosphate
Green synthesis
Ó 2014 Elsevier Ltd. All rights reserved.
Antimicrobial activity
Statistical analysis
Seed germination assay
During the last few years, organochalcogen (Se, S) compounds
have been the subject of constant scientific interest due to its broad
spectrum of applications in the field of synthesis as well as medic-
inal biology. The organoselenium compounds found its eligibility
as anticancer,1,2 anti-oxidant,3–7 anti-inflammatory, anti-allergic
agents.8–11 Organosulfur compounds proved its efficiency in the
field of medicinal chemistry as many health benefits have been as-
cribed to them. They possess several biological activities including
antimicrobial,12,13 antiatherosclerotic,14,15 lipid and cholesterol
lowering effects,16–18 inhibition of carcinogenesis,19–24 enhancing
the immune system. On the other hand, organophosphorus com-
pounds have important and multifaceted functions in biochemistry
of living system. These are associated with antiviral,25 anticancer,26
antimicrobial27–30 activities. They have been found to play an
important role in various areas of functional material sciences31
and particularly, in catalysis as ligands.32 As both chalcogen and
phosphorous scaffolds show strong biological activities, we
thought that chalcogenophosphate moieties having both the chal-
cogen and phosphorus molecules might also show interesting
pharmaceutical activities. Therefore, we made an attempt to syn-
thesize various chalcogenophosphate moieties and seek for its bio-
logical activities. However, only a few approaches are in literature
to synthesize these compounds.33–36 The major limitations of these
reported methods are use of reagents sensitive to air and moisture
or long and harsh reaction conditions. Herein, we report an envi-
ronmentally benign method for the synthesis of some novel S-aryl
and Se-aryl phosphorochalcogenoates using zinc dust in ethanol
solvent at room temperature within a very short time period
(Scheme 1).
We made a further antimicrobial study of these scaffolds in or-
der to establish our assumption, against some bacteria as well as
fungi. Furthermore, a thorough statistical analysis and seed germi-
nation assay was also performed to make our study effective.
Initially we started the reaction with diphenyl diselenide
(0.5 mmol) and diethyl phosphite (1 mmol) in presence of catalytic
amount of zinc dust (0.2 mmol) under neat condition at room tem-
perature. The desired phenylselenophosphate was obtained in low
yield (16%) after 30 min (entry 1, Table 1). The yield of the reaction
did not improve after longer time stirring also (24 h). But the for-
mation of the desired product inspired us to proceed further.
Increasing the amount of zinc dust from 0.2 to 0.5 mmol, the prod-
uct yield increased from 16% to 41% (entry 2, Table 1). No signifi-
cant increment of yield was observed on increasing the
O
P
O
P
R
Zn dust
X
X
R
H
X
+
OR1
R
OR1
R1O
EtOH, rt
R1O
30 mins.
R = Ph, Bn
X = S, Se
⇑
Corresponding author. Tel./fax: +91 3463 261526.
E-mail address: alakananda.hajra@visva-bharati.ac.in (A. Hajra).
Scheme 1. Synthesis of arylphosphorochalcogenoates.
http://dx.doi.org/10.1016/j.bmcl.2014.03.008
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

S. Mitra et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2198–2201
2199
Table 1
Table 2
Substrate scope
Optimization of the reaction conditionsa
O
P
O
P
Zn dust (0.5 mmol)
CH3
CH2
O
X
R
X
H
CH3
O
O
Se
O
+
OR1
R
R
OR1
R1O
X
EtOH (2 mL)
P
R1O
Catalyst
HP
O
CH2
O
room temperature
Se
+
Se
30 mins.
Solvent, Temp.
0.5 mmol
1 mmol
H2C CH3
H2C CH3
30 mins.
3a
1
2
O
H2
C
Se
Entry
Catalyst (mmol)
Solvent
Temp (°C)
Yieldb (%)
P
CH3
CH2
O
O
O
Se
Se
P
O
O
P
CH3
1
2
3
4
5
6
7
8
Zn (0.2)
Zn (0.5)
Zn (0.5)
Zn (1.0)
Zn (0.5)
Zn (0.5)
Zn (0.5)
Zn (0.5)
Zn (0.5)
Zn (0.5)
Zn (0.5)
Zn (0.5)
ZnCl2 (0.5)
Zn(OTf)2 (0.5)
FeCl3 (0.5)
CuI (0.5)
Cu (0.5)
Fe (0.5)
—
—
—
—
—
rt
rt
80
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
16
41
43
44
35
50
30
25
42
60
20
80 (81)c
<5
<5
—
<5
18
27
—
O
O
CH2
O
CH3
H2C CH3
3a, 80%
3b, 75%
CH3
3c, 71%
DCM
DMSO
Toluene
CH3CN
PEG-400
H2O
Dioxane
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
H3C
CH3
H2C
CH3
CH3
CH3
CH3
O
CH3
O
O
O
Se
O
P
P
Se
CH2
P
O
9
O
O
O
H2C Se
10
11
12
13
14
15
16
17
18
19
CH3
CH2
H3C
3d, 84%
3e, 78%
3f, 79%
H3C
O
H2
C
S
O
O
P
O
S
H2
C
P
Se
CH2
CH3
O
O
O
P
O
CH3
CH2
O
H2C CH3
a
b
c
Diphenyl diselenide (0.5 mmol), diethyl phosphite (1 mmol), solvent 2 mL.
Isolated yield.
Reflux condition.
3g, 71%
3h, 68%
3i, 65%
a Isolated yield.
temperature from room temperature to 80 °C (entry 3, Table 1).
Further we increased the amount of zinc to 1 mmol and similar
yield (44%) was obtained (entry 4, Table 1). Next we varied the
reaction conditions with various protic and aprotic solvents (en-
tries 5–12, Table 1) using 0.5 mmol zinc dust. A remarkable varia-
tion in yield was observed on going from aprotic to protic solvents.
Maximum yield was obtained in ethanol (80%). After choosing eth-
anol as suitable solvent for this reaction, other catalysts were sur-
veyed (entries 13–16, Table 1). However, no improvement of yield
was observed on varying these Zn, Fe and Cu catalysts. Lower
yields were obtained in case of Cu and Fe dust also (entries 17
and 18, Table 1). The reaction did not proceed at all in absence of
Zn dust even after 24 h (entry 19, Table 1). Finally the optimization
of the reaction condition was done using 0.5 mmol zinc dust in eth-
anol solvent under room temperature for 30 min (entry 12,
Table 1).
Under the optimized reaction conditions, various chalcogeno-
phosphates were synthesized and the results are summarized in
Table 2. Both diphenyl diselenide and dibenzyl diselenide reacted
well under the present reaction conditions. A wide range of phos-
phites such as dimethyl phosphite, diethyl phosphite, diisopropyl
phosphite and dibenzyl phosphite were tested and all the phos-
phites produced moderate to high yields. Phenylthiophosphates
were also obtained in moderate yields (3h and 3i, Table 2).
A plausible reaction pathway is outlined in Scheme 2. Initially
Zn reacts with diphenyl dichalcogenide to form Zn(XPh)2.37,38
Probably Zn(XPh)2 reacts rapidly with dialkyl phosphite to form
the desired product at room temperature.
allowed to solidify and 0.1% cell suspension (106 CFU/mL) of test
organisms were spread uniformly and kept for 15 min. After solid-
ification, the wells (6 mm diameter) were prepared. The samples
(100 lg/mL) were dissolved in DMSO and poured into the wells
prepared in the respective plates. After 24 h of incubation at 30–
35 °C, the zone of inhibition was calculated and all experiments
were conducted in parallel sets of triplicate. The values calculated
as mean standard deviation. Data were analyzed by the analysis
of variance (ANOVA) using the Origin pro 8 statistical software
and the mean differences were separated using Turkey’s studen-
tized range test at the 1% level of probability.
The investigation of the antimicrobial screening data along with
the statistical analysis shown in Table 3 revealed that the synthe-
sized compounds showed promising results against the microor-
ganisms. For gram positive bacteria B. subtilis, compound 3d acts
as the most effective one and is mostly comparable to the effective-
ness of the marketed antibacterial drug chlorampenicol. The phe-
nylthiophosphates (3h and 3i) show the antibacterial activity
with insignificant variation upto 1% level. Statistically insignificant
variation is shown by compound 3d, 3g and the marketed drug
chlorampenicol, though compound 3d shows greater activity than
X
X
X
Zn
Zn
Antimicrobial activity of the synthesized compounds was as-
sessed in vitro against two gram positive bacteria Bacilus subtilis
(MTCC121) and Staphylococcus aureus (MTCC1430), two gram neg-
ative bacteria Escherichia coli (MTCC1610) and Pseudomonas aeru-
ginosa (MTCC424) and one fungus Candida albicans (MTCC227).
For antimicrobial study, agar diffusion as say cup method was
used. The agar plates were prepared by pouring 20 mL of molten
nutrient agar medium into sterile petri plates. The plates were
X = Se, S
2
O
P
OR
OR
X
O
X
OR
Zn
2
+
2 HP
OR
2
Zn (II)
Scheme 2. Plausible mechanism.

2200
S. Mitra et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2198–2201
Table 3
Inhibitory activity of the compounds 3a–i expressed as zone of inhibition (mm)a
Compound
Gram positive
Staphylococcus aureus
Gram negative
Escherichia coli
Fungus
Bacilus subtilis
P. aeruginosa
C. albicans
3a
3b
3c
3d
3e
3f
3g
3h
31.6 0.2a
22.1 0.3b
23.4 0.4c
33.1 0.3d
29.9 0.3e
21.8 0.3b
32.8 0.1d
20.6 0.3f
19.8 0.6f
33.4 0.3d
—
30.2 0.3a
28.4 0.3b
20.3 0.1c
30.5 0.4a
25.4 0.4dg
25.9 0.3d
31.8 0.5e
22.5 0.2f
25.1 0.1g
30.9 0.4a
—
24.3 0.5a
22.5 0.3b
27.3 0.2c
35.1 0.1d
30.2 0.6e
25.7 0.3f
22.6 0.4b
21.0 0.3g
22.9 0.1b
35.0 0.1d
—
32.1 0.4a
25.6 0.6b
24.3 0.3dc
33.4 0.2d
27.5 0.1e
26.9 0.4f
25.4 0.2bc
23.6 0.2c
25.4 0.3bc
33.6 0.2d
—
34.0 0.3a
28.3 0.7b
25.9 0.1c
38.6 0.6d
30.2 0.2e
27.4 0.5bc
30.5 0.3e
26.4 0.4c
28.1 0.1b
—
3i
Chlorampenicol
Nystatin
39.2 0.6d
Each value represents a mean standard deviation (SD) of three replications. Values followed by the same letter(s) in each column are not statistically different according to
Turkey’s test (P < 0.01).
a
The zone diameters have been calculated in mm by digital vernier caliper.
Figure 1. Seed germination assay of compound 3a and 3h on gram, pea and mung seeds.
drugs chlorampenicol as well as nystatin are not statistically differ-
ent by its activities upto 1% level of significance, which we found
mostly supportive by our assumption on the synthesized products.
After screening for the antimicrobial properties, further seed
germination experiments were carried out to investigate the ad-
verse effects of the Se and S containing reference compounds (3a
and 3h) on plant system. To perform the toxicity test, compounds
3a and 3h were taken for study on gram, pea and mung seeds along
with a control experiment. The seeds were kept in the solution of
the test compounds and the control seed batch was kept in water.
The seedling fresh weight and seedling length was calculated after
Figure 2. Seed germination assessment: seedling fresh weight.
3g. Compounds 3a and 3d varies insignificantly with chlorampeni-
col though compound 3g shows the highest activity against
S. aureus. Compound 3d shows the highest zone of inhibition and
is statistically insignificant with the clinical drug against the refer-
ence strain of E. coli. Similar conclusion can be drawn that com-
pound 3d shows the best result against another gram negative
bacterial strain P. aeruginosa. For the study of antifungal activity,
C. albicans was chosen and compound 3d shows almost compara-
ble activity with the clinical drug nystatin. From analysis of vari-
ance it can be concluded that compound 3d (selenophosphoric
acid O,O0-diisopropyl ester Se-phenyl ester) and the marketed
Figure 3. Seed germination assay: seedling length.

S. Mitra et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2198–2201
2201
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Figures 2 and 3 display the results of the seed germination as-
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A.H. acknowledges the financial support from CSIR, New Delhi
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Supplementary data
Supplementary data (characterization data and NMR spectra)
associated with this article can be found, in the online version, at
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