Silica-supported fluoroboric acid (HBF4-SiO2) catalyzed highly productive synthesis of thiomorpholides as activators of l-asparaginase as well as the antioxidant agent

Article abstract of DOI:10.1016/j.bmc.2010.03.033

An efficient solvent-free procedure for the synthesis of thiomorpholides in the presence of a catalytic amount of solid-supported fluoroboric acid (HBF₄-SiO₂) is described. The advantages of this method are high yields, short reaction times, ease of product isolation, low cost, and the catalyst can be recycled for a number of times without significant loss of activity. Three thiomorpholides possessing electron-donating group (4c, 4g, and 4h) were exhibiting excellent stimulatory activities against Erwinia carotovora l-asparaginase. The most potent activator, compound 4h displayed the following kinetic parameters, K_m = 75 μM and V_max = 1000 μmol mg^-1 min^-1 and K_A = 0.985 μM. Furthermore, these compounds (4g, 4h, 4c, 4f, 4a, and 4d) have also shown promising 2,2′-diphenyl-1-picrylhydrazyl (DPPH) reducing antioxidant activity (21-36%) at 1 mM concentration as compared to standard butylated hydroxyl anisole (72% at 1 mM).

Full text of DOI:10.1016/j.bmc.2010.03.033

Bioorganic & Medicinal Chemistry 18 (2010) 3618–3624
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Silica-supported fluoroboric acid (HBF4–SiO2) catalyzed highly productive
synthesis of thiomorpholides as activators of L-asparaginase as well as the
antioxidant agent
b
c
d
Babasaheb P. Bandgar a,b, , Shrikant S. Gawande , Suchita C. Warangkar , Jalinder V. Totre
*
a Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur 413 255, India
b Organic Chemistry Research Laboratory, School of Chemical Sciences, Swami Ramanand Teerth Marathawada University, Nanded 431 606, India
c Biochemistry Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathawada University, Nanded 431 606, India
d Institute for Drug Research, Department of Medicinal Chemistry and Natural Products, School of Pharmacy, The Hebrew University of Jerusalem, 91120, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient solvent-free procedure for the synthesis of thiomorpholides in the presence of a catalytic
amount of solid-supported fluoroboric acid (HBF4–SiO2) is described. The advantages of this method
are high yields, short reaction times, ease of product isolation, low cost, and the catalyst can be recycled
for a number of times without significant loss of activity. Three thiomorpholides possessing electron-
donating group (4c, 4g, and 4h) were exhibiting excellent stimulatory activities against Erwinia carotovo-
Received 25 February 2010
Revised 12 March 2010
Accepted 13 March 2010
Available online 18 March 2010
ra
Km = 75
L-asparaginase. The most potent activator, compound 4h displayed the following kinetic parameters,
Keywords:
Silica-supported fluoroboric acid
Thiomorpholide
Solvent-free conditions
Willgerodt–Kindler reaction
Erwinia carotovora
l
M and Vmax = 1000 l lM. Furthermore, these compounds (4g,
mol mgꢀ1 minꢀ1 and KA = 0.985
4h, 4c, 4f, 4a, and 4d) have also shown promising 2,20-diphenyl-1-picrylhydrazyl (DPPH) reducing anti-
oxidant activity (21–36%) at 1 mM concentration as compared to standard butylated hydroxyl anisole
(72% at 1 mM).
Ó 2010 Published by Elsevier Ltd.
L-Asparaginase
Antioxidant activity
1. Introduction
thesis have posed a serious threat to the environment.
Consequently, methods that successfully minimize their use are
the focus of much attention.13,14 In recent years, the use of cata-
lysts immobilized on solid supports has received considerable
attention.15–19 Such catalysts not only simplify the purification
process but also help in preventing the release of reaction residues
into the environment. Thus, the solvent-less condition along with
supported catalyst provides a protocol for achieving environment
friendly organic synthesis.
Thiomorpholides are of importance in medicinal chemistry1 due
to their biological activity, for example, against bacterial infection,
as fungicides, herbicides, and antiulcerative agents.2 Apart from
these applications, thioamides are known to be versatile intermedi-
ates in organic synthesis,3 particularly in the field of peptide
chemistry4 as well as building blocks for the synthesis of five- and
six-membered heterocycles.5 A traditional approach to their synthe-
sis is the Willgerodt–Kindler reaction which has found only limited
applications, because of the high reaction temperatures and long
reaction periods required, and the low to moderate yields obtained.6
Recently, the synthesis of thiomorpholide has been carried out using
ionic liquid7 and microwave.8 However, some of the above-men-
tioned limitations are sill associated with both methods such as high
temperature,9,10 long reaction time,11 expensive and large amount
of catalyst.12 To the best of our knowledge, uptil now the synthesis
of thiomorpholides using the solid-supported catalyst as well as
temperature lower than 100 °C has not been reported so far.
L
-Asparaginase (
lyzes the hydrolysis of
nia.20–24
-asparaginase from Escherichia coli and Erwinia
chrysanthemii were used as chemotherapeutics in Acute Lympho-
blastic Leukemia (ALL) for the past three decades.25 The
-asparagin-
L
-asparagine amino hydrolase, E.C.3.5.1.1) cata-
L
-asparagine into -aspartic acid and ammo-
L
L
L
ases from E. coli and Erwinia carotovora possess different
immunological specificities and offer an important alternative ther-
apy if a patient becomes hypersensitive to one of the enzyme.26 The
comparison of two enzymes leads to the conclusion that E. coli-aspar-
aginase can be recommended for first-line therapy, reserving Erwi-
nia-asparaginase for allergic patients, because most patients
allergic to the former are not immediately allergic to the latter.27 En-
zyme activation by thiol group or thio group is not an uncommon
event in enzymology. It was reported that thiol compounds such as
N-acetyl cysteine and glutathione, in a fixed concentration potentate
The toxic and volatile nature of many organic solvents, particu-
larly chlorinated hydrocarbons that are widely used in organic syn-
* Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.03.033

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 3618–3624
3619
O
O
N
CH3
HBF4-SiO2 (5 mol%)
H
N
O
S
2
800C
S
R
R
1
4
3
Scheme 1.
Table 1
Reaction of acetophenone with sulfur and morpholine under various conditions
the activity of asparaginase from Cylindrocarpon obtusisporum MB-
10,28 Pseudomonas stutzeri MB-405,29 and E. carotovora.30 For in-
stance,catalytic activitiesofcysteineproteasefromSchistosomaman-
Entry
Solvent
Catalyst (mol %)
Temp (°C)
Time [h]/min
Yield (%)
x
soni,31 N -phosphoarginine hydrolase from rat liver,32 reticulocytic
1
2
3
4
5
6
7
8
9
Neat
Neat
Neat
Neat
Neat
Neat
Neat
Neat
Neat
CH2Cl2
THF
—
—
rt
[48]
[30]
[3]
[2]
90
60
45
45
45
2
35
60
65
75
85
95
95
95
60
55
70
protein kinase,33 glyoxylase,34 the family C G-protein coupled recep-
tors, that is, the metotropic glutamate receptors were profoundly
accelerated by the addition of natural thiol compound.35 The molec-
ular mechanism behind the activation effects of enzymes by thiol
compounds or thio group has been due to the redox changes.36 Erwi-
nia asparaginase activation considered because of the presence and
involvement of free thio group in the enzyme catalysis.37
As reactive oxidative species (ROS) are involved in the patho-
genesis of many diseases, finding efficient antioxidants as thera-
peutic agents have been one of the hottest areas in biomedicine.
However, only relatively trivial success has been attained in anti-
oxidant-based drug discovery over this period. According to the
MDL Drug Data Report (MDDR) database, only two antioxidant
drugs have been launched (i.e., idebenone and edaravone) in the
past 30 years. Thus, it seems that it is urgent to improve the trans-
lationally efficiency of antioxidant research.
80
80
80
80
80
80
80
80
80
80
80
HBF4–SiO2 (1)
HBF4–SiO2 (2)
HBF4–SiO2 (3)
HBF4–SiO2 (4)
HBF4–SiO2 (5)
HBF4–SiO2 (7)
HBF4–SiO2 (10)
HBF4–SiO2 (5)
HBF4–SiO2 (5)
HBF4–SiO2 (5)
10
11
12
[2]
[2]
[2]
CHCl3
5 mol % of catalyst is sufficient to produce an excellent yield of
the product (Table 1, entry 7). Whereas more than 5 mol % of the
catalyst did not improve the results (Table 1, entries 8 and 9). Infe-
rior results were obtained in the presence of solvents (Table 1, en-
tries 10–12).
Free radical induced oxidative stress is linked to the majority of
metabolic disorders such as cancers, inflammation, Parkinson’s dis-
eases, atherosclerosis, Alzheimer diseases, premature aging, and
stroke.38 The free radicals include superoxide anions, hydrogen per-
oxide, peroxyl radicals, reactive hydroxyl radicals, nitric oxide, and
peroxynitrite anions, causing oxidative damage to cell components.
Diseases caused by oxidative damage are prevented by antioxidant
reagents that react with free radicals and chelating catalytic
metals.39,40
In this communication, we wish to report simple and efficient
method for the synthesis of thiomorpholides from various aryl al-
kyl ketones with sulfur and morpholine using solid-supported
fluoroboric acid (HBF4–SiO2) and evaluated for their effect on ki-
netic parameters of E. carotovora asparaginase and antioxidant
activity (Scheme 1).
To establish the generality of this protocol, various aryl alkyl ke-
tones were reacted with sulfur and morpholine in the presence of a
catalytic amount of HBF4–SiO2 (5 mol %) at 80 °C under solvent-
free conditions. The results were represented (Table 2). Both elec-
tron-rich and electron-deficient acetophenones reacted well with
sulfur and morpholine to give thioamides in excellent yields. Bulky
and sterically hindered substrates such as 2-hydroxyacetophe-
none, 2-acetylnaphthalene, and 2-aminoacetophenone reacted
smoothly under similar conditions to produce the corresponding
thioamides (Table 2, entries g, i, and j). In case of halogenated ace-
tophenones, no side products were observed arising from nucleo-
philic displacement of halogen by morpholine under these
conditions (Table 2, entry b). Another substrate such as butyrophe-
none also gave the corresponding thioamide in 89% yield (Table 2,
entry l). In most of the cases, the products were obtained as solids.
In theclassical Willgerodt–Kindler reaction, morpholinewasgen-
erally used in excess solvent as well as reactant.6,8 The use of excess
ionic liquid as a solvent minimized the quantity of morpholine in the
reaction.7 Therefore, in this respect the present methodology using
solvent-free condition is superior. Furthermore, under the classical
conditions, the reactions require high temperature, longer reaction
time (8–12 h) and low yields of products.6,8 Yields are improved in
case of method using ionic liquid.7 However, longer reaction time
(3–6 h) and drastic reaction conditions (110 °C) are the limitations
associated with this green protocol. It is important to note that the
present solid-supported method is better in comparison with the re-
ported methods considering reaction time (0.75- 3.5 h), mild reac-
tion condition (80 °C), and excellent yields (82–95%).
2. Results and discussion
2.1. Chemistry
As part of our program aiming at developing new, selective, and
environmental friendly methodologies for the preparation of bio-
logically active medicinal scaffolds using HBF4–SiO2 as a catalyst,41
we wish to describe herein a highly effective protocol for the syn-
thesis of thiomorpholides from the reaction of various aryl alkyl
ketones with sulfur and morpholine under solvent-free conditions
(Scheme 1).
Initially, a systematic study was carried out for catalytic evalu-
ation of HBF4–SiO2 as a catalyst for the reaction of acetophenone
(1 mmol) with sulfur (1.2 mmol) and morpholine (1 mmol) under
various conditions (Table 1). The reaction was very slow with
low yield in the absence of catalyst at room temperature (Table 1,
entry 1) as well as 80 °C (Table 1, entry 2). Next, we optimized the
quantity of the HBF4–SiO2 at 80 °C under solvent-free conditions
(Table 1, entries 3–9) and it was observed that the use of just
The advantage of the use of a heterogeneous catalyst for this
transformation is the ease of catalyst/substrate separation. In our
process, when the catalytic reaction was completed, HBF4–SiO2
may be recovered conveniently from the reaction mixture and
used further for the next cycle without activation, only through fil-
tration and subsequent washing with ethyl acetate. Efforts were
made to examine the reusability of HBF4–SiO2 by using acetophe-

3620
B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 3618–3624
Table 2
Preparation of thiomorpholine using HBF4–SiO2 catalyst
Entry
Arylalkyl ketone (1)
Thiomorpholinea (4)
Yieldb (%)
Time [h]/min
45
COCH3
O
N
a
95
S
COCH3
O
N
b
c
d
e
f
92
87
90
87
92
91
[2.2]
[2.0]
[2.2]
[3.0]
[2.5]
[2.1]
Br
S
Br
COCH3
COCH3
COCH3
COCH3
O
N
MeO
Me
S
MeO
O
N
S
Me
O
N
O2N
S
O2N
O
N
O
BzO
S
BzO
OH
OH
N
COCH3
g
S
MeO
MeO
O
O
COCH3
MeO
MeO
N
h
i
88
82
93
[3.5]
[3.0]
[2.0]
S
COCH3
N
O
S
NH2
NH2
N
N
COCH3
j
S
S
O
N
COCH3
k
l
87
89
[2.5]
[2.6]
O
O
O
O
S
a
All products were characterized by 1H NMR, IR, and mass spectroscopy.
Yield refers to pure product after column chromatography.
b
none (1 mmol) with sulfur (1.2 mmol) and morpholine (1 mmol)
under solvent-free conditions at 80 °C as a model substrate and
the results are presented (Fig. 1). It is observed that, with the
increasing number of cycles of the reaction, the catalytic activity
of the HBF4–SiO2 decreases, and it was nearly lost after 5th cycle.
2.2. Biological evaluation
The -asparaginase was purified from E. carotovora by using sin-
gle-step chromatography. The final recovery of enzyme was re-
vealed as 68% with approximate 78% final purity (data not
L

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 3618–3624
3621
100
90
80
70
60
50
40
30
20
10
0
70
60
50
40
No. of cycle
% Yield
30
20
10
1
2
3
4
5
6
Figure 1. Effect of catalytic cycles on product yield.
0
200
400
600
800
1000
Time (S)
shown). The enzyme showed typical Michaelis–Menten kinetics at
substrate concentration (20–100 M) and the apparent Km value
for -asparagine is 85 M. In these studies, -asparaginase activa-
tion by thiomorpholides was fully characterized by performing
Lineweaver–Burk analysis and secondary replots. Hence, -aspara-
ginase activity was determined in the presence of different thio-
morpholide compounds. All thiomorpholide compounds contain
sulfur along with different substituents attached such as elec-
tron-donating, electron-withdrawing, bulky, and sterically hin-
dered. Of the different substituents attached, the compounds
having electron-donating substituents were excellent stimulators
Figure 2.
presence of increasing amounts of thiomorpholides (0–100
M; closed circles, 4b (50 M); open circles, 4d (50 M); crosses, 4j (50
closed squares, 4g (50 M); small closed circles, 4c (50 M); open triangles, 4h
(50 M). All are represented by solid lines, representing the best fitted lines,
showing increased activity in its presence. While, lower dashed lines, representing
decreased activity than control. Open squares, 4a (50 M); squares with lines, 4k
(50 M); small open circles, 4e (50 M).
L
-Asparaginase catalyzed production of
L
-aspartate and ammonia in the
M); closed triangles,
M);
l
l
L
l
L
0
l
l
l
l
l
l
l
L
l
l
l
thiomorpholide compound were determined (Figs. 3 and 4). The
constants and b refer to the fold change in Km and Vmax, respec-
a
of
tron-withdrawing, bulky, and sterically hindered substituents
had shown the significant activation of -asparaginase.
The pattern of overall activation of -asparaginase among elec-
tron-donating substituents, revealed as 4h > 4c > 4g > 4j. The com-
pound 4h containing two methoxy substituents and compound 4c
having a single methoxy substituent. Hence, the resulting activa-
L-asparaginase activity. None of the thiomorpholide with elec-
tively, in the presence of the thiomorpholides.
While, explaining the asparaginase activation by 4c and 4h, it is
to conceive that enzyme requires an electron-releasing ability of
the substituents present in the thiomorpholide compounds along
with the thio group. The activation in the presence of 4h was ob-
tained twofold higher than that of 4c, because of two methoxy sub-
stituents in 4h. Hence, as electron-donating ability of
L
L
tion of L-asparaginase by 4h and 4c was higher than other com-
thiomorpholide compound increases the fold activation of L-aspar-
pounds. This activation may be due to the electron-releasing
ability of 3,4-dimethoxy substituent so that it becomes stable dia-
magnetic molecule. In 4h and 4c, both the substituents attached
aginase also increases. Data suggest that relatively specific interac-
tion may take place in between free thio group and enzyme as a
critical determinant of these interactions as well as activation.
Nevertheless, it happens only when the interacting compound also
supported by electron-releasing substituents. The whole data of
present work indicate that compounds 4h and 4c may bind to
the site, which is more deficient in electrons. Through these inter-
actions, the apparent affinity of an enzyme for substrate has been
raised due to a thio group, when conformational stability attained
by interaction with electron-donating substituents.
and thio group were crucial for L-asparaginase activation due to re-
dox and antioxidant properties. The activation obtained in the
presence of 4g was relatively lower than 4h and 4c. It was due to
the presence of electron-donating substituents along with bulky
and sterically hindered substituents. The possible activation of L-
asparaginase has been explained by using kinetic experiments
and progress curve. The pattern of the lines obtained in the pres-
ence of thiomorpholides has shown direct evidence for L-asparagi-
The DPPH free radical scavenging activity of the synthesized
thiomorpholides is summarized (Table 4). The overall pattern of
the antioxidant activity was obtained as 4g > 4h > 4c > 4f > 4a > 4d
with good to moderate antioxidant activity (Fig. 5). The SAR study
revealed that the parent compound 4a showed moderate antioxi-
dant activity (24.32%) when compared with standard BHA
(72.34%). Compounds 4g, 4h, and 4c substituted with electronega-
nase activation (Fig. 2). From the summary of kinetic parameters, it
is evident that Vmax values were increased and Km values were de-
creased in the presence of thiomorpholide 4h, 4c, and 4g (0–
100
l
M) that corresponds to a nonessential mode of activation (Ta-
vs 1/[thiomorpholide com-
, b, and binding constants KA for each
ble 3). By using secondary replots (1/
pound]), the values of
D
a
Table 3
Summary of kinetic parameters and binding constants of L-asparaginase in the presence of thiomorpholides
Kinetic parameters
Control
4h
1.13 0.01
4.0 0.2
0.985 0.005
75 0.4
4c
1.062 0.02
2.0 0.8
0.0851 0.002
80 0.6
4g
a
b
—
—
—
1.030 0.04
1.34 0.02
0.625 0.003
82.5 0.5
333.4 0.8
4.04 0.4
KA
Km
Vmax
(
(
l
l
(
M)
M)
mol mgꢀ1 minꢀ1
85 0.5
250 5.0
2.94 0.04
l
)
1000 6.0
13.34 0.14
500 10
6.25 0.05
Vmax/Km
The data shown above were an average of three different experiments

3622
B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 3618–3624
Table 4
Percentage antioxidant activity of thiomorpholines
Compounds
% Antioxidant activityb (1 mM/mL)
4a
4b
4c
4d
4e
4f
4g
4h
4i
24.32 0.22
0
24.32 0.22
21.59 0.11
-1
µ
0
25.92 0.14
36.12 0.17
32.56 0.23
0
0
0
0
µ
µ
µ
4j
4k
4l
BHAa
72.34 0.51
a
Standard substance.
Mean SD, n = 3.
b
3. Conclusion
We report fluoroboric acid adsorbed on silica gel (HBF4–SiO2)
catalyzed highly efficient protocol, for the synthesis of thiomor-
pholides by the condensation of aryl alkyl ketones with sulfur
and morpholine under solvent-free conditions with excellent
yields. The remarkable catalytic activity that HBF4–SiO2 exhibited
is convincingly superior to other recently reported catalytic meth-
ods with respect to high conversions, operational simplicity, en-
hanced reaction rates, cleaner reaction profiles, ease of isolation
of products, inexpensive and ready availability of the catalyst
makes the procedure an attractive alternative to the existing meth-
ods for the synthesis of thiomorpholides. The antioxidant activity
of the compounds further confirmed them as therapeutically active
agents in the treatment of oxidative stress induced diseases.
Moreover, in the present study we have also introduced antiox-
µ
Figure 3. Lineweaver–Burk analysis of the activity shown by
Erwinia carotovora in the presence of different concentrations of thiomorpholide 4h.
In the inset the secondary replot of (1/ Slope vs 1/[4h] (
Mꢀ1)).
L-asparaginase from
D
l
tive groups (–OCH3 and –OH) further enhanced the activity up to
30–36% at the same concentration. The compounds 4d and 4f gave
intermediate results (21–25%). While the remaining thiomorpho-
lides (4b, 4e, 4i, 4j, 4k, and 4l) do not show any remarkable anti-
oxidant activity.
idant thiomorpholides as activators of therapeutically important
L-
asparaginase. In future, these findings may also find some alterna-
tive to attain thermodynamic stability of E. carotovora
L-
asparaginase.
4. Experimental
-1
µ
4.1. Chemistry—general aspects
µ
µ
All chemicals were purchased from Sigma–Aldrich and Merck
Chemical Companies. All reactions were monitored by thin layer
chromatography (TLC) using aluminum plates coated with silica
gel (Merck) using 10% ethyl acetate and 90% hexane as eluent.
Melting points was recorded on a Buchi R-535 apparatus and are
uncorrected. The products were characterized by making a com-
parison of their spectral and physical data with those of authentic
samples. The IR spectra were recorded on Nicolet (impact 400D
model) FTIR Spectrometer. The 1H NMR spectra were recorded on
a Bruker DRX-300 AVANCE at 300 MHz instrument using TMS as
an internal standard and DMSO-d6 or CDCl3 as a solvent. Mass
spectra were obtained by using a GC–MS Hewlett Packard (EI,
20 eV) instrument. All yields refer to isolated ones.
µ
µ
4.2. Preparation of catalyst
HBF4 (1.65 g, as a 40% aqueous solution) was added to the sus-
pension of silica gel (13.35 g, 230–400 mesh) in diethylether
(40 mL). The mixture was concentrated and the residue was dried
under vacuum at 100 °C for 72 h to afford HBF4–SiO2
(0.5 mmol gꢀ1) as a free flowing powder.41
µ
Figure 4. Lineweaver–Burk analysis of the activity shown by
Erwinia carotovora in the presence of different concentrations of thiomorpholide,
4h. In the inset the secondary replot of (1/ Slope vs 1/[4c] (
Mꢀ1)).
L-asparaginase from
D
l

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 3618–3624
3623
Antioxidant activity
80
70
60
50
40
30
20
10
0
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l BHA
Compounds
Figure 5. Graphical representation of antioxidant activity.
4.3. Experimental procedure
4.4.7. 2-(2-Hydroxy-phenyl)-1-morpholin-4-yl-ethanethione (4g)
Yellow solid; mp: 75–77 °C; IR (KBr): 3689, 1627, 1489, 1211,
A mixture of acetophenone (1 mmol) with sulfur (1.2 mmol)
and morpholine (1 mmol) was stirred at 80 °C in the presence of
a catalytic amount of HBF4–SiO2 (250 mg, 5 mol %) for the appro-
priate time (see Table 2). After completion of the reaction as indi-
cated by TLC, the reaction mixture was diluted with ethanol
(10 ml). The filtrate and the catalyst were washed with ethanol
(3 ꢁ 5 ml). The organic layer was concentrated under vacuum to
afford the crude product, which on further recrystallization from
ethanol afforded the pure product. The recovered catalyst was re-
used at least five times affording 94%, 92%, 91%, 90%, and 86%
yields, respectively.
1117, 764 cmꢀ1 1H NMR (300 MHz, CDCl3): d 11.3 (s, 1H), 7.43–
;
7.6 (m, 1H), 7.0 (d, 1H), 6.84 (d, 1H), 4.34 (s, 2H), 4.30 (t, 1H),
3.85 (t, 2H), 3.8 (t, 1H), 3.63 (m, 4H); MS: m/z 238 (M+1).
4.4.8. 2-(3,4-Dimethoxy-phenyl)-1-morpholin-4-yl-
ethanethione (4h)
Yellow oil; IR (neat): 3525, 2923, 2850, 2751, 2590, 2288, 2045,
1650, 1593, 1482, 1340, 1270, 1112, 1025 cmꢀ1
;
1H NMR
(300 MHz, CDCl3): d 6.90 (s, 1H), 6.75 (s,2H), 4.30 (t, 2H), 4.23
(s,2H), 3.85 (s,6H), 3.60–3.70 (m, 4H), 3.40 (t, 2H); MS: m/z 282
(M+1).
4.4. Spectral data of synthesized compounds
4.4.9. 1-Morpholin-4-yl-2-naphthalen-2-yl-ethanethione (4i)
Yellow solid; mp: 95–99 °C; IR (KBr): 3440, 3052, 2961, 2923,
4.4.1. 1-Morpholino-2-phenylethanethione (4a)
2854, 1662, 1622, 1491, 1435, 1271, 1110 cmꢀ1 1H NMR
;
Yellow solid; mp: 62–65 °C; IR (KBr): 3473, 3026, 2968, 2861,
(300 MHz, CDCl3): d 7.4–8.05 (m, 7H), 4.5 (s, 1H), 4.36 (t, 1H), 4.3
(s, 1H), 3.92 (t, 1H), 3.78 (t, 2H), 3.7 (t, 1H), 3.65 (t, 2H), 3.3 (t,
1H); MS: m/z 272 (M+1).
1960, 1711, 1488, 1279, 1109, 958,707, 622 cmꢀ1
;
1H NMR
(400 MHz, CDCl3): d 7.17–7.22 (m, 5H), 4.32 (t, 2H), 4.30 (s, 2H),
3.74 (t, 2H), 3.53 (t, 2H), 3.35 (t, 2H); MS: m/z 222 (M+1).
4.4.10. 2-(2-Amino-phenyl)-1-morpholin-4-yl-ethanethione (4j)
4.4.2. 2-(4-Bromophenyl)-1-morpholinoethanethione (4b)
Yellow oil, IR (neat): 3368, 3240, 2820, 1618, 1532, 1400, 1328,
Light yellow solid; mp: 94–95 °C; IR (KBr): 3416, 2922, 2858,
1262, 1208, 1029, 842, 668 cmꢀ1 1H NMR (300 MHz, CDCl3): d
;
1486, 1432, 1277, 1219, 1111, 1030, 961, 722, 614 cmꢀ1
;
1H NMR
7.25–7.80 (m, 4H), 4.20 (s, 2H, –NH2), 4.30–4.33 (m, 2H), 4.29 (s,
(400 MHz, CDCl3): d 7.45 (d, 2H), 7.20 (d, 2H), 4.35 (t, 2H), 4.22
(s, 2H), 3.75 (t, 2H), 3.60 (t, 2H), 3.40 (t, 2H); MS: m/z 300 (M+1).
2H), 3.73 (t, 2H), 3.53 (t, 2H), 3.35 (t, 2H); MS: m/z 238 (M+1).
4.4.11. 2-Furan-2-yl-1-morpholin-4-yl-ethanethione (4k)
4.4.3. 2-(4-Methoxy-phenyl)-1-morpholin-4-yl-ethanethione (4c)
Light yellow solid; mp: 86–89 °C; IR (KBr): 3397, 2935, 2720,
Oil; IR (neat): 3458, 2924, 2845, 1518, 1250, 1115, 1035,
2352, 1489, 1230, 1215, 1112, 774, 730 cmꢀ1 1H NMR (300 MHz,
;
769 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 7.10–7.30 (d, 2H), 6.80
CDCl3): d 7.0–7.2 (m, 3H), 4.27 (t, 4H), 3.65 (t, 2H), 3.55 (t, 2H),
3.32 (t, 2H); MS: m/z 228 (M+1).
(d, 2H), 4.35 (t, 2H), 4.25 (s, 2H), 3.80 (s, 3H), 3.70 (t, 2H), 3.63 (t,
2H), 3.32 (t, 2H); MS: m/z 252 (M+1).
4.4.12. 1-Morpholin-4-yl-4-phenyl-butane-1-thione (4l)
4.4.4. 1-Morpholin-4-yl-2-p-tolyl-ethanethione (4d)
Light yellow oil, IR (neat): 3473, 3112, 3011, 1642, 1490, 1210,
Colorless liquid; IR (neat): 2918, 1656, 1488, 1250, 1108, 1008,
1115 cmꢀ1 1H NMR (200 MHz, CDCl3): d 7.30–7.39 (m, 3H), 7.25
;
750, 578 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 7.10 (d, 2H), 6.90 (d,
(d, 2H), 6.90 (t, 1H), 6.05 (t, 1H), 3.80–3.86 (m, 4H), 3.13 (t, 4H),
2H), 4.45 (s, 2H), 4.30 (t, 2H), 3.75 (t, 4H), 3.40–3.50 (t, 2H), 2.20
2.02 (t, 2H), 1.39 (t, 2H); MS: m/z 283 (M+1).
(s, 3H); MS: m/z 236 (M+1).
4.5. Enzyme production and partial purification
4.4.5. 1-Morpholin-4-yl-2-(4-nitro-phenyl)-ethanethione (4e)
Yellow oil; IR (neat): 3359, 3222, 2921, 2856, 1628, 1585, 1313,
The E. carotovora (M.T.C.C. 1428) used as a source for L-aspara-
1272, 1237, 1173 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 7.75 (d, 2H),
ginase production42 and purified by known method.43 All other re-
agents used further were of analytical grade.
6.60 (d, 2H), 4.35 (t, 2H), 4.20 (s, 2H), 3.94 (t, 2H), 3.60 (t, 4H); MS:
m/z 267 (M+1).
4.5.1. Asparaginase activity assay
4.4.6. 2-(4-Benzyloxy-phenyl)-1-morpholin-4-yl-ethanethione (4f)
Yellow solid; mp: 107–110 °C; IR (KBr): 3465, 2923, 1649, 1583,
1510, 1431, 1118 cmꢀ1; 1H NMR (300 MHz, CDCl3): d 7.30–7.40 (m,
5H), 7.0 (d, 2H), 6.86 (d, 2H), 5.03 (s, 2H), 4.28 (s, 2H), 3.88 (t, 2H),
3.67 (t, 2H), 3.55–3.63 (m, 2H), 3.32 (t, 2H); MS: m/z 328 (M+1).
L
-Asparaginase activity was determined by a modified method
of Mashburn and Wriston.44 A 0.1 ml purified enzyme solution,
0.9 ml of 0.1 M. sodium borate buffer (0.1 M, pH 8.5) and 1 ml
L-
asparagine (0.04 M) solution were added and incubated the same
for 10 min at 37 °C. The reaction was terminated by adding

3624
B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 3618–3624
0.5 ml trichloroacetic acid (15% w/v). The reaction contents were
centrifuged at 8000 rpm. The supernatant was collected and
0.2 ml of the supernatant was diluted to 8 ml with distilled water.
The resulting mixture was treated with 1.0 ml of Nessler’s reagent
and 1.0 ml of NaOH (2.0 M). The color reaction allows to proceed
for 15 min. The release of ammonia was determined by using stan-
dard curve prepared from ammonium sulfate as the ammonia
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%antioxidant activity
¼ ½1 ꢀ OD of test compound=OD of control compoundꢂ ꢁ 100
Acknowledgment
The authors are thankful to the Director, School of Life Sciences,
Swami Ramanand Teerth Marathwada University, Nanded, for pro-
viding the necessary facilities.