Synthesis and xanthine oxidase inhibitory activity of 7-methyl-2- (phenoxymethyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one derivatives

Article abstract of DOI:10.1016/j.bmc.2010.11.034

An elevated level of blood uric acid (hyperuricemia) is the underlying cause of gout. Xanthine oxidase is the key enzyme that catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Allopurinol, a widely used xanthine oxidase inhibitor is the most commonly used drug to treat gout. However, a small but significant portion of the population suffers from adverse effects of allopurinol that includes gastrointestinal upset, skin rashes and hypersensitivity reactions. Moreover, an elevated level of uric acid is considered as an independent risk factor for cardiovascular diseases. Therefore use of allopurinol-like drugs with minimum side effects is the ideal drug of choice against gout. In this study, we report the synthesis of a series of pyrimidin-5-one analogues as effective and a new class of xanthine oxidase inhibitors. All the synthesized pyrimidin-5-one analogues are characterized by spectroscopic techniques and elemental analysis. Four (6a, 6b, 6d and 6f) out of 20 synthesized molecules in this class showed good inhibition against three different sources of xanthine oxidase, which were more potent than allopurinol based on their respective IC₅₀ values. Molecular modeling and docking studies revealed that the molecule 6a has very good interactions with the Molybdenum-Oxygen-Sulfur (MOS) complex a key component in xanthine oxidase. These results highlight the identification of a new class of xanthine oxidase inhibitors that have potential to be more efficacious, than allopurinol, to treat gout and possibly against cardiovascular diseases.

Full text of DOI:10.1016/j.bmc.2010.11.034

Bioorganic & Medicinal Chemistry 19 (2011) 211–220
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and xanthine oxidase inhibitory activity
of 7-methyl-2-(phenoxymethyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one
derivatives
K. R. Sathisha a, Shaukath A. Khanum b, J. N. Narendra Sharath Chandra c, F. Ayisha b, S. Balaji d,
c
Gopal K. Marathe e, Shubha Gopal a, , K. S. Rangappa
⇑
a Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore 570006, India
b Department of Chemistry and Biochemistry, Yuvaraja’s College, University of Mysore, Mysore 570005, India
c Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
d Department of Biotechnology, Manipal Institute of Technology, Manipal University, Manipal 576104, India
e Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore 570006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An elevated level of blood uric acid (hyperuricemia) is the underlying cause of gout. Xanthine oxidase is
the key enzyme that catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Allopu-
rinol, a widely used xanthine oxidase inhibitor is the most commonly used drug to treat gout. However, a
small but significant portion of the population suffers from adverse effects of allopurinol that includes
gastrointestinal upset, skin rashes and hypersensitivity reactions. Moreover, an elevated level of uric acid
is considered as an independent risk factor for cardiovascular diseases. Therefore use of allopurinol-like
drugs with minimum side effects is the ideal drug of choice against gout. In this study, we report the syn-
thesis of a series of pyrimidin-5-one analogues as effective and a new class of xanthine oxidase inhibitors.
All the synthesized pyrimidin-5-one analogues are characterized by spectroscopic techniques and
elemental analysis. Four (6a, 6b, 6d and 6f) out of 20 synthesized molecules in this class showed good
inhibition against three different sources of xanthine oxidase, which were more potent than allopurinol
based on their respective IC50 values. Molecular modeling and docking studies revealed that the molecule
6a has very good interactions with the Molybdenum–Oxygen–Sulfur (MOS) complex a key component in
xanthine oxidase. These results highlight the identification of a new class of xanthine oxidase inhibitors
that have potential to be more efficacious, than allopurinol, to treat gout and possibly against cardiovas-
cular diseases.
Received 24 September 2010
Revised 12 November 2010
Accepted 12 November 2010
Available online 18 November 2010
Keywords:
Pyrimidin-5-ones
Xanthine oxidase
Xanthine
Allopurinol
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
of pathophysiological processes including diabetes, post-ischemic
reperfusion injury and chronic heart failure.6
Xanthine oxidase (XO) is situated at the end of a catabolic
sequence of the purine nucleotide metabolism in humans and a
few other uricotelic species.1 This enzyme catalyzes the oxidation
of hypoxanthine to xanthine and then to uric acid.2 The overpro-
duction of uric acid termed as hyperuricemia is the underlying
cause of gout.3 The increase in uric acid concentration eventually
leads to the deposition of micro and macroscopic deposits of so-
dium hydrogen urate monohydrate crystals in joints of humans.
This deposition of urate crystals initiates an inflammatory reaction
at the joints that includes swelling, redness and pain, the hallmarks
of inflammation.4,5 XO also generates reactive oxygen species
(ROS). ROS generated by XO have been implicated in a wide variety
Allopurinol is the widely used drug for inhibiting XO in gout.7
Allopurinol blocks the synthesis of uric acid and it also prevents
the formation of ROS, there by protecting against post-ischemic
reperfusion injury.8,9 More recently, allopurinol is also known to
produce modest but beneficial adenosine A1 receptor-mediated
anti-nociceptive effects in mice.6 Thus action of XO inhibitors is be-
yond the mere inhibition of XO. However, a small but significant
portion of the population suffers from adverse effects of allopurinol
that includes gastrointestinal upset, skin rashes, and hypersensitiv-
ity reactions, liver dysfunction, exfoliative dermatitis, vasculitis,
eosinophilia and acute interstitial nephritis.8 Some of these symp-
toms are common in Stevens–Johnson syndrome and DRESS (Drug
Rash with Eosinophylia and Systemic Symptoms).10,11
Synthetic pyrimidine derivatives are known to possess several
beneficial biological activities that include antioxidant, immunotrop-
ic, anti-inflammatory and membrane stabilizing properties.12–14
⇑
Corresponding author. Tel.: +91 821 2419328.
E-mail address: sguom@yahoo.com (S. Gopal).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.034

212
K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
In this connection, several pyrimidine derivatives were synthesized
with condensing various pharmacologically active alkyl groups by
means of alkyl halides. The present study is undertaken with a view
tofindtheeffect of pyrimidine derivatives6a–tasXOinhibitors. Here,
we report four pyrimidine analogues (6a, 6b, 6d and 6f) that act as
potent xanthine oxidase inhibitors.
The inhibitory activity of the newly synthesized compounds
against XO was studied using the method of Kadam and Iyer15
and Litwack et al.16 Rate of formation of uric acid from the oxida-
tion of xanthine in presence of these inhibitors against different
sources of XO was studied. Pattern of XO inhibition, for all the
three sources of XO is shown in Figures 1–3. The inhibitory activity
of synthesized compounds was compared with the inhibitory
activity shown by the standard inhibitor allopurinol. The different
pyrimidine derivatives 6a–t were tested for their ability to block
the XO activity for the substrate xanthine. The order of potency
is found to be 6a > 6d > 6b > 6f. The other compounds screened
failed to elicit inhibition of XO from rat liver homogenate at such
a low concentration (Table 1).
Present study identifies a new class of XO inhibitor. It is possible
that the pyrimidine binds to the active site and substituent con-
taining heterocyclic rings bind to peripheral site of the enzyme
and transfers electrons to the molybdenum center (a two electron
acceptor from the MO site of XO). Accordingly, our docking studies
revealed that all inhibitors bind between two phenylalanine resi-
dues, Phe649 and Phe1013, what is believed to promote the stabil-
ization of the binding positions of the aromatic substrate and
might be important for the substrate recognition.17 The presence
of these residues makes the region closer to the MOS1327 complex,
which compromises the binding of larger substituted rings. This
was observed with all docked inhibitors taken for our study, in
which only phenyl ring was allowed to bind in that region. As
shown in Figure 4, compound 6a was expected to bind with XO
2. Result and discussion
The novel 7-methyl-2-(phenoxymethyl)-5H-[1,3,4]thiadiazolo
[3,2-a] pyrimidin-5-ones (6a–t) were synthesized by multiple step
synthesis as depicted in Scheme 1. The effect of different doses of
pyrimidine derivatives on XO in rat liver is shown in Table 1.
Activity was reduced to 50% at a dose of 289 5.7, 449 10.3,
362 11.1, 623 23.1 nM for compounds 6a, 6b, 6d, and 6f respec-
tively (Fig. 1). The significant inhibition was shown by 6a with
methoxy group at para-position, followed by 6d with methyl group
at ortho position, 6b with chloride group at para-position and 6f
with methyl group at meta-position and chloro group at para-
position. These compounds showed much more potent inhibitory
activity than allopurinol (IC50=753 33.1 nM). This was true even
with bovine milk, and microbial XO (Figs. 2 and 3). Compounds
6c, 6e, 6g, 6h, 6i, 6j, 6k, 6l, 6m, 6n, 6o, 6p, 6q, 6r, 6s and 6t showed
inhibitory activity at much higher concentration and hence were
not chosen further (Table 1). Compound 6a with methoxy group
at para-position is the most potent of all the inhibitory compounds
tested.
NH2
O
O
OH
O
O
NH
R1
O
ClCH2COOC2H5
K2CO3/Dry Acetone
R1
R2
H2N-NH2
C2H5OH
KSCN
R2
R1
R2
C2H5OH
R3
1a-t
R3
R3
2a-t
3a-t
S
CH3
N
NH2
N
H2N
NH
O
N
S
HN
O
N
CH3COCH2COOC2H5
CH3OH
H3PO4
N
S
O
O
R1
R1
R2
O
R1
R2
R2
R3
R3
5a-t
4a-t
R3
6a-t
a: R1=R2=H, R3=OCH3
c: R1=Br, R2=R3=H
e: R1=Cl, R2=R3=H
g: R1=R3=H, R2=CH3
i: R1=R2=H, R3=F
k: R1=R2=H, R3=NO2
m: R1=F, R2=R3=H
o: R1=R2=H, R3=CH3
q: R1=R3=H, R2=Br
s: R1=R2=R3=H,
b: R1=R2=H, R3=Cl
d: R1= CH3, R2=R3=H
f:: R1=H, R2=CH3, R3=Cl
h: R1=R2=H, R3=Br
j: R1=NO2, R2= R3=H
l: R1=R3=CH3, R2=H
n: R1=R3=H, R2=NO2
p: R1=R3=H, R2=Cl
r: R1=R3=H, R2=F
t: R1=R2=CH3, R3=H
Scheme 1.

K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
213
Figure 1. Percentage inhibition of rat liver Xanthine oxidase by pyrimidin-5-one derivatives.
Figure 2. Percentage inhibition of bovine milk Xanthine oxidase by pyrimidin-5-one derivatives.
Figure 3. Percentage inhibition of microbial Xanthine oxidase by pyrimidin-5-one derivatives.
in a similar way. The compound 6a was sandwiched between the
active site aminoacids Phe 649 and Phe 1013, respectively. The
ligand was surrounded by several hydrophobic amino acid
residues and was positioned at the entrance of the active site-
directed channel.18 The reference compound lumazine (an allopu-
rinol analogue) is also shown in Figure 4. Therefore the inhibitory

214
K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
Table 1
been evaluated. Compounds 6a, 6b, 6d and 6f demonstrated potent
inhibitory activities against XO. Further research in this area is in
progress in our laboratory.
Comparative inhibitory activities of pyrimidin-5-one derivatives (6a–t) against
different sources of xanthine oxidase (IC50, nM)
Compound
Rat liver
Bovine milk
Microbial source
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
289 5.7a
449 10.3d
413 10.1c
362 11.1b
1830 27.7j
623 23.1f
1070 32.1h
ND
269 21.1a
461 40.1c
603 45.5e
326 23.4b
1650 67.3
634 37.7d
1030 51.1g
ND
269 20.7a
423 25.5c
ND
4. Experimental part
4.1. Chemistry
346 30.1b
1821 55.3j
596 57.3d
1108 73.1g
ND
The synthesis of the hitherto unreported title compounds is as
outlined in Scheme 1. Substituted phenols 1a–t on reaction with
ethyl chloroacetate affords substituted ethyl phenoxy acetates
2a–t in excellent yield.19,20 which on treatment with hydrazine
hydrate yields corresponding phenoxy acetohydrazides 3a–t.21
Condensation of 3a–t with potassium isothiocyanate in dry ethanol
resulted in the formation of substituted phenoxy acetyl-N-
hydrazine carbothioamides 4a–t. Intramolecular cyclization of
4a–t with anhydrous orthophosphoric acid afforded 5-phenoxy
methyl-2-amino-1,3,4-thiadiazoles 5a–t.22 Finally compounds
5a–t on condensation with ethylacetoacetate in presence of meth-
anol afforded the title compounds 7-methyl-2-(phenoxymethyl)-
5H-[1,3,4]thiadiazolo[3,2-a] pyrimidin-5-ones 6a–t.
ND
ND
ND
ND
2240 87.8k
ND
2030 55.7k
1760 27.3i
590 21.3ef
ND
2501 53.4i
1800 48.7h
ND
ND
1730 73.6ij
ND
ND
ND
ND
ND
ND
ND
ND
ND
1020 47.3h
6180 100m
2560 76.1l
1980 57.8k
555 23.1e
ND
834 55.1f
ND
6q
6r
6s
6t
ND
1665 73.3h
ND
ND
Allopurinol
753 33.1g
730 37.4f
730 40.7e
Chemicals were purchased from Sigma Aldrich Chemical Co.
TLC was performed on aluminium-baked silica plates and visual-
ized by UV-light. Melting points were determined on a Thomas
Hoover capillary melting point apparatus with a digital thermom-
eter. IR spectra were recorded in Nujol on FT-IR Shimadzu 8300
spectrophotometer, 1H NMR spectra were recorded on a Bruker
300 MHz NMR spectrophotometer in CDCl3 and chemical shift
were recorded in parts per million down field from tetramethylsil-
ane. Mass spectra were obtained with a VG70-70H spectrophotom-
eter and important fragments are given with the relative
intensities in the brackets. Elemental analysis results are within
0.5% of the calculated value.
*
**
#
Values are Mean SD of triplicate determinations.
Values with different superscript letters a,b,c,d . . . differ significantly at p 6 0.001.
ND: not detected.
property of only pyrimidin-5-one derivatives 6a–t was investi-
gated in this work and we found that it effectively inhibited the
enzymatic formation of uric acid from xanthine. The level of XO
in the serum is significantly increased in several pathological sta-
tuses. Thus inhibition of this enzyme by compounds such as 6a,
6b, 6d and 6f may have properties that are of therapeutic interest.
3. Conclusion
4.1.1. Synthesis of ethyl-4-methoxy phenoxy acetate (2a)
A mixture of 1a (5 g, 0.04 mol), ethyl chloroacetate (4.8 g,
0.04 mol) in dry acetone (50 mL) and anhydrous potassium car-
bonate (5.6 g, 0.04 mol) was refluxed for 8 h then cooled and the
From the results of the present study, it is concluded that, a ser-
ies of novel pyrimidin-5-one derivatives 6a–t were synthesized
and their XO inhibitory activities from three different sources have
Figure 4. The active compound 6a is shown in sticks (inside the dotted Connolly surface) in comparison with the allopurinol binding site (complexed with Lumazine, an
allopurinol analogue inside the transparent green Connolly surface). The spherical balls closer to the compounds represent molybdenum (blue), Sulfur (yellow) and oxygens
O1 and O2 (red).

K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
215
solvent removed under reduced pressure. The residual mass was
triturated with ice water to remove potassium carbonate and ex-
tracted with ether (3 ꢀ 50 mL) and the ether layer was washed
with 10% sodium hydroxide solution (3 ꢀ 30 mL) followed by
water (3 ꢀ 30 mL) and then dried over anhydrous sodium sulphate
and evaporated to dryness to get crude solid, which on recrystalli-
zation with ethanol gave 2a (yield 80%) as colorless paste mass.
Compound 2a: IR (Nujol): 1735 cmꢁ1 (ester, C@O); 1H NMR
(CDCl3): d 1.22 (t, J = 7 Hz, 3H, –CH3), 3.71 (s, 3H, –OCH3), 4.22
(q, J = 6 Hz, 2H, –CH2–), 4.45 (s, 2H, –OCH2–), 6.92–7.40 (m, 4H,
Ar-H). Anal. Calcd for C11H14O4 (210): C, 62.85; H, 6.71. Found: C,
62.71; H, 6.41.
7.18 (m, 3H, Ar-H). Anal. Calcd for C12H16O3 (208): C, 69.21; H,
7.74. Found: C, 61.21; H, 7.59.
Compound 2m: Yield (79%); IR (Nujol): 1740 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.19 (t, J = 7 Hz, 3H, –CH3), 4.30 (q, J = 6 Hz, 2H,
–CH2–), 4.51 (s, 2H, –OCH2–), 6.65–7.09 (m, 4H, Ar-H). Anal. Calcd
for C10H11FO3 (198): C, 60.60; H, 5.59; F, 9.59. Found: C, 60.53; H,
5.72; F, 9.42.
Compound 2n: Yield (76%); IR (Nujol): 1770 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.25 (t, J = 7 Hz, 3H, –CH3), 4.41 (q, J = 6 Hz, 2H,
–CH2–), 4.53 (s, 2H, –OCH2–), 6.90–7.31 (m, 4H, Ar-H). Anal. Calcd
for C10H11NO5 (225): C, 53.33; H, 4.92; N, 6.22. Found: C, 53.15; H,
4.88; N, 6.38.
Compound 2b: Yield (79%); IR (Nujol): 1715 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.25 (t, J = 7 Hz, 3H, –CH3), 4.25 (q, J = 6 Hz, 2H,
–CH2–), 4.51 (s, 2H, –OCH2–), 6.82–7.33 (m, 4H, Ar-H). Anal. Calcd
for C10H11ClO3 (214.5): C, 55.96; H, 5.17; Cl, 16.52. Found: C, 55.79;
H, 5.29; Cl, 16.42.
Compound 2o: Yield (80%); IR (Nujol): 1700 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.18 (t, J = 7 Hz, 3H, –CH3), 2.15 (s, 3H, Ar-
CH3), 4.19 (q, J = 6 Hz, 2H, –CH2–), 4.48 (s, 2H, –OCH2–),
6.83–7.25 (m, 4H, Ar-H). Anal. Calcd for C11H14O3 (194): C, 68.02;
H, 7.27. Found: C, 68.19; H, 7.33.
Compound 2c: Yield (76%); IR (Nujol): 1730 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.30 (t, J = 7 Hz, 3H, –CH3), 4.31 (q, J = 6 Hz,
2H, –CH2–), 4.55 (s, 2H, –OCH2–), 6.95–7.45 (m, 4H, Ar-H). Anal.
Calcd for C10H11BrO3 (259): C, 46.36; H, 4.28; Br, 30.84. Found: C,
46.18; H, 4.44; Br, 30.71.
Compound 2p: Yield (79%); IR (Nujol): 1740 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.30 (t, J = 7 Hz, 3H, –CH3), 4.18 (q, J = 6 Hz, 2H,
–CH2–), 4.50 (s, 2H, –OCH2–), 6.91–7.15 (m, 4H, Ar-H). Anal. Calcd
for C10H11ClO3 (214.5): C, 55.96; H, 5.17; Cl, 16.52. Found: C, 55.83;
H, 5.31; Cl, 16.65.
Compound 2d: Yield (81%); IR (Nujol): 1740 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.25 (t, J = 7 Hz, 3H, –CH3), 2.25 (s, 3H, Ar-CH3),
4.29 (q, J = 6 Hz, 2H, –CH2–), 4.49 (s, 2H, –OCH2–), 6.99–7.35 (m,
4H, Ar-H). Anal. Calcd for C11H14O3 (194): C, 68.02; H, 7.27. Found:
C, 68.23; H, 7.42.
Compound 2q: Yield (77%); IR (Nujol): 1780 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.32 (t, J = 7 Hz, 3H, –CH3), 4.21 (q, J = 6 Hz, 2H,
–CH2–), 4.61 (s, 2H, –OCH2–), 7.08–7.41 (m, 4H, Ar-H). Anal. Calcd
for C10H11BrO3 (259): C, 46.36; H, 4.28; Br, 30.84. Found: C, 46.18;
H, 4.07; Br, 30.71.
Compound 2e: Yield (77%); IR (Nujol): 1720 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.24 (t, J = 7 Hz, 3H, –CH3), 4.32 (q, J = 6 Hz,
2H, –CH2–), 4.50 (s, 2H, –OCH2–), 6.89–7.32 (m, 4H, Ar-H). Anal.
Calcd for C10H11ClO3 (214.5): C, 55.96; H, 5.17; Cl, 16.52. Found:
C, 55.79; H, 5.29; Cl, 16.42.
Compound 2r: Yield (70%); IR (Nujol): 1775 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.25 (t, J = 7 Hz, 3H, –CH3), 4.30 (q, J = 6 Hz,
2H, –CH2–), 4.62 (s, 2H, –OCH2–), 6.99–7.42 (m, 4H, Ar-H). Anal.
Calcd for C10H11FO3 (198): C, 60.60; H, 5.59; F, 9.59. Found: C,
60.46; H, 5.39; F, 9.42.
Compound 2f: Yield (75%); IR (Nujol): 1720 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.30 (t, J = 7 Hz, 3H, –CH3), 2.38 (s, 3H, Ar-
CH3), 4.50 (q, J = 6 Hz, 2H, –CH2–), 4.60 (s, 2H, –OCH2–),
6.95–7.41 (m, 3H, Ar-H). Anal. Calcd for C11H13ClO3 (228.5): C,
57.78; H, 5.73; Cl, 15.50. Found: C, 57.54; H, 5.21; Cl, 15.22.
Compound 2g: Yield (80%); IR (Nujol): 1710 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.19 (t, J = 7 Hz, 3H, –CH3), 2.22 (s, 3H, Ar-
CH3), 4.19 (q, J = 6 Hz, 2H, –CH2–), 4.38 (s, 2H, –OCH2–),
6.75–7.25 (m, 4H, Ar-H). Anal. Calcd for C11H14O3 (194): C, 68.02;
H, 7.27. Found: C, 68.25; H, 7.42.
Compound 2s: Yield (72%); IR (Nujol): 1720 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.15 (t, J = 7 Hz, 3H, –CH3), 4.22 (q, J = 6 Hz,
2H, –CH2–), 4.45 (s, 2H, –OCH2–), 6.81–7.25 (m, 5H, Ar-H). Anal.
Calcd for C10H12O3 (180): C, 66.65; H, 6.71. Found: C, 66.80; H, 6.56.
Compound 2t: Yield (70%); IR (Nujol): 1735 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.26 (t, J = 7 Hz, 3H, –CH3), 2.32 (s, 6H, Ar-
2CH3), 4.18 (q, J = 6 Hz, 2H, –CH2–), 4.44 (s, 2H, –OCH2–),
6.80–7.15 (m, 3H, Ar-H). Anal. Calcd for C12H16O3 (208): C, 69.21;
H, 7.74. Found: C, 61.33; H, 7.32.
Compound 2h: Yield (78%); IR (Nujol): 1760 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.35 (t, J = 7 Hz, 3H, –CH3), 4.30 (q, J = 6 Hz, 2H,
–CH2–), 4.60 (s, 2H, –OCH2–), 7.00–7.45 (m, 4H, Ar-H). Anal. Calcd
for C10H11BrO3 (259): C, 46.36; H, 4.28; Br, 30.84. Found: C, 46.11;
H, 4.43; Br, 30.66.
4.1.2. Synthesis of 4-methoxy phenoxy acetohydrazide (3a)
To 2a (2 g, 9.5 mmol) in methanol (10 mL), 80% hydrazine hy-
drate (0.475 g, 9.5 mmol) was added in drops and stirred for 1 h
at room temperature. A white solid separated, which on recrystal-
lization with ethanol gave 3a (75%) as white needles.
Compound 3a: Mp 177–180 °C; IR (KBr): 1655 (amide, C@O),
3110–3215 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3): d 3.71 (br s,
2H, –NH2), 3.84 (s, 3H, –OCH3), 4.52 (s, 2H, –OCH2–), 6.91–7.43
(m, 4H, Ar-H), 9.25 (br s, 1H, –CO–NH–). Anal. Calcd for
C9H12N2O3 (196): C, 55.09; H, 6.16; N, 14.28. Found: C, 55.21; H,
6.27; N, 14.43.
Compound 3b: Yield (70%); mp 175–177 °C; IR (KBr): 1645
(amide, C@O), 3100–3205 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.55 (br s, 2H, –NH2), 4.55 (s, 2H, –OCH2–), 7.29–7.60 (m, 4H,
Ar-H), 9.41 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9ClN2O2
(200.5): C, 47.89; H, 4.52; Cl, 17.67; N, 13.96. Found: C, 47.77; H,
4.39; Cl, 17.51; N, 13.8.
Compound 3c: Yield (69%); mp 185–187 °C; IR (Nujol): 1660
(amide, C@O), 3115–3220 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.72 (br s, 2H, –NH2), 4.55 (s, 2H, –OCH2–), 7.09–7.51 (m, 4H,
Ar-H), 9.39 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9BrN2O2
(245): C, 39.21; H, 3.70; Br, 32.60; N, 11.43. Found: C, 39.42; H,
3.82; Br, 32.75; N, 11.69.
Compound 2i: Yield (72%); IR (Nujol): 1780 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.32 (t, J = 7 Hz, 3H, –CH3), 4.41 (q, J = 6 Hz,
2H, –CH2–), 4.65 (s, 2H, –OCH2–), 6.95–7.42 (m, 4H, Ar-H). Anal.
Calcd for C10H11FO3 (198): C, 60.60; H, 5.59; F, 9.59. Found: C,
60.49; H, 5.41; F, 9.39.
Compound 2j: Yield (82%); IR (Nujol): 1700 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.15 (t, J = 7 Hz, 3H, –CH3), 4.39 (q, J = 6 Hz,
2H, –CH2–), 4.59 (s, 2H, –OCH2–), 6.99–7.51 (m, 4H, Ar-H). Anal.
Calcd for C10H11NO5 (225): C, 53.33; H, 4.92; N, 6.22. Found: C,
53.55; H, 4.78; N, 6.41.
Compound 2k: Yield (74%); IR (Nujol): 1715 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.29 (t, J = 7 Hz, 3H, –CH3), 4.35 (q, J = 6 Hz, 2H,
–CH2–), 4.45 (s, 2H, –OCH2–), 6.88–7.45 (m, 4H, Ar-H). Anal. Calcd
for C10H11NO5 (225): C, 53.33; H, 4.92; N, 6.22. Found: C, 53.11; H,
4.83; N, 6.41.
Compound 2l: Yield (71%); IR (Nujol): 1725 cmꢁ1 (ester, C@O);
1H NMR (CDCl3): d 1.29 (t, J = 7 Hz, 3H, –CH3), 2.25 (s, 6H, Ar-
2CH3), 4.09 (q, J = 6 Hz, 2H, –CH2–), 4.39 (s, 2H, –OCH2–), 6.79–

216
K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
Compound 3d: Yield (71%); mp 179–181 °C; IR (Nujol): 1650
7.19–7.50 (m, 4H, Ar-H), 9.15 (br s, 1H, –CO–NH–). Anal. Calcd
for C9H12N2O2 (180): C, 59.99; H, 6.71; N, 15.55. Found: C, 59.85;
H, 6.58; N, 15.42.
Compound 3p: Yield (66%); mp 179–181 °C; IR (KBr): 1665
(amide, C@O), 3160–3265 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.45 (br s, 2H, –NH2), 4.5 (s, 2H, –OCH2–), 7.19–7.55 (m, 4H,
Ar-H), 9.31 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9ClN2O2
(200.5): C, 47.89; H, 4.52; Cl, 17.67; N, 13.96. Found: C, 47.81; H,
4.42; Cl, 17.77; N, 13.85.
Compound 3q: Yield (70%); mp 161–163 °C; IR (KBr): 1605
(amide, C@O), 3160–3255 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.55 (br s, 2H, –NH2), 4.55 (s, 2H, –OCH2–), 7.14–7.50 (m, 4H,
Ar-H), 9.11 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9BrN2O2
(245): C, 39.21; H, 3.70; Br, 32.60; N, 11.43. Found: C, 39.46; H,
3.56; Br, 32.49; N, 11.29.
Compound 3r: Yield (69%); mp 147–149 °C; IR (KBr): 1675
(amide, C@O), 3120–3230 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.54 (br s, 2H, –NH2), 4.55 (s, 2H, –OCH2–), 7.08–7.55 (m, 4H,
Ar-H), 9.21 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9FN2O2
(184): C, 52.17; H, 4.93; F, 10.32; N, 15.21. Found: C, 52.26; H,
4.78; F, 10.22; N, 15.15.
Compound 3s: Yield (70%); mp 149–151 °C; IR (Nujol): 1610
(amide, C@O), 3100–3200 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.31 (br s, 2H, –NH2), 4.54 (s, 2H, –OCH2–), 7.09–7.45 (m, 5H,
Ar-H), 9.07 (br s, 1H, –CO–NH–). Anal. Calcd for C9H12N2O2 (166):
C, 57.82; H, 6.07; N, 16.86. Found: C, 57.69; H, 6.18; N, 16.70.
Compound 3t: Yield (72%); mp 186–188 °C; IR (Nujol): 1660
(amide, C@O), 3125–3220 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 2.25 (s, 6H, –CH3), 3.53 (br s, 2H, –NH2), 4.53 (s, 2H, –OCH2–),
7.05–7.55 (m, 3H, Ar-H), 9.13 (br s, 1H, –CO–NH–). Anal. Calcd
for C10H14N2O2 (194): C, 61.84; H, 7.27; N, 14.42. Found: C,
61.70; H, 7.39; N, 14.30.
(amide, C@O), 3105–3210 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 2.23 (s, 3H, –CH3), 3.55 (br s, 2H, –NH2), 4.53 (s, 2H, –OCH2–),
7.29–7.61 (m, 4H, Ar-H), 9.28 (br s, 1H, –CO–NH–). Anal. Calcd
for C9H12N2O2 (180): C, 59.99; H, 6.71; N, 15.55. Found: C, 59.82;
H, 6.62; N, 15.44.
Compound 3e: Yield (68%); mp 167–169 °C; IR (KBr): 1615
(amide, C@O), 3140–3245 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.61 (br s, 2H, –NH2), 4.61 (s, 2H, –OCH2–), 6.92–7.35 (m, 4H,
Ar-H), 9.41 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9ClN2O2
(200.5): C, 47.89; H, 4.52; Cl, 17.67; N, 13.96. Found: C, 47.71; H,
4.33; Cl, 17.57; N, 13.82.
Compound 3f: Yield (70%); mp 161–163 °C; IR (Nujol): 1620
(amide, C@O), 3115–3220 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 2.19 (s, 3H, –CH3), 3.58 (br s, 2H, –NH2), 4.49 (s, 2H, –OCH2–),
6.91–7.35 (m, 3H, Ar-H), 9.19 (br s, 1H, –CO–NH–). Anal. Calcd
for C9H11ClN2O2 (214.5): C, 50.36; H, 5.17; Cl, 16.52; N, 13.05.
Found: C, 50.25; H, 5.28; Cl, 16.32; N, 13.15.
Compound 3g: Yield (69%); mp 172–174 °C; IR (Nujol): 1640
(amide, C@O), 3105–3210 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 2.25 (s, 3H, –CH3), 3.51 (br s, 2H, –NH2), 4.55 (s, 2H, –OCH2–),
7.17–7.45 (m, 4H, Ar-H), 9.06 (br s, 1H, –CO–NH–). Anal. Calcd
for C9H12N2O2 (180): C, 59.99; H, 6.71; N, 15.55. Found: C, 59.77;
H, 6.61; N, 15.49.
Compound 3h: Yield (72%); mp 161–163 °C; IR (KBr): 1605
(amide, C@O), 3160–3255 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.55 (br s, 2H, –NH2), 4.55 (s, 2H, –OCH2–), 7.12–7.43 (m, 4H,
Ar-H), 9.17 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9BrN2O2
(245): C, 39.21; H, 3.70; Br, 32.60; N, 11.43. Found: C, 39.46; H,
3.56; Br, 32.49; N, 11.29.
Compound 3i: Yield (68%); mp 155–157 °C; IR (KBr): 1635
(amide, C@O), 3150–3250 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.43 (br s, 2H, –NH2), 4.61 (s, 2H, –OCH2–), 6.95–7.35 (m, 4H,
Ar-H), 9.15 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9FN2O2
(184): C, 52.17; H, 4.93; F, 10.32; N, 15.21. Found: C, 52.29; H,
4.81; F, 10.19; N, 15.44.
4.1.3. Synthesis of 4-methoxy phenoxy acetyl-N-hydrazine
carbothioamides (4a)
A mixture of potassium isothiocyanate (0.44 g, 3.28 mmol) and
3a (0.6 g, 3.18 mmol) in dry ethanol (20 ml) was refluxed with stir-
ring for 4 h, the mixture was cooled, filtered, washed with ethanol,
dried and recrystallized with ethanol to give 4a (60%) as colorless
needles.
Compound 3j: Yield (71%); mp 180–182 °C; IR (KBr): 1640
(amide, C@O), 3130–3230 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.31 (br s, 2H, –NH2), 4.53 (s, 2H, –OCH2–), 6.92–7.33 (m, 4H,
Ar-H), 9.16 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9FN3O4
(211): C, 45.50; H, 4.30; N, 19.90. Found: C, 45.63; H, 4.41; N, 19.81.
Compound 3k: Yield (67%); mp 187–189 °C; IR (KBr): 1630
(amide, C@O), 3140–3230 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.60 (br s, 2H, –NH2), 4.62 (s, 2H, –OCH2–), 6.82–7.25 (m, 4H,
Ar-H), 9.23 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9FN3O4
(211): C, 45.50; H, 4.30; N, 19.90. Found: C, 45.59; H, 4.46; N, 19.78.
Compound 3l: Yield (71%); mp 166–168 °C; IR (Nujol): 1670
(amide, C@O), 3105–3210 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 2.39 (s, 6H, 2-CH3), 3.51 (br s, 2H, –NH2), 4.45 (s, 2H, –OCH2–),
7.18–7.40 (m, 3H, Ar-H), 9.13 (br s, 1H, –CO–NH–). Anal. Calcd
for C10H14N2O2 (194): C, 61.84; H, 7.27; N, 14.42. Found: C,
61.71; H, 7.42; N, 14.29.
Compound 3m: Yield (72%); mp 150–152 °C; IR (KBr): 1645
(amide, C@O), 3110–3230 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.51 (br s, 2H, –NH2), 4.65 (s, 2H, –OCH2–), 7.09–7.45 (m, 4H,
Ar-H), 9.23 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9FN2O2
(184): C, 52.17; H, 4.93; F, 10.32; N, 15.21. Found: C, 52.22; H,
4.83; F, 10.22; N, 15.32.
Compound 3n: Yield (69%); mp 187–189 °C; IR (KBr): 1655
(amide, C@O), 3110–3260 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 3.35 (br s, 2H, –NH2), 4.55 (s, 2H, –OCH2–), 6.95–7.35 (m, 4H,
Ar-H), 9.32 (br s, 1H, –CO–NH–). Anal. Calcd for C8H9FN3O4
(211): C, 45.50; H, 4.30; N, 19.90. Found: C, 45.39; H, 4.44; N, 19.99.
Compound 3o: Yield (71%); mp 170–172 °C; IR (Nujol): 1655
(amide, C@O), 3100–3200 cmꢁ1 (NH–NH2); 1H NMR (CD3COCD3):
d 2.19 (s, 3H, –CH3), 3.41 (br s, 2H, –NH2), 4.45 (s, 2H, –OCH2–),
Compound 4a: Yield (58%); mp 210–213 °C; IR (Nujol): 1235
(C@S), 1700 (amide, C@O), 3200–3300 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 3.78 (s, 3H, –OCH3), 4.62 (s, 2H, –OCH2–),
7.01–7.55 (m, 4H, Ar-H), 9.52 (br s, 2H, 2-NH–), 10.11 (s, 2H,
–NH2). Anal. Calcd for C10H13N3O3S (255): C, 47.05; H, 5.13; N,
16.46; S, 12.56. Found: C, 47.25; H, 5.23; N, 16.36; S, 12.36.
Compound 4b: Yield (61%); mp 200–203 °C; IR (Nujol): 1230
(C@S), 1710 (amide, C@O), 3250–3350 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.56 (s, 2H, –OCH2–), 6.99–7.51 (m, 4H, Ar-
H), 9.48 (br s, 2H, 2-NH–), 10.08 (s, 2H, –NH2). Anal. Calcd for
C9H10ClN3O2S (259.5): C, 41.62; H, 3.88; Cl, 13.65; N, 16.18; S,
12.35. Found: C, 41.51; H, 3.43; Cl, 13.49; N, 16.26; S, 12.24.
Compound 4c: Yield (59%); mp 218–220 °C; IR (Nujol): 1225
(C@S), 1710 (amide, C@O), 3150–3360 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.55 (s, 2H, –OCH2–), 6.95–7.41 (m, 4H, Ar-
H), 9.37 (br s, 2H, 2-NH–), 10.21 (s, 2H, –NH2). Anal. Calcd for
C9H10BrN3O2S (304): C, 35.54; H, 3.31; Br, 26.27; N, 13.81; S,
10.54. Found: C, 35.39; H, 3.48; Br, 26.18; N, 13.65; S, 10.41.
Compound 4d: Yield (60%); mp 205–208 °C; IR (Nujol): 1205
(C@S), 1720 (amide, C@O), 3210–3325 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 2.18 (s, 3H, –CH3), 4.65 (s, 2H, –OCH2–),
6.95–7.52 (m, 4H, Ar-H), 9.46 (br s, 2H, 2-NH–), 10.15 (s, 2H,
–NH2). Anal. Calcd for C10H13N3O2S (239): C, 50.19; H, 5.48; N,
17.56; S, 13.40. Found: C, 50.29; H, 5.31; N, 17.38; S, 13.32.
Compound 4e: Yield (62%); mp 195–197 °C; IR (Nujol): 1240
(C@S), 1740 (amide, C@O), 3260–3360 cmꢁ1 (NH–NH, NH); 1H

K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
217
NMR (CD3COCD3): d 4.70 (s, 2H, –OCH2–), 7.06–7.55 (m, 4H,
Ar-H), 9.45 (br s, 2H, 2-NH–), 10.21 (s, 2H, –NH2). Anal. Calcd for
C9H10ClN3O2S (259.5): C, 41.62; H, 3.88; Cl, 13.65; N, 16.18; S,
12.35. Found: C, 41.73; H, 3.61; Cl, 13.82; N, 16.28 S, 12.48.
Compound 4f: Yield (60%); mp 220–222 °C; IR (Nujol): 1270
(C@S), 1760 (amide, C@O), 3260–3355 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.62 (s, 2H, –OCH2–), 7.02–7.60 (m, 3H,
Ar-H), 9.47 (br s, 2H, 2-NH–), 10.16 (s, 2H, –NH2). Anal. Calcd for
NMR (CD3COCD3): d 4.91 (s, 2H, –OCH2–), 7.11–7.55 (m, 4H,
Ar-H), 9.51(br s, 2H, 2-NH–), 10.30 (s, 2H, –NH2). Anal. Calcd for
C9H10ClN3O2S (259.5): C, 41.62; H, 3.88; Cl, 13.65; N, 16.18; S,
12.35. Found: C, 41.76; H, 3.99; Cl, 13.88; N, 16.32; S, 12.48.
Compound 4q: Yield (60%); mp 260–262 °C; IR (Nujol): 1245
(C@S), 1790 (amide, C@O), 3170–3385 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.77 (s, 2H, –OCH2–), 7.09–7.42 (m, 4H,
Ar-H), 9.52 (br s, 2H, 2-NH–), 10.51 (s, 2H, –NH2). Anal. Calcd for
C9H10BrN3O2S (304): C, 35.54; H, 3.31; Br, 26.27; N, 13.81; S,
10.54. Found: C, 35.65; H, 3.43; Br, 26.19; N, 13.93; S, 10.43.
Compound 4r: Yield (63%); mp 235–237 °C; IR (Nujol): 1240
(C@S), 1770 (amide, C@O), 3270–3385 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.75 (s, 2H, –OCH2–), 7.02–7.41 (m, 4H,
Ar-H), 9.65 (br s, 2H, 2-NH–), 10.51 (s, 2H, –NH2). Anal. Calcd for
C9H10FN3O2S (243): C, 44.44; H, 4.14; F, 7.81; N, 17.27; S, 13.18.
Found: C, 44.51; H, 4.04; F, 7.69; N, 17.10; S, 13.06.
Compound 4s: Yield (61%); mp 183–185 °C; IR (Nujol): 1245
(C@S), 1720 (amide, C@O), 3200–3300 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.73 (s, 2H, –OCH2–), 7.07–7.44 (m, 5H,
Ar-H), 9.43 (br s, 2H, 2-NH–), 10.33 (s, 2H, –NH2). Anal. Calcd for
C9H11N3O2S (225): C, 47.99; H, 4.92; N, 18.65; S, 14.23. Found: C,
47.83; H, 4.80; N, 18.51; S, 14.19.
Compound 4t: Yield (58%); mp 155–157 °C; IR (Nujol): 1295
(C@S), 1780 (amide, C@O), 3240–3345 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 2.03 (s, 6H, 2-CH3), 4.85 (s, 2H, –OCH2–),
6.78–7.15 (m, 3H, Ar-H), 9.31 (br s, 2H, 2-NH–), 10.22 (s, 2H,
–NH2). Anal. Calcd for C11H15N3O2S (253): C, 52.15; H, 5.97; N,
16.59; S, 12.66. Found: C, 52.22; H, 5.86; N, 16.47; S, 12.54.
C10H12ClN3O2S (273.5): C, 43.88; H, 4.42; Cl, 12.95; N, 15.35; S,
11.71. Found: C, 43.62; H, 4.21; Cl, 12.76; N, 15.51; S, 11.59.
Compound 4g: Yield (56%); mp 215–217 °C; IR (Nujol): 1235
(C@S), 1735 (amide, C@O), 3230–3335 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 2.34 (s, 3H, –CH3), 4.56 (s, 2H, –OCH2–),
6.92–7.45 (m, 4H, Ar-H), 9.34 (br s, 2H, 2-NH–), 10.32 (s, 2H,
–NH2). Anal. Calcd for C10H13N3O2S (239): C, 50.19; H, 5.48; N,
17.56; S, 13.40. Found: C, 50.31; H, 5.61; N, 17.41; S, 13.28.
Compound 4h: Yield (63%); mp 228–230 °C; IR (Nujol): 1235
(C@S), 1730 (amide, C@O), 3160–3365 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.62 (s, 2H, –OCH2–), 6.92–7.35 (m, 4H,
Ar-H), 9.17 (br s, 2H, 2-NH–), 10.19 (s, 2H, –NH2). Anal. Calcd for
C9H10BrN3O2S (304): C, 35.54; H, 3.31; Br, 26.27; N, 13.81; S,
10.54. Found: C, 35.41; H, 3.22; Br, 26.38; N, 13.70; S, 10.68.
Compound 4i: Yield (61%); mp 190–192 °C; IR (Nujol): 1275
(C@S), 1750 (amide, C@O), 3260–3365 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.71 (s, 2H, –OCH2–), 6.92–7.34 (m, 4H,
Ar-H), 9.55 (br s, 2H, 2-NH–), 10.34 (s, 2H, –NH2). Anal. Calcd for
C9H10FN3O2S (243): C, 44.44; H, 4.14; F, 7.81; N, 17.27; S, 13.18.
Found: C, 44.58; H, 4.22; F, 7.69; N, 17.11; S, 13.30.
Compound 4j: Yield (59%); mp 196–198 °C; IR (Nujol): 1280
(C@S), 1760 (amide, C@O), 3255–3365 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.75 (s, 2H, –OCH2–), 6.81–7.23 (m, 4H, Ar-
H), 9.42 (br s, 2H, 2-NH–), 10.21 (s, 2H, –NH2). Anal. Calcd for
C9H10N4O4S (270): C, 40.00; H, 3.73; N, 20.73; S, 11.86. Found: C,
40.11; H, 3.88; N, 20.63; S, 11.77.
Compound 4k: Yield (56%); mp 178–180 °C; IR (Nujol): 1285
(C@S), 1765(amide, C@O), 3265–3355 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.73 (s, 2H, –OCH2–), 6.91–7.25 (m, 4H,
Ar-H), 9.34 (br s, 2H, 2-NH–), 10.11 (s, 2H, –NH2). Anal. Calcd for
C9H10N4O4S (270): C, 40.00; H, 3.73; N, 20.73; S, 11.86. Found: C,
40.17; H, 3.62; N, 20.66; S, 11.85.
Compound 4l: Yield (61%); mp 161–163 °C; IR (Nujol): 1275
(C@S), 1740 (amide, C@O), 3230–3345 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 2.31 (s, 6H, 2-CH3), 4.65 (s, 2H, –OCH2–),
6.65–7.17 (m, 3H, Ar-H), 9.23 (br s, 2H, 2-NH–), 10.01 (s, 2H,
–NH2). Anal. Calcd for C11H15N3O2S (253): C, 52.15; H, 5.97; N,
16.59; S, 12.66. Found: C, 52.28; H, 5.82; N, 16.49; S, 12.52.
Compound 4m: Yield (55%); mp 230–132 °C; IR (Nujol): 1200
(C@S), 1720 (amide, C@O), 3230–3345 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.80 (s, 2H, –OCH2–), 7.10–7.45 (m, 4H,
Ar-H), 9.62 (br s, 2H, 2-NH–), 10.43 (s, 2H, –NH2). Anal. Calcd for
C9H10FN3O2S (243): C, 44.44; H, 4.14; F, 7.81; N, 17.27; S, 13.18.
Found: C, 44.61; H, 4.28; F, 7.73; N, 17.40; S, 13.28.
Compound 4n: Yield 62%); mp 150–152 °C; IR (Nujol): 1235
(C@S), 1740 (amide, C@O), 3270–3385 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 4.65 (s, 2H, –OCH2–), 6.95–7.43 (m, 4H,
Ar-H), 9.42 (br s, 2H, 2-NH–), 10.15 (s, 2H, –NH2). Anal. Calcd for
C9H10FN3O2S (243): C, 44.44; H, 4.14; F, 7.81; N, 17.27; S, 13.18.
Found: C, 44.61; H, 4.27; F, 7.66; N, 17.38; S, 13.28.
4.4.4. Synthesis of 5(4-methoxy) phenoxy-2-amino-1,3,4-
thiadiazoles (5a)
Compound 4a (0.3 g, 0.89 mmol) was added gradually to anhy-
drous orthophosphoric acid (8 ml) in about 5 min. The reaction
mixture was heated in an oil bath at 120 °C for 2 h. The slurry thus
obtained was poured on crushed ice. The solid was filtered, washed
with cold distilled water (3 ꢀ 15 ml), dried and recrystallized with
ethanol to give 5a (55%) as brown solid.
Compound 5a: Yield (52%); mp 130–133 °C; IR (Nujol): 750
(C–S–C), 1620 (C@N), 3150 cmꢁ1 (NH); 1H NMR (CDCl3): d 3.75
(s, 3H, –OCH3), 4.68 (s, 2H, –OCH2–), 4.85 (br s, 1H, –NH, D2O
exchangeable), 7.02–7.51 (m, 4H, Ar-H); EI-MS: m/z 237 (M+, 45).
Anal. Calcd for C10H11ClN3O2S (237): C, 50.62; H, 4.67; N, 17.71;
S, 13.51. Found: C, 50.47; H, 4.51; N, 17.85; S, 13.75.
Compound 5b: Yield (50%); mp 128–130 °C; IR (Nujol): 750
(C–S–C), 1615 (C@N), 3140 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.52
(s, 2H, –OCH2–), 4.85 (br s, 1H, –NH, D2O exchangeable),
7.15–7.64 (m, 4H, Ar-H); EI-MS: m/z 241.5 (M+, 40). Anal. Calcd
for C9H8ClN3OS (241.5): C, 44.72; H, 3.34; Cl, 14.67; N, 17.39; S,
13.27. Found: C, 44.63; H, 3.41; Cl, 14.53; N, 17.49; S, 13.37.
Compound 5c: Yield (54%); mp 135–137 °C; IR (Nujol): 745
(C–S–C), 1615 (C@N), 3145 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.59
(s, 2H, –OCH2–), 4.82 (br s, 1H, –NH, D2O exchangeable),
6.92–7.31 (m, 4H, Ar-H); EI-MS: m/z 286 (M+, 43). Anal. Calcd for
C9H8BrN3OS (286): C, 37.78; H, 2.82; Br, 27.92; N, 14.68;
S, 11.21. Found: C, 37.88; H, 2.72; Br, 27.81; N, 14.76; S,
11.35.
Compound 5d: Yield (50%); mp 125–127 °C; IR (Nujol): 755
(C–S–C), 1625 (C@N), 3155 cmꢁ1 (NH); 1H NMR (CDCl3): d 2.18
(s, 3H, –CH3), 4.62 (s, 2H, –OCH2–), 4.91 (br s, 1H, –NH, D2O
exchangeable), 7.01–7.52 (m, 4H, Ar-H); EI-MS: m/z 207 (M+, 47).
Anal. Calcd for C10H11N3OS (221): C, 54.28; H, 5.01; N, 18.99; S,
14.49. Found: C, 54.38; H, 5.11; N, 18.80; S, 14.61.
Compound 4o: Yield (59%); mp 240–242 °C; IR (Nujol): 1225
(C@S), 1725 (amide, C@O), 3240–3355 cmꢁ1 (NH–NH, NH); 1H
NMR (CD3COCD3): d 2.25 (s, 3H, –CH3), 4.82 (s, 2H, –OCH2–),
7.01–7.43 (m, 4H, Ar-H), 9.32 (br s, 2H, 2-NH–), 10.22 (s, 2H,
–NH2). Anal. Calcd for C10H13N3O2S (239): C, 50.19; H, 5.48; N,
17.56; S, 13.40. Found: C, 50.30; H, 5.60; N, 17.05; S, 13.50.
Compound 4p: Yield (55%); mp 253–255 °C; IR (Nujol): 1290
(C@S), 1740 (amide, C@O), 3280–3360 cmꢁ1 (NH–NH, NH); 1H
Compound 5e: Yield (55%); mp 120–122 °C; IR (Nujol): 755
(C–S–C), 1625 (C@N), 3155 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.62
(s, 2H, –OCH2–), 4.86 (br s, 1H, –NH, D2O exchangeable),
7.03–7.42 (m, 4H, Ar-H); EI-MS: m/z 241.5 (M+, 40). Anal. Calcd

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K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
for C9H8ClN3OS (241.5): C, 44.72; H, 3.34; Cl, 14.67; N, 17.39; S,
13.27. Found: C, 44.65; H, 3.42; Cl, 14.51; N, 17.44; S, 13.41.
Compound 5f: Yield (53%); mp 116–117 °C; IR (Nujol): 765
(C–S–C), 1645 (C@N), 3175 cmꢁ1 (NH); 1H NMR (CDCl3): d 2.32
(s, 3H, –CH3), 4.65 (s, 2H, –OCH2–), 4.83 (br s, 1H, –NH, D2O
exchangeable), 7.01–7.35 (m, 3H, Ar-H); EI-MS: m/z 255.5 (M+,
40). Anal. Calcd for C10H10ClN3OS (255.5): C, 46.97; H, 3.94; Cl,
13.86; N, 16.43; S, 12.54. Found: C, 46.83; H, 3.85; Cl, 13.73; N,
16.36; S, 12.67.
Compound 5g: Yield (50%); mp 141–143 °C; IR (Nujol): 725
(C–S–C), 1655 (C@N), 3175 cmꢁ1 (NH); 1H NMR (CDCl3): d 2.28
(s, 3H, –CH3), 4.75 (s, 2H, –OCH2–), 4.85 (br s, 1H, –NH, D2O
exchangeable), 7.13–7.55 (m, 4H, Ar-H); EI-MS: m/z 207 (M+, 47).
Anal. Calcd for C10H11N3OS (221): C, 54.28; H, 5.01; N, 18.99; S,
14.49. Found: C, 54.36; H, 5.13; N, 18.82; S, 14.64.
6.95–7.32 (m, 4H, Ar-H); EI-MS: m/z 241.5 (M+, 48). Anal. Calcd
for C9H8ClN3OS (241.5): C, 44.72; H, 3.34; Cl, 14.67; N, 17.39; S,
13.27. Found: C, 44.84; H, 3.26; Cl, 14.77; N, 17.26; S, 13.21.
Compound 5q: Yield (57%); mp 175–177 °C; IR (Nujol): 790
(C–S–C), 1620 (C@N), 3140 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.62
(s, 2H, –OCH2–), 4.81 (br s, 1H, –NH, D2O exchangeable),
6.95–7.33 (m, 4H, Ar-H); EI-MS: m/z 286 (M+, 43). Anal. Calcd for
C9H8BrN3OS (286): C, 37.78; H, 2.82; Br, 27.92; N, 14.68; S,
11.21. Found: C, 37.87; H, 2.71; Br, 27.99; N, 14.56; S, 11.09.
Compound 5r: Yield (54%); mp 192–194 °C; IR (Nujol): 780
(C–S–C), 1650 (C@N), 3140 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.65
(s, 2H, –OCH2–), 4.82 (br s, 1H, –NH, D2O exchangeable),
6.95–7.34 (m, 4H, Ar-H); EI-MS: m/z 225 (M+, 45). Anal. Calcd for
C9H8FN3OS (225): C, 47.99; H, 3.58; F, 8.43; N, 18.66; S, 14.24.
Found: C, 47.85; H, 3.44; F, 8.56; N, 18.76; S, 14.11.
Compound 5h: Yield (56%); mp 105–107 °C; IR (Nujol): 715
(C–S–C), 1655 (C@N), 3155 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.62
(s, 2H, –OCH2–), 4.81 (br s, 1H, –NH, D2O exchangeable),
6.95–7.34 (m, 4H, Ar-H); EI-MS: m/z 286 (M+, 43). Anal. Calcd for
C9H8BrN3OS (286): C, 37.78; H, 2.82; Br, 27.92; N, 14.68; S,
11.21. Found: C, 37.83; H, 2.75; Br, 27.84; N, 14.75; S, 11.34.
Compound 5i: Yield (52%); mp 151–153 °C; IR (Nujol): 775
(C–S–C), 1660 (C@N), 3140 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.62
(s, 2H, –OCH2–), 4.78 (br s, 1H, –NH, D2O exchangeable),
6.92–7.34 (m, 4H, Ar-H); EI-MS: m/z 225 (M+, 45). Anal. Calcd for
C9H8FN3OS (225): C, 47.99; H, 3.58; F, 8.43; N, 18.66; S, 14.24.
Found: C, 47.78; H, 3.67; F, 8.36; N, 18.52; S, 14.33.
Compound 5s: Yield (51%); mp 100–102 °C; IR (Nujol): 735
(C–S–C), 1655 (C@N), 3145 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.63
(s, 2H, –OCH2–), 4.75 (br s, 1H, –NH, D2O exchangeable),
6.92–7.25 (m, 5H, Ar-H); EI-MS: m/z 207 (M+, 50). Anal. Calcd for
C9H9N3OS (207): C, 52.16; H, 4.38; N, 20.27; S, 15.47. Found: C,
52.28; H, 4.21; N, 20.39; S, 15.35.
Compound 5t: Yield (49%); mp 198–200 °C; IR (Nujol): 755
(C–S–C), 1655 (C@N), 3155 cmꢁ1 (NH); 1H NMR (CDCl3): d 2.32
(s, 6H, 2-CH3), 4.75 (s, 2H, –OCH2–), 4.95 (br s, 1H, –NH, D2O
exchangeable), 7.01–7.43 (m, 3H, Ar-H); EI-MS: m/z 235 (M+, 43).
Anal. Calcd for C11H13N3OS (235): C, 56.15; H, 5.57; N, 17.86; S,
13.63. Found: C, 56.29; H, 5.69; N, 17.95; S, 13.51.
Compound 5j: Yield (51%); mp 136–138 °C; IR (Nujol): 795
(C–S–C), 1670 (C@N), 3160 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.63
(s, 2H, –OCH2–), 4.75 (br s, 1H, –NH, D2O exchangeable),
7.01–7.42 (m, 4H, Ar-H); EI-MS: m/z 252 (M+, 40). Anal. Calcd for
C9H8N4O3S (252): C, 42.85; H, 3.20; N, 22.21; S, 12.71. Found: C,
42.97; H, 3.28; N, 22.33; S, 12.63.
Compound 5k: Yield (58%); mp 160–162 °C; IR (Nujol): 750
(C–S–C), 1620 (C@N), 3140 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.71
(s, 2H, –OCH2–), 4.82 (br s, 1H, –NH, D2O exchangeable),
7.02–7.42 (m, 4H, Ar-H); EI-MS: m/z 252 (M+, 40). Anal. Calcd for
C9H8N4O3S (252): C, 42.85; H, 3.20; N, 22.21; S, 12.71. Found: C,
42.98; H, 3.31; N, 22.34; S, 12.79.
Compound 5l: Yield (54%); mp 169–171 °C; IR (Nujol): 730
(C–S–C), 1640 (C@N), 3150 cmꢁ1 (NH); 1H NMR (CDCl3): d 2.25
(s, 6H, 2-CH3), 4.73 (s, 2H, –OCH2–), 4.92 (br s, 1H, –NH, D2O
exchangeable), 7.04–7.45 (m, 3H, Ar-H); EI-MS: m/z 235 (M+, 43).
Anal. Calcd for C11H13N3OS (235): C, 56.15; H, 5.57; N, 17.86; S,
13.63. Found: C, 56.23; H, 5.45; N, 17.74; S, 13.52.
Compound 5m: Yield (50%); mp 110–112 °C; IR (Nujol): 780
(C–S–C), 1635 (C@N), 3185 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.68
(s, 2H, –OCH2–), 4.76 (br s, 1H, –NH, D2O exchangeable),
7.05–7.35 (m, 4H, Ar-H); EI-MS: m/z 225 (M+, 45). Anal. Calcd for
C9H8FN3OS (225): C, 47.99; H, 3.58; F, 8.43; N, 18.66; S, 14.24.
Found: C, 47.80; H, 3.66; F, 8.55; N, 18.51; S, 14.16.
Compound 5n: Yield (56%); mp 180–182 °C; IR (Nujol): 715
(C–S–C), 1615 (C@N), 3150 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.68
(s, 2H, –OCH2–), 4.69 (br s, 1H, –NH, D2O exchangeable),
7.05–7.35 (m, 4H, Ar-H); EI-MS: m/z 252 (M+, 42). Anal. Calcd for
C9H8N4O3S (252): C, 42.85; H, 3.20; N, 22.21; S, 12.71. Found: C,
42.96; H, 3.25; N, 22.32; S, 12.61.
Compound 5o: Yield (55%); mp 146–148 °C; IR (Nujol): 730
(C–S–C), 1670 (C@N), 3160 cmꢁ1 (NH); 1H NMR (CDCl3): d 2.15
(s, 3H, –CH3), 4.75 (s, 2H, –OCH2–), 4.85 (br s, 1H, –NH, D2O
exchangeable), 7.05–7.45 (m, 4H, Ar-H); EI-MS: m/z 207 (M+, 46).
Anal. Calcd for C10H11N3OS (221): C, 54.28; H, 5.01; N, 18.99; S,
14.49. Found: C, 54.35; H, 5.15; N, 18.82; S, 14.36.
4.1.5. A typical procedure is described for the synthesis of 7-
methyl-2(4-methoxy) phenoxymethyl-[1,3,4]thiadiazolo[3,2-
a]pyrimidin-5-one (6a)
To a solution of ethyl acetoacetate (0.075 g, 0.58 mmol) in
methanol (10 ml) 5a (0.15 g, 0.58 mmol) was added and refluxed
for 5 h and poured into cold water. The separated solid was filtered,
dried and recrystallized form ethanol to afford 6a (69%) as brown
solid.
Compound6a:Yield(65%);mp120–122 °C;IR(Nujol):1670 cmꢁ1
(C@O); 1HNMR(CDCl3):d 1.71 (s, 3H, –CH3), 3.73(s, 3H, –OCH3), 4.45
(s, 2H, –OCH2–), 6.32 (s, 1H, –CH–), 6.94–7.32 (m, 4H, Ar-H); EI-MS:
m/z 303 (M+, 55). Anal. Calcd for C14H13N3O3S (303): C, 55.43; H,
4.32; N, 13.85; S, 10.57. Found: C, 55.32; H, 4.47; N, 13.71; S,
10.69.
Compound 6b: Yield (62%); mp 129–131 °C; IR (Nujol): 1660 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.72 (s, 3H, –CH3), 4.44 (s, 2H, –OCH2–),
6.31 (s, 1H, –CH–), 6.95–7.43 (m, 4H, Ar-H); EI-MS: m/z 307.5 (M+,
53). Anal. Calcd for C13H10ClN3O2S (307.5): C, 50.73; H, 3.28; Cl,
11.52; N, 13.65; S, 10.42. Found: C, 50.61; H, 3.38; Cl, 11.70; N,
13.79; S, 10.31.
Compound 6c: Yield (61%); mp 135–137 °C; IR (Nujol): 1675 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.74 (s, 3H, –CH3), 4.56 (s, 2H, –OCH2–),
6.25 (s, 1H, –CH–), 7.06–7.44 (m, 4H, Ar-H); EI-MS: m/z 352 (M+,
57). Anal. Calcd for C13H10BrN3O2S (352): C, 44.33; H, 2.86; Br,
22.69; N, 11.93; S, 9.10. Found: C, 44.22; H, 2.73; Br, 22.79; N,
11.81; S, 9.19.
Compound 6d: Yield (60%); mp 144–146 °C; IR (Nujol): 1680 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.75 (s, 3H, –CH3), 2.11 (s, 3H, Ar-CH3),
4.55 (s, 2H, –OCH2–), 6.32 (s, 1H, –CH–), 7.05–7.43 (m, 4H, Ar-H);
EI-MS: m/z 287 (M+, 60). Anal. Calcd for C14H13N3O2S (287): C,
58.52; H, 4.56; N, 14.62; S, 11.16. Found: C, 58.41; H, 4.69; N,
14.72; S, 11.28.
Compound 6e: Yield (67%); mp 151–153 °C; IR (Nujol): 1680 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.69 (s, 3H, –CH3), 4.53 (s, 2H, –OCH2–),
6.25 (s, 1H, –CH–), 6.93–7.31 (m, 4H, Ar-H); EI-MS: m/z 307.5 (M+,
55). Anal. Calcd for C13H10ClN3O2S (307.5): C, 50.73; H, 3.28; Cl,
11.52; N, 13.65; S, 10.42. Found: C, 50.85; H, 3.17; Cl, 11.39; N,
13.78; S, 10.56.
Compound 5p: Yield (53%); mp 156–158 °C; IR (Nujol): 760
(C–S–C), 1605 (C@N), 3105 cmꢁ1 (NH); 1H NMR (CDCl3): d 4.65
(s, 2H, –OCH2–), 4.90 (br s, 1H, –NH, D2O exchangeable),

K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
219
Compound 6f: Yield (60%); mp 160–162 °C; IR (Nujol): 1685 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.74 (s, 3H, –CH3), 2.26 (s, 3H, Ar-CH3),
4.64 (s, 2H, –OCH2–), 6.35 (s, 1H, –CH–), 7.05–7.37 (m, 3H, Ar-H);
EI-MS: m/z 321.5 (M+, 52). Anal. Calcd for C14H12ClN3O2S (321.5):
C, 52.26; H, 3.76; Cl, 11.02; N, 13.06; S, 9.97. Found: C, 52.41; H,
3.64; Cl, 11.16; N, 13.19; S, 9.86.
56). Anal. Calcd for C13H10BrN3O2S (352): C, 44.33; H, 2.86; Br,
22.69; N, 11.93; S, 9.10. Found: C, 44.44; H, 2.76; Br, 22.55; N,
11.86; S, 9.19.
Compound 6r: Yield (61%); mp 98–100 °C; IR (Nujol): 1670 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.73 (s, 3H, –CH3), 4.57 (s, 2H, –OCH2–),
6.31 (s, 1H, –CH–), 6.95–7.32 (m, 4H, Ar-H); EI-MS: m/z 291 (M+,
54). Anal. Calcd for C13H10FN3O2S (291): C, 53.60; H, 3.46; F,
6.52; N, 14.42; S, 11.01. Found: C, 53.77; H, 3.37; F, 6.43; N,
14.57; S, 11.18.
Compound 6g: Yield (65%); mp 178–180 °C; IR (Nujol): 1665 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.71 (s, 3H, –CH3), 2.15 (s, 3H, Ar-CH3),
4.52 (s, 2H, –OCH2–), 6.29 (s, 1H, –CH–), 7.05–7.47 (m, 4H, Ar-H);
EI-MS: m/z 287 (M+, 58). Anal. Calcd for C14H13N3O2S (287): C,
58.52; H, 4.56; N, 14.62; S, 11.16. Found: C, 58.65; H, 4.42; N,
14.51; S, 11.05.
Compound 6s: Yield (69%); mp 139–141 °C; IR (Nujol): 1680 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.75 (s, 3H, –CH3), 4.53 (s, 2H, –OCH2–),
6.25 (s, 1H, –CH–), 7.04–7.36 (m, 5H, Ar-H); EI-MS: m/z 273 (M+,
59). Anal. Calcd for C13H11N3O2S (273): C, 57.13; H, 4.06; N,
15.37; S, 11.73. Found: C, 57.24; H, 4.20; N, 15.48; S, 11.61.
Compound 6t: Yield (65%); mp 194–196 °C; IR (Nujol): 1680 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.75 (s, 3H, –CH3), 2.35 (s, 6H, Ar-2CH3),
4.65 (s, 2H, –OCH2–), 6.43 (s, 1H, –CH–), 7.05–7.45 (m, 3H, Ar-H);
EI-MS: m/z 301 (M+, 62). Anal. Calcd for C15H15N3O2S (301): C,
59.78; H, 5.02; N, 13.94; S, 10.64. Found: C, 59.65; H, 5.22; N,
13.81; S, 10.73.
Compound 6h: Yield (62%); mp 165–167 °C; IR (Nujol): 1675 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.73 (s, 3H, –CH3), 4.55 (s, 2H, –OCH2–),
6.32 (s, 1H, –CH–), 6.95–7.31 (m, 4H, Ar-H); EI-MS: m/z 352 (M+,
55). Anal. Calcd for C13H10BrN3O2S (352): C, 44.33; H, 2.86; Br,
22.69; N, 11.93; S, 9.10. Found: C, 44.17; H, 2.97; Br, 22.58; N,
11.81; S, 9.22.
Compound 6i: Yield (63%); mp 185–187 °C; IR (Nujol): 1690 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.75 (s, 3H, –CH3), 4.54 (s, 2H, –OCH2–),
6.31 (s, 1H, –CH–), 6.92–7.25 (m, 4H, Ar-H); EI-MS: m/z 291 (M+,
51). Anal. Calcd for C13H10FN3O2S (291): C, 53.60; H, 3.46; F,
6.52; N, 14.42; S, 11.01. Found: C, 53.69; H, 3.31; F, 6.67; N,
14.31; S, 11.19.
5. Biology
5.1. Assay of xanthine oxidase activity
Compound 6j: Yield (61%); mp 190–192 °C; IR (Nujol): 1670 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.72 (s, 3H, –CH3), 4.54 (s, 2H, –OCH2–),
6.35 (s, 1H, –CH–), 6.91–7.38 (m, 4H, Ar-H); EI-MS: m/z 318 (M+,
54). Anal. Calcd for C13H10N4O4S (318): C, 49.05; H, 3.17; N,
17.60; S, 10.07. Found: C, 49.15; H, 3.28; N, 17.48; S, 10.21.
Compound 6k: Yield (61%); mp 111–113 °C; IR (Nujol): 1655 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.69 (s, 3H, –CH3), 4.45 (s, 2H, –OCH2–),
6.25 (s, 1H, –CH–), 7.05–7.35 (m, 4H, Ar-H); EI-MS: m/z 318 (M+,
53). Anal. Calcd for C13H10N4O4S (318): C, 49.05; H, 3.17; N,
17.60; S, 10.07. Found: C, 49.16; H, 3.06; N, 17.71; S, 10.15.
Compound 6l: Yield (68%); mp 116–118 °C; IR (Nujol): 1690 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.72 (s, 3H, –CH3), 2.34 (s, 6H, Ar-2CH3),
4.65 (s, 2H, –OCH2–), 6.35 (s, 1H, –CH–), 7.10–7.41 (m, 3H, Ar-H);
EI-MS: m/z 301 (M+, 60). Anal. Calcd for C15H15N3O2S (301): C,
59.78; H, 5.02; N, 13.94; S, 10.64. Found: C, 59.87; H, 5.19; N,
13.83; S, 10.55.
The XO inhibitory activity was monitored spectrophotometri-
cally following the absorbance of uric acid at 292 nm under aerobic
condition. Rat liver was homogenized in 0.01 M Tris–HCl pH (8.0)
containing 1 mM EDTA. The homogenate was centrifuged and the
supernatant was used as a source of enzyme. It was stored at
ꢁ80 °C until use.15,16 Bovine milk crude XO was prepared accord-
ing to Ball EG method.23 Microbial XO was obtained from Sigma
Laboratory and similar procedure was employed for the assay as
that of rat liver XO. The protein content was determined by the
Lowry’s method,24 using bovine serum albumin as standard.
Xanthine oxidase activity was recorded using a Shimadzu Spec-
trophotometer. The enzyme assay mixture consisted of 50 mM
potassium phosphate buffer (pH 7.5) containing 0.3 mM EDTA
and the enzyme source in a total volume of 2 ml. In dose-
dependent inhibition studies, the reaction was initiated by the
Compound 6m: Yield (60%); mp 198–200 °C; IR (Nujol):
1660 cmꢁ1 (C@O); 1H NMR (CDCl3): d 1.72 (s, 3H, –CH3), 4.55 (s,
2H, –OCH2–), 6.35 (s, 1H, –CH–), 6.91–7.32 (m, 4H, Ar-H); EI-MS:
m/z 291 (M+, 51). Anal. Calcd for C13H10FN3O2S (291): C, 53.60;
H, 3.46; F, 6.52; N, 14.42; S, 11.01. Found: C, 53.77; H, 3.59; F,
6.42; N, 14.56; S, 11.15.
addition of xanthine (50
lM) as the substrate to the above assay
mixture and the test compounds. The absorption rate at
a
wavelength of 292 nm indicates the formation of uric acid at
10 min intervals at ambient temperature. Duplicate assays were
repeated three to four times. Allopurinol was used as positive
control. The inhibitory activity of each test compound against XO
was indicated by their IC50 values. The percentage inhibition of
XO activity was calculated using the following formula.
Compound 6n: Yield (69%); mp 102–104 °C; IR (Nujol): 1660 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.73 (s, 3H, –CH3), 4.47 (s, 2H, –OCH2–),
6.27 (s, 1H, –CH–), 7.05–7.42 (m, 4H, Ar-H); EI-MS: m/z 318 (M+,
53). Anal. Calcd for C13H10N4O4S (318): C, 49.05; H, 3.17; N,
17.60; S, 10.07. Found: C, 49.20; H, 3.31; N, 17.49; S, 10.19.
Compound 6o: Yield (62%); mp 155–157 °C; IR (Nujol): 1655 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.75 (s, 3H, –CH3), 2.32 (s, 3H, Ar-CH3),
4.52 (s, 2H, –OCH2–), 6.29 (s, 1H, –CH–), 7.01–7.39 (m, 4H, Ar-H);
EI-MS: m/z 287 (M+, 61). Anal. Calcd for C14H13N3O2S (287): C,
58.52; H, 4.56; N, 14.62; S, 11.16. Found: C, 58.63; H, 4.43; N,
14.51; S, 11.29.
Abs control ꢁ Abs sample
Xanthine oxidase Inhibitionð%Þ ¼
ꢀ 100
Abs control
Abs control = Absorbance of the control reaction (containing all
reagents except the test compound).
Abs sample = Absorbance of the test compound.
Compound 6p: Yield (60%); mp 125–127 °C; IR (Nujol): 1670 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.73 (s, 3H, –CH3), 4.55 (s, 2H, –OCH2–),
6.29 (s, 1H, –CH–), 6.99–7.32 (m, 4H, Ar-H); EI-MS: m/z 307.5 (M+,
51). Anal. Calcd for C13H10ClN3O2S (307.5): C, 50.73; H, 3.28; Cl,
11.52; N, 13.65; S, 10.42. Found: C, 50.62; H, 3.39; Cl, 11.66; N,
13.55; S, 10.31.
5.2. Molecular modeling and docking studies
The crystal structure of XO from human milk was retrieved
from the PDB database with the corresponding entry code 2CKJ
(resolution of 3.59 Å). The protein was prepared by removing all
water molecules and adding all hydrogen atoms using the Accelrys
Discovery Studio ver 1.7 (Accelrys, Inc., San Diego.). The ligands,
once built were docked into the active site using the molecular
docking software SYBYL ver 7.3 (Tripos, L.P.) Surflex-Dock
Compound 6q: Yield (66%); mp 201–203 °C; IR (Nujol): 1670 cmꢁ1
(C@O); 1H NMR (CDCl3): d 1.75 (s, 3H, –CH3), 4.55 (s, 2H, –OCH2–),
6.35 (s, 1H, –CH–), 6.95–7.34 (m, 4H, Ar-H); EI-MS: m/z 352 (M+,

220
K. R. Sathisha et al. / Bioorg. Med. Chem. 19 (2011) 211–220
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(BioPharmics LLC) with the default parameters. Both of these pro-
prietary software are licensed to Manipal Institute of Technology,
Manipal University, India. Surflex-Dock is a program for calculating
the docking modes of small molecules into protein-binding sites. In
this study we have used ChemScore, a scoring function that is
derived from regression against ligand-receptor binding free
energies. In the docking process the active site was defined along
with MOS1327 complex. For each ligand 20 conformations were
generated and docked into XO.
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5.3. Data analysis
Data was analyzed by ANOVA followed by Tukey’s multiple
comparisons test for significant differences and the correlations
were calculated by Pearson’s correlation using SPSS 14.0 software.
IC50 values were calculated by Boltzmann’s dose–response analysis
using Origin 6.1 software.
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Acknowledgment
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The authors are grateful to University of Mysore for providing
laboratory facility at Department of Chemistry and Biochemistry,
Mysore.
References and notes
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