Synthesis and biological evaluation of diamide derivatives of (S)-BINOL and biphenyl as potential anti-inflammatory/anti-arthritic agents

Article abstract of DOI:10.1007/s00044-012-0410-6

A series of (S)-BINOL and biphenyl based diamides have been synthesized and characterized from analytic and spectral data. Anti-arthritic activity was studied by inhibition of protein denaturation method (Bovine serum albumin). All the diamides exhibit excellent anti-arthritic activity. Anti-inflammatory activity of the diamides synthesized was investigated using human red blood cells membrane stabilization method, and some of the diamides exhibit good anti-inflammatory activity.

Full text of DOI:10.1007/s00044-012-0410-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0410-6
ORIGINAL RESEARCH
Synthesis and biological evaluation of diamide derivatives
of (S)-BINOL and biphenyl as potential anti-inflammatory/
anti-arthritic agents
•
Raj Rajadurai Ramar Padmanabhan
•
Sarkkarai Ananthan
Received: 29 July 2012 / Accepted: 8 December 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of (S)-BINOL and biphenyl based
diamides have been synthesized and characterized from
analytic and spectral data. Anti-arthritic activity was studied
by inhibition of protein denaturation method (Bovine serum
albumin). All the diamides exhibit excellent anti-arthritic
activity. Anti-inflammatory activity of the diamides syn-
thesized was investigated using human red blood cells
membrane stabilization method, and some of the diamides
exhibit good anti-inflammatory activity.
(Angell et al., 2008a, 2008b). Synthesis of new chiral
receptors containing (S)-BINOL and thiourea units, and
their enantioselective recognition ability for chiral car-
boxylate anions has been recently reported (Hu et al.,
2009).
Inflammation comprises a complex series of biochemi-
cal events that can be initiated by a number of stimuli
including noxious chemical agents, pathogens, and auto-
immune responses, and in pathological situations, may lead
to the destruction of cells, tissues, and organs. Non-ste-
roidal anti-inflammatory drugs (NSAIDs) alleviate pain by
counteracting the cyclooxygenase (COX) enzyme that
converts arachidonic acid into prostaglandins in inflam-
matory processes (Tozokoparan et al., 2007; Cena et al.,
2003). NSAID are used for the treatment of pain, fever, and
inflammation particularly arthritis (Bhandari et al., 2008).
The common NSAIDs such as aspirin, ibuprofen, naproxen,
and fenbufen have side effects like upper GI irritation,
ulceration, dyspepsia, bleeding, in some cases deaths and
gives only temporary relief for short periods (Zadrazil,
2006; Husain et al., 2009). To overcome the GI ulceration
side effect of drug, more COX-2 selective inhibitor NSAID
is preferred, which does not significantly inhibit cycloox-
ygenase in the stomach and appears to be less likely to
cause GI ulceration (Laine, 1996). Unfortunately, very
effective COX-2 selective inhibitor drugs, i.e., rofecoxib
and celecoxib, were withdrawn from the market because of
the increased risk of heart attack and stroke associated with
long-term, high-dose use. Hence, it is of interest to syn-
thesize and study the anti-arthritic and anti-inflammatory
activity of novel amides. However, to the best of our
knowledge, such BINOL, biphenyl based diamides have
not been reported in the literature. Herein, we report the
synthesis, characterization of anti-arthritic and anti-
inflammatory activities of amides 1–8.
Keywords Diamide ꢀ Cysteamine ꢀ Anti-inflammatory ꢀ
Anti-arthritic
Introduction
Difluorobiphenyl amides have been reported as potential
anti-inflammatory agents recently (Zhong et al., 2009). The
diamides prepared from 2-phenylimidazopyridines are
effective in animal models of asthma, cancer, inflamma-
tion, and infectives (antiviral) (Banie et al., 2007). Biaryl
diamides exhibit as potent melanin concentrating hormone
receptor 1 antagonists (Palani et al., 2005). The biphenyl
diamides are novel series of p38 MAP kinase inhibitor
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0410-6) contains supplementary
material which is available to authorized users.
R. Rajadurai ꢀ R. Padmanabhan
Research and Development Centre, Orchid Chemicals and
Pharmaceuticals Ltd., Shozhinganallur, Chennai 600119, India
R. Rajadurai ꢀ S. Ananthan (&)
Department of Chemistry, Presidency College, Chennai 600005,
India
e-mail: sarkkaraiananthan@gmail.com
123

Med Chem Res
Results and discussion
chloride 3.0 equiv. in the presence of triethyl amine in
dichloromethane at 0–5 °C for 2 h. After usual workup
followed by recrystallization from acetone afforded the bis-
oxy mesyl compounds 17 and 18 in about 71 and 80 %
yields, respectively. The structure of the bis-oxy mesyl
compounds 17 and 18 was confirmed from the spectral and
The synthetic pathway leading to diamines 9 and 10 is
outlined in Scheme 1. Reaction of 1.0 equiv. of bisphenol
11 and (S)-BINOL 12 with 2.2 equiv. of ethyl bromoace-
tate in the presence of 3.0 equiv. potassium carbonate in
acetonitrile in the presence of catalytic amount of KI at
reflux for 8 h resulted in the formation of bis-oxy esters 13
and 14 in about 73 and 70 % yields, respectively, after
purification by column chromatography. The structure of
the bis-oxy esters 13 and 14 was confirmed from the
1
analytic data. The H NMR spectrum of bis-oxy mesyl
compound 17 displayed the O-methylene protons as a
multiplet between d 4.21–4.23 and d 4.37–4.40. The rest of
the aromatic protons appeared between d 6.98 and 7.34.
The mass spectrum showed the molecular ion peak
([M?NH4]?) at m/z 448.1. Similarly, the structure of bis-
oxy mesyl compound 18 was confirmed from spectral and
analytic data. Bis-oxy mesyl compounds 17 and 18 were
brominated with LiBr 4.0 equiv. in dry THF at room
temperature. After usual workup, bis-oxy bromides 19 and
20 were isolated in about 77 and 78 % yields, respectively,
after purification by column chromatography. The structure
of the bis-oxy bromide 19 and 20 was confirmed from the
1
spectral and analytic data. The H NMR spectrum of bis-
oxy ester 13 displayed the O-methylene protons as a
quartet at d 4.10 and singlet at d 4.70. The rest of the
aromatic protons appeared between d 6.94 and 7.31. The
mass spectrum showed the molecular ion peak ([M?NH4]?)
at m/z 376.1. Similarly, the structure of bis-oxy ester 14
was confirmed from spectral and analytic data. The bis-oxy
esters 13 and 14 were reduced using LiAlH4 (4.0 equiv.) in
dry THF at reflux for 3 h. The reaction mixture was
quenched with saturated ammonium chloride solution to
give the bis-oxy alcohols 15 and 16 with 65 and 70 %
yields, respectively. The structure of the bis-oxy alcohols
15 and 16 was confirmed from the spectral and analytic
1
spectral and analytic data. The H NMR spectrum of bis-
oxy bromide compound 19 displayed the O-methylene
protons as a triplet at d 3.62 and d 4.25. The rest of the
aromatic protons appeared between d 6.98 and 7.32. The
mass spectrum showed the molecular ion peak ([M?NH4]?)
at m/z 416.0. Similarly, the structure of bis-oxy bromide 20
was confirmed from spectral and analytic data. The bis-oxy
bromides 19 and 20 were treated with cysteamine 10 equiv.
in dry THF in the presence of sodium methoxide at room
temperature for 4 h. The bis-oxy amines 9 and 10 were
purified by column chromatography in about 65 and 72 %
yields, respectively. The structure of the bis-oxy amines 9
and 10 was confirmed from the spectral and analytic data.
1
data. The H NMR spectrum of bis-oxy alcohol 15 dis-
played the O-methylene protons as a multiplet between
d 3.55–3.60 and triplet at d 3.94. The rest of the aromatic
protons appeared between d 6.93 and 7.30. The mass
spectrum showed the molecular ion peak ([M?NH4]?) at
m/z 292.1. Similarly, the structure of bis-oxy alcohol 16
was confirmed from spectral and analytic data. The bis-oxy
alcohols 15 and 16 were treated with methane sulfonyl
Scheme 1 Reagents and
conditions: i ethyl bromo
acetate, K2CO3, KI, ACN,
reflux, 8 h, 13 (73 %) and 14
(70 %); ii LiAlH4, THF, reflux,
3 h, 15 (65 %) and 16 (70 %);
iii mesyl chloride, DCM,
O
O
O
R
R
i
ii
O
H
H
R
HO
OH
CH3
H3C
15, 16
13, 14
0–5 °C, 2 h, 17 (71 %) and 18
(80 %); iv LiBr, THF, rt, 4 h, 19
(78 %) and 20 (78 %) and
v cysteamine, sodium
methoxide, THF, rt, 4 h, 9
(65 %) and 10 (72 %)
iii
O
O
O
O
R
=
,
R
O O
O O
12
11
S
O
O
S
CH3
H3C
17, 18
R
iv
S
S
R
v
Br
Br
NH2
19, 20
H2N
9, 10
123

Med Chem Res
Scheme 2 Reagents and
conditions: i TEA, DCM, rt,
3 h, 1 (67 %), 2 (68 %), 3
(71 %) and 4 (71 %)
N
O
O
N
N
O
O
N
N
S
S
S
S
N
H
N
H
O
O
O
O
H
N
H
N
N
N
N
2
1
COCl
COCl
N
N
N
N
i
i
S
S
NH2
NH2
O
O
9
O
COCl
S
COCl
i
i
O
O
O
S
S
O
N
H
S
S
N
H
O
O
O
O
O
H
N
H
N
S
S
O
3
4
1
The H NMR spectrum of bis-oxy amine 9 displayed the
N-CH2 protons as a triplet at d 2.68, O-methylene protons
as a triplet at d 4.07, and S-CH2 protons as a triplet at
d 2.40 and d 2.57. The rest of the aromatic protons
appeared between d 6.95 and 7.32. In the 13C NMR
spectrum of 9, the N-CH2 carbons appeared at d 41.1,
O-methylene carbons appeared at d 68.4, and S-CH2 carbons
appeared at d 29.9 and 34.8. The mass spectrum showed the
molecular ion peak ([M?H]?) at m/z 393.1. Similarly, the
structure of bis-oxy amine 10 was confirmed from spectral
and analytic data. All the new compounds gave satisfactory
FT-IR, 1H and 13C NMR, and mass spectral analysis.
temperature. The reaction mixture after usual work-up
afforded the diamides 1–4 in about 67, 68, 71, and 71 %
yields, respectively (Scheme 2). The structure of the bis-
oxy diamides 1–4 was confirmed from the spectral and
1
analytic data. The H NMR spectrum of diamide 1 dis-
played the N-CH2 protons as a AB quartet at d 3.12, a
singlet at d 5.22, O-methylene protons as a triplet at d 4.07,
S-CH2 protons as a triplet at d 2.43, 2.70, and NH protons
as a triplet at d 8.46. The rest of the aromatic protons
appeared between d 6.95 and 9.33. In the 13C NMR
spectrum of 8, the N-CH2 carbons appeared at d 38.6, 49.4;
O-methylene carbons appeared at d 68.4; S-CH2 carbons
appeared at d 29.9, 31.0; and carbonyl carbon at d 164. The
FT-IR spectrum of 1 showed the carbonyl carbon stretching
frequency at 1,671 cm-1, and the mass spectrum showed
the molecular ion peak ([M?H]?) at m/z 613.1. Similarly,
the structures of diamides 2–4 were confirmed from
In order to test the synthetic utility of bis-oxy amine 9
for the synthesis of diamides, 1.0 equiv. of bis-oxy amine 9
was coupled with 2.2 equiv. of each tetrazole acetyl chlo-
ride, benzoyl chloride, furoyl chloride, and thiophene
acetyl chloride in the presence of TEA in dry DCM at room
123

Med Chem Res
Scheme 3 Reagents and
conditions: i TEA, DCM, rt,
3 h, 5 (71 %), 6 (66 %), 7
(67 %) and 8 (62 %)
N
N
O
O
N
N
O
N
S
S
S
S
N
H
N
H
O
O
O
O
H
N
H
N
N
N
N
O
6
COCl
5
COCl
N
N
N
N
i
i
S
S
NH2
NH2
O
O
10
O
COCl
S
COCl
i
i
O
O
S
O
S
S
S
O
N
H
N
H
O
O
O
O
O
H
H
N
N
O
S
S
8
7
Table 1 In vitro anti-arthritic activity of diamides 1–8 by inhibition
of protein denaturation method (Bovine serum albumin)
spectral and analytic data. All the new compounds gave
1
satisfactory FT-IR, H and 13C NMR, mass spectral, and
Diamide
Activity (% inhibition of protein denaturation)
elemental analysis.
In order to test the synthetic utility of bis-oxy amine 10
for the synthesis of diamides, 1.0 equiv. of bis-oxy amine
10 was coupled with 2.2 equiv. of each tetrazole acetyl
chloride, benzoyl chloride, furoyl chloride, and thiophene
acetyl chloride in the presence of TEA in dry DCM at room
temperature. The reaction mixture, after usual work-up,
afforded the diamides 5–8 in about 71, 66, 67, and 62 %
yields, respectively (Scheme 3). The structure of the bis-
oxy diamides 5–8 was confirmed from the spectral and
50 lg/ 100 lg/ 200 lg/ 400 lg/ 800 lg/
mL
mL
mL
mL
mL
1
2
3
4
5
6
7
8
21.6
15.8
25.7
28.3
19.6
19.5
29.5
23.6
5.6
42.2
38.2
46.6
34.9
39.6
30.3
50.8
42.2
10.9
65.1
53.0
68.8
41.1
67.1
52.3
71.5
59.1
15.2
76.3
67.8
83.1
69.6
74.1
65.8
87.8
73.3
21.6
91.9
81.8
97.2
84.8
94.2
82.7
98.7
87.4
76.7
1
analytic data. The H NMR spectrum of diamide 5 dis-
played the N-CH2 protons as a multiplet between d 2.90
and 2.97, a singlet at d 5.20, O-methylene protons as a
triplet at d 4.14, S-CH2 protons as a multiplet between d
2.13–2.23, at d 2.50 (merged with DMSO), and NH protons
Diclofenac
sodium
Each value represents mean SD of three observations
123

Med Chem Res
Fig. 1 Anti-arthritic activity of
diamides 1–8
Anti-arthritic activity
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
Concentration in µg/mL
Diclofenac Sodium
800
50
100
200
400
as a triplet at d 8.38. The rest of the aromatic protons appeared
between d 6.92 and 9.34. In the 13C NMR spectrum of 5, the
N-CH2 carbonsappearedatd38.2, 49.3;O-methylenecarbons
appeared at d 69.7; S-CH2 carbons appeared at d 29.9, 30.7;
and carbonyl carbon at d 164.5. The FT-IR spectrum of 5
showed the carbonyl carbon stretching frequency at
1,676 cm-1, and the mass spectrum showed the molecular ion
peak ([M?H]?) at m/z 713.2. Similarly, the structures of
diamides 6–8 were confirmed from spectral and analytic data.
protein denaturation results showed that the amides are
more stable in BSA than diclofenac sodium. It is possible
that the stability related to the strong binding of the amides
on BSA is due to the increased hydrophobicity as com-
pared to diclofenac sodium. The degree of anti-arthritic
activity of amides 7, 3, 5, 1, 8, 4, 6 and 2 at 800 lg/mL is
found to be 98.7, 97.2, 94.2, 91.9, 87.4, 84.8, 82.7, and
81.8 %, respectively (Table 1; Fig. 1).
All the new compounds gave satisfactory FT-IR, 1H and 13
NMR, mass spectral, and elemental analysis.
C
Anti-inflammatory activity
Further anti-inflammatory activity of compounds 1–8 was
investigated using human red blood cells (HRBCs) mem-
brane stabilization (Sangeetha et al., 2011; Gandhidsan
et al., 1991; Sadique et al., 1989) with prednisolone as the
standard, and the results are shown in Table 2. The lyso-
somal enzymes released during inflammatory condition
produce a variety of disorders. The extra cellular activity of
these enzymes is said to be related to acute or chronic
Anti-arthritic activity
In order to study the anti-arthritic activity of the diamides
1–8, inhibition of protein denaturation method [Bovine
serum albumin (BSA)] (Sangeetha et al., 2011; Mizushima
and Kobayashi, 1968; Mizushima, 1965) was employed,
and diclofenac sodium was used as a standard. The dena-
turation of protein as one of the causes for rheumatoid
arthritis was well documented (Mizushima, 1966). Pro-
duction of auto-antigens in certain rheumatic diseases may
be due to in vivo denaturation of proteins (Brown and
Mackey, 1968). The mechanism of denaturation probably
involves alteration in electrostatic, hydrogen, hydrophobic,
and disulfide bonding (Grant et al., 1970). The anti-arthritic
activities of all the diamides are concentration-dependent.
At higher concentration, the amides exhibit better anti-
arthritic activity than at lower concentration. The amides 7,
3, 5, 1, 8, 4, 6 and 2 were found to possess the maximum
anti-arthritic activity (98.7, 97.2, 94.2, 91.9, 87.4, 84.8,
82.7, and 81.8 % at 800 lg/mL) when compared to the
reference drug diclofenac sodium (76.7 % at 800 lg/mL),
which clearly shows that amides are superior to the refer-
ence drug diclofenac sodium. The superior inhibition of
Table 2 In vitro anti-inflammatory activity of diamides 1–8 by
HRBC membrane stabilization method
Diamide
Activity (% prevention of lysis)
10 lg/mL 50 lg/mL 100 lg/mL 200 lg/mL
1
2
3
4
5
6
7
8
54.0 0.2 63.3 0.4 75.3 0.5 89.4 0.3
35.7 0.7 49.0 0.4 60.7 0.7 77.5 0.5
55.2 0.6 62.9 0.3 84.7 0.5 94.1 0.8
47.3 0.8 58.6 0.5 67.2 0.7 83.3 0.6
61.9 0.9 69.7 0.5 82.5 0.6 91.6 0.3
34.5 0.5 51.4 0.4 63.6 0.6 79.4 0.8
56.5 0.4 65.5 0.3 88.9 0.1 96.2 0.4
50.2 0.6 61.7 0.4 72.8 0.6 86.0 0.8
Prednisolone 46.0 0.1 57.9 0.3 84.8 0.4 91.0 0.5
Each value represents mean SD of three observations
123

Med Chem Res
Fig. 2 Anti-inflammatory
activity of diamides 1–8
Anti-inflammatry activity
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
ne
o
l
so
i
n
i
Concentration in µg/mL
d
re
P
10
50
100
200
inflammation. The anti-inflammatory agent acts by either
inhibiting the lysosomal enzymes or by stabilizing the
lysosomal membranes.
diamides with different biologically important substituent to
improve the solubility and efficacy, in vivo anti-arthritic,
anti-inflammatory assay, and molecular recognition toward
various biologically important anions are under investi-
gation.
The anti-inflammatory activities of all the amides are
concentration-dependent. At higher concentration, the
amides exhibited better anti-inflammatory activity than at
lower concentration. The amides 7, 3 and 5 were found to
possess the maximum anti-inflammatory activity (96.2,
94.1, and 91.6 % at 200 lg/mL) when compared to the
reference drug prednisolone (91.0 % at 200 lg/ml), which
clearly shows that anti-inflammatory activity of the amides
is superior to the reference drug prednisolone. The better
anti-inflammatory activity of amides could be due to their
binding on to the erythrocyte membranes with subsequent
alteration of the charges on the membrane surface of the
cells. This might have prevented physical interaction with
aggregating agents or promote dispersal by mutual repul-
sion of like charges which are involved in the hemolysis of
red blood cells. The degree of anti-inflammatory activity of
cyclophane amides 7, 3, 5, 1, 8, 4, 6 and 2 at 200 lg/mL is
found to be 96.2, 94.1, 91.6, 89.4, 86.0, 83.3, 79.4, and
77.5 %, respectively (Table 2; Fig. 2).
Experimental
All the reagents and solvents employed were of the best
grade available and were used without further purification.
The melting points were determined using a Metler Toledo
melting point apparatus by open capillary tube method and
were uncorrected. Spectroscopic data were recorded by the
following instruments: UV/Vis: Shimadzu 2550 spectro-
photometer. IR: Perkin-Elmer series 2000 FT-IR spectro-
photometer. NMR: Bruker Avance 400 MHz. Mass:
ESI—PerkinElmer Sciex, API 3000 mass spectrometer and
FAB—mass spectra Jeol SX 102/DA-6000 mass spec-
trometer. The elemental analysis for the compounds was
carried out using the Elementar Vario EL III elemental
analyzer (SIPRA LABS LTD., Hyderabad, India). Pre-
coated silica gel plates from Merck were used for TLC.
Column chromatography was carried out using silica gel
(100–200 mesh) purchased from ACME.
Conclusions
In conclusion, diamides were 1–8 synthesized and show
better anti-arthritic activity than the reference drug diclofe-
nac sodium. All the diamides 3, 5 and 7 show good anti-
inflammatory activity at (200 lg/mL) than the reference
drug prednisolone. The diamides 1–8 may be developed as
anti-arthritic drug as they showed better activity than the
reference drug diclofenac sodium. The diamides 3, 5 and 7
may be developed as anti-inflammatory drug (NSAID) as
they show better activity than the reference drug predniso-
lone. Further studies are required to determine their toxicity,
bioavailability, mode of action etc. Synthesis of similar
General procedure for the synthesis of diamines
To a solution of cysteamine (200 mmol), sodium meth-
oxide (210 mmol) in dry THF (100 mL) and dibromide
(20 mmol) were added to the reaction mixture and stirred
for further 4 h. The solvent was removed at reduced
pressure; and the residue obtained was then dissolved in
DCM (50 mL), washed with water (2 9 200 mL) and then
dried over anhydrous potassium carbonate. Removal of the
DCM under reduced pressure gave the corresponding
123

Med Chem Res
amine as a crude material which was purified by column
chromatography (SiO2).
J = 6.7 Hz); 13C NMR (100 MHz, DMSO-d6) d 164.6,
155.7, 145.1, 131.2, 128.6, 127.5, 120.4, 112.20, 68.4,
49.4, 38.6, 31.0, 29.9; MS (ES) m/z: 613.1 [M?H]?; Ele-
mental Anal. Calc. for. C26H32N10O4 S2: C, 50.97; H, 5.26;
N, 22.86; S, 10.47 %; Found: C, 50.78; H, 5.35; N, 22.72;
S, 10.38 %.
2,20-(2,20-(Biphenyl-2,20-diylbis(oxy))bis(ethane-2,
1-diyl))bis(sulfanediyldiethanamine (9)
1
Yield 65 %; mp 93 °C; IR (KBr, cm-1) 1669, 3360; H
NMR (400 MHz, DMSO-d6)d 7.27–7.32 (m, 2H), 7.16 (dd,
2H, J = 7.4 and 1.6 Hz), 7.04 (d, 2H, J = 8.1 Hz), 6.95 (t,
2H J = 7.4 Hz), 4.07 (t, 4H, J = 6.5 Hz), 2.68 (t, 4H,
J = 6.5 Hz), 2.57 (t, 4H, J = 6.6 Hz), 2.40 (t, 4H, J =
6.6 Hz); 13C NMR (100 MHz, DMSO-d6) d 155.7, 131.2,
128.6, 127.6, 120.3, 112.2, 68.4, 41.1, 34.8, 29.9; MS (ES)
m/z: 393.1 [M?H]?; Elemental Anal. Calc. for. C20H28N2
O2S2: C, 61.19; H, 7.19; N, 7.14; S, 16.34 %; Found: C,
61.43; H, 7.35; N, 7.31; S, 16.18 %.
N,N0-(2,20-(2,20-(Biphenyl-2,20-diylbis(oxy))bis
(ethane-2,1-diyl))bis(sulfanediyl)bis(ethane-2,1-diyl))
dibenzamide (2)
1
Yield 68 %; mp 118 °C; IR (KBr, cm-1) 1633, 3317; H
NMR (400 MHz, DMSO-d6) d 8.50 (bs, 2H), 7.81 (d, 4H,
J = 7.4 Hz), 7.50 (d, 2H, J = 7.0 Hz), 7.43 (t, 4H, J =
7.6 Hz), 7.22 (t, 2H, J = 7.9 Hz), 7.16 (d, 2H, J =
7.4 Hz), 7.01 (d, 2H, J = 8.2 Hz), 6.92 (t, 2H J = 7.4 Hz),
4.10 (t, 4H, J = 6.3 Hz), 3.29 (d, 4H, J = 7.4 Hz), 2.74 (t,
4H, J = 6.2 Hz), 2.55 (t, 4H, J = 6.7 Hz),; 13C NMR
(100 MHz, DMSO-d6) d 166.1, 155.7, 134.4, 131.2, 131.1,
128.5, 128.3, 127.5, 127.1, 120.3, 112.2, 68.3, 40.1*, 31.1,
29.9, *Merged with DMSO peak; MS (ES) m/z: 600.9
[M?H]?; Elemental Anal. Calc. for. C34H36N2O4 S2: C,
67.97; H, 6.04; N, 4.66; S, 10.67 %; Found: C, 67.78; H,
6.22; N, 4.72; S, 10.48 %.
(S)-2,20-(2,20-(1,10-Binaphthyl-2,20-diylbis(oxy))bis
(ethane-2,1-diyl))bis(sulfanediyl) diethanamine (10)
1
Yield 72 %; mp 96 °C; IR (KBr, cm-1) 1634, 3350; H
NMR (400 MHz, DMSO-d6) d 8.06 (m, 2H), 7.95 (d, 2H,
J = 8.1 Hz), 7.62 (d, 2H, J = 9.1 Hz), 7.33–7.37 (m, 2H),
7.23–7.27 (m, 2H), 6.93 (d, 2H J = 8.4 Hz), 4.14–4.24 (m,
4H), 2.62–2.71 (m, 4H), 2.53–2.61 (m, 4H), 2.40–2.50 (m,
4H); 13C NMR (100 MHz, DMSO-d6) d 153.7, 133.4,
129.5, 128.9, 128.0, 126.4, 124.6, 123.6, 119.3, 115.7,
69.2, 37.9, 29.7, 28.2; MS (ES) m/z: 493.3 [M?H]?; Ele-
mental Anal. Calc. for. C28H32N2O2S2: C, 68.26; H, 6.55;
N, 5.69; S, 13.02 %; Found: C, 68.11; H, 6.45; N, 5.81; S,
13.18 %.
N,N0-(2,20-(2,20-(Biphenyl-2,20-diylbis(oxy))bis
(ethane-2,1-diyl))bis(sulfanediyl)bis(ethane-2,1-diyl))
bis(2-(thiophen-2-yl)acetamide)(3)
1
Yield 71 %; mp 100 °C; IR (KBr, cm-1) 1649, 3277; H
NMR (400 MHz, DMSO-d6)d 8.09 (t, 2H, J = 5.5 Hz),
7.32 (dd, 2H, J = 5.12 and 1.2 Hz), 7.25–7.29 (m, 2H),
7.15 (dd, 2H, J = 7.5 and 1.7 Hz), 7.01 (d, 2H, J =
8.0 Hz), 6.92–6.97 (m, 4H), 6.89–6.90 (m, 2H), 4.06 (t, 4H,
J = 6.4 Hz), 3.62 (s, 4H), 3.08 (q, 4H, J = 6.6 Hz), 2.68
(t, 4H, J = 6.4 Hz), 2.42 (t, 4H, J = 6.9 Hz); 13C NMR
(100 MHz, DMSO-d6) d 169.0, 155.7, 137.6, 131.2, 128.5,
127.5, 126.5, 126.0, 124.8, 120.3, 112.2, 68.3, 38.6*, 36.5,
31.1, 30.0, *Merged with DMSO peak; MS (ES) m/z: 640.8
[M?H]?; Elemental Anal. Calc. for. C32H36N2O4 S4: C,
59.97; H, 5.66; N, 4.37; S, 20.01 %; Found: C, 59.82; H,
5.53; N, 4.60; S, 20.23 %.
General procedure for the synthesis of diamides
To a solution of diamine (1.0 mmol), TEA (2.5 mmol) in
dry DCM (20 mL) and acid chloride (2.2 mmol) were
added to the reaction mixture and stirred for 3 h. The
reaction mixture was washed with water to remove trieth-
ylammonium chloride and then dried over anhydrous
sodium sulfate. Removal of the DCM under reduced
pressure gave the corresponding amide as a crude material
which was purified by column chromatography (SiO2).
N-(2-(2-(20-(2-(2-(2-Oxo-2-(1H-tetrazol-1-yl)
ethylamino)ethylthio)ethoxy)biphenyl-2-yloxy)
ethylthio) ethyl)-2-(1H-tetrazol-1-yl)acetamide (1)
N,N0-(2,20-(2,20-(Biphenyl-2,20-diylbis(oxy))
bis(ethane-2,1-diyl))bis(sulfanediyl)bis(ethane-2,1-diyl))
difuran-2-carboxamide (4)
Yield 67 %; mp 167 °C; IR (KBr, cm-1) 1671, 3304; H
Yield 71 %; mp 115 °C; IR (KBr, cm-1) 1642, 3287; H
1
1
NMR (400 MHz, DMSO-d6) d 9.33 (s, 2H), 8.46 (t, 2H,
J = 5.1 Hz), 7.27 (t, 2H, J = 7.9 Hz), 7.16 (t, 4H, J =
7.4 Hz), 7.02 (d, 2H, J = 8.3 Hz), 6.95 (t, 2H, J = 7.3 Hz),
5.22 (s, 4H), 4.07 (t, 4H, J = 6.2 Hz), 3.12 (q, 4H,
J = 6.2 Hz), 2.70 (t, 4H, J = 6.2 Hz), 2.43 (t, 4H,
NMR (400 MHz, DMSO-d6)d 8.31 (t, 2H, J = 5.6 Hz), 7.82
(s, 2H), 7.21 (t, 2H, J = 7.4 Hz), 7.15 (d, 2H, J = 7.3 Hz),
7.07–7.08 (m, 2H), 7.01 (d, 2H, J = 8.2 Hz), 6.92 (d, 2H,
J = 7.4 Hz), 6.61–6.62 (m, 2H), 4.09 (t, 4H, J = 6.4 Hz),
3.23 (t, 4H, J = 6.5 Hz), 2.72 (t, 4H, J = 6.4 Hz), 2.53–2.54
123

Med Chem Res
(m, 4H)*, *merged with DMSO peak; 13C NMR (100 MHz,
DMSO-d6) d 157.6, 155.7, 147.9, 144.9, 131.2, 128.5, 127.5,
120.3, 113.3, 112.2, 111.9, 68.3, 38.1, 31.1, 29.9; MS (ES)
m/z: 580.9 [M?H]?; Elemental Anal. Calc. for. C30H32N2O6
S2: C, 62.05; H, 5.55; N, 4.82; S, 11.04 %; Found: C, 62.32;
H, 5.47; N, 4.66; S, 11.23 %.
DMSO-d6) d 168.9, 153.8, 137.6, 133.4, 129.4, 128.9,
128.0, 126.5, 126.3, 126.0, 124.8, 124.6, 123.6, 119.3,
115.7, 69.5, 38.3, 36.4, 30.9, 29.9; MS (ES) m/z: 740.8
[M?H]?; Elemental Anal. Calc. for. C40H40N2O4S4: C,
64.83; H, 5.44; N, 3.78; S, 17.31 %; Found: C, 64.69; H,
5.56; N, 3.61; S, 17.22 %.
(S)-N,N0-(2,20-(2,20-(1,10-Binaphthyl-2,20-diylbis(oxy))
bis(ethane-2,1-diyl))bis (sulfanediyl)bis(ethane-2,1-diyl))
bis(2-(1H-tetrazol-1-yl)acetamide (5)
(S)-N,N0-(2,20-(2,20-(1,10-Binaphthyl-2,20-diylbis(oxy))
bis(ethane-2,1-diyl))bis (sulfanediyl)bis(ethane-2,1-diyl))
difuran-2-carboxamide (8)
1
Yield 75 %; mp 170 °C; IR (KBr, cm-1) 1676, 3289; H
1
Yield 67 %; mp 90 °C; IR (KBr, cm-1) 1650, 3290; H
NMR (400 MHz, DMSO-d6)d 9.34 (s, 2H), 8.38 (t, 2H,
J = 5.24 Hz), 8.03 (d, 2H, J = 9.04 Hz), 7.93 (d, 2H, J =
8.20 Hz), 7.57 (d, 2H, J = 9.08 Hz), 7.31 (t, 2H, J =
7.16 Hz), 7.21 (t, 2H, J = 7.88 Hz), 6.92 (d, 2H,
J = 8.44 Hz), 5.20 (s, 4H), 4.14 (t, 4H, J = 6.24 Hz),
2.90–2.97 (m, 4H), 2.13–2.23 (m, 4H), 4-CH2 Protons
merged with DMSO peak; 13C NMR (100 MHz, DMSO-
d6) d 164.5, 153.8, 145.1, 133.4, 129.4, 128.9, 127.9, 126.3,
124.6, 123.6, 119.2, 115.6, 69.7, 49.3, 38.2, 30.7, 29.9; MS
(ES) m/z: 713.2 [M?H]?; Elemental Anal. Calc. for.
C34H36N10O4S2: C, 57.29; H, 5.09; N, 19.65; S, 9.00 %;
Found: C, 57.41; H, 5.23; N, 19.76; S, 9.13 %.
NMR (400 MHz, DMSO-d6)d 8.21 (t, 2H, J = 5.6 Hz),
8.00 (d, 2H, J = 9.0 Hz), 7.90 (d, 2H, J = 8.1 Hz),
7.81–7.82 (m, 2H), 7.56 (d, 2H, J = 9.1 Hz), 7.30 (d, 2H,
J = 7.1 Hz), 7.20 (t, 2H, J = 7.6 Hz), 7.05–7.06 (m, 2H),
6.92 (d, 2H, J = 8.4 Hz), 6.60–6.62 (m, 2H), 4.17 (t, 4H,
J = 6.5 Hz), 3.03–3.13 (m, 4H), 2.53 (m, 4H)*, 2.22–2.36
(m, 4H), *merged with DMSO peak; 13C NMR (100 MHz,
DMSO-d6) d 157.6, 153.7, 147.9, 144.9, 133.4, 129.4,
128.9, 128.0, 126.3, 124.6, 123.5, 119.2, 115.6, 113.3,
118.8, 69.5, 37.8, 30.80, 29.8; MS (ES) m/z: 740.8
[M?H]?; Elemental Anal. Calc. for. C38H36N2O6S4: C,
67.04; H, 5.33; N, 4.11; S, 9.42 %; Found: C, 67.19; H,
5.46; N, 4.26; S, 9.26 %.
(S)-N,N0-(2,20-(2,20-(1,10-Binaphthyl-2,20-diylbis(oxy))
bis(ethane-2,1-diyl))bis (sulfanediyl)bis(ethane-2,1-diyl))
dibenzamide (6)
Biology
1
Yield 66 %; mp 107 °C; IR (KBr, cm-1) 1651, 3318; H
In vitro anti-arthritic study
NMR (400 MHz, DMSO-d6) d 8.41–8.42 (m, 2H), 8.00 (d,
2H, J = 8.9 Hz), 7.90 (d, 2H, J = 7.8 Hz), 7.80 (d, 4H,
J = 7.2 Hz), 7.57 (d, 2H, J = 9.0 Hz), 7.43–7.51 (m, 6H),
7.14–7.33 (m, 4H), 6.93 (d, 2H, J = 8.3 Hz), 4.17–4.20 (m,
4H), 3.12–3.15 (m, 4H), 2.53–2.56 (m, 4H)*, 2.29–2.39 (m,
4H), *merged with DMSO peak; 13C NMR (100 MHz,
DMSO-d6) d 166.4, 134.8, 133.8, 131.5, 129.8, 129.3,
128.6, 128.4, 127.5, 126.7, 125.0, 123.9, 119.7, 116.0, 69.9,
39.0, 31.2, 30.3; MS (ES) m/z: 700.9 [M?H]?; Elemental
Anal. Calc. for. C32H40N2O4S2: C, 71.97; H, 5.75; N, 4.00;
S, 9.15 %; Found: C, 71.85; H, 5.57; N, 4.12; S, 9.22 %.
The test solution (0.5 mL) consists of Bovine Serum Albu-
min (0.45 mL, 5 % w/v aqueous solution) and a solution of
amides in DMSO (800, 400, 200, 100, 50 lg in 0.05 mL).
Test control solution (0.5 mL) consists of Bovine Serum
Albumin (0.45 mL, 5 % w/v aqueous solution) and distilled
water (0.05 mL). Product control solution (0.5 mL) consists
of distilled water (0.45 mL) and a solution of amides in
DMSO (800, 400, 200, 100, 50 lg in 0.05 mL). Standard
solution (0.5 mL) consists of bovine serum albumin
(0.45 mL, 5 % w/v aqueous solution) and diclofenac sodium
(800, 400, 200 lg/mL in 0.05 mL). All the above solutions
were adjusted to pH 6.3 using 1 N HCl. The samples were
incubated at 37 °C for 20 min, and the temperature was
increased to keep the samples at 57 °C for 3 min. After
cooling, phosphate buffer (2.5 mL) was added to the above
solutions. The absorbance was measured using UV–Visible
spectrophotometer at 416 nm. The percentage inhibition of
protein denaturation can be calculated as follows:
(S)-N,N0-(2,20-(2,20-(1,10-Binaphthyl-2,20-diylbis(oxy))
bis(ethane-2,1-diyl))bis (sulfanediyl)bis(ethane-2,1-diyl))
bis(2-(thiophen-2-yl)acetamide) (7)
1
Yield 62 %; mp 115 °C; IR (KBr, cm-1) 1646, 3408; H
NMR (400 MHz, DMSO-d6)d 8.00–8.04 (m, 4H), 7.92 (d,
2H, J = 8.2 Hz), 7.55 (d, 2H, J = 9.0 Hz), 7.30–7.34 (m,
4H), 7.20 (t, 2H, J = 7.9 Hz), 6.91–6.93 (m, 4H),
6.87–6.88 (m, 2H), 4.12 (t, 4H, J = 6.5 Hz), 3.58 (s, 4H),
2.89–2.92 (m, 4H), 2.15–2.22 (m, 4H), 4-CH2 pro-
tons merged with DMSO peak; 13C NMR (100 MHz,
%Inhibition ¼100 ꢁ ½optical density of test solution
ꢁopticaldensityofproduct controlÞ
ꢂ optical density of test controlÞꢃ ꢄ 100
123

Med Chem Res
Angell RM, Aston NM, Bamborough P, Buckton JB, Cockerill SG,
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In vitro anti-inflammatory study
Blood samples were collected from healthy volunteers, and
the collected blood was mixed with equal volume of ster-
ilized Alsever’s solution (2 % dextrose, 0.8 % sodium
citrate, 0.05 % citric acid, and 0.42 % sodium chloride).
The blood was centrifuged at 1,500 rpm, and the packed
cells were washed with isotonic sodium chloride (0.85 %,
pH 7.2) and a 10 % v/v suspension of the packed cells was
made with isotonic sodium chloride. The assay mixture
contained the amide concentration in DMSO (10, 50, 100,
200, and 400 lg/mL), Phosphate buffer (1 mL, 0.15 M, pH
7.4), hypotonic sodium chloride (2 mL, 0.36 %), and
HRBC suspension (0.5 mL). Prednisolone (100 lg) was
used as the reference drug. Instead of hypotonic sodium
chloride, distilled water (2 mL) was used in the control. All
the assay mixtures were incubated at 37 °C for 30 min and
centrifuged. The hemoglobin content in the supernatant
solution was estimated using spectrophotometer (Systronic
UV–Vis Spectrophotometer 118) at 560 nm. The percent-
age hemolysis was calculated by assuming the hemolysis
produced in the presence of distilled water as 100 %. The
percentage of HRBC membrane stabilization or protection
was calculated using the following formula:
%Protection ¼ 100 ꢁ f½ðoptical density of test solution
ꢁ optical density ofproduct controlÞ
ꢂ ðoptical density of test controlÞꢃg
ꢄ 100
Mizushima Y (1965) Simple screening test for Antirheumatic drugs.
The Lancet 285:169
Acknowledgments The authors thank DST, New Delhi, for financial
assistance; RSIC, CDRI, Lucknow for MS, DST-FIST for providing
facility for the Department of Chemistry, Presidency college, Uni-
versity of Madras; and Prof. D. Chamundeeswari, Ph.D., Department
of Pharmacognosy, Sri Ramachandra College of Pharmacy, Sri
Ramachandra University, Chennai-600 116, for in vitro studies. R. R.
thanks Orchid Chemicals and Pharmaceuticals Ltd., for providing the
laboratory and analytic facility.
Mizushima Y (1966) Screening test for antirheumatic drugs. The
Lancet 288:443
Mizushima Y, Kobayashi M (1968) Interaction of anti-inflammatory
drugs with serum proteins, especially with some biologically
active proteins. J Pharm Pharmacol 20:169
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bioactivity of certain medicinal plants on the stabilization of
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Sangeetha M, Kousalya K, Lavanya R, Sowmya C, Chamundeeswari
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and anti-arthritic activity of leaves of Clerodendron inerme. Res
J Pharm Biol Chem Sci 2:822
Tozokoparan B, Ku¨peli E, Yes¸ilada E, Ertan M (2007) Preparation of
5-aryl-3-alkythio-1;2,4-triazoles and corresponding sulfones
with antiinflammatory-analgesic activity. Bioorg Med Chem
15:1808
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