Synthesis, study on anti-arthritic, anti-inflammatory activity and toxicity of some novel bis-oxy cyclophane diamides

Article abstract of DOI:10.1016/j.bmcl.2012.04.010

A series of bis-oxy cyclophane diamides with bis(aminomethyl)m-terphenyl as spacer have been synthesized and characterized from spectral and analytical data. All the cyclophane diamides exhibit better anti-arthritic activity than the reference drug viz. d

Full text of DOI:10.1016/j.bmcl.2012.04.010

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3770–3775
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Bioorganic & Medicinal Chemistry Letters
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Synthesis, study on anti-arthritic, anti-inflammatory activity and toxicity
of some novel bis-oxy cyclophane diamides
Perumal Rajakumar a, , Ramar Padmanabhan b, Navin Rajesh c
⇑
a Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, India
b Research and Development Centre, Orchid Chemicals and Pharmaceuticals Ltd, Sozhanganallur, Chennai 600 119, India
c Toxicology–New Drug Discovery, Orchid Research Laboratories Ltd, Sozhanganallur, Chennai 600 119, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bis-oxy cyclophane diamides with bis(aminomethyl)m-terphenyl as spacer have been synthe-
sized and characterized from spectral and analytical data. All the cyclophane diamides exhibit better anti-
arthritic activity than the reference drug viz. diclofenac sodium. Some of the cyclophane diamides exhibit
good anti-inflammatory activity. The cyclophane amide 4 and 5 do not show any evidence of mutagenic-
ity and cytotoxicity.
Received 23 November 2011
Revised 18 March 2012
Accepted 3 April 2012
Available online 9 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cyclophane diamides
Bis-oxy cyclophane amides
Anti-inflammatory activity
Anti-arthritic activity
Toxicity
The aza-crown macrocycles are used as synthetic receptors and
in anion complexation process that are similar to those in biological
systems.1 Introduction of functional groups like amide and ester in
the aza-crown macrocyclic system would make them as models for
protein-metal binding sites in biological systems.2 Synthesis of
mixed aza-oxo-thia macrocyclic tetra amides and their antimicro-
bial, cytotoxicity studies were recently reported.3 Cyclic peptides
with fluorophore-tag are useful for the design of mercuric ion sen-
sors.4 Synthesis of amide-based supramolecular systems has been
reported in the literature.5,6 Supramolecular amides are also used
as molecular clefts,7 molecular receptors8 and in molecular recogni-
tion9 of biologically interacting substrates including anti-HIV active
macrocyclic amides.10 Various chiral macrocyclic tetra amides and
their transition metal complexes have also been reported.11
Non-steroidal anti-inflammatory drugs (NSAIDs) alleviate pain
by counteracting the cyclooxygenase (COX) that converts arachi-
donic acid into prostaglandins in inflammatory processes.12 NSAIDs
are used for the treatment of pain, fever, and inflammation partic-
ularly arthritis.13 The common NSAIDs such as aspirin, ibuprofen,
naproxen and fenbufen gives only temporary relief14 and have side
effects like upper GI irritation, ulceration, dyspepsia, bleeding, in
some cases deaths. To overcome the GI ulceration of the drug, more
COX-2 selective inhibiting NSAID is preferred.15 Unfortunately very
effective COX-2 selective inhibitor drugs, such as rofecoxib and
celecoxib, were withdrawn from the market because of the
increased risk of heart attack and stroke associated with long-term,
high-dose usage. The COX-2 inhibition activity of terphenyl ana-
logues was reported by Li et al.16 Hence considerable attention
has been focused to synthesize and study the anti-arthritic and
anti-inflammatory activity of novel cyclophane amides.
We have recently reported the synthesis of cyclophane amides
with anti-bacterial efficacy, anti-inflammatory activity17 and car-
bazole based macrocyclic amides with antimicrobial activity.18 In
continuation of our on-going investigation, novel bis-oxy cyclo-
phane diamides were synthesized and their biological activities
have been investigated. However, to the best of our knowledge,
bis-oxy cyclophane diamides with bis(aminomethyl)m-terphenyl
as spacer have not been reported in the literature. Herein, we re-
port the synthesis, in vitro anti-inflammatory, anti-arthritic activ-
ity, systemic bioavailability and toxicity of bis-oxy cyclophane
amides 1–9 (Fig. 1).
Cyclophane amides (1–9) (Fig. 1) were synthesized from
bis(aminomethyl)m-terphenyl (19)19 and possible combinations
of precyclophane acid chlorides 10–18 (Fig. 2). Reaction of one
equivalent of each extended bis-oxyacetyl chlorides 10–18 with
1 equiv of bis(aminomethyl)m-terphenyl 19 gave the bis-oxy
cyclophane amides (1–9) in 35–80% yield (Schemes 1 and 2). All
the new compounds gave satisfactory FT-IR, 1H and 13C NMR, mass
spectral and elemental analysis.
In order to study the anti-arthritic activity of the cyclophane
amides 1–9 inhibition of protein denaturation method (Bovine
Serum Albumin)20 was employed and diclofenac sodium was used
as a standard. The denaturation of protein is one of the causes for
⇑
Corresponding author. Tel.: +91 44 2220 2810.
E-mail address: perumalrajakumar@gmail.com (P. Rajakumar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.010

P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3770–3775
3771
NO2
N
O
O
O
O
O
O
HN
O
O
NH
HN
O
O
NH
HN
O
O
NH
1
= ortho
2
3
1, 1a, 1b
1a = meta
1b = para
O
O
O
O
O
O
O
O
NH
HN
O
O
HN
NH
O
N
H
N
O
H
6
5
4
O
O
O
O
NH
NH
O
O
NH
O O
O
O
O O
HN
NH
HN
7
8
9
Figure 1. Structure of cyclophane diamides 1–9.
rheumatoid arthritis was well documented.21 Production of
auto-antigens in certain rheumatic diseases may be due to
in vivo denaturation of proteins.22 The mechanism of denaturation
probably involves alteration in electrostatic and hydrophobic
interactions, and hydrogen and disulfide bonding.23
The anti-arthritic activities of all the diamide cyclophanes are
concentration dependent. The amide cyclophanes 7, 6, 2, 1, 8, 1a,
4, 3, 9, 5 and 1b was found to possess the maximum anti-arthritic
activity (84.3%, 83.3%, 82.1%, 81.9%, 81.4%, 78.3%, 75.7%, 70.0%,
tory agent acts by either inhibiting the lysosomal enzymes or by
stabilizing the lysosomal membranes.
The anti-inflammatory activities of all the amide cyclophanes
are concentration dependent. At higher concentration, the amide
cyclophanes exhibited better anti-inflammatory activity than at
lower concentration. All the amide cyclophanes 1–9 was found to
possess the maximum anti-inflammatory activity (92.37%,
93.03%, 91.80%, 95.50%, 95.10%, 93.30%, 91.59%, 96.40%, 97.20%,
96.03% and 97.70% at 200
drug prednisolone (91.01% at 200 l
l
g/mL) when compared to the reference
g/ml) which clearly shows that
69.9%, 68.6% and 67.7% at 400
lg/mL) when compared to the refer-
ence drug diclofenac sodium (21.3% at 400
l
g/mL) which clearly
anti-inflammatory activity of the cyclophane amides are better
than the reference drug prednisolone. The better anti-inflamma-
tory activity of amide cyclophanes 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 repulsion of like charges which are involved in
the hemolysis of red blood cells. The degree of anti-inflammatory
activity of cyclophane amides 9, 7, 6, 8, 2, 3, 4, 1a, 1, 1b and 5 at
200 lg/mL is found to be 97.70%, 97.20%, 96.40%, 96.03%, 95.50%,
95.10%, 93.30%, 93.03%, 92.37%, 91.80% and 91.59% respectively
(Table 2 and Fig. 4).
Cyclophane diamide 4 and 5 was chosen to study the toxicity
because they have more number of phenyl rings than other cyclo-
phane diamides. All the strains of Salmonella typhimurium, TA98
shows that cyclophane amides are better than the reference drug
diclofenac sodium. The inhibition of protein denaturation results
showed that the amide cyclophanes are more stable in BSA than
dicofenac sodium. The degree of anti-arthritic activity of cyclo-
phane amides 8, 6, 2, 1, 7, 1a, 4, 1b, 3, 9 and 5 at 800 lg/mL is found
to be 98.2%, 96.8%, 94.4%, 92.4%, 90.6%, 90.5%, 87.8%, 83.9%, 82.4%,
81.3% and 78.2% respectively (Table 1 and Fig. 3).
Further in the present study the anti-inflammatory activity of
compounds 1–9 was investigated using human red blood cells
(HRBC) membrane stabilization24 and with prednisolone as the
standard and the results are shown in Table 2. The lysosomal en-
zymes 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 inflammation. The anti-inflamma-

3772
P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3770–3775
NO2
O
O
O
O
O
O
Cl
O
O
Cl
Cl
O
O
Cl
Cl
O
O
Cl
11
10,10a,10b
12
10
= ortho
10a meta
=
10b =
para
O
O
O
O
O
O
O
O
Cl
Cl
O
O
O
Cl
O
O
Cl
Cl
Cl
15
13
14
O
O
Cl
O
O
O
O
O
O
O
O
O
Cl
Cl
Cl
Cl
Cl
16
17
18
Figure 2. Structure of diacid chlorides 10–18.
NO2
N
O
O
O
O
HN
O
O
NH
HN
O
O
NH
i
11
3
12
2
i
O
O
i
i
O
O
13
10,10a,10b
NH
HN
O
O
O
O
NH2
NH2
HN
NH
19
i
14
1,1a, 1b
4
1
= ortho
1a = meta
1b
= para
O
O
O
N
H
N
O
H
5
Scheme 1. Reagents and conditions: (i) TEA, DCM, rt, 24 h, 1 (35%), 1a (40%, 1b (52%), 2 (42%), 3 (43%), 4 (61%) and 5 (80%).

P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3770–3775
3773
O
O
O
O
O
O
O
O
NH
NH
NH
HN
7
6
15
16
i
i
NH2
NH2
19
i
i
18
17
O
O
O
O
NH
O
HN
O
O
O
HN
NH
8
9
Scheme 2. Reagents and conditions; (i) TEA, DCM, 24 h, 6 (30%), 7 (40%), 8 (35%), and 9 (40%).
Table 1
In vitro anti-arthritic activity of cyclophane diamides 1–9 by inhibition of protein denaturation method (bovine serum albumin)
Cyclophane amide
Activity (% inhibition of protein denaturation)
50
lg/mL
100
lg/mL
200
lg/mL
400
lg/mL
800 lg/mL
1
1a
1b
2
3
4
5
6
25.4
19.4
13.1
23.6
22.0
39.2
12.6
14.5
20.1
40.5
11.7
5.3
50.1
44.8
36.8
49.6
38.2
49.5
25.2
25.4
49.5
61.9
37.0
10.9
72.1
63.4
55.3
73.1
56.3
67.8
48.5
60.5
67.9
76.2
59.7
15.0
81.9
78.3
67.7
82.1
70.0
75.7
68.6
83.3
84.3
81.4
69.9
21.3
92.4
90.5
83.9
94.4
82.4
87.8
78.2
96.8
90.6
98.2
81.9
76.5
7
8
9
Diclofenac Sodium
Each value represents mean SD of three observations.
Anti-arthritic activity
120
100
80
60
40
20
0
50
100
200
Concentration in µg/mL
400
800
1
1a
1b
2
3
4
5
6
7
8
9
Diclofenac Sodium
Figure 3. In vitro anti-arthritic activity of cyclophane diamides 1–9.

3774
P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3770–3775
Table 2
In vitro anti-inflammatory activity of cyclophane diamides 1–9 by HRBC membrane stabilization method
Cyclophane amide
Activity (% prevention of lysis)
g/mL 100 g/mL
10
l
g/mL
50
l
l
200 lg/mL
1
1a
1b
2
3
4
5
6
64.03 0.20
64.27 0.63
57.63 0.21
66.71 0.73
70.23 0.55
70.27 0.75
61.89 0.87
74.50 0.46
76.50 0.44
74.20 0.60
77.30 0.24
46.03 0.13
73.33 0.39
72.53 0.32
66.70 0.46
75.97 0.37
76.93 0.35
82.60 0.47
71.74 0.51
85.40 0.369
85.50 0.25
85.65 0.42
86.20 0.05
57.94 0.39
82.30 0.50
83.07 0.45
82.20 0.37
85.66 0.74
84.73 0.53
91.20 0.66
82.51 0.65
3.63 0.58
90.90 0.08
93.77 0.59
90.60 0.27
84.87 0.36
92.37 0.34
93.03 0.25
91.80 0.62
95.50 0.46
95.10 0.80
93.30 0.56
91.59 0.31
96.40 0.75
97.20 0.37
96.03 0.75
97.70 0.45
91.01 0.45
7
8
9
Predini Solone
Each value represents mean SD of three observations.
Anti-inflammatory activity
120
100
80
60
40
20
0
10
50
100
200
Concentration in µg/mL
1
1a
1b
2
3
4
5
6
7
8
9
Predinisolone
Figure 4. In vitro anti-inflammatory activity of cyclophane diamides 1–9.
Table 3
Table 4
AMES mutagenicity screening of cyclophane diamide 4 using Salmonella typhimurium
AMES mutagenicity screening of cyclophane diamide 5 using Salmonella typhimurium
Dose levels (mg/plate)
Cyclophane amide 4
TA 100
+S9
180
Dose levels (mg/plate)
Cyclophane amide 5
TA 100
+S9
196
TA 98
+S9
23
TA 98
+S9
19
ÀS9
ÀS9
184
ÀS9
ÀS9
184
5.000
2.500
1.250
0.625
0.312
0.156
0.078
0.039
NC
23
21
21
22
21
23
22
23
22
823
2
4
2
3
4
2
4
2
4
2
2
2
3
3
1
4
1
2
4
4
1
4
1
4
1
1
1
3
6
4
3
3
1
6
1
3
5
8
1.660
0.830
0.410
0.200
0.100
0.051
0.025
0.012
NC
20
24
23
27
22
20
21
20
22
823
1
1
2
1
1
1
4
1
4
2
1
3
1
1
0
2
1
0
4
4
4
6
1
6
6
3
4
3
3
3
1
3
3
1
4
4
5
8
24
22
21
20
24
23
24
23
199
199
199
191
193
197
198
194
201
199
190
197
194
195
196
195
2126
21
20
19
20
24
23
24
23
195
185
195
188
192
196
181
194
194
197
194
190
185
195
194
195
2126
PC
1531 14
2023 11
PC
1531 14
2023 11
PC = Positive control; NC = Negative control (DMSO).
PC = Positive control; NC = Negative control (DMSO).
and TA100 when exposed to different concentrations of cyclo-
phane amide 4 and 5, did not show two-fold or greater increase
in the mean number of revertants as compared to the negative con-
trol group as given in Tables 3 and 4. All strains used in the study
exhibited marked increase (>10-fold) in the number of revertants
when treated with positive control agents. The results confirmed
the sensitivity of the tester strains to mutagens and thus the valid-
ity of the assay. The results indicated that the mean number of his-
tidine revertants in the treatment groups were comparable to the
mean number of revertants in the negative control group in all S.
typhimurium tester strains TA98 and TA100 both in the absence
and presence of metabolic activation. cyclophane amide 4 upto
5 mg/plate in the presence and absence of metabolic activation
was found to be non-mutagenic where as cyclophane amide 5
was found to be non- mutagenic up to 1.66 mg/plate in two five
S. typhimurium tester strains.
In conclusion, cyclophane amides 1, 1a, 2, 6, 7 and 8 show supe-
rior anti-arthritic activity than the reference drug diclofenac so-
dium. All the cyclophane amides 1–9 show good anti-
inflammatory activity at low concentration (200 lg/mL) than the
reference drug prednisolone. Cyclophane amide 4 and 5 do not
show any evidence of mutagenicity both with and without meta-

P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3770–3775
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The authors thank DST, New Delhi, for financial assistance, RSIC,
CDRI, Lucknow for MS, DST-FIST for providing NMR facility for the
Department of Organic Chemistry, University of Madras, Prof. D.
Chamundeeswari, Ph.D., Department of Pharmacognazy, Sri
Ramachandra College of Pharmacy, Sri Ramachandra University,
Chennai-600 116, for in vitro studies and Dr. Shridhar Narayanan,
Ph.D., Executive Vice President, Discovery Biology and Drug Devel-
opment, Orchid Research Laboratories Limited for his support to
carry out the in vivo and toxicity studies. R. P. thanks Orchid Chem-
icals and Pharmaceuticals Ltd for providing the laboratory and ana-
lytical facility.
Supplementary data
Supplementary data (experimental and spectral data for
bis-oxy cyclophane amides 1–9) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2012.04.010.
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