Synthesis, anti-arthritic, and anti-inflammatory activity of n-tosyl aza cyclophanes

Article abstract of DOI:10.1071/CH11435

The synthesis of novel N-tosyl tetraaza cyclophanes and N-tosyl diaza cyclophane incorporating m-terphenyl as spacer units is described. Anti-arthritic activity was studied by inhibition of the protein denaturation method (bovine serum albumin). All the N

Full text of DOI:10.1071/CH11435

CSIRO PUBLISHING
Aust. J. Chem. 2012, 65, 186–192
Full Paper
http://dx.doi.org/10.1071/CH11435
Synthesis, Anti-Arthritic, and Anti-Inflammatory
Activity of N-Tosyl aza Cyclophanes
A C
,
B
Perumal Rajakumar
and Ramar Padmanabhan
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
Corresponding author. Email: perumalrajakumar@gmail.com
The synthesis of novel N-tosyl tetraaza cyclophanes and N-tosyl diaza cyclophane incorporating m-terphenyl as spacer
units is described. Anti-arthritic activity was studied by inhibition of the protein denaturation method (bovine serum
albumin). All the N-tosyl aza cyclophanes exhibit excellent anti-arthritic activity. Anti-inflammatory activity of the
synthesized cyclophanes was investigated using the human red blood cells (HRBC) membrane stabilization method and
some of the N-tosyl aza cyclophanes exhibited good anti-inflammatory activity.
Manuscript received: 13 November 2011.
Manuscript accepted: 9 January 2012.
Published online: 1 February 2012.
Introduction
(e.g. celecoxib, etriocoxib, rofecoxib, parecoxib and valde-
coxib) and acetazolamide.
Transfer of information between molecules in living systems
and in supramolecular structures takes place mainly through
non-covalent interactions. Molecular recognition in biology
plays an important role in the development of supramolecular
chemistry. The aza-crown macrocycles are used as synthetic
receptors in anion complexation processes that are similar to
those in biological systems.[1] Furthermore, in supramolecular
chemistry the polyaza macrocycles are highly interesting sys-
tems as they can be used for the binding of both organic and
inorganic ions.[2,3] The intra annular functionality present in the
macrocycles enhances the molecular recognition, metal binding
properties and biological activity.[4] Introducing functional
groups such as amides and esters in the aza-crown macrocyclic
system would make them models of protein-metal binding sites
in biological systems.[5–7] Sulfone derivatives[8] are well
known for their biological activities such as antimicrobial,[9]
anti-inflammatory,[10] and inhibition of HIV-1 reverse tran-
scription.[11] Synthesis and characterization of macrocyclic
aromatic tetrasulfonates and polyaza macrocycles were recently
reported.[12]
Sulfonamides incorporated with acylic receptors and their
complexation with anionic guests by hydrogen bonding have
also been reported.[13–15] Supramolecular triazadisulfonamides
and their anti-HIV studies have been also recently reported by
Pinheiro et al.[16] Sulfonamides are the basis of several groups of
drugs. The sulfonamide chemical moiety is present in antibac-
terials (sulfa drugs), thiazide diuretics (including hydrochloro-
thiazide, metolazone, and indapamide), loop diuretics
(including furosemide, bumetanide and torsemide) sulfonylur-
eas (including glipizide, glyburide), some COX-2 inhibitors
Non-steroidal anti-inflammatory drugs (NSAIDs) alleviate
pain by counteracting the cyclooxygenase (COX) enzyme that
converts arachidonic acid into prostaglandins in inflammatory
processes.[17] NSAIDs are used for the treatment of pain, fever,
and inflammation, particularly arthritis.[18] Thecommon NSAIDs
such as aspirin, ibuprofen, naproxen and fenbufen have side
effects such as upper gastrointestinal (GI) irritation, ulceration,
dyspepsia, bleeding, in some cases death and give only tempo-
rary relief.[19] To overcome the GI ulceration side effect of these
drugs, more COX-2-selective inhibitor NSAIDs are preferred,
which do not significantly inhibit cyclooxygenase in the
stomach and appear to be less likely to cause GI ulceration.[20]
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. The COX-2 inhibition
activity of terphenyl analogues was reported by Li et al.[21]
Hence considerable attention has been focussed to synthesize
and study the anti-arthritic and anti-inflammatory activity of
novel cyclophane sulfonamides.
We have recently reported the synthesis of cyclophane
amides with anti-inflammatory and anti-bacterial efficacy,[22]
and carbazole based macrocyclic amides with antimicrobial
activity.[23] In continuation of our ongoing investigation on the
synthesis of various bioactive cyclophanes, attempts were made
to synthesize novel N-tosyl tetraaza cyclophanes and N-tosyl
diaza cyclophanes by coupling between suitable dibromides and
bis(tosylaminomethyl)m-terphenyl, and then to study the bio-
activity of the synthesized cyclophanes. However, to the best of
Journal compilation Ó CSIRO 2012
www.publish.csiro.au/journals/ajc

N-Tosyl aza Cyclophanes
187
CH3
CH3
NO2
H3C
H3C
O
O
O
O
S
S
S
S
O
O
O
O
N
N
N
N
N
N
N
N
O
O
O
S
O
S
S
S
O
O
O
O
H3C
CH3
H3C
CH3
NO2
1, 1a
ϭ ortho
2
1
1a ϭ meta
CH3
CH3
H3C
O
O
O
O
S
N
N
S
S
O
O
N
N
N
S
N
N
O
O
S
S
O
O
N
O
O
H3C
CH3
3
CH3
4
Fig. 1. Structure of N-tosyl tetraaza cyclophanes 1–3 and N-tosyl diaza cyclophane 4.
our knowledge, such cyclophanes with bis(tosylaminomethyl)m-
terphenyl as the spacer have not been reported in the literature.
Herein, we report the synthesis, anti-arthritic and anti-
inflammatory activity of N-tosyl tetraaza cyclophanes 1–3 and
N-tosyl diaza cyclophane 4 (Fig. 1).
m-terphenyl 6[24] and possible combinations of o-xylylene
dibromide, m-xylylene dibromide, m-nitro xylylene dibromide,
2,6-(bisbromomethyl)pyridine and m-terphenyl dibromide as
building units. Bis(tosylaminomethyl)m-terphenyl 6 required
for the synthesis of N-tosyl tetraaza cyclophanes 1–3 and N-tosyl
diaza cyclophane, 4 was obtained in 85 % yield by the reaction
of 1 equiv m-terphenyl diamine 5 with 2.1 equiv p-toluene
sulfonyl chloride in the presence of 2.2 equiv pyridine in dry
chloroform for 5 h at 0–58C (Scheme 1). The 1H NMR spectrum
of precyclophane 6 displayed a singlet for the methyl protons at
Results and Discussion
N-tosyl tetraaza cyclophanes (1–3) and N-tosyl diaza cyclo-
phane (4) (Fig. 1) were synthesized with bis(tosylaminomethyl)

188
P. Rajakumar and R. Padmanabhan
1, 1a
1
ϭ ortho
1a ϭ meta
O2N
(ii)
Br
Br
2
Br
Br
(ii)
(i)
O
O
HN
NH
O
S
S
O
NH2
NH2
5
Br
N
CH3
CH3
6
Br
(ii)
3
(ii)
Br
Br
4
Scheme 1. Reagents and conditions: (i) p-toluene sulfonyl chloride, pyridine, CHCl3, 0–58C, 5 h, 6 (85 %); (ii) K2CO3, ACN, rt,
48 h, 1 (68 %), 1a (68 %), 2 (50 %), 3 (40 %) and 4 (80 %).
d 2.44, a doublet for the N-methylene protons at d 4.19 and a
triplet for NH protons at d 4.63. The remaining aromatic protons
resonated between d 7.18 and 7.80. The structure of pre-
cyclophane 6 was further confirmed from the spectral and
analytical data.
In vitro anti-arthritic activity was studied by inhibition of the
protein denaturation method (bovine serum albumin [BSA]).[25]
The denaturation of protein is one of the causes of rheumatoid
arthritis.[26] Production of auto-antigens in certain rheumatic
diseases may be owing to in vivo denaturation of proteins.[27]
The mechanism of denaturation probably involves alteration in
electrostatic, hydrogen, hydrophobic and disulfide bonding.[28]
The observed data showing the anti-arthritic activity of the
compounds and the control drug are given in Table 1 and
Fig. 2. In order to study the anti-arthritic activity of the N-tosyl
tetraaza cyclophanes 1, 1a, 2, 3 and N-tosyl diaza cyclophane 4
inhibition of the protein denaturation method was employed
(BSA) and diclofenac sodium was used as a standard. The
results are shown in Table 1. The anti-arthritic activities of all
of the N-tosyl tetraaza and diaza macrocycles are concentration
dependent. At the higher concentration, the N-tosyl tetraaza and
diaza macrocycles 1a, 2, 3 and 4 exhibited better anti-arthritic
activity than at lower concentration. All the compounds 1, 1a, 2,
3 and 4 were found to possess the maximum anti-arthritic
One equiv of precyclophane 6 was coupled with 1 equiv of
o-xylylene dibromide, m-xylylene dibromide, m-nitro xylylene
dibromide, 2,6-(bisbromomethyl)pyridine or m-terphenyl
dibromide in the presence of anhydrous potassium carbonate
in dry acetonitrile at room temperature under high dilution
conditions. The reaction afforded the N-tosyl tetraaza cyclo-
phanes 1, 1a, 2, 3 and N-tosyl diaza cyclophane 4 in 68, 68, 50,
40 and 80 % yields respectively, after purification by column
chromatography (Scheme 1). The structures of the N-tosyl
tetraaza and diaza cyclophanes 1–4 were confirmed using
spectral and analytical data. The 1H NMR spectrum of N-tosyl
tetraaza cyclophane 1a displayed a singlet for the methyl
protons at d 2.45 and a singlet for the N-methylene protons at
d 4.17. The rest of the aromatic protons resonated between
d 6.67 and 7.77. In the 13C NMR spectrum of N-tosyl tetraaza
cyclophane 1a, the N-methylene carbons appeared at d 50.7
and 51.1. The FT-IR spectrum of 1a displayed the sulfonyl
stretching frequency at 1597 cmꢀ1 and the mass spectrum
showed the molecular ion peak [(MþNH4)þ] at m/z 1414.6.
Similarly the structure of the N-tosyl tetraaza and diaza cyclo-
phanes 1, 2, 3 and 4 was also confirmed from spectral and
analytical data.
activity (74.42, 82.02, 93.75, 86.85 and 78.63 % at 800 mg mLꢀ1
)
when compared with the reference drug diclofenac sodium
(76.46 % at 800 mg mLꢀ1), which clearly shows that N-tosyl
tetraaza and diaza macrocycles are superior to the reference drug
diclofenac sodium. The superior inhibition of protein denatur-
ation results showed that the N-tosyl tetraaza and diaza macro-
cycles are more stable than dicofenac sodium in BSA. It is
possible that the stability related to the strong binding of the

N-Tosyl aza Cyclophanes
189
Table 1. In vitro anti-arthritic activity of N-tosyl tetraaza and diaza cyclophanes 1–4 by inhibition of protein denaturation method (bovine
serum albumin)
Each value represents mean ꢁ s.d. of three observations
Cyclophane sulfonamide
Activity (% inhibition of protein denaturation)
200 mg mLꢀ1
50 mg mLꢀ1
100 mg mLꢀ1
400 mg mLꢀ1
800 mg mLꢀ1
1
13.60
23.68
22.63
21.42
17.05
5.25
24.59
40.95
45.21
38.96
35.50
10.86
47.09
57.90
68.48
59.40
53.93
14.95
62.12
78.17
81.16
70.62
66.28
21.26
74.42
82.02
93.75
86.85
78.63
76.46
1a
2
3
4
Diclofenac sodium
100
90
80
70
60
50
40
30
20
10
0
Anti-arthritic activity
50
100
200
400
800
Concentration [µg mLϪ1
]
1
1a
2
3
4
Diclofenac sodium
Fig. 2. Anti-arthritic activity of N-tosyl tetraaza and diaza cyclophanes 1–4.
Table 2. In vitro anti-inflammatory activity of N-tosyl tetraaza and diaza cyclophanes 1–4 by HRBC membrane stabilization
Each value represents mean ꢁ s.d. of three observations
Cyclophane sulfonamide
Activity (% prevention of lysis)
10 mg mLꢀ1
50 mg mLꢀ1
100 mg mLꢀ1
200 mg mLꢀ1
1
39.63 ꢁ 0.54
34.18 ꢁ 0.35
57.38 ꢁ 0.46
43.20 ꢁ 0.71
54.23 ꢁ 0.35
46.03 ꢁ 0.13
53.38 ꢁ 0.31
47.01 ꢁ 0.56
72.99 ꢁ 0.07
51.97 ꢁ 0.83
65.93 ꢁ 0.48
57.94 ꢁ 0.39
73.11 ꢁ 0.63
61.36 ꢁ 0.72
89.65 ꢁ 0.37
74.79 ꢁ 0.52
89.73 ꢁ 0.85
84.87 ꢁ 0.36
85.63 ꢁ 0.25
74.54 ꢁ 0.48
96.88 ꢁ 0.26
82.34 ꢁ 0.35
97.23 ꢁ 0.40
91.01 ꢁ 0.45
1a
2
3
4
Prednisolone
cyclophanes on BSA is owing to the increased hydrophobicity
compared with diclofenac sodium. The degree of anti-arthritic
activity of cyclophane amides are 2 . 3 . 1a . 4 . 1 at
800 mg mLꢀ1 is found to be 93.75, 86.85, 82.02, 78.63 and
74.42 %, respectively (Fig. 2).
In vitro anti-inflammatory activity was studied by the human
red blood cells (HRBC) membrane stabilization method.[29] The
lysosomal enzymes released during inflammatory condition
produce a variety of disorders. The extracellular activity of
these enzymes is said to be related to acute or chronic inflam-
mation. The anti-inflammatory agent acts by either inhibiting
the lysosomal enzymes or by stabilizing the lysosomal mem-
branes. Since the HRBC membrane are similar to lysosomal
membrane components, the prevention of hypotonicity-induced
HRBC membrane lysis is taken as a measure of anti-
inflammatory activity of the drug. The observed data showing
the anti-inflammatory activity of the compounds and the control
drug are given in Table 2 and Fig. 3. Further, in the present study
the anti-inflammatory activity of compounds 1, 1a, 2, 3 and 4
was investigated using HRBC membrane stabilization and with
prednisolone as the standard, the results of which are shown in
Table 2.
The anti-inflammatory activities of all the N-tosyl tetraaza
and diaza cyclophanes are concentration dependent. At the
higher concentration, the aza cyclophanes 1a, 2, 3 and 4
exhibited better anti-inflammatory activity than at lower

190
P. Rajakumar and R. Padmanabhan
120
100
80
60
40
20
0
Anti-inflammatory activity
10
50
100
200
Concentration [µg mLϪ1
]
1
1a
2
3
4
Prednisolone
Fig. 3. Anti-inflammatory activity of N-tosyl tetraaza and diaza cyclophanes 1–4.
concentration. The sulfonamide macrocycles 4 and 2 were found
to possess the maximum anti-inflammatory activity (97.23 and
96.88 % at 200 mg mLꢀ1, respectively) when compared with the
reference drug prednisolone (91.01 % at 200 mg mLꢀ1), which
clearly shows that anti-inflammatory activity of synthesized
cyclophanes is superior to the reference drug. The better anti-
inflammatory activity of sulfonamide macrocycles could be
owing to their binding 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 that are involved in the haemolysis of red blood
cells. The degree of anti-inflammatory activity of N-tosyl
cyclophane amines 4 . 2 . 1 . 3 . 1a at 200 mg mLꢀ1 is found
to be 97.23, 96.88, 85.63, 82.34 and 74.54 %, respectively
(Fig. 3).
SX 102/DA-6000 mass spectrometer. Elemental analyses for the
compounds were carried out using the Elementar Vario EL III
elemental analyzer. Pre-coated silica gel plates from Merck
were used for TLC analysis. Column chromatography was
carried out using silica gel (100–200 mesh) purchased from
ACME.
Procedure for the Synthesis of Precyclophane (6)
A solution of p-toluenesulfonyl chloride (29.2 mmol) in dry
chloroform (50 mL) was added dropwise to a well-stirred
solution of m-terphenyl diamine 5 (13.9 mmol) and pyridine
(30.2 mmol) in dry chloroform (150 mL) at 0–58C. The resulting
yellow suspension was stirred for 5 h after which time the sol-
vent was removed under reduced pressure. The obtained solid
was washed well with water to remove pyridine hydrochloride
and then purified by column chromatography (SiO2) with
chloroform as the eluting solvent. Yield 85 %; mp 2058C. nmax
(KBr)/cmꢀ1 1598, 1567. dH (400 MHz, CDCl3) 7.79 (d, J 8.1,
4H), 7.70 (s, 1H), 7.54 (t, J 8.1, 5H), 7.28–7.32 (m, 9H), 7.19 (d,
J 7.5 Hz, 1H), 4.63 (t, J 3.4, 2H), 4.19, 4.20 (dd, J 3.5, 3.4, 4H),
2.44 (s, 6H). dC (100 MHz, CDCl3) 142.8, 140.9, 139.1, 137.9,
137.3, 130.1, 129.6, 128.7, 128.0, 127.6, 127.1, 126.6, 48.4,
21.6. m/z (ES) 614.1 [(MþNH4)þ].
Conclusion
In conclusion, all the cyclophanes 1, 1a, 2, 3 and 4 show superior
anti-arthritic activity than the reference drug diclofenac sodium.
However, the N-tosyl tetraaza cyclophanes 4 and 2 show
superior anti-inflammatory activity at lower concentration
(200 mg mLꢀ1) than the reference drug prednisolone, which
could lead to their development as anti-inflammatory drugs
(NSAIDs). Further studies are required to determine their tox-
icity, bioavailability, mode of action etc. The syntheses of
similar N-tosyl tetraaza cyclophanes with different biologically
important spacer units to improve the solubility and efficacy,
in vivo anti-arthritic, anti-inflammatory assay and molecular
recognition towards various biologically important anions are
under investigation.
General Procedure for the Synthesis of N-Tosyl Tetraaza
and Diaza Cyclophanes (1–4)
A solution of N-tosyl m-terphenyl diamine 6 (8.0 mmol) in dry
acetonitrile (125 mL) and a solution of the corresponding
o-xylylene dibromide, m-xylylene dibromide, m-nitro xylylene
dibromide, 2,6-(bisbromomethyl)pyridine or m-terphenyl
dibromide (8.4 mmol) in dry acetonitrile (125 mL) were simul-
taneously added dropwise to a well-stirred mixture of anhydrous
potassium carbonate (80 mmol) in dry acetonitrile (250 mL) for
8 h. After the addition was complete, the reaction mixture was
stirred for a further 48 h. The reaction mixture was filtered and
the solvent removed under reduced pressure to give a solid,
which was washed with water (2 ꢂ 100 mL) and purified by
column chromatography (SiO2) with chloroform as the eluting
solvent.
Experimental
General
All reagents and solvents employed were of the best grade
available and were used without further purification. The
melting points were determined using a Mettler Toledo melting
point apparatus by the open capillary tube method and were
uncorrected. Spectroscopic data were recorded by the following
instruments: UV-vis: Shimadzu 2550 spectrophotometer;
IR: Perkin-Elmer series 2000 FTIR spectrophotometer;
NMR: Bruker Avance 400 MHz; Mass: ESI – PerkinElmer
Sciex, API 3000 mass spectrometer and FAB-mass spectra Jeol
5,9,15,19-N-p-Toluenesulfonyltetraaza
1,3,11,13(1,4),2,12(1,3),7,17(1,2)
octabenzenacycloeicosaphane (1)
Yield 68 %, mp 2528C. nmax (KBr)/cmꢀ1 1598, 1597. dH
(400 MHz, CDCl3) 7.67 (d, J 8.2, 8H), 7.46 (s, 2H),

N-Tosyl aza Cyclophanes
191
7.37 (d, J 8.2, 2H), 7.31 (d, J 8.1, 4H), 7.27 (d, merged with
CHCl3, J 8.1, 8H), 7.19 (d, J 8.2, 8H), 7.02 (d, J 8.1, 8H), 6.83
(ABq, J 8.2, 8H), 4.19 (s, 16H), 2.44 (s, 12H). dC (100 MHz,
DMSO-d6) 158.2, 140.8, 139.3, 138.7, 137.2, 136.6, 131.3,
129.5, 128.9, 128.3, 127.9, 127.2, 124.8, 50.9, 48.2, 21.4. m/z
(ES) 1414.9 [(MþNH4)þ]. Anal. Calc. for C84H76N4O8S4:
C 72.18, H 5.48, N 4.01. Found: C 72.46, H 5.57, N 4.08 %.
200, 100, 50 mg in 0.05 mL DMSO). The test control solution
(0.5 mL) consists of BSA (0.45 mL, 5 % w/v aqueous solution)
and distilled water (0.05 mL). The product control solution
(0.5 mL) consists of distilled water (0.45 mL) and aza cyclo-
phane solutions (800, 400, 200, 100, 50 mg in 0.05 mL DMSO).
The standard solution (0.5 mL) consists of BSA (0.45 mL, 5 %
w/v aqueous solution) and diclofenac sodium (800, 400,
200 mg mLꢀ1 in 0.05 mL water). All of the above solutions were
adjusted to pH 6.3 using 1N HCl. The samples were incubated at
378C for 20 min and the temperature was increased to keep the
samples at 578C for 3 min. After cooling, phosphate buffer
(2.5 mL) was added to the above solutions. The absorbance was
measured using a Systronic UV-Vis Spectrophotometer 118 at
416 nm. The percentage inhibition of protein denaturation can
be calculated as:
5,9,15,19-N-p-Toluenesulfonyltetraaza1,3,11,13(1,4),
2,7,12,17(1,3)octabenzenacycloeicosaphane (1a)
Yield 68 %, mp 2678C. nmax (KBr)/cmꢀ1 1597, 1516. dH
(400 MHz, CDCl3) 7.75 (d, J 8.0, 8H), 7.40 (d, J 8.1, 8H), 7.35
(d, J 8.0, 8H), 7.25 (d, merged with CHCl3, J 8.0, 4H), 7.09 (d, J
7.3, 2H), 7.05 (s, 4H), 6.99 (d, J 7.3, 4H), 6.95 (d, J 8.0, 8H), 6.68
(d, J 8.2, 2H), 4.18 (s, 16H), 2.45 (s, 12H); dC (100 MHz,
DMSO-d6) 158.3, 140.9, 139.2, 138.8, 137.6, 137.0, 136.7,
129.6, 129.1, 128.7, 128.2, 127.7, 127.1, 124.9, 51.1, 50.7,
21.6. m/z (ES) 1414.6 [(MþNH4)þ]. Anal. Calc. for
C84H76N4O8S4: C 72.18, H 5.48, N 4.01. Found: C 72.45,
H 5.56, N 4.07 %.
% Inhibition ¼ 100 ꢀ f½ðOptical density of test solution
ꢀ Optical density of product controlÞ
C ðOptical density of test controlÞꢃg ꢂ 100
In Vitro Anti-Inflammatory Studies
5,9,15,19-N-p-Toluenesulfonyltetraaza1,3,11,13(1,4),
2,7,12,17(1,3)phane7,17-bis(m-nitrobenzeno)
hexabenzenacycloeicosaphane (2)
The HRBC membrane stabilization has been used as a method to
study the anti-inflammatory activity using the drug prednisolone
as a standard. Blood was collected from three healthy volunteers
and the collected blood was mixed with an equal volume of
sterilized Alsever’s solution (2 % dextrose, 0.8 % sodium cit-
rate, 0.05 % citric acid and 0.42 % sodium chloride). The blood
was centrifuged at 1500 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 contain the aza cyclophanes dis-
solved in DMSO (200, 400 and 800 mg mLꢀ1), phosphate buffer
(1 mL, 0.15 M, pH 7.4), hypotonic sodium chloride (2 mL,
0.36 %) and HRBC suspension (0.5 mL). Prednisolone (10, 50,
100 and 200 mg mLꢀ1 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 378C for
30 min and centrifuged. The haemoglobin content in the
supernatant solution was estimated using a Systronic UV-Vis
Spectrophotometer 118 at 560 nm. The percentage haemolysis
was calculated by assuming the haemolysis produced in the
presence of distilled water 100 %. The percentage of HRBC
membrane stabilization or protection was calculated using the
formula:
Yield 50 %, mp 1638C. nmax (KBr)/cmꢀ1 1597, 1533. dH
(400 MHz, CDCl3) 7.78 (d, J 7.8, 8H), 7.61 (d, J 7.6, 4H), 7.36–
7.50 (m, 16H), 7.27 (d, merged with CHCl3, J 8.2, 2H), 7.22 (s,
4H), 7.09 (d, J 7.8, 4H), 7.02 (d, J 7.8, 4H), 6.98 (d, J 7.8, 4H),
4.25 (s, 16H), 2.47 (s, 12H). dC (100 MHz, DMSO-d6) 158.5,
141.2, 139.4, 138.9, 137.7, 137.1, 136.6, 129.6, 129.1, 128.7,
128.3, 127.6, 127.2, 124.8, 50.8, 50.1, 21.9. m/z (ES) 1504.6
[(MþNH4)þ]. Anal. Calc. for C84H74N6O12S4: C 67.81, H 5.01,
N 5.65. Found: C 67.98, H 5.09, N 5.72 %.
5,9,15,19-N-p-Toluenesulfonyltetraaza1,3,11,
13(1,4),2,7,12,17(1,3),7,17-dipyridino
hexabenzenacycloeicosaphane (3)
Yield 40 %, mp 1358C. nmax (KBr)/cmꢀ1 1596, 1516. dH
(400 MHz, CDCl3) 7.70 (d, J 8.1, 8H), 7.48 (d, J 7.8, 2H), 7.42
(d, J 8.1, 2H), 7.36 (d, J 8.1, 8H), 7.29 (d, J 8.1, 8H), 7.24 (d, J
8.1, 8H), 7.13 (d, J 7.8, 4H), 7.09 (d, J 8.1, 4H), 6.98 (d, J 8.1,
4H), 4.25 (s, 16H), 2.42 (s, 12H). dC (100 MHz, CDCl3) 155.8,
144.8, 141.3, 140.6, 137.1, 134.9, 129.9, 129.2, 128.7, 128.4,
127.5, 127.1, 126.2, 125.6, 121.8, 52.7, 51.3, 21.7. m/z (ES)
1416.5 [(MþNH4)þ]. Anal. Calc. for C82H74N6O8S5: C 70.36,
H 5.33, N 6.00 %. Found: C 70.50, H 5.41, N 6.08 %.
% Protection ¼ 100 ꢀ f½ðOptical density of test solution
ꢀ Optical density of product controlÞ
5,11-N-p-Toluenesulfonyldiaza1,3,7,9(1,4),2,8(1,3)
hexabenzenacyclododecaphane (4)
C ðOptical density of test controlÞꢃg ꢂ 100
Yield 80 %, mp 2618C. nmax (KBr)/cmꢀ1 1598, 1583, 1518.
dH (400 MHz, CDCl3) 7.85 (d, J 8.2, 4H), 7.42 (d, J 8.1, 6H),
7.34 (d, J 0.9, 6H), 7.27 (d, merged with CHCl3, J 8.1, 8H), 7.14
(d, J 8.1, 8H), 4.36 (s, 8H), 2.50 (s, 6H). dC (100 MHz, CDCl3)
143.5, 139.3, 137.7, 137.2, 135.5, 129.9, 129.4, 128.7, 128.4,
127.9, 126.9, 126.3, 50.8, 21.5. m/z (ES) 851.3 [(MþH)þ]. Anal.
Calc. for C54H46N2O4S2: C 76.21, H 5.45, N 3.29 %. Found:
C 76.44, H 5.52, N 3.35 %.
The lysosomal enzyme released during inflammation pro-
duces a variety of disorders. This extracellular activity of
this enzyme is related to acute or chronic inflammation. Since
the HRBC membrane are similar to lysosomal membrane
components, the prevention of hypotonicity induced HRBC
membrane lysis is taken as a measure of anti-inflammatory
activity of the drug.
In Vitro Anti-Arthritic Studies
Acknowledgements
The test solution (0.5 mL) consists of BSA (0.45 mL, 5 % w/v
aqueous solution) and a solution of aza cyclophane (800, 400,
The authors thank DST, New Delhi, for financial assistance, RSIC,
CDRI, Lucknow for MS, DST-FIST for providing the NMR facility for the

192
P. Rajakumar and R. Padmanabhan
Department of Organic Chemistry, University of Madras and Prof. D.
Chamundeeswari, Ph. D., Department of Pharmacognazy, Sri Ramachandra
College of Pharmacy, Sri Ramachandra University, Chennai-600 116, for in
vitro studies. R.P. thanks Orchid Chemicals and Pharmaceuticals Ltd, for
providing the laboratory and analytical facility.
[14] (a) C.-F. Chen, Q.-Y. Chen, Tetrahedron Lett. 2004, 45, 3957.
doi:10.1016/J.TETLET.2004.03.108
(b) S.-i. Kondo, T. Suzuki, T. Toyama, Y. Yano, Bull. Chem. Soc. Jpn.
2005, 78, 1348. doi:10.1246/BCSJ.78.1348
(c) S.-i. Kondo, T. Suzuki, Y. Yano, Tetrahedron Lett. 2002, 43, 7059.
doi:10.1016/S0040-4039(02)01543-5
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