Synthesis and study of anti-inflammatory activity of some novel cyclophane amides

Article abstract of DOI:10.1016/j.bmcl.2006.08.124

Macrocyclic di- and tetra-amides with thia- and oxylinkages were synthesized and screened for in vitro anti-inflammatory activity. Cyclophane diamide 15 showed a dose-dependent activity, while the other cyclophane amides 16-20 exhibited mild activity.

Full text of DOI:10.1016/j.bmcl.2006.08.124

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6019–6023
Synthesis and study of anti-inflammatory activity
of some novel cyclophane amides
Perumal Rajakumar,a,* A. Mohammed Abdul Rasheed,a
A. Iman Rabiab and D. Chamundeeswarib
aDepartment of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, India
bSri Ramachandra College of Pharmacy, Sri Ramachandra Medical College and Research Institute, Chennai 600 116, India
Received 31 May 2006; revised 17 August 2006; accepted 30 August 2006
Available online 25 September 2006
Abstract—Macrocyclic di- and tetra-amides with thia- and oxylinkages were synthesized and screened for in vitro anti-inflammatory
activity. Cyclophane diamide 15 showed a dose-dependent activity, while the other cyclophane amides 16–20 exhibited mild activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Synthesis of new supramolecules architecturally novel
and of potential importance in the context of designing
simple models for studying biomolecular interactions
stimulates the imaginative skill of synthetic chemists.
The basic crown ether has been modified by substituting
the oxygen donor atom by sulfur and/or nitrogen atom
and introducing functional groups, viz., amide, ester in
the ring to use them as models of protein–metal binding
sites in biological systems,1 synthetic ionophores, thera-
peutic reagents in chelate therapy, cyclic antibiotics,2
and to study host–guest interactions.3 Cyclic amides
play important role in various biological systems.4 Cys-
tine based cyclic peptide has the unique ability of form-
ing double-helical structure.5 Cyclic peptides with open
pores are useful as transport vehicles for biologically
important ions or neutral molecules.6 The self-assembly
of acyclic peptides and their ability to form b-sheet
structures have been demonstrated.7,8 Adamantane-
based supramolecular systems also form double-helical
cyclic structures.8,9 Macrocyclic hexa-amides, which
can effectively bind peptides, have been reported.10
Supramolecular amides have been also used as molecu-
lar receptors and in molecular recognition of biological-
ly interacting substrates11 including anti-HIV active
macrocyclic amides.12 Copper complexes of macrocyclic
compounds exhibit increased antibiotic and antifungal
activity than the uncomplexed macrocyclic com-
pounds.13 Thus macrocyclic compounds containing
amide linkages are found to be biologically active.
Hence we are interested in the synthesis and study of
anti-inflammatory activity of cyclophane amides.
Diamines 1–6 were synthesized and used for the prepa-
ration of cyclophane amides.
Diamine 1 and 214 were prepared by the reaction of their
corresponding dihalides 7 and 8 with 2-aminothiophenol
in methanolic KOH at room temperature. Dichloride
815 was obtained by the reaction of o-phenylenediamine
with 2 equiv of chloroacetyl chloride in the presence of
triethylamine in methylene chloride. Diamine 3 was pre-
pared by the reaction of the corresponding dibromide16
9 with 2-aminothiophenol in toluene and aq KOH in the
presence of tetrabutylammonium bromide at reflux.
Nitro compounds 10 and 11 were obtained by the
alkylation of 4-nitrobenzyl bromide and 1-(4-nitrophen-
oxy)-2-bromo ethane with 2-aminothiophenol, respec-
tively. Reduction of the nitro compounds 10 and 11
with iron and dil HCl gave the diamine 417 and 5 in
62% and 70% yields, respectively (Scheme 1).
In order to synthesize the cyclic tetra-amide with oxy
linkages, diamine 6 was obtained in 65% yield by the
reaction of diacid chloride 12 with 2 equiv of N-pheny-
lethylenediamine (Scheme 2).
Keywords: Cyclophane; Anti-inflammatory; HRBC stabilization.
*
Diacid chloride 1418 was obtained by the reaction of the
corresponding diacid 13 with excess thionyl chloride in
Corresponding author. Tel.: +914422351269; fax: +914422352494;
e-mail: perumalrajakumar@hotmail.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.124

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P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6019–6023
O
O
NH HN
S
S
S
S
S
S
H2N
NH2
NH2
H2N
H2N
NH2
2
3
1
NH2
S
O
O
O
O
S
NH2
NH
HN
O
NH2
HN
Ph
NH
NH2
Ph
4
5
6
Scheme 1. Reagents and conditions: (a) 2-aminothiophenol (2 equiv), KOH, methanol, room temperature; (b) 2-aminothiophenol (2 equiv), aq
KOH, toluene, TBAB, reflux, 4 h; (c) KOH, methanol, room temperature; (d) activated iron powder, dil HCl, reflux, 3 h.
molecular ion appeared at m/z 798 in the FAB mass
spectrum for cyclophane amide 15, which further con-
firms the proposed structure. Cyclophane amides 1620
and 1721 were also completely characterized by spectral
and analytical data.
Cyclophane amide 1822 in 1H NMR spectrum displayed
the aromatic methyl protons as two singlets at d 2.25
and d 2.27, SCH2 protons as singlet at d 3.65, OCH2
Scheme 2. Reagents and conditions: (a) N-phenylethylenediamine
(2 equiv), TEA, CH2Cl2, room temperature, 1 h.
protons as two singlets at d 5.08 and d 5.17, and the
amide protons as a broad singlet at d 9.69 in addition
to the aromatic protons. In 13C NMR spectrum cyclo-
phane amide 18 displayed two aromatic methyl carbons
the presence of triethyl amine in methylene chloride.
Thus diacid chloride 14 was reacted with diamines 1–6
to give cyclophane amides 15–20 in 50%, 45%, 35%,
40%, 42%, and 40% yields, respectively (Scheme 3).
at d 19.6 and d 19.8, SCH2 carbon at d 43.3, OCH2 car-
bons at d 69.5 and d 70.8, and carbonyl carbons at d
162.8 and d 165.7 in addition to the aromatic carbons.
In the mass spectrum the molecular ion appeared at
m/z 600.
1
In H NMR spectrum cyclophane amide 1519 displayed
the aromatic methyl, SCH2, and OCH2 protons as sin-
glets at d 2.16, d 3.95, and d 4.80, respectively, in addi-
tion to aromatic protons. The protons attached to the
amide nitrogen appeared at d 9.68 as a broad singlet.
In 13C NMR spectrum aromatic methyl, SCH2, OCH2,
and carbonyl carbons appeared at d 19.4, 41.9, 67.9,
and 164.0 in addition to the aromatic carbons. The
Cyclophane amide 1923 in 1H NMR spectrum displayed
the aromatic methyl protons as two singlets at d 2.25
and d 2.33, SCH2CH2O protons as two triplet at d
2.97 and d 3.77 with J = 4.8 Hz, OCH2 protons as two
singlet at d 5.01 and d 5.06 in addition to the aromatic
protons, and the amide protons as a broad singlet at d
9.13. In 13C NMR spectrum cyclophane amide 19

P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6019–6023
6021
Scheme 3. Reagents and conditions: (a) SOCl2, TEA, CH2Cl2, reflux, 3 h; (b) TEA, CHCl3, room temperature, 6 h.
Table 1. Effect of cyclophane amides 15–20 on HRBC membrane
stabilization
displayed two aromatic methyl carbons at d 19.2 and d
19.8, SCH2CH2O carbons at d 37.7 and d 64.9, OCH2
carbons at d 66.5 and d 70.9, and carbonyl carbons at
d 162.5 and d 165.0 in addition to the aromatic carbons.
In the FAB mass spectrum, the molecular ion appeared
at m/z 630. Similarly cyclophane amide 2024 was also
completely characterized by spectral and analytical data.
Compound
Activity (% prevention of lysis)
50 lg/ml 100 lg/ml 200 lg/ml
10 lg/ml
15
16
17
18
19
20
65.17 0.95 82.43 0.48 89.67 0.68 91.00 0.58
40.53 0.75 42.60 0.95 48.72 0.31 49.65 0.49
48.27 0.61 49.37 0.54 51.42 0.59 54.14 0.38
50.31 0.73 52.27 0.39 56.04 0.18 60.86 0.46
44.28 0.11 45.63 0.64 50.25 0.63 51.27 0.63
38.84 0.44 41.72 0.61 45.25 0.86 47.97 0.09
Anti-inflammatory activity. Cyclophane amides 15–20
were screened for in vitro anti-inflammatory activity.
The HRBC membrane stabilization25 has been used as
a method to study the anti-inflammatory activity using
prednisolone as standard drug.
Prednisolone —
59.34 0.44 86.37 0.49 —
Blood was 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 1500 rpm and the
packed cells were washed with isotonic NaCl (0.85%,
pH 7.2) and a 10% v/v suspension of the packed cells
was made with isotonic NaCl. The assay mixture
contained the drug (concentration as mentioned in
Table 1), 1 ml phosphate buffer (0.15 M, pH 7.4),
2 ml of hypotonic NaCl (0.36%), and 0.5 ml HRBC
suspension. Prednisolone 100 lg was used as the refer-
ence drug. Instead of hypotonic NaCl, 2 ml of distilled
water 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 (systronics
model) at 500 nm. The percentage hemolysis was cal-
culated 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 formula:

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P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6019–6023
9. Ranganathan, D.; Haridas, V.; Nagaraj, R.; Karle, I. L.
J. Org. Chem. 2000, 65, 4415.
Activity ð% prevention of lysisÞ
OD of drug treated sample
10. Eun, J. J.; Soo, Y. S. Bull. Korean Chem. Soc. 2002, 23,
1483.
¼ 100 ꢀ
ꢁ 100
OD of control
11. Cheng, S.-K.; Van Ergen, D.; Fan, E.; Hamilton, A. D.
J. Am. Chem. Soc. 1991, 113, 7640.
12. (a) Jhaumeer-Laulloo, B. S.; Witvrouw, M. Indian J.
Chem. 2000, B, 842; (b) Jhaumeer-Laulloo, B. S. Asian J.
Chem. 2000, 12, 775.
13. Sulekh, C.; Sangeetika, T.; Shalini, T. Transition Met.
Chem. 2004, 29, 925.
14. Cheng, C.-C.; Rokito, S. E.; Burrows, C. J. Angew. Chem.,
Int. Ed. Engl. 1993, 32, 277.
The lysosomal enzymes released during inflammation
produce a variety of disorders. This extracellular activity
of these enzymes is related to acute or chronic inflamma-
tion. Since the HRBC membranes are similar to lyso-
somal membrane components, the prevention of
hypotonicity induced HRBC membrane lysis is taken
as a measure of anti-inflammatory activity of drug.
15. Halit, K.; Guelsev, D.; Yasar, G.; Uemmuehan, O.; Riza,
A. Transition Met. Chem. 2003, 28, 51.
16. Du, C.-J. F.; Hart, H.; Ng, K. K. D. J. Org. Chem. 1986,
51, 3162.
17. Harada, T.; Takayama, M.; Oohashi, H.; Koike, W.;
Yazawa, C. Jpn. Kokai Tokkyo Koho 80 33,348; Chem.
Abstr. 1980, 93, p96593q).
18. Rajakumar, P.; Rasheed, A. M. A. Tetrahedron 2005, 61,
5351.
The degree of anti-inflammatory activity in cyclo-
phane amide 15 at 50 lg/ml was found to be
82.43%, whereas that of the reference drug predniso-
lone, at the same concentration, was 59.34%. The
activity of cyclophane amide 15 was found to be more
than that of prednisolone which clearly shows that
cyclophane amide 15 is superior to the reference drug
prednisolone. Though the degree of anti-inflammatory
activity in cyclophane amide 18 at 50 lg/ml was
52.27% which is some what nearer to the reference
drug prednisolone, at 100 lg/ml it was only 56.04%
which indicates that cyclophane amide 18 is inferior
to prednisolone. Cyclophane amides 16, 17, 19, and
20 did not show much activity. The results are sum-
marized in Table 1.
19. A solution of the diacid chloride 14 (0.5 mmol) in dry
chloroform (100 mL) and a solution of the diamine 1
(0.5 mmol) and triethylamine (1.1 mmol) in dry chloro-
form (100 mL) were added simultaneously dropwise to
chloroform (500 mL) with vigorous stirring during 6 h.
After the addition was complete, the reaction mixture
was stirred for another 6 h. The solvent was removed at
reduced pressure and the residue obtained was then
dissolved in chloroform (300 mL), washed with water
(2· 100 mL) to remove triethylamine hydrochloride and
then dried over magnesium sulphate. Removal of the
chloroform gave the cyclophane 15 as a crude material,
which was purified by column chromatography (SiO2)
using hexane/chloroform (1:1) as eluting agent to give
pure cyclophane amide 15 as a violet crystalline solid.
Yield: 50%; Rf 0.66 (toluene/ethyl acetate, 9:1); mp: 234–
236 ꢁC; IR (KBr, cmꢀ1) 3379, 1662, 1508; 1H NMR
(400 MHz, CDCl3) d 2.16 (s, 6H), 3.95 (s, 4H), 4.80 (s,
4H), 6.75–8.58 (m, 26H), 9.68 (s, 2H); 13C NMR
(100.4 MHz, CDCl3) d 19.4, 41.9, 67.9, 113.7, 121.1,
121.2, 123.9, 123.9, 124.4, 126.6, 128.2, 128.5, 129.8,
130.4, 130.5, 135.6, 136.1, 137.3, 138.7, 140.7, 156.3,
164.0; FAB Mass spectrum: m/z 798 (M+); Elemental
analysis calcd for C50H42N2O4S2: C, 75.18; H, 5.26; N,
3.50. Found: C, 75.35; H, 5.32; N, 3.61.
Cyclophane amides 15–20 were synthesized by the acyl-
ation of the corresponding diamines with acid chloride
14. Cyclophane amides 15–20 showed a dose-dependent
effect in HRBC membrane stabilization. Anti-inflamma-
tory activity was more in cyclophane amide 15 than the
cyclophane amides 16–20.
Acknowledgments
The authors thank CSIR, New Delhi, for financial assis-
tance, UGC SAP, for providing facility to the Depart-
ment, SAIF, IIT, Chennai, for NMR spectra and
RSIC, CDRI, Lucknow, for FAB MS.
20. Cyclophane amide 16. White solid. Eluent for column
chromatography: chloroform to chloroform/methanol
(99:1); yield: 45%; Rf 0.54 (chloroform/methanol, 9:1);
mp: 280–283 ꢁC; IR (KBr, cmꢀ1) 3280, 1672, 1581, 1517;
1H NMR (400 MHz, DMSO-d6) d 2.08 (s, 6H), 3.38 (s,
4H), 5.62 (s, 4H), 7.10–8.29 (m, 22H), 9.47(s, 2H), 10.52 (s,
2H); 13C NMR (100.4 MHz, DMSO-d6) d 19.0, 40.1, 68.2,
113.5, 120.9, 122.2, 122.6, 124.8, 125.5, 127.9, 130.3, 131.2,
131.6, 132.0, 133.0, 135.9, 138.0, 155.7, 163.4, 166.7; FAB
Mass spectrum: m/z 808 (M+); Elemental analysis calcd for
C46H40N4O6S2: C, 68.31; H, 4.95; N, 6.93. Found: C,
68.42; H, 5.12; N, 6.84.
21. Cyclophane amide 17. Beige crystalline solid. Eluent for
column chromatography: hexane to hexane/chloroform
(1:1); yield: 35%; Rf 0.45 (toluene/ethyl acetate, 9:1); mp:
150–154 ꢁC; IR (KBr, cmꢀ1) 3280, 1662, 1581, 1517; 1H
NMR (400 MHz, CDCl3) d 1.94 (s, 6H), 4.03 (s, 4H), 5.05
(s, 4H), 6.75–8.43 (m, 30H), 9.73 (s, 2H); 13C NMR
(100.4 MHz, CDCl3) d 19.3, 41.5, 68.1, 114.3, 121.4, 121.9,
123.9, 124.5, 125.4, 126.8, 127.4, 128.7, 128.8, 128.9, 129.3,
130.4, 130.8, 132.7, 134.6, 136.5, 137.2, 140.0, 140.2, 140.9,
156.4, 163.9; FAB Mass spectrum: m/z 874 (M+); Elemen-
References and notes
1. Hosseini, M. W.; Lehn, J. M.; Duff, S. R.; Gu, K.; Mertes,
M. P. J. Org. Chem. 1987, 52, 1662.
2. Dobler, M.. In Comprehensive Supramolecular Chemistry;
Gokul, G. W., Ed.; Pergamon: New York, 1996; Vol. 1, p
267.
3. Bradshaw, J. S.; Izatt, R. M.; Bordunov, A. V.; Zhu, C.
Y.; Hathway, J. K.. In Comprehensive Supramolecular
Chemistry; Gokel, G. W., Ed.; Pergamon: New York,
1996; Vol. 1, p 35.
4. Sprengard, U.; Schudok, M.; Schmidt, W.; Kretzschmar,
G.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1996, 35, 321.
5. Karle, I. L.; Ranganathan, D.; Haridas, V. J. Am. Chem.
Soc. 1996, 118, 10916.
6. Kataoka, H.; Katagi, T. Tetrahedron 1987, 43, 4519.
7. Karle, I. L.; Ranganathan, D.; Kurur, S. J. Am. Chem.
Soc. 1999, 121, 7156.
8. Karle, I. L.; Ranganathan, D.; Haridas, V. J. Am. Chem.
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P. Rajakumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6019–6023
6023
tal analysis calcd for C56H46N2O4S2: C, 76.88; H, 5.30; N,
3.20. Found: C, 76.79; H, 5.41; N, 3.28.
(100.4 MHz, CDCl3) d 19.2, 19.8, 37.7, 64.9, 66.5, 70.9,
113.4, 113.8, 120.1, 120.4, 120.5, 120.6, 122.4, 122.7, 123.1,
124.5, 125.7, 127.5, 127.7, 128.2, 129.0, 130.0, 131.4, 132.0,
132.1, 132.2, 132.9, 133.0, 134.0, 135.5, 136.1, 138.5, 141.6,
154.0, 156.1, 162.5, 165.0; FAB Mass spectrum: m/z 630
(M+); Elemental analysis calcd for C38H34N2O5S: C,
72.38; H, 5.39; N, 4.44. Found: C, 72.55; H, 5.59; N, 4.53.
24. Cyclophane amide 20. White crystalline solid. Eluent for
column chromatography: chloroform/methanol (99:1);
yield: 40%; Rf 0.65 (chloroform/methanol, 9:1); mp:
160–162 ꢁC; IR (KBr, cmꢀ1) 3234, 1672, 1664, 1590; 1H
NMR (400 MHz, CDCl3) d 2.19 (s, 6H), 3.07–3.95 (m,
8H), 4.47 (s, 4H), 5.11 (s, 4H), 6.58–7.66 (m, 24H), 8.00
(br s, 2H); 13C NMR (100.4 MHz, CDCl3) d 19.3, 38.5,
47.6, 65.4, 67.6, 112.5, 114.2, 120.8, 121.8, 127.1, 127.3,
127.7, 128.3, 128.5, 128.7, 130.2, 130.8, 132.2, 132.5,
136.6, 141.6, 147.1, 154.3, 169.0, 170.1; FAB Mass
spectrum: m/z 832 (M+); Elemental analysis calcd for
C50H48N4O8: C, 72.10; H, 5.81; N, 6.73. Found: C,
72.28; H, 5.74; N, 6.66.
22. Cyclophane amide 18. Beige crystalline solid. Eluent for
column chromatography: hexane to hexane/chloroform
(1:1); yield: 40%; Rf 0.55 (toluene/ethyl acetate, 9:1); mp:
1
206–208 ꢁC; IR (KBr, cmꢀ1) 3340, 1677, 1591; H NMR
(400 MHz, CDCl3) d 2.25 (s, 3H), 2.27 (s, 3H), 3.65 (s,
2H), 5.08 (s, 2H), 5.17 (s, 2H), 6.54–8.50 (m, 18H), 9.69 (br
s, 2H); 13C NMR (100.4 MHz, CDCl3) d 19.6, 19.8, 43.3,
69.5, 70.8, 113.0, 116.3, 121.0, 122.1, 122.2, 122.4, 124.2,
125.2, 127.7, 128.0, 128.5, 128.9, 130.5, 131.4, 131.7, 132.2,
133.1, 133.6, 134.4, 135.5, 136.7, 137.5, 137.5, 138.9, 141.6,
156.0, 156.8, 162.8, 165.7; Mass spectrum: m/z 600 (M+);
Elemental analysis calcd for C37H32N2O4S: C, 74.00; H,
5.33; N, 4.66. Found: C, 73.85; H, 5.49; N, 4.78.
23. Cyclophane amide 19. Yellow crystalline solid. Eluent for
column chromatography: hexane to hexane/chloroform
(1:1); yield: 42%; Rf 0.60 (toluene/ethyl acetate, 9:1); mp:
1
202–204 ꢁC; IR (KBr, cmꢀ1) 3355, 1674, 1595; H NMR
(400 MHz, CDCl3) d 2.25 (s, 3H), 2.33 (s, 3H), 2.97 (t, 2H,
J = 4.8 Hz), 3.77 (t, 2H, J = 4.8 Hz), 5.01 (s, 2H), 5.06 (s,
2H), 6.17–8.97 (m, 18H), 9.13 (br s, 2H); 13C NMR
25. Gandhidasan, R.; Thamaraichelvan, A.; Baburaj, S.
Fitoterapia 1991, 62, 81.