Synthesis and In-vitro anti-inflammatory activity of novel glycodendrimers with benzene 1,3,5 carboxamide core and triazole as branching unit

Article abstract of DOI:10.1016/j.ejmech.2011.06.017

Novel glycodendrimers scaffolds containing twelve and eighteen α-D-glycopyranoside at the periphery and triazole as branching unit have been synthesized using click chemistry. Higher generation dendrimers exhibit better anti-inflammatory activity than the

Full text of DOI:10.1016/j.ejmech.2011.06.017

European Journal of Medicinal Chemistry 46 (2011) 4687e4695
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and In-vitro anti-inflammatory activity of novel glycodendrimers with
benzene 1,3,5 carboxamide core and triazole as branching unit
*
Perumal Rajakumar , Ramasamy Anandhan
Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai-600025, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel glycodendrimers scaffolds containing twelve and eighteen a-D-glycopyranoside at the periphery
Received 8 May 2011
Received in revised form
9 June 2011
Accepted 13 June 2011
Available online 26 June 2011
and triazole as branching unit have been synthesized using click chemistry. Higher generation den-
drimers exhibit better anti-inflammatory activity than the lower generation due to the presence of more
triazole branching units and multivalent glycopyranoside units.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Glycodendrimers
Click chemistry
PTC
HRBC membrane
anti-inflammatory
1. Introduction
inflamed regions might be expected to provide an important and
viable strategy for suppressing inflammation with minimal side
Cu(I)-catalyzed 1,3-dipolar cycloaddition of an azide to an alkyne
has been extensively used in carbohydrate research for chemical
labelling of biomolecules as well as preparation of oligosaccharide
analogues [1], glycodendrimers [2], scaffolds and carbohydrate
microarrays [3]. Click chemistry is an efficient way for the design of
new dendrimers and the synthesis of 1,2,3-Triazoles has also
attractedwide spreadattention due tothe diversebiological activity.
Glycodendrimers play an important role in a vast array of biological
processes such as cell recognition [4], cell-protein interactions [5],
the targeting of hormones, antibodies [6] and toxins. The impor-
tance of carbohydrate binding with proteins or lectins [7] in regu-
lating biological systems is widely recognized [8].
The commonly used NSAIDs (Non Steroidal Anti-Inflammatory
Drugs) are always associated with many side effects such as
dyspepsia, gastrointestinal bleeding, perforation in addition to
renal side effects and distinct salicylate intoxication [9]. Further
high dosages of NSAIDs are required for acute and chronic condi-
tions. Surfactant encapsulated systems have drawbacks concerning
premature and incomplete release of drug owing to their sensitivity
to the biological environment. Apart from discovering a safer
molecular level target, site-specific targeting of NSAIDs into
effects and better bioavailability.
Many macromolecular drug delivery systems have been devel-
oped to enhance the activity of NSAIDs and limit their side effects,
which promise to be safer than their traditional NSAID counter-
parts over the years [10]. A macromolecular drug delivery system is
a complex material in which a drug is attached to a carrier molecule
such as a synthetic polymer, antibody, hormone or liposome. As the
absorption and distribution of the drug in such system depended
on the properties of the macromolecular carrier, parameters such
as site specificity, protection from degradation and minimization of
side effects can be altered by modifying the properties of the carrier
[11]. Incorporation of NSAIDs into delivery systems such as lipo-
somes or traditional polymers has been reported to significantly
reduce such side effects. Poly(amidoamine) (PAMAM) dendrimers
were shown to be effective as nanoscale vectors for the controlled
delivery of a variety of NSAID and steroidal type anti-inflammatory
drugs [12,13].
There is increasing recognition of the importance of polyvalent
receptor-ligand interactions between carbohydrates and proteins
in many aspects of cell surfaceemediated immuno-regulation [14].
Hence any new drug that aims to effectively modulate these
interactions must possess multiple and cooperative receptor
binding properties. The surface group of dendrimers can be
modified with novel biological properties that exploit polyvalent
and cooperative receptor-ligand interactions [15]. Polyvalent D(þ)-
* Corresponding author. Tel.: þ91 44 2220 2810; fax: þ91 44 235 24 94.
E-mail address: perumalrajakumar@gmail.com (P. Rajakumar).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.017

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P. Rajakumar, R. Anandhan / European Journal of Medicinal Chemistry 46 (2011) 4687e4695
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
N
N
O
N
N
N
N
O
O
S
N
O
N
O
NH
O
N
O
N
N
OAc
AcO
AcO
N
OAc
OAc
OAc
S
H
N
O
AcO
O
OAc
O
HN
S
O
O
N
O
N
OAc
N
N
N
N
AcO
OAc
AcO
AcO
OAc
OAc
O
OAc
1
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
AcO
N
N
N
O
OAc
OAc
N
N
OAc
O
AcO
AcO
AcO
N
N
N
N
N
O
AcO
N
N
AcO
O
N
O
O
O
O
O
S
N
O
NH
O
N
OAc
N
N
AcO
AcO
N
OAc
OAc
S
OAc
H
N
O
AcO
O
OAc
O
HN
S
O
O
O
N
O
N
N
N
AcO
N
N
OAc
OAc
AcO
N
AcO
OAc
N
N
OAc
O
OAc
OAc
O
OAc
OAc
OAc
2
Fig. 1. Structure of the glycodendrimers.

P. Rajakumar, R. Anandhan / European Journal of Medicinal Chemistry 46 (2011) 4687e4695
4689
AcOAcO
AcO
AcO
AcO
AcO
O
AcO
O
N
AcOAcO
AcO
N
N
N
AcO
AcO
AcO
N
AcO
O
AcO
O
N
AcO
O
N
N
N
N
O
N
O
N
O
N
N
N
N
N
N
O
O
OAc
OAc
N
AcO
O
N
S
N
N
N
N
N
OAc
OAc
O
N
NH
O
N
AcO
AcO
O
O
N
N
O
O
AcO
N
S
H
N
O
O
N
O
O
HN
OAc
OAc
OAc
AcO
O
N
N
N
OAc
N
S
N
AcO
AcO
O
AcO
O
O
N
N
N
N
N
N
O
N
O
N
N
N
N
N
O
OAc
O
O
OAc
OAc
OAc
OAc
O
N
N
N
N
OAc
OAc
OAc
N
N
OAc
O
OAc
OAc
OAc
O
OAc
OAc
OAc
OAc
3
Fig. 1. (continued)
glucosamine dendrimers were synthesized and used as immuno-
modulatory, antiangiogenic, and anti-inflammatory agents [16].
We report herein the straightforward synthesis and anti-
inflammatory activity of glycodendrimers 1, 2, 3 and 4 from the
corresponding azidocarbohydrates and acetylene-bearing dendritic
arms through click chemistry (Fig. 1).
released during inflammatory condition produce a variety of
disorders. The extracellular activity of these enzymes is said to be
related to acute or chronic inflammation. The anti-inflammatory
agent acts by either inhibiting the lysosomal enzymes or by stabi-
lizing the lysosomal membranes. The observed data showing the
anti-inflammatory activity of the compounds and the control drug
are given in Table 1.
2. Chemistry
4. Results and discussion
Glycodendrimers (1e4) were synthesized by utilizing the step-
wise strategy as shown in the Scheme 1 and 2. Azido reaction,
phase transfer catalysed S-alkylation, amide coupling reaction and
click chemistry were employed to achieve the target glycoden-
drimers. All the new compounds gave satisfactory 1H and 13C NMR,
mass spectral and elemental analysis.
Reaction of 3, 5-bis(propargyloxy)benzyl chloride 6 with two
equivalents of 2,3,4,6-tetra-O-acetyl-
a
-D-glucopyranoazide
5
under the Cu (I) catalyzed click reaction condition gave the first
generation dendritic chloride 7 in 96% yield (AB2-type). The use of
NaN3 in mixture of acetone/water (4:1) at 60 ꢀC allowed the
transformation of the dendritic chloride 7 to the corresponding
dendritic azide 8 in 98% yield. In 1H NMR spectrum compound 8
3. Biology
displayed four singlets at
glyco-acetoxy protons, a singlet at
a singlet at
7.86 for triazole proton. The 13C NMR spectrum of
d
1.78, 1.96 & 2.00 for the four different
In vitro anti-inflammatory activity was studied by human red
blood cell membrane stabilization method. The lysosomal enzymes
d
5.13 for the eO-CH2 proton and
d

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P. Rajakumar, R. Anandhan / European Journal of Medicinal Chemistry 46 (2011) 4687e4695
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
AcO
AcO
AcO
O
AcO
N
AcO
AcO
O
N
AcO
OAc
OAc
O
N
N
OAc
N
AcO
N
AcO
N
N
N
N
O
AcO
AcO
O
N
O
N
N
N
O
O
N
AcO
N
N
N
OAc
O
O
OAc
OAc
N
N
N
OAc
O
N
N
AcO
AcO
AcO
O
N
O
O
N
N
N
O
N
N
AcO
N
N
N
N
N
O
O
OAc
N
O
O
N
O
S
N
NH
O
O
N
N
N
OAc
N
N
AcO
OAc
OAc
N
O
O
N
N
N
N
O
N
S
OAc
O
H
N
AcO
O
AcO
AcO
O
HN
S
O
O
OAc
N
AcO
N
OAc
OAc
O
N
N
N
N
OAc
O
AcO
AcO
AcO
O
O
O
N
N
N
N
N
N
O
N
N
N
N
N
N
N
O
N
O
OAc
N
OAc
OAc
OAc
O
O
O
OAc
OAc OAc
OAc
N
N
N
N
O
O
N
N
O
OAc
N
N
N
N
OAc
OAc
OAc
N
N
O
OAc
OAc OAc
OAc
OAc
OAc
O
O
OAc
OAc
OAc
OAc
OAc
OAc
4
Fig. 1. (continued)
compound 8 displayed four different gluco-acetoxy carbons at
19.1, 19.5 19.5 and 19.6, the eO-CH2 carbon resonated at 84.8, the
triazole carbon resonated at 158.6 and the four different carbonyl
carbon appeared at 167.9, 168.4, 168.9 and 169.5, the acetylene
and methylene carbons at 75.3 and at 57.3. The appearance of
aminothiophenol 11 with benzene-1,3,5-tricarboxylic acid chlo-
ride in the presence of Et3N in dry dichloromethane at room
temperature under N2 afforded the amide core based propargylated
dendritic wedge 12 and 13 in 75% and 71% yields respectively. The
1H NMR spectrum of propargylated dendritic wedge with amide
core 13 displayed a two different triplet for the two different
d
d
d
d
d
d
molecular ion peak at m/z 987 also confirmed the structure of the
dendritic azide 8 (Scheme 1).
acetylene proton at
protons at 3.89 and NH protons as a singlet at
NMR spectrum, propargylated dendritic wedge 13, displayed the
two different acetylene carbons at 75.3 and 75.9, S-methylene
d
2.34 and
d
2.45, a singlet for S-methylene
Reaction of 2-aminothiophenol with 3,5-bis(propargyloxy)
benzyl chloride 6/3,4,5-tris(propargyloxy)benzyl chloride 9 in
toluene and aqueous KOH in presence of catalytic amount tetra-
butylammonium bromide (TBAB) as phase transfer catalyst at
reflux furnished 3,5-bis(propargyloxy)benzyl-2-aminothiophenol
10/3,4,5-tris(propargyloxy)benzyl-2-aminothiophenol 11 in 86%
and 79% yields, respectively. Reaction of 3,5-bis(propargyloxy)
benzyl-2-aminothiophenol 10/3,4,5-tris(propargyloxy)benzyl-2-
d
d
9. 31. In the 13C
d
carbon at d 41.8 and carbonyl carbon at d 168.7. FT-IR spectrum also
shows the carbonyl stretching frequency at 1639 cmꢁ1 for prop-
argylated dendritic wedge 13 (Scheme 2).
The click chemistry functionalizaton reaction of the acetylene
amide core 12 and 13 with 2.1 equivalents of 2,3,4,6-tetra-O-acetyl-

P. Rajakumar, R. Anandhan / European Journal of Medicinal Chemistry 46 (2011) 4687e4695
4691
Scheme 1. Reagents and conditions: (i) CuSO4 (5 mol %), sodium ascorbate (10 mol %), H2O/THF (1:1); r.t., 10 h; (ii) 1.5 equiv NaN3, CH3COCH3/H2O, 60 ꢀC, 3 h.
a
-D-mannopyranosyl azide 5/dendritic azide G1-N3 8 in the pres-
[Mþ þ Na] in the mass spectrum of glycodendrimer 4 and from
ence of the Cu (I) catalyzed click reaction afforded the glycoden-
spectral and analytical data, the structure of the glycodendrimer 4
has been also confirmed (Fig. 2).
drimers 1e4 in 90e96% yields, respectively. The 1H NMR spectrum
glycodendrimer
different glyco-acetoxy protons at
singlet at 3.78 for eS-CH2 protons, a sharp singlet at
CH2 protons, a sharp singlet at 8.09 for the triazole protons and
NH protons as a singlet at
9.54. The 13C NMR spectrum of glyco-
dendrimer 3 displayed four different gluco-methoxy carbons at
20.1, 20.5 and 20.6 (2C), the eO-CH2 carbon resonated at 85.6,
the amide carbonyl and four different gluco-methoxy carbonyl
3
displayed three different singlets for four
1.78, 2.02 and 2.07, a sharp
5.32 for eO-
d
4.1. Anti-inflammatory activity
d
d
d
The lysosomal enzymes released during inflammation produced
a variety of disorders. The extracellular activity of these enzymes is
related to acute or chronic inflammation. Hence lysis of human red
blood cell membrane is taken as a measure of anti-inflammatory
activity of the drug.
d
d
d
carbons appeared at
d 163.2, 168.9, 169.4, 170.0 and 170.5. The
The anti-inflammatory activity of all the glycodendrimers are
concentration dependent. At the lower concentration, all the gly-
codendrimers 1, 2, 3 and 4 exhibited better anti-inflammatory
activity that at higher concentration. Glycodendrimers 1 showed
appearance of molecular ion peak at m/z 7076 [Mþ þ Na] also
confirmed the structure of the glycodendrimer 3 (Scheme 2).
Similarly, from the appearance of molecular ion peak at m/z 10202
O
R
O
H2N
R
O
S
Cl
O
O
NH
S
i
ii
S
H
N
O
O
O
R
O
O
O
HN
R
S
6
9
R = H
10
R = H
O
R =
O
11
R =
O
O
R
12
13
R = H
O
R =
iii
1 2 3 4
&
Glycodendrimers
,
,
Scheme 2. Reagents and conditions: (i) 2-aminothiophenol, cat. TBAB, toluene/H2O, reflux, 12 h; (ii) benzene-1,3,5-tricarboxylic acid chloride, TEA, DCM (dry), 24 h; (iii) 2,3,4,6-
tetra-O-acetyl- -D-mannopyranosyl azide 5 and dendritic azide 8 CuSO4 (5 mol%), sodium ascorbate (10 mol%), H2O/THF (1:1); r.t., 10 h.
a

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P. Rajakumar, R. Anandhan / European Journal of Medicinal Chemistry 46 (2011) 4687e4695
Table 1
Effect of glycodendrimers 1e4 on HRBC membrane stabilization.
Conc.
mg/ml
Activity (% prevention of lysis)
Glyco-dendrimer 1
Glyco-dendrimer 2
Glyco-dendrimer 3
Glyco-dendrimer 4
Prednisolone
2.5
5
10
86.41 ꢂ 0.16
95.25 ꢂ 0.45
60.61 ꢂ 0.62
63.50 ꢂ 0.22
89.71 ꢂ 0.23
60.50 ꢂ 0.19
79.64 ꢂ 0.68
66.25 ꢂ 0.12
58.59 ꢂ 0.12
94.79 ꢂ 0.32
98.63 ꢂ 0.02
87.01 ꢂ 0.19
95.77 ꢂ 0.18
63.38 ꢂ 0.29
74.50 ꢂ 0.20
95.31 ꢂ 0.10
82.30 ꢂ 0.19
73.74 ꢂ 0.24
98.95 ꢂ 0.33
93.36 ꢂ 0.70
97.12 ꢂ 0.59
99.49 ꢂ 0.25
85.63 ꢂ 0.31
90.72 ꢂ 0.25
85.34 ꢂ 0.30
96.71 ꢂ 0.21
86.33 ꢂ 0.25
92.90 ꢂ 0.07
96.62 ꢂ 3.00
99.21 ꢂ 0.11
98.24 ꢂ 0.87
98.67 ꢂ 0.19
95.00 ꢂ 0.28
94.24 ꢂ 0.20
94.32 ꢂ 0.16
96.07 ꢂ 0.35
15.27 ꢂ 0.46
25.16 ꢂ 0.25
40.05 ꢂ 0.39
50
59.34 ꢂ 0.44
100
200
400
800
1000
86.37 ꢂ 0.49
e
e
e
e
the haemolysis of red blood cells. Glycodendrimer 4 shows better
anti-inflammatory activity than all other dendrimers at all concen-
trations (Table 1) which shows that its binding on to the membrane
of the erythrocyte is very effective which could be due to the pres-
ence of more number of triazole units and multivalent effect of the
glycopyranoside surface unit. Hence synthesis of further higher
generation dendrimers and with excessive branching points than
dendrimer 4 could show still better anti-inflammatory activity.
7000
6000
5000
4000
3000
2000
1000
0
10202 [M++Na]
5. Conclusion
In conclusion, glycodendrimers 1e4 with amide core and tri-
azole branching unit were synthesized through click chemistry and
exhibited significant anti-inflammatory activity. This anti-
inflammatory activity is concentration dependent as well as
depends on the dendrimers generation.
10100
10200
10300
10400
m/z
6. Experimental section
Fig. 2. MALDI-TOF-MS spectra of glycodendrimers 4.
6.1. Chemistry
higher activity at 5
mg/mL. However, glycodendrimers 2, 3 and 4
exhibited higher activity at 5
respectively. Moreover the anti-inflammatory activity increases
with increases in dendrimer generation. Glycodendrimer
exhibited better anti-inflammatory activity at all concentrations.
Higher generation dendrimers has more number of branching tri-
mg/mL, 50 mg/mL and 100 mg/mL
All reagents were commercially available and used as such unless
otherwise stated. 3,5-Bis(propargyloxy)benzyl chloride [17] (6),
3,4,5-Tris(propargyloxy)benzyl chloride [18] (11) and azido-
functionalized sugar [19] (5) were prepared according to the repor-
ted procedure. Analytical TLC was performed on commercial Merk
plates coated with Silica Gel GF254. Analytical samples were
obtained from flash silica gel chromatography, using silica gel of
100e200 mesh and elution with the solvent system as mentioned
undereachexperiment section. Themelting points weredetermined
by using a Metler Toledo melting point apparatus by open capillary
tube method and were uncorrected. 1H and 13C NMR spectra were
recorded on a 300 MHz BRUKER AVANCE (75 MHz for 13C NMR)
4
azole unit and a-D-glycopyranosyl at the surface and hence showed
higher activity to adsorb and interact at the membrane. Percentage
stabilization of the membrane in the presence of glycodendrimers
1, 2, 3 and 4 at various concentration levels of 2.5, 5,10, 50,100, 200,
400, 800 and 1000 mg/mL is shown in Fig. 3.
Anti-inflammatory activity glycodendrimers 1e4 could be due to
their binding on to the erythrocyte membranes with subsequent
alteration of the charges on the 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
spectrometer. All chemical shifts values are reported in
tive to internal standard tetramethylsilane (TMS,
0.00). 13C chem-
ical shifts are reported in relative to CDCl3 (center of triplet, 77.23)
or relative to DMSO-d6 (center of septet, 39.51). The spin multi-
d ppm rela-
d
d
d
d
plicities are indicated by the symbols s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet) and br (broad), dd (doublet of
doublets). The coupling constants J, are reported in Hertz (Hz). Mass
spectra (MS) were recorded using Voyager-DE PRO mass spectrom-
eter by either Fast Atom Bombardment or MALDI-TOF technique.
6.2. First generation dentritic azide 8
Dendritic chloride 7 (1.0 mmol, 1 eq.) was dissolved in acetone/
water (4:1, 20 mL). NaN3 (1.5 mmol, 1.5 eq.) was added, and the
mixture was heated to 60 ꢀC for 3 h. The mixture was cooled to
room temperature, acetone evaporated, diluted with H2O (100 mL)
and extracted with EtOAc (250 mL). The organic layer was washed
with saturated NaCl, dried over Na2SO4 and evaporated to give the
Fig. 3. Anti-inflammatory activity of glycodendrimers 1e4.

P. Rajakumar, R. Anandhan / European Journal of Medicinal Chemistry 46 (2011) 4687e4695
4693
dendritic azide 8 as a white solid. which was purified by column
6.4.1. Dendritic wedge 12
Pale yellow solid; yield: 75%; Rf ¼ 0.71 (CHCl3:Methanol, 99:1);
mp.: 101 ꢀC; 1H NMR (300 MHz, CDCl3):
chromatography (SiO2) with Hexane: Ethylacetate, 1:1 as eluvent
(Rf ¼ 0.6); mp: 133 ꢀC; 1H NMR: (300 MHz, CDCl3):
d
1.78, 1.96, 2.00
d
2.34 (t, J ¼ 2.1 Hz, 6 H),
(3 X s, 24 H), 3.92e3.97 (m, 2 H), 4.04e4.11 (m, 2 H), 4.21e4.26 (m,
4 H), 5.13 (s, 4 H), 5.18e5.21 (m, 2 H), 5.32e5.43 (m, 4 H), 5.84 (d,
J ¼ 2.7 Hz, 2 H). 6.50 (d, J ¼ 2.1 Hz, 2 H), 6.57 (d, J ¼ 2.1 Hz, 1 H), 7.86
3.89 (s, 6 H), 4.37 (d, J ¼ 2.1 Hz, 12 H), 6.24 (s, 6H), 6.28 (s, 3H), 7.15
(t, J ¼ 7.5 Hz, 3 H), 7.47 (t, J ¼ 7.8 Hz, 3 H), 7.57 (d, J ¼ 7.8 Hz, 3 H),
8.46 (s, 3 H), 8.60 (d, J ¼ 8.1 Hz, 3 H), 9.33 (s, 3H) ppm. 13C NMR
(s, 2 H) ppm. 13C NMR: (75 MHz, CDCl3):
d
19.1, 19.5 19.5, 19.6, 53.6,
(75 MHz, CDCl3):
d
¼ 42.1, 55.8, 75.8, 78.2, 101.5, 108.1, 120.3, 122.9,
60.6, 60.9, 66.8, 69.4, 71.7, 74.2, 84.8, 100.6,106.8,120.3, 137.0, 143.6,
158.6, 167.9, 168.4, 168.9, 169.5 ppm. MS (FAB): m/z ¼ 987.
Elemental Anal.Calcd for C41H49N9O20: C, 49.85; H, 5.00; N, 12.76%.
Found: C, 49.81; H, 4.96; N, 12.51%.
124.8, 128.6, 130.6, 135.7, 136.5, 140.0, 158.6, 162.7 ppm. MS (FAB):
m/z ¼ 1125 [Mþ]. Elemental Anal.Calcd for C66H51N3O9S3: C, 70.38;
H, 4.56; N, 3.73%. Found: C, 70.21; H, 4.51; N, 3.68%.
6.4.2. Dendritic wedge 13
6.3. General procedure for the S-alkylation
Pale yellow solid; yield: 71%; Rf ¼ 0.54 (CHCl3:Methanol, 99:1);
mp.: 79 ꢀC; 1H NMR (300 MHz, CDCl3):
d
2.34 (t, J ¼ 2.4 Hz, 3 H),
A
mixture of propargyloxybenzyl chloride derivatives
2.45 (t, J ¼ 2.4 Hz, 6 H), 3.90 (s, 6 H), 4.49 (d, J ¼ 2.4 Hz, 12 H), 4.55
(d, J ¼ 2.4 Hz, 6 H), 6.36 (s, 6 H), 7.13 (dt, J ¼ 7.5 Hz J ¼ 0.9 Hz, 3 H),
7.44 (dt, J ¼ 7.8 Hz J ¼ 1.2 Hz, 3 H), 7.52 (dd, J ¼ 7.5 Hz J ¼ 1.2 Hz, 3 H),
8.47 (d, J ¼ 7.8 Hz, 3 H), 8.70 (d, J ¼ 1.5 Hz, 3 H), 9.31 (s, 3H) ppm. 13C
(1.0 mmol, 1 eq.), 2-aminothiophenol (1.2 mmol, 1.2 eq.), TBAB
(5 mg), KOH (1.5 mmol, 1.5 eq.), in mixture of toluene/water (1:1,
40 mL)was refluxed for 4 h. Toluene layer was separated, washed
with 5% KOH solution (2 ꢃ 10 mL), water (20 mL) and then dried
over Na2SO4. Toluene was evaporated under vacuum and the
residue obtained was purified by column chromatography with
hexane-chloroform as eluent to give the corresponding prop-
argyloxybenzyl-2-aminothiophenol.
NMR (75 MHz, CDCl3): d 41.8, 56.9, 60.2, 75.3, 75.9, 78.3, 79.0, 108.8,
120.8, 123.5, 125.0, 130.3, 130.6, 131.1, 131.4, 133.8, 135.8, 136.3,
139.9, 151.5, 163.1, 168.7 ppm. MS (FAB): m/z ¼ 1287 [Mþ].
Elemental Anal.Calcd for C75H57N3O12S3: C, 69.91; H, 4.46; N, 3.26%.
Found: C, 69.85; H, 4.40; N, 3.21%.
6.3.1. 3,5-Bis(propargyloxy)benzyl-2-aminothiophenol 10
Colourless liquid; yield: 86%; Rf ¼ 0.45 (Hexane: CHCl3, 4:1); 1H
6.5. General procedure for the Cu-catalyzed Huisgen ‘click reaction’
NMR (300 MHz, CDCl3):
d
2.52 (t, J ¼ 2.4 Hz, 2 H), 3.82 (s, 2 H), 4.25
(bs, 2 H), 4.57 (d, J ¼ 2.4 Hz, 2 H), 6.37 (d, J ¼ 2.4 Hz, 2 H), 6.47 (t,
J ¼ 2.4 Hz,1 H), 6.37 (d, J ¼ 2.4 Hz, 2 H), 6.63 (dt, J ¼ 7.5 Hz J ¼ 1.5 Hz,
1 H), 6.70 (dd, J ¼ 9.0 Hz J ¼ 1.2 Hz,1 H), 7.11 (dt, J ¼ 9.0 Hz J ¼ 1.8 Hz,
1 H), 7.23 (dd, J ¼ 7.8 Hz J ¼ 1.5 Hz, 1 H) ppm. 13C NMR (75 MHz,
A mixture of alkyne (1.0 mmol, 1.0 eq.), azide (6.0 mmol, 6.0 eq./
9.0 mmol, 9.0 eq.), CuSO4.5H2O (5 mol %) and NaAsc. (10 mol %) in
a mixture of THF/H2O (1:1, 20 mL) was stirred for 12 h at room
temperature. The residue obtained after evaporation of the solvent
was dissolved in CHCl3 (150 mL) and washed with brine (150 mL),
water (100 mL), dried over Na2SO4 and evaporated to give the crude
triazole, which was purified by column chromatography (SiO2).
CDCl3):
d 39.7, 55.9, 75.6, 78.4, 101.5, 108.4, 114.9, 117.2, 118.5, 130.1,
136.6, 140.8, 148.7, 158.5 ppm. MS (FAB): m/z ¼ 323 [Mþ].
Elemental Anal.Calcd for C19H17NO2S: C, 70.56; H, 5.30; N, 4.33%.
Found: C, 70.49; H, 5.25; N, 4.31%.
6.5.1. First generation dentritic chloride 7
6.3.2. 3,4,5-Tris(propargyloxy)benzyl-2-aminothiophenol 11
Colourless liquid; yield: 79%; Rf ¼ 0.60 (CHCl3, 100%); 1H NMR
White solid; Yield: 96%; Rf ¼ 0.6 (Ethylacetate: Hexane, 1:1);
mp.: 115 ꢀC; 1H NMR: (300 MHz, CDCl3):
d 1.85, 2.03, 2.07 (3 X s,
(300 MHz, CDCl3):
d
2.45 (t, J ¼ 2.4 Hz, 1 H), 2.48 (t, J ¼ 2.4 Hz, 2 H),
24 H), 4.00e4.04 (m, 2 H), 4.13e4.17 (m, 2 H), 4.28e4.32 (m, 2H),
4.51 (s, 2 H), 5.20 (s, 4 H), 5.22e5.28 (m, 2 H), 5.39e5.49 (m, 4 H),
5.92 (d, J ¼ 8.7 Hz, 2 H), 6.64 (s, 3 H), 7.92 (s, 2 H) ppm. 13C NMR:
3.81 (s, 2 H), 4.20 (bs, 2 H), 4.61 (d, J ¼ 2.4 Hz, 4 H), 4.70 (d,
J ¼ 2.4 Hz, 2 H), 6.45 (s, 2H), 6.62 (dt, J ¼ 7.5 Hz J ¼ 1.2 Hz, 1 H), 6.67
(dd, J ¼ 8.1 Hz J ¼ 1.2 Hz, 1 H), 7.11 (dt, J ¼ 8.1 Hz J ¼ 1.5 Hz, 1 H), 7.20
(75 MHz, CDCl3):
d 20.1, 20.5, 20.5, 20.6, 46.0, 61.5, 61.9, 67.7, 70.3,
(dd, J ¼ 7.5 Hz J ¼ 1.5 Hz,1 H) ppm. 13C NMR (75 MHz, CDCl3):
d
39.9,
72.6, 75.2, 85.8, 101.7, 108.2, 121.4, 139.9, 144.6, 159.4, 168.9, 169.4,
169.9, 170.5 ppm. MS (FAB): m/z ¼ 980 [Mþ]. Elemental Anal.Calcd
for C41H49Cl1N6O20: C, 50.18; H, 5.03; N, 8.56%. Found: C, 50.13; H,
5.95; N, 8.60%.
57.0, 60.3, 75.1, 75.8, 78.5, 79.3, 109.1,114.8, 116.8,118.5,130.2, 134.6,
136.1, 137.0, 149.01, 151.3 ppm. MS (FAB): m/z ¼ 377 [Mþ].
Elemental Anal.Calcd for C22H19NO3S: C, 70.00; H, 5.07; N, 3.71%.
Found: C, 69.97; H, 5.04; N, 3.52%.
6.5.2. Glycodendrimer 1
6.4. General procedure for the amide core based propargylated
dendritic wedge
Glycodendrimer 1 (0.23 g, 96%) was obtained as pale brown
solid from propargylated dendritic wedge 12 (0.08 g, 0.07 mmol)
and 2,3,4,6-tetra-O-acetyl-
a
-D-glucopyranoazide
5
(0.19 g,
A solution of the benzene-1,3,5-tricarboxylic acid chloride
(1.0 mmol) in dry DCM (100 mL) and a solution of the propargyloxy
benzyl-2-aminothiophenol derivative (3.0 mmol) and triethyl-
amine (3.1 mmol) in dry dichloromethane (100 mL) were simul-
taneously added drop wise to a well-stirred solution of dry
dichloromethane (500 mL) during 6 h. After the addition was
complete, the reaction mixture was stirred for another 6 h. The
solvent was removed under reduced pressure and the residue
obtained was then dissolved in dichloromethane (300 mL), washed
with water (2 ꢃ 100 mL) to remove triethylamine hydrochloride
and then dried over Na2SO4. Dichloromethane was evaporated
under vacuum and the obtained residue was purified by column
chromatography with chloroform-methanol as eluent to give the
corresponding amide core based propargylated dendritic wedge.
0.50 mmol) using the general procedure of click chemistry.
Rf ¼ 0.68 (CHCl3:Methanol, 9:1); mp.: 130 ꢀC; 1H NMR (300 MHz,
CDCl3): d 1.77, 2.02, 2.02 (3xs, 72 H), 3.85(s, 6 H), 4.01e4.04 (m, 6 H),
4.08e4.11 (m, 6 H), 4.15e4.30 (m, 6 H), 4.86 (s, 12 H), 5.27 (t,
J ¼ 9.6 Hz, 6 H), 5.41 (t, J ¼ 9.3 Hz, 6 H), 5.51 (t, J ¼ 9.6 Hz, 6 H), 5.90
(d, J ¼ 9.3 Hz, 6 H), 6.24 (s, 6 H), 6.29 (s, 3 H), 7.16 (t, J ¼ 7.5 Hz, 3 H),
7.45 (t, J ¼ 7.5 Hz, 3 H), 7.54 (d, J ¼ 6.3 Hz J ¼ 1.2 Hz, 3 H), 7.94 (s, 6H),
8.46 (d, J ¼ 8.1 Hz, 3 H), 8.59 (s, 3 H), 9.43 (s, 3H) ppm. 13C NMR
(75 MHz, CDCl3):
d 20.1, 20.5, 20.6, 20.6, 40.6, 61.7, 67.8, 70.3, 71.8,
72.8, 75.0, 85.6, 101.1, 107.9, 121.1, 121.8, 124.4, 125.2, 128.8, 130.3,
130.9, 132.4, 135.9, 140.1, 144.4, 159.3, 163.1, 168.9, 169.4, 170.0,
170.5 ppm. MS (MALDI-TOF): m/z ¼ 3388 [MþþNa]. Elemental
Anal.Calcd for C150H165N21O63S3: C, 53.52; H, 4.94; N, 8.74%. Found:
C, 53.50; H, 4.86; N, 8.67%.

4694
P. Rajakumar, R. Anandhan / European Journal of Medicinal Chemistry 46 (2011) 4687e4695
6.5.3. Glycodendrimer 2
Glycodendrimer 2 (0.17 g, 91%) was obtained as pale brown solid
from propargylated dendritic wedge 13 (0.08 g, 0.04 mmol) and
equal volume of sterilised Alsever solution (2% dextrose, 0.8%
sodium citrate, 0.05% citric acid and 0.42% sodium chloride in
water). The blood was centrifuged at 3000 rpm and packed cells
were washed with isotonic sodium chloride (0.85%, pH 7.2) and
a 10% (v/v) suspension was made with isotonic sodium chloride.
The assay mixture contained the drug (concentration as mentioned
in Table 1), phosphate buffer (1 ml, 0.15 M, pH 7.4), hypotonic NaCl
2,3,4,6-tetra-O-acetyl-
using the general procedure of click chemistry. Rf
(CHCl3:Methanol, 9:1); mp.: 148 ꢀC; 1H NMR (300 MHz, CDCl3):
1.74, 1.80, 1.96, 1.99, 2.02, 2.04, 2.09 (7xs, 108 H), 3.89 (s, 6 H),
a
-D-glucopyranoazide 5 (0.15 g, 0.38 mmol)
¼
0.47
d
4.07e4.17 (m, 18 H), 4.27e4.33 (m, 9 H), 5.01 (s, 12 H), 5.03 (s, 6 H),
5.38e5.456 (s, 18 H), 5.57e5.65 (s, 9 H), 5.98 (d, J ¼ 9.3 Hz, 6 H), 6.17
(d, J ¼ 9.3 Hz, 3 H), 6.35 (s, 6 H), 7.13 (t, J ¼ 7.8 Hz, 4 H), 7.43 (t,
J ¼ 7.8 Hz, 4 H), 8.31 (s, 3H), 8.43 (s, 6 H), 8.46 (t, J ¼ 8.1 Hz, 4 H), 8.71
(2 ml, 0.36%) and HRBC suspension (0.5 mL). Prednisolone, (100 mg)
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 UVeVis Spectrophotometer
118) at 560 nm. The percentage 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 formula.
(s, 3H), 9.57 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3):
d 20.0, 20.1,
20.5, 20.6, 20.6, 40.7, 61.7, 62.8, 66.0, 67.8, 70.1, 70.4, 72.9, 73.1, 74.7,
74.9, 85.3, 85.6, 107.7, 114.1, 121.2, 122.2, 122.8, 124.1, 125.1, 129.0,
130.3, 133.9, 135.9, 136.4, 139.7, 145.0, 151.9, 163.2, 168.9, 169.5,
169.6, 170.0, 170.6 ppm. MS (MALDI-TOF): m/z ¼ 4670 [MþþNa].
Elemental Anal.Calcd for C201H228N30O93S3: C, 51.94; H, 4.94; N,
9.04%. Found: C, 51.86; H, 4.96; N, 9.02%.
% protection ¼ 100 ꢁ O.D of drug treated samples/O.D of control
ꢃ 100 instead of %.
6.5.4. Glycodendrimer 3
Glycodendrimer 3 (0.13g, 94%) was obtained as pale brown solid
from propargylated dendritic wedge 12 (0.02 g, 0.02 mmol) and
dendritic azide 8 (0.11 g, 0.13 mmol) using the general procedure of
click chemistry. Rf ¼ 0.51 (CHCl3:Methanol, 9:1); mp.: 158 ꢀC; 1H
Acknowledgment
The author thanks CSIR, New Delhi, India, for financial assis-
tance and DST-FIST for providing NMR facility to the department.
RA thanks CSIR, New Delhi, for fellowship and Prof. D. Cha-
mundeeswari, Deptartment of Pharmacognosy, Faculty of phar-
macy, Sri Ramachandra Medical College and Research Institute,
Chennai-116 for anti-inflammatory studies.
NMR (300 MHz, CDCl3):
d 1.78, 2.02,2.07 (3xs, 144 H), 3.78(s, 6 H),
4.08e4.16 (m, 24 H), 4.27e4.30 (m, 12 H), 4.82e4.97 (m, 36 H),
5.30e5.33 (s, 24 H), 5.44 (t, J ¼ 9.0 Hz, 12 H), 5.54e5.56 (m, 12 H),
5.97 (s, 12 H), 6.33 (s, 3 H), 6.42e6.52 (m, 27 H), 7.07 (d, J ¼ 3.3 Hz,
6 H), 7.29 (m, 3 H), 7.67e7.79 (m, 6 H), 8.09 (s, 12 H), 8.51 (s, 3 H),
9.54 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3):
d 20.1, 20.5, 20.6, 20.6,
References
53.9, 61.7, 67.7, 70.3, 72.8, 74.9, 75.0, 85.6, 85.6, 101.7, 101.8, 107.7,
114.1, 121.8, 122.1, 122.2, 123.4, 123.8, 137.3, 137.3, 137.4, 137.6, 139.3,
143.9, 144.2, 151.8, 151.9, 159.5, 159.6, 168.9, 169.5, 167.0, 170.6 ppm.
MS (MALDI-TOF): m/z ¼ 7076 [Mþ þ Na]. Elemental Anal.Calcd for
C312H345N57O129S3: C, 53.13; H, 4.93; N, 11.32%. Found: C, 53.08; H,
4.86; N, 11.34%.
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Glycodendrimer 4 (0.16 g, 90%) was obtained as pale brown solid
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216 H), 3.86 (s, 6 H), 4.05e4.16 (m, 36 H), 4.26 (d, J ¼ 3.9 Hz, 12 H),
4.30 (d, J ¼ 5.1 Hz, 6 H), 4.77 (s, 12 H), 5.07 (s, 24 H), 5.11 (s, 18 H),
5.26e2.32 (m, 18 H), 5.43 (t, J ¼ 9.6 Hz, 36 H), 5.48e5.56 (m, 18 H),
5.94 (d, J ¼ 3.6 Hz, 12 H), 5.97 (d, J ¼ 3.3 Hz, 6 H), 6.18 (d, J ¼ 4.8 Hz,
6 H), 6.46 (s, 12 H), 6.50 (s, 6 H), 6.56 (s, 6 H), 6.60 (s, 3 H), 7.10 (d,
J ¼ 8.4 Hz, 3 H), 7.27e7.38 (m, 3 H), 7.49 (d, J ¼ 7.5 Hz, 3 H), 7.58 (s,
3H), 7.60 (s, 6 H), 8.01 (s, 6 H), 8.03 (s, 12 H), 8.30 (d, J ¼ 7.8 Hz, 3 H),
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20.6, 20.6, 40.8, 53.9, 61.6, 61.7, 67.7, 70.3, 72.6, 72.7, 75.0, 75.1, 85.6,
85.7, 100.9, 101.6, 101.7, 121.8, 121.9, 122.0, 123.2, 123.4, 135.4, 137.3,
140.1, 143.9, 144.2, 144.3, 159.3, 159.6, 159.7, 163.2, 168.9, 169.4,
170.0, 170.5 ppm. MS (MALDI-TOF): m/z ¼ 10202 [MþþNa].
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11.56%. Found: C, 52.39; H, 4.84; N, 11.55%.
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