Synthesis and analysis of in vitro anti-arthritic activity of dendrimers with methyl, ethyl and isopropyl salicylates as surface groups

Article abstract of DOI:10.1039/c4ra07992b

Frchet type dendrimers with salicylates as surface groups have been synthesized using the divergent approach and their in vitro anti-arthritic activity was analyzed by inhibition of protein denaturation.

Full text of DOI:10.1039/c4ra07992b

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Graphical Abstract
Dendrimers containing methyl, ethyl and isopropyl salicylates at the surface were
synthesized by divergent approach starting from a simple core unit benzene 1,3,5-
tricarboxylic acid.
COOR
ROOC
ROOC
O
O
O
ROOC
COOR
O
COOR
O
O
O
O
O
O
O
COOR
COOR
O
O
O
O
ROOC
O
COOR
Br
O
O
O
O
O
COOR
O
O
O
Br
COOR
COOR
O
O
O
Br
ROOC
O
O
O
O
O
O
COOR
COOR
O
O
O
ROOC
COOR
O
O
O
O
O
COOR
O
O
COOR
ROOC
COOR
O
O
ROOC
COOR
i
R = Me, Et, Pr

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ARTICLE TYPE
Synthesis of In-vitro anti-arthritic activity of dendrimers with methyl,
ethyl and isopropyl salicylates as surface units
a
a
Mani Jayanthi and perumal Rajakumar*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Fretchet type dendrimers with salicylates as surface groups have been synthesized by divergent approach and their In-vitro anti-
arthritic acitivities were carried out by inhibition of protein denaturation method.
Introduction
Results and discussion
1
1
2
2
0
5
0
5
For years, methyl salicylate (Wintergreen Oil) has been found
to be the active ingredient of most of the topical analgesics; it is
Chemistry
the major ingredient (about 30 %) in Bengay (pain relieving 55 In order to synthesize the required dendrimers with methyl, ethyl
cream, Johnson & Johnson). Methyl salicylate is approved by the
and isopropyl salicylate surface units, a multifunctional core unit
has to be synthesized. For which, 1,3,5ꢀTris(bromomethyl)
benzene 16 was chosen as core unit and was synthesized from
benzene 1,3,5ꢀ tricarboxylic acid 13 by esterification in ethanol in
U.S. Food and Drug Administration (FDA) and finds many
1ꢀ3
applications along with the analgesic property such as stimulant
or surrogant for sulfur mustard and also finds application in
3
3
immunohistochemistry , triboluminescence ; hence the synthesis 60 the presence of thionyl chloride to give the triester 14 followed
of dendrimers with methyl and other salicylate analogous were
by the reduction with LAH (Lithium Aluminium Hydride) in
4
ꢀ5
15
focused. Dendrimers is one of the most exciting classes of
macromolecules that have sparked significant interest in recent
years from synthetic, structural and functional points of view.
Recently, conjugated dendrimers and bioactive molecules show
increased therapeutic efficacy and the delivery of bioactive
molecules, is of great importance. Synthesis of dendrimers with
THF to give the corresponding triol 15 and finally brominating
the triol 15 with PBr (Scheme 1).
3
Zeroꢀgeneration dendrimers (G ) 1, 2 and 3 were synthesized
0
6
5
by reacting three equivalents of methyl, ethyl and isopropyl
salicylate with one equivalent of 1,3,5ꢀtris(bromomethyl)benzene
16 in the presence of potassium carbonate in dry acetone
(Scheme 1). In the H NMR spectrum of denerimer 1, showed a
sharp singlet at δ 3.88, 5.22 for the ester methyl and Oꢀmethylene
6
antiꢀbacterial activity has been reported from our group .
1
7
Synthesis and Inꢀvitro antiꢀarthritic as well as antiꢀinflammatory
activities of glycodendrimer bearing αꢀDꢀglycopyranosyl surface
8
70 protons respectively in addition to the signals for the aromatic
unit was reported from our laboratory .
13
protons. The C NMR spectrum of dendrimer 1 showed the ester
methyl, Oꢀmethylene carbon peaks at δ 52.0, 70.6 respectively.
The carbonyl carbon appeared at δ 166.8 in addition to the signals
for the aromatic carbons. The structure of zeroꢀgeneration
dendrimers 2 and 3 was also confirmed from the spectral and
analytical data.
3
3
4
4
5
0
5
0
5
0
In general salicylic acid and its derivatives find a versatile
place in pharmaceutical chemistry because of their bioactivity in
vitro system. Salicylate derivatives gains the tendency to build up
electrical charge when crushed or rubbed with sugar, which can
be observed by crushing wintergreen Life Savers candy in a dark
75
9
room . Further, metal binding properties of salicylate dendrimers
Initially 5ꢀhydroxy isophthalic acid was chosen as building
unit in order to synthesis dendrimers with higher generation by
synthesize dendrimers with methyl, ethyl and isopropyl salicylate 80 convergent approach. In this strategy, deprotection of hydroxyl
1
0
has been also reported in 2001 . Hence it is of great interest to
11ꢀ12
group of the dendron (which contains the two salicylates groups
at 1,3 positions and the hydroxyl group protected as acetyl/ trityl/
TBDMS) either by acidic or basic condition leads to
complications as the reaction condition also hydrolyse the ester
groups of salicylates. Hence, the Oꢀalkylation method was
adapted to synthesis dendrimers 1-12 which eliminates the
protection and deprotection protocol in the divergent approach.
units at the periphery by means of Oꢀalkylation methodology
.
Herein, we report the synthesis and inꢀvitro antiꢀarthritic activity
of Frechet type dendrimers 1-12 (Figure 1).
85
In order to study the antiꢀarthritic activity of salicylate
dendrimers 1-12 inhibition of protein denaturation method (using
BSA ꢀ Bovine Serum Albumin) was employed and diclofenac
1
3
sodium was used as standard . It was reported that the
denaturation of protein is one of the causes for rheumatic arthritic
and the production of autoꢀantigens in certain rheumatoid may be
In order to synthesize the firstꢀgeneration (G ) dendrimer the
1
90
hexaester 17 was synthesized by reacting three equivalents of
diethyl 5ꢀhydroxyisophthalate with one equivalent 1,3,5ꢀ
tris(bromomethyl)benzene 16 in dry acetone in the presence of
potassium carbonate. The alcohol 18 obtained by the LAH
reduction of the ethyl hexaester 17 was converted into the
1
4
due to in vivo denaturation of proteins and probably involves
alteration in electrostatic and hydrophobic interactions, hydrogen
1
5
and disulfide bonding .
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COOR
O
COOR
ROOC
20
O
O
O
COOR
COOR
COOR
O
O
COOR
O
O
O
5
O
COOR
COOR
O
ROOC
O
O
O
O
COOR
25
O
O
O
O
O
O
ROOC
COOR
O
O
O
O
ROOC
COOR
O
Go Dendrimer
O
10
ROOC
ROOC
O
1
2
3
R = Me
R = Et
R = Pr
G1 Dendrimer
i
COOR
O
30
4
5
6
R = Me
R = Et
R = Pr
O
COOR
O
O
ROOC
i
COOR
15
COOR
ROOC
ROOC
G2 Dendrimer
O
7
8
R = Me 3
O
O
O
ROOC
COOR
5
R = Et
COOR
i
O
9 R = Pr
O
O
O
O
O
O
O
O
COOR
COOR
O
O
O
ROOC
O
COOR
40
45
50
55
O
O
COOR
O
O
O
COOR
COOR
O
O
O
ROOC
O
O
O
O
O
O
COOR
COOR
O
O
O
O
ROOC
COOR
O
O
O
O
O
COOR
O
O
ROOC
O
COOR
COOR
O
O
ROOC
COOR
G3 Dendrimer
1
1
1
0 R = Me
1 R = Et
2 R = Pr
i
Figure 1 Frechet type salicylate dendrimers
O
OR
OH
Br
hexabromide 19 using PBr3 in CHCl3 (Scheme 2). A similar
reaction procedure was repeated with the hexabromide 19 to give
the dodeca bromide 22 and further the tetracosa bromide 25 in 75
% and 73 % yields respectively (Scheme 2).
(
ii)
(iii)
RO
(
O
HO
Br
80
O
OR
OH
Br
0
1
5
16
6
1
1
3 R=H
4 R=Et
i)
(iv)
The firstꢀgeneration (G ) dendrimers 4, 5 and 6 were obtained
1
in 71 %, 73% and 69 % respectively by the etherification of six
COOR
85 equivalents of methyl, ethyl and isopropyl salicylates with one
O
65
equivalent of the hexabromide 19 in dry acetone in the presence
1
of potassium carbonate (Scheme 3). In the H NMR spectrum,
COOR
O
dendrimer 4 showed a sharp singlet at δ 3.89, 5.13 and 5.17 for
O
ester methyl and for two distinct methylene protons respectively.
The C NMR spectrum of dendrimer 4 showed the ester methyl
carbon at δ 52.0 and the two distinct methylene carbons at δ 69.8,
13
90
ROOC
R=Me
70
1
2
3
7
0.2 respectively and the carbonyl carbon appeared at δ 166.7.
R=Et
i
R= Pr
Similarly the structure of dendrimers 5 and 6 were also confirmed
Scheme 1
Reagents and conditions: (i) EtOH, SOCl , Reflux, 12 h (ii)
LAH, THF, 50 C, 8 h (iii) PBr , CH Cl , 0 CꢀRT, 6 h (iv)
3 2 2
from the spectral and analytical data.
2
95
o
o
75
Methyl / ethyl / isopropyl salicylate, K CO , dry acetone, RT, 24h
2
3
2
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Br
Br
Br
Br
X
X
O
O
OEt
OEt
O
O
5
0
5
0
5
0
5
0
5
0
5
Br
O
Br
O
O
ii)
i), ii) & iii)
i)
Br
Br
20, 21
O
O
Br
O
O
X
O
Br
EtO
O
16
O
O
Br
O
X
O
OEt
X
O
1
1
2
2
3
3
4
4
5
5
EtO
O
X
O
O
OEt
1
8 X=OH
19 R=Br
iii)
17
Br
Br
2
2
Br
Br
Br
Br
Br
Br
Br
Br
O
O
i), ii) & iii)
Br
Br
O
O
23, 24
O
O
Br
Br
Br
O
O
Br
O
O
O
Br
O
O
O
O
Br
Br
O
O
Br
O
O
Br
O
O
Br
Br
Br
Br
Br
Br
Br
2
5
Scheme 2
Reagents and conditions: (i) Diethyl 5ꢀhydroxy isophthalate, K CO , dry acetone, RT, 24 h (ii) LAH, THF, 50 C, 8 h (iii)
PBr , CH Cl , 0 CꢀRT, 6 h.
o
2
3
o
3
2
2
COOR
O
O
Br
Br
COOR
O
O
i)
COOR
O
O
O
Br
O
O
O
Br
COOR
O
Br
ROOC
1
9
Br
O
ROOC
4
5
6
R= Me
R= Et
R=Pri
Scheme 3
s
1
The secondꢀgeneration dendrimers (G ) 7, 8 and 9 were
obtained in 68 %, 69 % and 65 % yields respectively by the 60 compound 7 showed sharp singlet at 3.89, 5.21 and 5.23 for ester
potassium carbonate (Scheme 4). In the H NMR spectrum,
2
etherification of twelve equivalents of methyl, ethyl and
isopropyl salicylate with one equivalents of the secondꢀ
generation dodecabromide 22 in dry acetone in the presence of
methyl and two distinct Oꢀmethylene protons respectively in
addition to the signals for the aromatic protons. The C NMR
spectrum of compound 7 showed ester methyl and two distinct
1
3
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1
Oꢀmethylene carbons at δ 52.0, 70.4 and 70.6 respectively. The
carbonyl carbon appeared at δ 165.7 in addition to the signals for
other aromatic carbons. The structure of secondꢀgeneration
carbonate (Scheme 5). In the H NMR spectrum, compound 10
showed signals at δ 3.85, and 5.09, 5.12 for ester methyl and two
distinct Oꢀmethylene protons respectively in addition to the
dendrimers (G ) 8 and 9 was also confirmed from spectral and
13
2
1
5
signals for the aromatic protons. The C NMR spectrum of
5
0
5
analytical data.
compound 10 shows ester methyl and two distinct Oꢀmethylene
carbons at δ 51.8, 70.5 and 70.7 respectively. The carbonyl
carbon appeared at δ 163.2 in addition to the signals for the
aromatic carbons. The structure of thirdꢀgeneration dendrimers
The thirdꢀgeneration dendrimers (G ) 10, 11 and 12 were
3
obtained in 67 %, 69 % and 64 % yields respectively by the
etherification of twenty four equivalents of methyl, ethyl and
isopropyl salicylate with one equivalent of the third generation
tetracosa bromide 25 in dry acetone in the presence of potassium
20
(G ) 11 and 12 was also confirmed from spectral and analytical
3
1
data.
COOR
ROOC
Br
Br
O
O
2
COOR
COOR
Br
Br
O
O
O
O
O
O
ROOC
Br
O
O
O
COOR
Br
O
i)
30
Br
O
O
COOR
O
O
O
O
Br
O
O
O
O
O
O
ROOC
O
COOR
O
35
40
45
50
Br
Br
O
ROOC
COOR
22
Br
Br
O
O
COOR
7
R= Me
8 R= Et
9
i
R=Pr
Scheme 4
Reagents and conditions: i) Methyl / ethyl / isopropyl salicylate, K CO , dry acetone, RT, 24 h.
2
3
COOR
ROOC
ROOC
O
Br
Br
Br
Br
O
O
ROOC
COOR
O
COOR
O
Br
Br
O
O
O
O
O
O
O
Br
Br
Br
O
O
O
COOR
O
COOR
O
O
O
O
ROOC
O
Br
O
COOR
Br
O
O
O
O
O
O
O
COOR
Br
O
O
O
i)
O
COOR
O
O
COOR
O
Br
O
O
O
ROOC
O
O
O
O
O
Br
O
O
O
Br
O
O
COOR
55
60
65
COOR
O
O
Br
O
O
ROOC
COOR
O
O
O
O
Br
O
O
O
O
Br
COOR
O
Br
O
Br
Br
ROOC
O
COOR
COOR
Br
Br
Br
2
3
O
O
ROOC
COOR
1
1
1
0 R= Me
1 R= Et
2 R=
i
Pr
Scheme 5
Reagents and conditions: i) Methyl / ethyl / isopropyl salicylate, K CO , dry acetone, RT, 24 h
2
3
4
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Biologicalactivity
Anti-Arthritic Activity
The degree of antiꢀarthritic activity of salicylate dendrimers 10,
12, 11, 6, 7 and 8 at 1000 ꢁg/mL are found to be 99.1 %, 97.9 %,
5
The antiꢀarthritic activities of all the synthesized dendrimers 20 97.5 %, 92.3 %, 89.1 % and 80.6 % respectively (Table 1 and
are concentration dependent and by adapting the standard
Figure 2).
13
protocol the salicylate dendrimers 7-12 were found to possess
the maximum antiꢀarthritic activity (85.4 %, 79.3 %, 70.1 %, 94.3
%, 94.0 % and 92.1 % at 400 ꢁg/mL) when compared to the
reference drug diclofenac sodium (58.4 % at 400ꢁg/mL) which
Conclusion
1
0
In conclusion, methyl, ethyl and isopropyl salicylate dendrimers
were synthesized by divergent approach in moderate to good
yield. Those dendrimers synthesized were subjected to Inꢀvitro
antiꢀarthritic activity by inhibition of protein denaturation
method. The second and third generation dendrimers 7-12 shows
superior antiꢀarthritic activity than the reference drug diclofenac
sodium at a concentration 100 ꢁg/mL and above. Especially the
thirdꢀgeneration (G3) dendrimer 10 shows better activity even at
low concentration.
25
1
00
80
30
60
Experimental section
4
0
3
4
4
5
0
5
Chemistry
20
General: All chemicals and solvents were purchased
commercially and used as such without further purification. All
melting points of those synthesized compounds are uncorrected
0
1
0
50
100
200
400
800
1000
Concentration in µg/mL
1
13
and the H NMR and C NMR spectra were recorded on Bruker
00ꢀMHz instrument in CDCl3 and DMSOꢀd6 solvent with
(1)
(2)
( 3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
DiclofenacSodium
3
Figure 2 Antiꢀarthritic activities of salicylate dendrimers 1-12
reveals that the salicylate dendrimers especially the dendrimer 10
tetramethylsilane (TMS) as an internal reference. Elemental
analyses were carried out on a PerkinꢀElmer CHNS 2400
instrument. Column chromatography was performed on silica gel
(
shows maximum activity even at a concentration of 10 ꢁm/gL)
(ACME, 100–200 mesh). Routine monitoring of the reaction was
1
5
5
0
shows better antiꢀarthritic activity than the reference drug
diclofenac sodium. These results also reveal that the salicylate
dendrimer are more stable in BSA than the diclofenac sodium.
made using thinꢀlayer chromatography (TLC) developed on 0.25
mm glass plates coated with silica gelꢀG (ACME) and visualized
with iodine.
Table 1 In vitro antiꢀarthritic activity of salicylate dendrimers 1–12 by inhibition of protein denaturation method (Bovine
Serum Albumin)
Dendrimer
Activity (% Inhibition of protein denaturation)
1
0 µg/ml
50 µg/ml
100 µg/ml
200 µg/ml
400 µg/ml
800 µg/ml
1000 µg/ml
1
2
3
4
5
6
7
8
9
3.66±0.86
2.98±0.99
2.72±1.58
7.07±1.32
3.97±0.97
3.33±0.86
12.72±0.82 16.33±0.80
11.09±1.23 15.83±0.92
10.75±0.95 13.32±0.81
70.92±0.91 80.64±0.54
27.93±0.94 52.12±0.42
27.71±1.01 40.13±0.47
3.97±1.02
2.97±0.84
2.84±1.92
9.81±0.45
6.18±0.63
6.03±1.38
7.07±0.56
3.14±1.06
2.92±0.72
15.79±0.94
13.09±0.62
12.84±0.55
48.78±0.56
42.16±0.67
41.34±0.78
91.09±0.19
73.98±0.45
71.65±0.95
13.61±0.42
11.51±1.04
11.13±0.94
25.90±0.91
21.11±0.84
19.97±0.91
57.74±1.03
54.80±0.59
48.05±0.62
92.38±0.68
88.14±0.84
87.50±0.78
22.21±1.08
19.87±2.00
18.84±0.53
46.01±0.74
37.13±0.93
35.23±0.94
85.44±0.76
79.26±0.62
70.08±0.93
94.29±1.08
93.98±0.82
92.11±0.87
31.93±0.98
25.90±1.93
26.68±0.82
55.24±0.97
48.81±1.31
42.61±2.01
86.62±0.93
83.21±0.81
72.34±0.75
96.64±1.04
94.28±1.13
95.08±0.91
45.52±0.64
31.03±0.54
30.86±1.54
64.10±1.05
56.62±0.79
49.62±0.91
92.31±0.54
89.08±0.29
80.64±0.34
99.11±0.97
97.52±1.54
97.91±0.53
1
1
1
0
1
2
Diclofenac
Sodium
9.50±1.21
16.32±0.64
22.20±0.95
39.37±0.64
58.42±0.87
64.49±1.08
81.42±0.98
5
5
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General procedure for esterification
1,3,5-Tris((3,5-bis((3,5-bis(bromomethyl)phenoxy)methyl)
phenoxy)methyl) benzene (22)
o
1
To a solution of the carboxylic acid (5 g) in ethanol (50 ml),
Offꢀwhite solid. mp: 148ꢀ152 C; Yield: 85 %; H NMR (300
MHz, CDCl ): δ 5.04 (s, 30H), 5.09 (s, 12H), 6.87ꢀ6.90 (m,
o
5
0
5
0
5
0
5
0
5
0
5
0
5
SOCl (about 1 ml) was added at 0 C. The reaction mixture was
2
3
H
then refluxed for 12 h. The solvent was removed under reduced 70 12H), 7.10ꢀ7.13 (m, 3H), 7.40ꢀ7.47 (m, 6H), 7.86 (s, 3H), 7.87ꢀ
13
pressure. The residue, thus obtained was dissolved in CHCl (200
7.88 (m, 6H); C NMR (75 MHz, CDCl ) δ 58.4, 58.5, 115.6,
3
3 C
ml), washed with water (2x 100 ml), brine (100 ml) and dried
over anhydrous Na SO . Removal of the solvent afforded the
121.3, 124.2, 139.6, 139.7, 156.9.
2
4
17aꢀe
1
1
2
2
3
3
4
4
5
5
6
6
esters
which was used as such without further purification.
1,3,5-Tris((3,5-bis((3,5-bis((3,5-bis(bromomethyl)phenoxy)
75
methyl)phenoxy)methyl) phenoxy)methyl)benzene (25)
o
1
General procedure for LAH reduction
Offꢀwhite solid. mp: 172ꢀ174 C; Yield: 83 %; H NMR (300
MHz, CDCl ): δ 5.02 (s, 48H), 5.03 (s, 42H), 6.79 (s, 24H),
3
H
To a stirred suspension of LAH (1.2 eq.,/ester group) in dry
6.87ꢀ6.90 (m, 12H), 7.02ꢀ7.13 (m, 12H), 7.40ꢀ7.46 (m, 12H),
13
THF, the ester compound in THF was added slowly under N2
7.80ꢀ7.86 (m, 6H); C NMR (75 MHz, CDCl ) δ 69.4, 69.7,
3 C
o
atmosphere at 0ꢀ5 C. The above reaction mixture was then 80 115.4, 115.5, 121.8, 126.1, 136.4, 137.4, 157.4
o
allowed to reach room temperature and heated at 50 C for 8 h. It
o
was then cautiously quenched with 10% NaOH solution at 0 C.
The reaction mixture was then filtered, and the residue obtained
was agitated with THF (3x50ml) and the combined THF layers
General Procedure for etherification
A solution containing the diethyl 5ꢀhydroxy isophthalate /
were evaporated. The residue obtained was dissolved in CHCl3 85 salicylates (2.1 mmol/4.2 mmol/8.4 mmol/16.8 mmol) and the
and extracted with CHCl (3x100ml), washed with brine (100
tribromide 16/ hexabromide 19/ dodecabromide 22/
tetracosabromide 25 (0.7 mmol) was stirred with K CO (5.1
3
ml), dried over Na SO and evaporated to give the hydroxyl
2
4
2
3
compound as crude product, which was purified using silica gel
mmol/10.1 mmol/15.1 mmol/ 20.1 mmol) in dry acetone at room
temperature for 24 h after which the reaction mixture was
filteredꢀoff and the solvent was evaporated to dryness to give the
multifunctional ester compound / dendrimer as residue which was
then purified by column chromatography using hexane: CHCl3
(3:2) as eluent.
100ꢀ200 using CHCl :hexane (3:5) as eluent.
3
90
1
,3,5-tris(Hydroxy methyl) benzene (15)
o
1
White solid. mp: 123ꢀ125 C; Yield: 73 %; H NMR (300 MHZ,
DMSOꢀd ): δH 4.47ꢀ4.49 (d, J=5.7 Hz, 6H), 5.18ꢀ5.22 (m, 3H),
6
13
7.13 (s, 3H); C NMR (75 MHZ, DMSOꢀd ): δ 62.9, 122.9,
6
C
1
42.0
95 Hexaethyl5,5',5''-(benzene-1,3,5-triyltris(methylene))tris(oxy)
triisophthalate (17)
o
1
(5,5',5''-(benzene-1,3,5-triyltris(methylene)) tris(oxy)tris(benzene-
White solid. mp: 122ꢀ124 C; Yield: 83 %; H NMR (300 MHz,
CDCl ): δ 1.39ꢀ1.44 (m, 18H), 4.37ꢀ4.44 (m, 12H), 5.21 (s, 6H),
5
,3,1-triyl))hexamethanol (18)
3
H
o
1
13
Offꢀwhite solid. mp: 138ꢀ142 C; Yield: 71 % ; H NMR (300
7.57 (s, 3H), 7.85 (d, J=0.9 Hz, 6H), 8.31 (s, 3H); C NMR (75
MHz, DMSOꢀd ): δ 3.38 (s, 12H), 4.39ꢀ4.49 (m, 6H), 6.37ꢀ6.38 100 MHz, CDCl ) δC 14.4, 61.5, 70.1, 119.9, 123.4, 126.4, 132.2,
6
H
3
1
3
(
m, 9H), 6.56ꢀ6.60 (m, 3H); C NMR (75 MHZ, DMSOꢀd ): δC
137.4, 158.6, 165.7
6
6
1.9, 68.9, 114.5, 122.2, 125.8, 137.3, 138.7, 153.7; Anal. calcd
for C H O : C, 68.74; H, 6.29; found: C, 68.72; H, 6.18.
Dodecaethyl 5,5',5'',5''',5'''',5'''''-(5,5',5''-(benzene-1,3,5-triyltris
(methylene)) tris(oxy)tris(benzene-5,3,1-triyl))hexakis(methylene)
105 hexakis(oxy)hexaisophthalate (20)
33
36
9
General procedure for bromination
o
1
Offꢀwhite solid. mp: 174ꢀ177 C; Yield: 84 % ; H NMR (300
MHz, CDCl ): δ 1.36ꢀ1.43 (m, 36H) , 4.34ꢀ4.43 (m, 24H), 5.09
To a solution of triol / hexa / dodeca / tetracosa hydroxy
3
H
compound (9.4 mmol) in CHCl (50 ml) was added an excess of
(s, 12H), 5.12 (s, 6H), 6.95 (s, 6H), 7.07 (s, 3H), 7.14 (s, 3H),
3
o
13
PBr at 0 C and stirred for 1 h. The reaction mixture was then
7.81 (s, 12H), 8.21ꢀ8.28 (m, 6H); C NMR (75 MHz, CDCl ): δ
3
3
C
allowed to reach room temperature and stirred for further 6 h. 110 16.5, 59.3, 69.5, 69.7, 115.0, 115.4, 115.5, 112.2, 126.1, 137.4,
After the completion of the reaction, it was extracted with CHCl3
3x100 ml), washed with aq., NaHCO (100 ml), brine (100 ml),
139.7, 158.9; Anal. calcd for C105H108O : C, 66.45; H, 5.74;
found: C, 66.30; H, 5.63.
33
(
3
dried over Na SO and evaporated. The residue obtained was
2
4
purified by column chromatography using CHCl /hexane (2:3) as
eluent.
Compound 23
3
o
1
115 Offꢀwhite solid. mp: 185ꢀ187 C; Yield: 81 % ; H NMR (300
MHz, CDCl ): δ 1.38ꢀ1.41 (m, 72H), 4.36ꢀ4.43 (m, 48H), 5.08
3
H
1
,3,5-tris(Bromomethyl)benzene (16)
(s, 24H), 5.12 (s, 18H), 6.99 (s, 6H), 7.09 (s, 12H), 7.12 (s, 6H),
7.75 (s, 6H), 7.81 (s, 24H), 8.28 (s, 12H); C NMR (75 MHz,
o
1
13
White solid. mp: 79ꢀ81 C; Yield: 88 %; H NMR (300 MHZ,
CDCl ): δH 4.46 (s, 6H), 7.36 (s, 3H); C NMR (75 MHz,
1
3
CDCl ): δC 14.3, 61.5, 69.9, 70.1, 114.4, 120.0, 120.9, 122.1,
3
3
CDCl ): δ 32.2, 129.6, 139.1; Anal. calcd for C H Br : C, 30.27; 120 123.2, 132.1, 138.3, 158.7, 165.9.
3
C
9
9
3
H, 2.54; found: C, 30.21; H, 2.48; m/z: 357 [M+1]
Dendrimer 1
o
1
1
,3,5-tris((3,5-bis(Bromomethyl)phenoxy)methyl)benzene (19)
Offꢀwhite solid. mp: 75ꢀ77 C; Yield: 74 % ; H NMR (300
MHZ, CDCl ): δ 3.88 (s, 9H), 5.22 (s, 6H), 6.98ꢀ7.02 (m, 6H),
o
1
White solid. mp: 135ꢀ138 C; Yield : 85 % ; H NMR (300 MHZ,
3
H
13
CDCl ): δ 4.43 (s, 12H), 5.11 (s, 6H), 6.96 (s, 6H), 7.03 (s, 3H), 125 7.39ꢀ7.47 (m, 3H), 7.59 (s, 3H), 7.81ꢀ7.85 (m, 3H); C NMR (75
3
H
13
7
.48 (s, 3H); C NMR (75 MHz, CDCl ) δ 69.5, 69.7, 115.4,
MHZ, CDCl ): δ 52.0, 70.6, 114.1, 120.8, 124.8, 125.9, 131.8,
3
C
3 C
1
22.2, 126.1, 137.4, 139.7, 158.9; Anal. calcd for C H Br O :
133.5, 137.7, 158.0, 166.8; Anal. calcd for C H O : C, 69.45;
33 30 9
3
3
30
6
3
C, 41.55; H, 3.17; found: C, 40.98; H, 3.10.
H, 5.30; found: C, 69.23; H, 5.22; m/z: 571 [M+1]
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

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DOI: 10.1039/C4RA07992B
Dendrimer 2
6.87ꢀ6.89 (m, 6H), 6.93ꢀ7.09 (m, 12H), 7.13ꢀ7.28 (m, 24H),
7.68ꢀ7.85 (m, 12H), 7.98ꢀ8.13 (m, 24H); Anal. calcd for
o
1
Offꢀwhite solid. mp: 81ꢀ83 C; Yield: 75 % ; H NMR (300
MHZ, CDCl ): δ 1.26ꢀ1.31 (m, 9H), 4.30ꢀ4.37 (m, 6H), 5.19 (s,
C201H204O : C, 72.29; H, 6.16; found: C, 72.12; H, 6.02.
3
H
45
6
H), 6.97ꢀ7.02 (m, 6H), 7.38ꢀ7.43 (m, 3H), 7.58 (s, 3H), 7.79ꢀ
13
5
0
5
0
5
0
5
0
5
0
5
0
5
7.82 (m, 3H); C NMR (75 MHZ, CDCl ): δ 14.3, 60.9, 70.6, 70 Dendrimer 10
3 C
1
1
14.0, 120.7, 121.3, 125.1, 131.7, 133.3, 137.6, 157.9, 166.4;
Brown Gum; Yield: 67 %; H NMR (300 MHz, CDCl ): δ 3.85
3 H
Anal. calcd for C H O : C, 70.57; H, 5.92; found: C, 70.13; H,
5
(s, 72H), 5.09 (s, 48H), 5.12 (s, 42H), 6.95 (s, 36H), 7.07 (s, 6H),
7.74 (s, 24H), 7.81 (s, 24H), 8.12ꢀ8.21 (m, 48H), 8.28 (s, 24H);
36
36
9
.67; m/z: 613 [M+1]
13
C NMR (75 MHz, CDCl ): δC 51.8, 70.5, 70.7, 116.4, 117.5,
3
1
1
2
2
3
3
4
4
5
5
6
6
Dendrimer 3
75 117.7, 118.9, 121.7, 126.4, 131.3, 132.9, 133.7, 133.9, 137.0,
148.5, 161.7, 163.2; Anal. calcd for C393H372O : C, 71.70; H,
5.70; found: C, 71.30; H, 5.56.
o
1
Offꢀwhite solid. mp: 67ꢀ69 C; Yield : 73 % ; H NMR (300
MHZ, CDCl ): δ 1.27ꢀ1.29 (d, J= 6.0 Hz, 18H), 5.19 (s, 6H),
5
3
68.2, 70.6, 113.9, 120.7, 121.9, 125.4, 131.4, 133.0, 137.6, 157.8,
1
7
93
3
H
.21ꢀ5.27 (m, 6H), 6.97ꢀ7.02 (m, 6H), 7.37ꢀ7.42 (m, 3H), 7.58 (s,
1
3
H), 7.76ꢀ7.79 (m, 3H); C NMR (75 MHZ, CDCl ): δC 21.9,
3
Dendrimer 11
1
8
8
9
9
0
5
0
5
Brown Gum; Yield: 69 %; H NMR (300 MHz, CDCl ): δ 1.21ꢀ
3
H
66.0; Anal. calcd for C H O : C, 71.54; H, 6.47; found: C,
3
9
42
9
1
6
(
.26 (m, 72H), 4.34ꢀ4.45 (m, 24H), 5.04 (s, 48H), 5.09 (s, 42H),
.88 (s, 48H), 6.96ꢀ6.99 (m, 36H), 7.10ꢀ7.13 (m, 24H), 7.39ꢀ7.48
m, 24H), 7.79 (s, 8H), 7.84ꢀ7.87 (m, 22H); Anal. calcd for
C H O93: C, 71.70; H, 5.70; found: C, 71.30; H, 5.56.
393 372
1.46; H, 6.42.
Dendrimer 4
o
1
Offꢀwhite solid. mp: 110ꢀ112 C; Yield : 71 % ; H NMR (300
MHz, CDCl ): δ 3.87 (s, 18H), 5.13 (12 H, s), 5.17 (s, 6H), 6.96
3
H
Dendrimer 12
Dark Brown Gum; Yield: 64 % ; H NMR (300 MHz, CDCl
(br, 6H), 6.99 (br, 6H) , 7.14 (s, 6H), 7.18 (s, 3H), 7.38ꢀ7.43 (m,
1
1
3
3
): δ
.34ꢀ1.36 (m, 144H), 5.21 (s, 48 H), 5.22 (s, 42H), 5.66ꢀ5.78 (m,
4H), 6.64ꢀ6.66 (m, 23H), 7.01 (s, 34H), 7.09ꢀ7.12 (m, 32H),
H
6
H), 7.48 (s, 3H), 7.80ꢀ7.82 (m, 6H); C NMR (75 MHz,
1
2
CDCl ): δ 51.4, 69.5, 69.7, 111.8, 113.9, 115.4, 122.2, 126.1,
137.4, 139.7, 147.8, 158.9, 167.4; Anal. calcd for C H O : C,
7
3
C
8
1
72 21
7.26ꢀ7.38 (m, 22H), 7.54 (s, 31H), 7.85ꢀ7.88 (m, 12H), 7.94ꢀ7.97
m, 8H) ; C NMR (75 MHz, CDCl ): δ 21.8, 67.6, 68.9, 116.2,
16.7, 117.5, 117.6, 118.9, 120.7, 126.1, 131.3, 133.0, 133.7,
133.8, 137.0, 150.4, 161.7, 163.2; Anal. calcd for C417
2.38; H, 6.12; found: C, 72.30; H, 6.03.
0.42; H, 5.25; found: C, 70.36; H, 5.18; m/z: 1382 [M+1]
13
(
3 C
1
Dendrimer 5
Offꢀwhite solid; Yield
CDCl ): δH 1.24ꢀ1.33 (m, 18H), 4.32ꢀ4.36 (m, 12H), 5.10 (s,
1
1
H O93: C,
420
: 73 % ; H NMR (300 MHz,
7
3
2H), 5.14 (s, 6H), 6.95 (s, 6H), 6.98 (s, 6H), 7.11 (s, 3H), 7.16
Anti-Arthritic Studies
(
s, 6H), 7.39 (s, 6H), 7.48 (s, 3H), 7.78ꢀ7.81 (d, J= 7.5 Hz, 6H) ;
1
3
C NMR (75 MHz, CDCl ): δC 14.3, 60.9, 70.2, 70.4, 112.8,
3
Test solutions consist of 0.45 ml of bovine serum albumin
5% w/v aqueous) and 0.05 ml of dendrimers 1-12 in various
00 concentrations were prepared and the pH was adjusted to 6.3 by
adding a small amount of 1 N HCl. In a similar manner test
control and standard solutions were prepared by adding 0.05 ml
of distilled water and 0.05 ml diclofenac sodium respectively to
.45 ml of bovine serum albumin. Product control was prepared
05 by adding 0.05 ml of test solutions in various concentrations to
0.45 ml of distilled water. The samples prepared were incubated
at 37 C for 20 minutes and then heated to 57 C for 3 minutes.
After cooling, 2.5 ml of phosphate buffer (pH 6.3) was added to
each solution. The optical densities (OD) of the samples were
10 measured from a spectrophotometer at 660 nm. Each experiment
was done in triplicate and taken the average.
1
13.8, 113.9, 120.7, 120.8, 121.3, 131.6, 131.7, 133.3, 138.3,
(
157.7, 166.4; Anal. calcd for C H O : C, 71.30; H, 5.78; found:
C, 70.89; H, 5.42.
8
7
84 21
1
1
1
Dendrimer 6
Offꢀwhite solid; Yield
CDCl ): δ 1.24ꢀ1.33 (m, 36H), 4.32ꢀ4.44 (m, 6H), 5.09 (s, 12H),
5
7
NMR (75 MHz, CDCl ): δ 21.9, 68.2, 69.8, 70.6, 113.3, 120.1,
1
C H O : C, 72.08; H, 6.24; found: C, 71.84; H, 6.12.
1
: 69 % ; H NMR (300 MHz,
0
3
H
.14 (s, 6H), 5.21ꢀ5.27 (m, 6H), 6.95ꢀ6.98 (m, 6H), 7.11 (s, 6H),
13
.16 (s, 3H), 7.39 (s, 6H),7.48 (s, 3H), 7.79ꢀ7.81 (m, 6H);
C
o
o
3
C
22.2, 125.4, 131.4, 133.8, 136.9, 158.8, 166.6; Anal. calcd for
9
3
96 21
Dendrimer 7
Pale Brown Gum; Yield: 68 % ; H NMR (300 MHz, CDCl ): δ ;
3
1
3
H
The percentage inhibition of Protein denaturation was calculated
as follows.
.89 (s, 36H), 5.21 (s, 24), 5.23 (s, 18H), 6.96 (s, 6H), 6.99ꢀ7.01
(m, 24H), 7.36ꢀ7.39 (m, 12H), 7.42ꢀ7.43 (m, 12H), 7.57ꢀ7.76 (m,
1
3
115
1
7
1
5
2H), 7.78ꢀ7.79 (m, 12H); C NMR (75 MHz, CDCl ): δ 52.0,
3 C
1
00 – [OD of test solution – OD of product
0.4, 70.6, 109.4, 114.1, 120.8, 124.8, 125.9, 131.8, 133.5, 137.7,
Percent
control]
42.1, 158.0, 165.7; Anal. calcd for C177H156O : C, 70.79; H,
45
Inhibition = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ X 100
OD of test control
.24; found: C, 70.64; H, 5.13.
1
20
Dendrimer 8
Pale Brown Gum; Yield: 69 %; H NMR (300 MHz, CDCl ): δ
1
1
7.88 (m, 12H), 8.12ꢀ8.17 (m, 24H); Anal. calcd for C189H180O45:
C, 71.58; H, 5.72; found: C, 71.34; H, 5.48.
1
The control represents 100 % protein denaturation and the
result was compared with diclofenac sodium treated samples.
3
H
.21ꢀ1.32 (m, 36H), 4.31ꢀ4.39 (m, 24H), 6.98 (s, 24H), 70.2 (s,
8H), 7.04ꢀ7.11 (m, 12H), 7.14 (s, 6H), 7.20ꢀ7.31 (m, 24H), 7.78ꢀ
Acknowledgements
125
The authors thank DSTꢀPURSE, New Delhi for financial
assistance, DSTꢀFIST for providing NMR facility to the
department and Faculty of Pharmacy, Sri Ramachandra
University, Chennaiꢀ600 116 for inꢀvitro antiꢀarthritic studies.
Dendrimer 9
1
Brown Gum; Yield: 64 % ; H NMR (300 MHz, CDCl ): δ 1.23ꢀ
1.29 (m, 72H), 5.12 (s, 24H), 5.16 (s, 18H), 5.48ꢀ5.52 (m, 12H),
3
H
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

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RSC Advances
View Article Online
DOI: 10.1039/C4RA07992B
1
3. Mizushima, Y. Lancet 1966, 2, 443.
14. Brown, J.H.; Mackey, H.K. Proc. Soc. Exp. Biol. Med.
968, 128, 225.
References and notes
a
Address: Department of Organic Chemistry, University of Madras,
1
Chennai-600025.
perumalrajakumar@gmail.com
† Electronic Supplementary Information (ESI) available: The H and
spectra of all the synthesized compounds and dendrimers. See
DOI: 10.1039/b000000x/
Telephone:
044-22202810:
E-mail:
1
1
1
5. Grant, N.H.; Alburn, H.E.; Kryzanauskas, C. Biochem.
Pharmacol. 1970, 19, 715.
1
13
40
5
0
5
0
5
0
C
6. Freeman, A.W.; Chrisstoffels, L.A.J.; Frechet, J.M.J, J. Org.
Chem. 2000, 65, 7612–7617.
1
7. (a) Methyl Salicylate: Yield: 93 %; H NMR (300 MHz,
1
.
James, D.G.; Price, T.S., J. Chem. Ecol. 2004, 30 (8), 1613ꢀ
CDCl
1H), 6.97ꢀ7.0 (dd, J
=5.2 Hz, J =1.2 Hz, 1H), 7.82ꢀ7.86 (dd, J
Hz, 1H), 10.77 (s, 1H); C NMR (75 MHz, CDCl
12.4, 117.6, 119.2, 129.9, 135.7, 161.6, 170.6 ;
3
): δ
H
3.95 (s, 3H), 6.86ꢀ6.91 (dt, J
=7.2 Hz, J =1.2 Hz, 1H), 7.43ꢀ7.49 (dt,
=4.8 Hz, J =1.8
): δ 52.3,
1 2
=5.2 Hz, J =1.2 Hz,
2
8.
4
5
5
6
6
5
0
5
0
5
1
2
1
1
2
2
3
2.
Harvey, E.N., Science. 1939, 90 (2324), 35ꢀ36.
BartletꢀHunt, S.L.; Knappe, R.U. Detlef et al., Critical
Reviews in Environmental Science and Technology. 2008, 38
J
1
2
1
2
3
.
13
3
C
1
(2), 112ꢀ136.
1
(
b) Ethyl Salicylate: Yield: 95 % ; H NMR (300 MHz, CDCl
3
):
4
.
Hawker, C. J.; Frechet, J. M. J. New Methods of Polymer
Synthesis, Vol. 2; Ebdon, J.R.; Eastmond G.C., (Eds). New
methods of polymer synthesis. Chap. 2. Blackie Academic
and Professional: Glasgow, UK (1995).
δ
H
1.30ꢀ1.34 (t, J= 7.2 Hz, 3H), 4.27ꢀ4.35 (q, J1= 7.2 Hz,
J2=14.4 Hz, 2H), 6.75ꢀ6.81 (m, 1H), 6.87ꢀ6.90 (dd, J1=0.9
Hz, J2=8.4 Hz, 1H), 7.32ꢀ7.38 (m, 1H), 7.74ꢀ7.77 (dd, J1=3.6
13
Hz, J2=8.1 Hz, 1H), 10.78 (s, 1H); C NMR (75 MHz,
5
.
Frechet, J.M.J.; Tomalia, D.A., Dendrimers and Other
Dendritic Polymers. John Wiley & Sons, Ltd.: Chichester
(2001).
CDCl
3
): δ
C
13.4, 61.7, 111.6, 115.4, 117.9, 128.8, 134.2,
160.9, 170.4;
1
(
c) Isopropyl Salicylate: Yield: 93 %; H NMR (300 MHz,
6
7
.
.
Rajakumar, P.; Ganesan, K.; Jayavelu, S.; Murugesan, K.,
Synthesis 2006, 3, 528ꢀ532.
Rajakumar, P.; Anandhan, R.; Kannan, A., Supramolecular
Chemistry 2013, 25 (6), 362–370.
CDCl
3
): δ
H
1.34ꢀ1.36 (d, J=6.0 Hz, 6H), 5.19ꢀ5.26 (m, 1H),
6
1
.61ꢀ6.66 (m, 2H), 7.22ꢀ7.28 (m, 1H), 7.85ꢀ7.88 (m, 1H),
0.50 (s, 1H); C NMR (75 MHz, CDCl ) δ 22.0, 67.6,
3 C
13
111.6, 116.2, 116.6, 131.3, 133.49, 150.4, 167.7;1
8.
Rajakumar, P.; Anandhan, R., European Journal of
Medicinal Chemistry 2011, 46, 4687ꢀ4695.
(
d) Diethyl 5-hydroxy isophthalate: Yield: 92 %; H NMR (300
3 H
MHz, CDCl ): δ 1.39ꢀ1.44 (t, J=7.2 Hz, 6H), 4.37ꢀ4.45 (q,
9
.
Altman, J.S.; Tyrer, N.M.; Strausfeld, N.J.; Miller, T.A,
Neuroanatomical Techniques, SpringerꢀVerlag (1980) 373ꢀ
J1=7.2 Hz, J2=14.4 Hz, 4H), 7.15 (s, 1H), 7.81 (d, J=1.5 Hz,
13
2
H), 8.24 (1H, s); C NMR (75 MHz, CDCl
3
): δ
C
+14.2, 61.7,
4
02.
121.0, 122.6, 132.0, 156.5, 166.2; MS: m/z 239 (M );
10. Cohen, S.M.; Petoud, S.; Raymond, K.N, Chem. Eur.
J. 2001, 7, 272ꢀ279.
1
(
e) Triethyl benzene-1,3,5-tricarboxylate(14): Yield: 93 %; H
NMR (300 MHz, CDCl ): δ 1.42ꢀ1.47 (t, J=7.2 Hz, 9H),
.42ꢀ4.49 (q, J1=7.2 Hz, J2=14.4 Hz, 6H), 8.85 (s, 3H);
3
H
1
1. Hawker, C.J.; Frechet, J.M.J, J. Chem. Soc., Chem. Commun.
990, 1010ꢀ1013
13
4
C
1
3 C
NMR (75 MHz, CDCl ) δ 14.3, 61.7, 131.4, 134.4, 165.1
1
2. Hawker, C.J.; Frechet, J.M.J, J. Am. Chem. Soc. 1990, 112,
3
5
0
7638ꢀ7647.
7
75
80
85
90
95
1
00
8
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