Synthesis and antioxidant properties of enone core based dendrimers with carbazole as surface group

Article abstract of DOI:10.1016/j.ejmech.2009.11.051

Synthesis of enone core based dendrimers with carbazole as surface group has been achieved. All the synthesized dendrimers showed excellent antioxidant behavior with commercially available 1,1-diphenyl-2-picryl hydrazyl (DPPH).

Full text of DOI:10.1016/j.ejmech.2009.11.051

European Journal of Medicinal Chemistry 45 (2010) 1220–1224
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antioxidant properties of enone core based dendrimers with
carbazole as surface group
,
a
a
b
Perumal Rajakumar a , Nagarathinam Venkatesan , Karuppannan Sekar , Subramani Nagaraj ,
*
Ramasamy Rengasamy b
a Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, India
b Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of enone core based dendrimers with carbazole as surface group has been achieved. All the
synthesized dendrimers showed excellent antioxidant behavior with commercially available
1,1-diphenyl-2-picryl hydrazyl (DPPH).
Received 5 September 2009
Received in revised form
9 November 2009
Accepted 27 November 2009
Available online 11 December 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Dendrimers
Enone
Carbazole
Antioxidant activity
DPPH
1. Introduction
application in medicinal field [12,13]. Synthesis of permanent
fluorescence sensing hyper branched dendritic architecture [14] as
Dendrimers are monodisperse macromolecules possessing
well-defined branched structure that can be precisely tailored into
discrete and designated functionality to create multifunctional
materials. Dendrimers are also often referred to as artificial proteins
[1]. The size, shape, branching length and density and their surface
functionality of dendrimers make them as ideal carriers in
biomedical applications such as drug delivery [2], gene transfection
[3], and imaging [4] and as catalyst in homogeneous as well as
heterogeneous medium [5]. Some of the surface modified den-
drimers themselves may act as nano-drugs [6,7] against cancer,
tumors, bacteria and viruses. Recent successes in simplifying and
optimizing the synthesis of dendrimers such as ‘lego’ [8] and ‘click’
[9] techniques, provide a large variety of structural variation while
at the same time reducing the cost of their production. The surface
of dendrimers provides as excellent platform for drug delivery.
Very recently, PAMAM dendrimers have been successfully used as
carriers of boron isotopes in boron neutron-capture treatment of
cancer tumors [10] and in nonsteroidal anti-inflammatory drugs
[11]. Hybrid dendrimers such as glycodendrimers exhibits potential
well as bactericidal efficacy of novel dendrimers [15] and axially
chiral enantiopure dendrimers [16] has been reported from our
laboratory recently. Very recently the bio and pharmaceutical
application of the dendrimers are well-defined in the literature
[17–19]. The enone nucleus is present in various natural products
[20] and known to be a very important organic residue in many
potentially bioactive compounds [21]. However the synthesis and
biological activity of hexanone core based bioactive carbazole [22]
hyper branched dendritic architecture are yet to be investigated. To
the best of our knowledge the antioxidant study of the carbazole
based dendrimer is still a rare observation. Therefore, we report
herein for the first time the synthesis of hexanone core based
dendritic architecture 1a, 1b, 1c and 1d with carbazole as surface
group and their antioxidant activity tested with commercially
available DPPH.
2. Chemistry
Synthesis of bioactive enone based dendrimers (1a–d) up to
third generation was achieved by simple O-alkylation method using
convergent synthetic route which include LAH reduction, benzylic
chlorination and NBS bromination starting from
carbazole moiety scheme (1–4). 2.1 Equiv. of dendritic bromide 3,
a bioactive
* Corresponding author. Tel.: þ91 4422351269; fax: þ91 4422352494.
E-mail address: perumalrajakumar@hotmail.com (P. Rajakumar).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.051

P. Rajakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 1220–1224
1221
Fig. 2. DPPH scavenging capacities (IC50) of dendrimer 1c and 1d with standard drugs.
Fig. 1. DPPH scavenging activity spectrophotometric assay of various concentrations of
the dendrimers 1a, 1b, 1c and 1d.
methylene protons in addition to the olefinic and aromatic protons
and in 13C NMR spectrum the two type of hexanone methylene, O-
chlorides 7, 10, and 13 react with 1 equiv. of 2,6-bis(4-hydroxy
benzylidene)cyclohexanone 4 in the presence of K2CO3 to give the
dendrimers 1a–d in 65–80% yield and the rest of the yield con-
tained mono alkylated product. All the new compounds gave
satisfactory IR, 1H and 13C NMR, mass spectral and elemental
analysis.
methylene carbons and the carbonyl carbon appeared at
d 23.1,
d
28.6, 69.6, and 190.2 respectively in addition to the olefinic and
d
d
aromatic carbons. The structure of the dendrimer 1a was further
confirmed from mass spectrum and elemental analysis.
Similarly first generation dendrimer 1b was obtained from the
first generation dendritic chloride 7 [G1]-Cl. Reaction of methyl-
3,5-dihydroxy benzoate with 2.1 equiv. of dendritic bromide 3
afforded methyl-carboxylate 5 in 72% yield. Reduction of 5 with
lithium aluminum hydride in dry THF afforded the dendritic
alcohol 6 [G1]-OH, which on further treatment with SOCl2 in dry
DCM at 0 ꢀC, gave the dendritic chloride 7 [G1]-Cl in 83% yield.
Reaction of 1.0 equiv. of hexanone 4 with 2.1 equiv. of the dendritic
chloride 7 [G1]-Cl in dry DMF afforded the first generation den-
drimer 1b in 76% yield (Scheme 2).
3. Biology
Antioxidant activity of all the dendrimers was tested by DPPH
free radical scavenging activity by spectrometric assay method [23].
Samples at different concentration were prepared and tested for
DPPH scavenging activity. The percentage of inhibition was calcu-
lated and the antioxidant activity of the dendrimers and standard
antioxidants like Ascorbic acid,
a-Tocopherol and Butylated
In the 1H NMR spectrum, dendrimer 1b displayed two broad
Hydroxyanisole (BHA) were recorded and compared. The antioxi-
dant activity and IC50 value of the dendrimers with standard drugs
are shown in the Figs. 1 and 2 respectively.
singlet each integrating for two and four proton at
for two types of methylene protons of hexanone unit and two sharp
singlet at 5.09 and 5.17 each integrating for four and eight
d 1.72 and d 2.84
d
d
protons respectively for O-methylene protons in addition to the
olefinic and the aromatic protons, and in 13C NMR spectrum the
two type of methylene carbon of hexanone and two type of O-
4. Results and discussion
Synthesis of dendrimers 1a–d was achieved by simple O-alkyl-
ation method using convergent synthetic route starting from
a bioactive carbazole moiety. Alkylation of 2,6-bis(4-hydroxy ben-
zylidene)cyclohexanone 4 with carbazole dendritic bromide 3 in
the presence of K2CO3 and 18-crown-6 in DMF afforded the zeroth
generation dendrimer 1a in 80% yield. Dendritic bromide 3 was
obtained in 84% yield by the radical bromination of 9-p-tolyl
carbazole 2 which in turn was obtained by the known procedure
[24] (Scheme 1).
methylene carbon and the carbonyl carbon appeared at
d 22.9,
d
28.5, 69.8, 70.0 and at 190.1 respectively in addition to the
d
d
d
olefinic and the aromatic carbons. In the FAB – mass spectrum, the
molecular ion peak appeared at m/z 1572 (Mþ) and the structure of
the dendrimer 1b was further confirmed from satisfactory
elemental analysis. Application of similar sequence afforded the
second generation dendrimer 1c in 65% yield from the second
generation dendritic chloride 10 [G2]-Cl (Scheme 3).
In the 1H NMR spectrum of dendrimer 1c displayed two broad
In the 1H NMR spectrum, 1a displayed a quintet at
a triplet at 2.95 for two type of methylene protons of hexanone
d 1.80 and
singlet at
d 1.68 and d 2.79 integrating for two and four protons for
d
methylene protons of hexanone moiety and three sharp singlet at
moiety and a sharp singlet appeared at
d
5.21 integrating for four
O
R
O
O
O
ii
N
N
+
N
1a
HO
OH
2 ( R = CH3 )
3 ( R = CH2Br )
4
i
Scheme 1. Reagents and Conditions: (i) NBS, BZ2O2, CCl4, reflux, 6 h, 3 (84%); (ii) K2CO3, 18-crown-6, DMF, 60 ꢀC, 48 h, 1a (80%).

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P. Rajakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 1220–1224
R
O
N
N
N
O
O
CO2Me
OH
O
O
O
O
O
O
3
4
i
i
N
N
HO
N
1b
R = CO2Me 5 [G1]-CO2Me
R = CH2OH 6 [G2]-OH
R = CH2Cl 7 [G2]-Cl
ii
iii
Scheme 2. Reagents and Conditions: (i) K2CO3, 18-crown-6, DMF, 60 ꢀC, 48 h, 5 (72%), 1b (76%); (ii) LAH, THF, reflux, 6 h, 6 (70%); (iii) SOCl2, Py, DCM, 0 ꢀC to rt, 3 h, 7 (83%).
d
5.01,
d
5.05,
d
5.16 for O-methylene protons each integrating four,
has been used for the determination of antioxidant property. DPPH
has violet colour and has absorption band at 517 nm in ethanol.
When DPPH is mixed with the dendrimers which can donate free
electron, DPPH looses its colour and undergoes reduction to give
diphenyl picryl hydrazine [25]. Dendrimers 1a–d were dissolved in
CHCl3 with concentration of 20, 40, 60, 80, 100, 250, 500 and
eight and sixteen protons in addition to the olefinic and aromatic
protons. In the 13C NMR spectrum, methylene carbon in hexanone
moiety appeared at
and carbonyl carbon appeared at
d
22.7,
d
28.5, the three different O-methylene
69.8, 69.9, 70.1 and 189.9
d
d
d
d
respectively in addition to the twenty one aromatic and olefinic
carbon. In the mass spectrum, the molecular ion peak appeared at
m/z ¼ 3105 (M þ Naþ) and the structure of dendrimer 1c was
further confirmed from satisfactory elemental analysis. Third
generation dendrimer 1d was obtained in 70% yield by similar
methodology from the dendritic chloride 13 [G3]-Cl (Scheme 4).
In the 1H NMR spectrum, dendrimer 1d showed two broad
singlet at
hexanone moiety and four singlet at
for four different methylene proton integrating for four, eight,
sixteen and thirty two protons in addition to the olefinic and
aromatic protons. In the 13C NMR spectrum, a set of methylene
1000 mg/ml and used for the study. The test solution of the den-
drimers 1a–d (0.1 ml) in CHCl3 was added to DPPH solution (0.5 ml)
in ethanol and absorption was measured at 517 nm. Percentage
activity was calculated from the following equation. Percentage
Activity ¼ [As ꢁ Ab/Ac ꢁ Ab] ꢂ 100, where As ¼ absorbance of DPPH
test solution. Ab ¼ absorption of DPPH solution with blank without
DPPH and dendrimers and Ac ¼ control with DPPH solution
without adding the dendrimer. Fig. 1 shows the percentage of DPPH
reduction with change in the concentration of the dendrimers 1a–
d. The second generation dendrimer 1c showed the excellent
d
1.67 and
d
2.78 for two type of methylene protons of
4.94, 4.96, 5.00 and 5.10
d
d
d
d
activity at all the concentration ranging from 20 to 1000 mg/ml and
carbon present in hexanone moiety appeared at
methylene and carbonyl carbons appeared at
and 189.1 in addition to the olefinic and aromatic carbons. The
structure of the dendrimer 1d was further confirmed by
the appearance of a molecular ion peak at m/z ¼ 6124 (M þ Naþ) in
the MALDI-TOF mass spectrum as well as from elemental analysis.
d
21.9,
d
28.7, the O-
hence dendrimer 1c has the efficient antioxidant property.
However the antioxidant property of first and third generation
dendrimer 1b and 1d alters depending on the concentration of the
dendrimers in the test solution. In fact zeroth generation den-
drimer 1a has the lowest antioxidant property. The antioxidant
property gradually increases with increasing the generation of
dendrimers and hence 1c shows better antioxidant property than
1b and 1a, which could be due to more number of carbazole groups
on the surface generally termed as multivalent effect [13]. Based on
similar trend 1d should show much greater antioxidant property.
However, high carbazole loading will cause serious steric hindrance
due to crowding effect which in turn decrease the availability of
d
68.6,
d
68.8, 68.9
d
d
4.1. Antioxidant studies of the dendrimers 1a–d
Free radical scavenging assay methodology has been employed
to study the antioxidant property of the dendrimers 1a–d. DPPH
(1,1-diphenyl-2-picryl hydrazyl) known as stable free radical source
N
N
R
O
O
O
O
O
N
N
N
N
O
O
CO2Me
OH
O
O
O
O
O
O
O
O
O
O
7
4
O
O
N
i
i
HO
N
N
O
O
N
1c
R = CO2Me 8 [G2]-CO2Me
R = CH2OH 9 [G2]-OH
R = CH2Cl 10 [G2]-Cl
ii
N
N
iii
Scheme 3. Reagents and Conditions: (i) K2CO3, 18-crown-6, DMF, 60 ꢀC, 48 h, 8 (74%), 1c (65%); (ii) LAH, THF, reflux, 6 h, 9 (67%); (iii) SOCl2, Py, DCM, 0 ꢀC to rt, 3 h, 10 (80%).

P. Rajakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 1220–1224
1223
N
N
O
N
N
R
O
O
O
O
N
N
O
O
O
O
N
O
N
O
O
O
O
O
O
O
O
CO2Me
OH
O
O
O
O
O
O
O
N
N
O
O
N
N
O
O
10
4
O
N
O
N
O
i
i
O
O
HO
O
O
N
N
O
O
O
O
N
N
O
O
O
N
N
R = CO2Me 11 [G3]-CO2Me
R = CH2OH 12 [G3]-OH
R = CH2Cl 13 [G3]-Cl
O
ii
O
1d
iii
N
N
N
N
Scheme 4. Reagents and Conditions: (i) K2CO3, 18-crown-6, DMF, 60 ꢀC, 48 h, 11 (72%), 1d (70%); (ii) LAH, THF, reflux, 6 h, 12 (68%); (iii) SOCl2, Py, DCM, 0 ꢀC to rt, 3 h, 13 (78%).
carbazole to exhibit antioxidant property. IC50 values were deter-
mined in order to confirm the reducing activity of the dendrimers
synthesized. All the dendrimers exhibited variation of free radical
scavenging activity with respect to the variation of concentration.
Scavenging activity of 50% was observed at 48 mg/ml and 66 mg/ml
for dendrimers 1c and 1d respectively (Fig. 2). The activity is
by open capillary tube method and are uncorrected. Spectroscopic
data were recorded by the following instruments IR: FTIR-8300
spectrometer; NMR Bruker 300 MHz; MS: FAB-MS spectra Jeol SX
102/DA-600 mass spectrometer and MALDI-TOF mass recorded
using Voyager DE-PRO Biospectrometry workstation (applied Bio-
systems) MALDI-TOF-MS instruments. The elemental analysis of
the dendrimers was carried out using the Perkin–Elmer 240B
elemental analyzer.
significant when compared with the standard antioxidant drugs
[26] like Ascorbic acid,
a-Tocopherol and BHA (with IC50 as 48 mg/
ml, 25 g/ml and 29 g/ml respectively). The antioxidant property
m
m
6.2. General procedure for the synthesis of dendrimers (1a–d)
of the dendrimers derived from syringaldehyde [27] is due to the
presence of phenolic unit where as the antioxidant effect of the
dendrimers reported herein is due to the presence of the bioactive
carbazole functionality. Though the antioxidant property of syrin-
galdehyde dendrimer reported is comparable to that of carbazole
dendrimer, the acidic and corrosive nature of phenolic group could
prevent their use in biological system and hence the dendrimer
reported in the current investigation will have more applicability.
In general some of the dendrimers have good water-solubility,
bioavailability and biocompatibility, and hence they can be
administrated by intravenous, oral, transdermal, and ocular
delivery systems [28]. Hence, in conclusion the carbazole based
dendrimers reported herein may have good biocompatibility,
cytotoxicity and bioavailability.
A mixture of the corresponding dendritic bromide (or) chlo-
ride (2.1 equiv.), 2,6-bis(4-hydroxy benzylidene)cyclohexanone
(1.0 equiv.), 18-crown-6 (0.1 equiv.) and potassium carbonate
(5.0 equiv.) in dry DMF (25 ml) was heated at 60 ꢀC with stirring
for 48 h under nitrogen. The reaction mixture was then allowed
to cool to room temperature and poured into ice water. The
resulting precipitate was filtered, washed thoroughly with water
and dissolved in CHCl3 (150 ml). The filtrate was evaporated
under vacuum and the residue was extracted with CHCl3
(2 ꢂ 50 ml), washed with brine (1 ꢂ50 ml) and then dried over
anhydrous sodium sulphate. Removal of the solvent under
reduced pressure gave the dendrimer as a crude material, which
was purified by column chromatography (SiO2).
5. Conclusion
6.2.1. Dendrimer 1a
Yield 80%; mp 222 ꢀC; IR (KBr, cmꢁ1):1644 (C]O); 1H NMR
In conclusion, enone core based dendrimers with carbazole as
surface group up to third generation were synthesized and all of
which showed better antioxidant activity. Among the dendrimers,
dendrimer 1c exhibited better antioxidant property and IC50 values
are significant for the second and third generation dendrimers 1c
and 1d when compared with the standard antioxidant drugs. The
second generation dendrimer 1c may be developed as antioxidant
drug as it showed better activity compared with commercial
available drugs. The cytotoxicity, biocompatibility and bioavail-
ability and in-vivo studies of the dendrimers synthesized are under
investigation and will be communicated latter as a full paper.
(300 MHz, CDCl3)
d
: 1.80 (quin, 2H, J ¼ 5.4 Hz), 2.95 (t, 4H, J ¼ 4.8 Hz),
5.21 (s, 4H), 7.08 (d, 4H, J ¼ 9.0 Hz), 7.27–7.34 (m, 4H), 7.41–7.44 (m,
8H), 7.50 (d, 4H, J ¼ 8.7 Hz), 7.60 (d, 4H, J ¼ 6.6 Hz), 7.67 (d, 4H,
J ¼ 8.4 Hz), 7.80 (s, 2H), 8.15 (d, 4H, J ¼ 7.8 Hz); 13C NMR (75 MHz,
CDCl3) d: 23.1, 28.6, 69.6, 109.7, 114.8, 120.1, 120.4, 123.5, 126.0, 127.3,
129.0, 129.3, 132.3, 134.6, 135.8, 136.5, 137.6, 140.8, 159.0, 190.2; m/z
(FAB-MS): 817 (Mþ). Elemental Anal. Calcd for C58H44N2O3: C, 85.27;
H, 5.43; N, 3.43. Found: C, 85.19; H, 5.52; N, 3.36.
6.2.2. Dendrimer 1b
Yield 76%; mp 148 ꢀC; IR (KBr, cmꢁ1):1643 (C]O); 1H NMR
(300 MHz, CDCl3) d: 1.72 (bs, 2H), 2.84 (bs, 4H), 5.09 (s, 4H), 5.17 (s,
6. Experimental
8H), 6.71 (s, 2H), 6.79 (s, 4H), 7.26 (t, 8H, J ¼ 7.5 Hz), 7.36–7.45 (m,
24H), 7.58 (d, 8H, J ¼ 8.1 Hz), 7.66 (d, 8H, J ¼ 8.1 Hz), 7.74 (s, 2H), 8.13
6.1. Chemistry
(d, 8H, J ¼ 7.8 Hz), 13C NMR (75 MHz, CDCl3)
d: 22.9, 28.5, 69.8, 70.0,
101.8, 106.6, 109.7, 114.2, 114.9, 120.1, 120.4, 123.5, 126.0, 127.3, 129.1,
132.3, 134.5, 135.9, 136.4, 137.6, 139.5, 140.8, 159.0, 160.3, 190.1; m/z
(FAB-MS):1572 (Mþ). Elemental Anal. Calcd for C110H82N4O7: C,
84.05; H, 5.26; N, 3.56. Found: C, 83.91; H, 5.35; N, 3.43.
All the reagents and solvents employed were of the best grade
available and were used without further purification. The melting
points were determined using a Toshniwal melting point apparatus

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P. Rajakumar et al. / European Journal of Medicinal Chemistry 45 (2010) 1220–1224
6.2.3. Dendrimer 1c
Acknowledgements
Yield 65%; mp 154 ꢀC; IR (KBr, cmꢁ1): 1646 (C]O); 1H NMR
(300 MHz, CDCl3)
d
: 1.68 (bs, 2H), 2.79 (bs, 4H), 5.01 (s, 4H), 5.05
The authors thank CSIR India, for financial assistance, DST-FIST
for providing NMR facility to the department. SAIF, CDRI Lucknow
and SAIF, IIT Madras for mass spectral data. NV thanks DST, New
Delhi for fellowship.
(s, 8H), 5.16 (s, 16H), 6.65(s, 2H), 6.70–6.78 (m, 16H), 6.92 (d, 4H,
J ¼ 8.7 Hz), 7.25–7.28 (m, 12H), 7.34–7.39 (m, 36H), 7.56–7.58 (m,
20H), 7.65 (d, 16H, J ¼ 8.1 Hz), 7.71 (s, 2H), 8.11 (d, 16H, J ¼ 7.5 Hz);
13C NMR (75 MHz, CDCl3)
d: 22.7, 28.5, 69.8, 69.9, 70.1, 101.8, 106.8,
109.8, 114.6, 114.8, 120.1, 120.4, 123.5, 126.0, 127.2, 128.5, 129.1,
132.3, 134.4, 135.9, 136.4, 137.6, 139.5, 14 0.8, 158.9, 160.2, 189.9; m/z
(MALDI-TOF-MS): 3105 (M þ Naþ). Elemental Anal. Calcd for
C214H158N8O15: C, 83.41; H, 5.17; N, 3.64. Found: C, 83.27; H, 5.04; N,
3.78.
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6.3.1. Antioxidant studies
Antioxidant activity of all the dendrimers studied by the method
used to determine the free radical scavenging activity. Samples
were prepared at various concentrations of 20, 40, 60, 80, 100, 250,
500, 750 and 1000 mg/ml and 0.1 ml of this solution is mixed with
0.5 ml of DPPH solution in ethanol. After 30 min the absorption was
measured at the wavelength 517 nm on Beckman UV–vis spec-
trometer. Every measurement was repeated three times. From the
absorption value, percentage of activity was calculated using
the formula. Percentage Activity ¼ [As ꢁ Ab/Ac ꢁ Ab] ꢂ 100, where
As – absorbance of DPPH solution with a tested solution (test),
Ab – absorbance of a DPPH solution with a blank without sample
and DPPH, Ac – Control with DPPH solution without sample solu-
tion. The antiradical activity was defined as the concentration of
a sample showing the DPPH radical scavenging activity and
determined from the graph (Fig. 1) in which concentration and
reduction activity were plotted against each other.
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