Synthesis and characterization of polyamide metallodendrimers and their anti-bacterial and anti-tumor activities

Article abstract of DOI:10.1007/s00044-011-9715-0

Pt(II) and Pd(II) complexes with aromatic amine terminated first generation (G1) and second generation (G2) polyamide dendrimers [PAD-(NH2)n] (n = 6 for G1 and 12 for G2) were synthesized. All the synthesized dendrimers and metallodenrimers were character

Full text of DOI:10.1007/s00044-011-9715-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2023–2031
DOI 10.1007/s00044-011-9715-0
ORIGINAL RESEARCH
Synthesis and characterization of polyamide metallodendrimers
and their anti-bacterial and anti-tumor activities
•
Tansir Ahamad S. F. Mapolie Saad M. Alshehri
•
Received: 26 September 2010 / Accepted: 16 June 2011 / Published online: 10 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Pt(II) and Pd(II) complexes with aromatic
amine terminated first generation (G1) and second gener-
ation (G2) polyamide dendrimers [PAD-(NH2)n] (n = 6 for
G1 and 12 for G2) were synthesized. All the synthesized
dendrimers and metallodenrimers were characterized by
elemental and spectral analysis. These novel dendrimers
and their metallodendrimers (metal complexes of dendri-
mers) were screened for their anti-bacterial activity against
Bacillus subtilis and Staphylococcus aureus (Gram posi-
tive) and Escherichia coli and Salmonella typhi (Gram
negative) using agar disk diffusion method. The cytotox-
icity assay of the dendrimers and metallodendrimers has
been performed against MFC-7 cell lines using MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay. Anti-bacterial evaluation indicates that the
second generation dendrimer exhibit stronger biological
activity against all the pathogenic bacterial strains in
comparison to the first generation dendrimers. PdG2
exhibited lowest value of MIC 70 lg/ml against E. coli,
while MIC value of PtG2 was 78 lg/ml. PdG2 and PtG2
show lowest anti-bacterial activity against S. typhi with 102
and 110 lg/ml (MIC value), respectively. In general, the
metallodendrimers showed lower cytotoxicity than cis-
platin (standard drug) and Pt(II) containing metalloden-
drimers were found to be more efficient in the induction of
MFC-7 death than Pd(II) containing metallodendrimers.
Keywords Metallodendrimers Á Polyamide Á
Spectral analysis Á Cytotoxicity assay
Introduction
Metallodendrimers are well-defined, highly branched, three
dimensional macromolecules with characteristic globular
structures for the larger systems (Martinovic et al., 2008).
These novel macromolecules functionalized with transition
metals have inspired many researchers to develop new
materials for pharmaceutical application (Yang and Kao,
2006). The recent impressive strides in synthetic proce-
dures increased the accessibility of functionalized metallo-
dendrimers, resulting in a rapid development of dendrimer
chemistry. The potential interests of metallodendrimers as
anti-microbial and anti-tumor agents, arise mainly from the
control of size and shape along with the placement of
functional groups (Emilio et al., 2009; Refat et al., 2009;
Machado et al., 2001). Recently, many dendrimers have
also been reported and several of them are subjected as
preclinical trial as useful additives in drug formulations for
increasing the solubility, stability, bio-availability, cellular
uptake, targeting ability, and patient compliance of the
administrated drugs and for decreasing the drug resistance
and irritation (Hou et al., 2009). The periphery-function-
alized dendrimers have their ligand systems and able to
coordinate with metal ions. Metal coordination to biolog-
ically active molecules can be used as a strategy to enhance
their activity and overcome resistance. For instance, metal
complexes of thiosemicarbazones can be more active than
the free ligand, or they can be employed as a vehicle for
activation of the ligand as the cytotoxic agent (Patil et al.,
2010; Pavan et al., 2010). Pd(II) and Pt(II) complexes with
T. Ahamad Á S. M. Alshehri (&)
Department of Chemistry, King Saud University,
P.O. Box 2455, Riyadh 11451, Kingdom of Saudi Arabia
e-mail: alshehri@ksu.edu.sa
S. F. Mapolie
Department of Chemistry and Polymer Science,
University of Stellenbosch, Private Bag X1,
Matieland 7602, South Africa
123

2024
Med Chem Res (2012) 21:2023–2031
antibiotics of the tetracycline family are more potent
against Escherichia coli a bacterial strain resistant to tet-
racycline (Guerra et al., 2005; Zerzankova et al., 2010).
Moamen et al. (2005) have reported the novel Cu(II) and
Zn(II) complexes of first and second generation poly(pro-
pylene amine) dendrimers containing 1,8-napthalimide unit
on periphery, and represent that the metal coordinating
dendrimers is more potent against several pathogenic
bacteria then dendritic ligands. The excellent biological
activity of the dendrimers inspires us to synthesize
periphery-functionalized metallodendrimer and used as
anti-tumor and anti-bacterial agents. Herein we selected
amino functionalized dendrimers as a ligand support these
dendrimers were modified into metallodendrimers with
transition metal ions. The present article, we have describes
the preparation and characterization of the Pd(II) and Pt(II)
metallodendrimers were synthesized using [PAD-(NH)2]6
and [PAD-(NH)2]12 and abbreviated as PdG1, PtG1 (com-
plexes of first generation) and PdG2, PtG2 (complexes of
second generation), respectively. In addition, the anti-
bacterial activity of the synthesized dendrimers and their
metallodendrimers has been carried out against against
Bacillus subtilis, Staphylococcus aureus (Gram positive)
and Escherichia coli, Salmonella typhi (Gram negative)
using agar disk diffusion method. The cytotoxicity assay of
the synthesized compounds has been performed on MFC-7
cell lines using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay and compared with
cisplatin as a standard drug.
Results and discussion
A general synthetic route for first and second generation
dendrimeric ligands G1 and G2 is shown in Schemes 1 and
2. G1 and G2 were prepared using ammonium formate and
commercial zinc dust as a reducing agent from their cor-
responding precursor [PAD-(NO2)6 and PAD-(NO2)12]
according to the literature (Zhou et al., 2006). The yellow
color powder of 1,3,5-tris(3,5-dinitrophenyl)benzamide
[PAD-(NO2)6] was prepared by the reaction of 1,3,5-ben-
zene tricarboxylchloride with 3,5-dinitroaniline in acetone
in 1:3 molar ratio in good yield. PAD-(NO2)6 has been
purified simply by precipitation in aqueous NaHCO3 in
quantitative yield. The formation and isolation of PAD-
(NO2)6 were confirmed by IR, elemental analysis, 1H
NMR, and 13C NMR spectroscopy. The IR spectrum of
PAD-(NO2)6 showed strong absorption at 1650 cm-1
characteristic to C=O stretching of amide groups and ter-
minal nitro groups at 1588 and 1290 cm-1. The reduction
of PAD-(NO2)6 by employing SnCl2 and concentrated
hydrochloric acid successfully afforded the desired 1,3,5-
tris(3,5-diaminophenyl)benzamide (G1). The FT-IR spec-
trum of G1 shows strong peak at 3410 cm-1 due to the
presence of terminal amino groups (Ueda et al., 1988). The
nitro groups band at 1588 cm-1 has disappeared com-
pletely after reduction. The proton signals at d 4.36 indicate
the presence of NH2 protons. The 12 nitro-terminated
second generation polyamide PAD-(NO2)12 was synthe-
sized by the reaction of G1 with 3,5-dinitrobenzolyl
Scheme 1 Synthesis of first
generation dendrimeric ligand
O N
NO
2
2
Cl
O
NH
O
O N
NO
2
2
acetone
reflux
+
3
Cl
NH
O
NO
2
O
NH
2
O
O
Cl
O N
NH
2
NO
2
NO
PAD(NO )
2
2 6
H N
NH
2
2
NH
O
SnCl /HCl
2
PAD(NO )
2 6
NH
NH
2
O
O
H N
NH
2
NH
2
NH
2
G
1
123

Med Chem Res (2012) 21:2023–2031
2025
NO2
NO2
O
NO2
NH
NH
O2N
NO2
O
NO2
NH
O
O2N
NO2
O
NO2
+
G
1
NH
NH
O
acetone
reflux
O
NH
NH
O
Cl
O2N
O
NH
O
NH
O
O2N
NO2
O2N
NO2
PAD(NO )
2 12
NH2
NH2
O
NH2
NH
NH
H2N
NH2
O
NH2
NH
SnCl /HCl
O
2
PAD(NO )
2 12
O
NH2
NH
NH
O
O
NH
NH
H2N
O
NH
O
NH
O
H2N
NH2
H2N
NH2
G
2
Scheme 2 Synthesis of second generation denrimeric ligand
Scheme 3 Synthesis of
metalodendrimers
Cl
Cl
NH2
NH2
NH2
NH2
NH
M
K2(MCl4)
reflux
H3C
NH
O
H3C
O
Where M= Pt and Pd
= dendretic part for G1 and G2
chloride in acetone under nitrogen atmosphere in 75%
yield. The structure of PAD-(NO2)12 was confirmed by
elemental analysis, IR, 1H NMR, and 13C NMR. The FTIR
spectra of PAD-(NO2)12 show a strong band due to the
presence of terminal nitro groups at 1588 and 1290 cm-1
.
The 1H NMR spectra of PAD-(NO2)12 show proton signals
for the internal branches amide and outer branch amide at
10.20 and 10.01 ppm, respectively (Okazaki et al., 2003).
123

2026
Med Chem Res (2012) 21:2023–2031
The amino terminated second generation dendrimer G2 was
synthesized using the reaction process similar to the
reduction of PAD-(NO2)6 as employed in the synthesis of
first generation dendrimer G1. The overall route for the
synthesis of second generation dendrimers G2 is illustrated
in Scheme 2. In the FTIR spectrum of G2, the nitro peak on
1342 cm-1 disappeared, meaning that completely reduc-
tion of all the nitro group into corresponding amino groups,
it is also supported by the appearance of new resonance
Therefore, these metallodendrimers have good chemo-
therapeutic potential for use as antimicrobial agent against
these bacteria.
The minimum inhibition concentration (MIC) of syn-
thesized compounds and standard antibiotics streptomycin
are given in Table 2. The data revealed that the MIC value
of G1 against E. coli, S. typhi, B. subtilis, and S. Sureus was
160, 172, 110, and 112 lg/ml, respectively, while the anti-
bacterial activities of streptomycin against these bacteria
was 72, 105, 82, and 80 lg/ml, respectively. The MIC
value result also showed that PdG2 is ore active than all
other compounds against all the pathogens used in this
study. Although the MIC value of PdG2 against Gram
negative bacteria were less that streptomycin. However,
slightly higher in the case of Gram positive bacteria.
The anti-tumor activity of G1 and G2 and their metal-
lodendrimers against MFC-7 (Human breast adenocarci-
noma) cell lines have carried out using MTT cell
proliferation assay and the results are summarized in
Fig. 5. Studies were undertaken with different concentra-
tions (10, 20, 30, and 50 lM). In parallel, the influence of
widely used anticancer drug (cisplatin) was assayed. While
in cell lines cultures, C50 was about 30 lM for PtG1 and
nearly 50 lM for the PdG1. In our experimental system
cisplatin was found to have a much higher anti-proliferat-
ing activity than that of newly synthesized compound with
IC50 2.33 lM in MFC-7 cell cultures. On the other hand,
dendrimers had much lower effect on cellular proliferation
than the Pt(II) and Pd(II) metallodendrimers, because cell
death cannot be distinguished using the MTT assay, the
number of dead and alive cells was measured using the
trypan blue exclusion test. PdG2 and PtG2 showed a much
higher cytotoxicity than PdG1 and PtG2, respectively.
50 lM PtG2 caused almost all cells whereas PtG1, PdG2,
PdG1, G1, and G2 at the same concentration give 85, 60, 55,
40 and 30% of dead cells, respectively.
1
signal at 4.82 ppm, when compared with H NMR spec-
trum of the precursor PAD-(NO2)12.
The metallodendrimers have been synthesized via sim-
ple complexation reaction of ethanolic solution of metal
salts with G1 and G2 in 3:1 and 6:1 molar ratio, respec-
tively. The Pd(II) and Pt(II) complexes were isolated as
brown and yellowish brown powders, respectively, in good
yields (Scheme 3). The formations of metal complexes
were also supported by FTIR and elemental analysis. The
results of elemental analysis revealed that the metal ions
attached to the terminal aromatic amine groups via coor-
dination-covalent bonds and containing two chloride ions
with each metal ion. The FT-IR spectra of the complexes
revealed that, the NH stretching at 3405–3410 cm-1 was
shifted to the lower frequency when compared with their
parental dendrimeric ligands (Okazaki et al., 2001). 13C
1
NMR spectra and H NMR spectra were also used to fol-
low the coordination of metal ions with G1 and G2. The
terminal NH2 groups peaks was shifted from 4.88 and
4.86 ppm to 4.81 and 4.80 ppm after coordination with
metal ions as shown in Figs. 1 and 2. All other peaks of
dendrimers protones were observed in corresponding
metallodendrimer with slightly shift. The MALDI-TOF
MS spectra of G1 and G2 showed the expected [M ? H]?
peak at 526.23, 1330.51, respectively. While MS mea-
surement of PdG2 and PtG2 showed the expected
[M ? H]? peak at 2371.14, 2903.04, respectively, as
shown in Figs. 3 and 4 and indicated the formation of
matellodendrimers.
The results of anti-bacterial activity of dendrimers,
metallodendrimers, and standard drug (streptomycin)
against four pathaogens [Bacillus subtilis and Staphylo-
coccus aureus (Gram positive) and Escherichia coli and
Salmonella typhi (Gram negative)] are summarized in
Table 1. Both dendrimers exhibit sufficient anti-bacterial
activity against all the pathogens. G1 and G2 show higher
activity 17.5 and 22 mm, respectively, against S. typhi and
lower activity 12 and 14 mm against E. coli, respectively.
The anti-bacterial activity of PdG1 against B. subtilis with
higher zone of inhibition 16.5 mm, it is higher than that of
standard antibiotics 16 mm. The PdG2 had shows far better
anti-bacterial activities against all the pathogens bacteria
than streptomycin. The activity of PtG2 and streptomycin
against B. subtilis show similar zone of inhibition 16 mm.
Conclusion
In this study the first and second generation amino ter-
minated polyamide dendrimers and their metallodendri-
mers with Pt(II) and Pd(II) have been synthesized. The
entire compounds have been characterized using elemental
and spectral technique. It was confirmed that both the
dendrimers (G1 and G2) and metallodendrimers showed
promising antimicrobial activities against all the bacteria.
The wider spectrum of the antimicrobial activities
revealed that PdG2 exhibit better anti-bacterial activity
than PtG2. Although the Pt(II) metallodendrimers were
found to be more cytotoxic than their corresponding Pd(II)
metallodendrimers.
123

Med Chem Res (2012) 21:2023–2031
2027
Fig. 1 1H NMR spectrum of PdG1
Fig. 2 1H NMR spectrum of
PdG2
Experimental section
MALDI mass spectrometer (Shimadzu Kratos) in positive
mode.
Materials and methods
Synthesis
1,3,5-Benzene tricarboxylchloride, 3,5-dinitro benzoyl
chloride, 3,5-dinitroaniline, SnCl2, hydrochloric acid
(Sigma-Aldrich). The reagents and solvents were used as
received. All manipulations of air and moisture sensitive
compounds were performed under a nitrogen atmosphere
using standard Schlenk techniques. The elemental analysis
of the dendrimers and metallodendrimers were determined
with a PerkinElmer elemental analyzer. FTIR spectra were
taken on a PerkinElmer IR spectrophotometer model 621
Synthesis of [1,3,5-tris(3,5-dinitrophenyl) benzamide)]
[PAD-(NO2)6]
In
a 250 ml three-neck round-bottom flask, 2.65 g
(0.1 mol) 1,3,5-bezene tricarboxylchloride and 2.74 g
(0.3 mol) 3,5-dinitroaniline were mixed in 100 ml ethanol.
The mixture was refluxed for 6 h under the flow of nitro-
gen. The resulting yellow mixture was cooled and excess
amount of solvent was removed under reduced pressure.
The residue was re-precipitated in hexane, resulting pre-
cipitate was filtrated and washed off several times. The
desired compound [PAD-(NO2)6] was obtained as a yellow
solid powder in 70% yield after purification by column
1
by using KBr pellets in the range of 400–4000 cm-1. H
NMR spectra were recorded on a JOEL-FX-100 FT-NMR
instrument in dimethylsulfoxide (DMSO) solution and
tetramethyl silane (TMS) as an internal standard. MALDI-
TOF mass spectra were recorded using a KMPACT
123

2028
Med Chem Res (2012) 21:2023–2031
Table 1 Antibacterial activity of dendritic ligands and
metallodendrimers
Abbreviation
Zone of inhibition (100 lg/ml)
E. coli
S. typhi
B. subtilis
S. aureus
PAD-(NO2)6
PAD-(NO2)12
PdG1
12.0
14.0
24.5
22.0
26.0
24.0
28.5
–
17.5
22.0
27.0
25.5
32.0
28.0
33.5
–
13.0
14.5
16.5
13.5
18.0
16.0
16.0
–
14.0
16.2
19.3
15.5
21.0
18.0
19.5
–
PtG1
PdG2
PtG2
Streptomycin
DMSO
Zone of inhibition in millimeters
Streptomycin as a standard drug
DMSO solvent of positive control
Fig. 3 MALDI-TOF spectra of PdG2
chloroform. The organic part of the mixture was separated
out and washed off several times with water. The solvent
was evaporated at reduced pressure, and the desired com-
pound PAD-(NH2)6] was obtained as a brown colored
1
powder in 54% yield. Mp: 245°C. H NMR (400 MHz,
DMSO, TMS, d): 9.84 (s, 3H, NH-CO), 6.81 (s, 3H, ArH),
6.52 (s, 6H, ArH), 6.21 (s, 3H, ArH), and 4.88 (s, 12H,
Ar-NH2). 13C NMR (100 MHz, DMSO, TMS): 102.3,
128.4, 134.9, 136.8, and 174.8 ppm. FTIR (KBr, cm-1):
1511, 1556, 1596, 1622, 1680, 3409. ESI MS (m/z): 526.23
(M ? H?). Anal. Calcd for C27H27N9O3 (525.56): C,
61.70; H, 5.18; N, 23.99. Found: C, 61.71; H, 5.17; N,
23.98.
Fig. 4 MALDI-TOF spectra of PtG2
Synthesis of nitro group terminated dendrimers
[PAD-(NO2)12]
chromatography (silica gel, 3/1 (v/v) petroleum ether/ethyl
1
A solution of 5.26 g (10 mmol) [PAD-(NH2)6] in chloro-
form (100 ml) was mixed with 13.99 g (60 mmol) 1,3-
dinitrobenzoyl chloride and refluxed under nitrogen at
70°C for 5 h. Resulting yellow color mixture was obtained.
The process of reaction was monitored by TLC using
CH2Cl2 and hexane as eluent. After completion of the
reaction, the excess amounts of the solvents were removed
under reduced pressure and washed off several times using
water. A yellow brown powder of [PAD-(NO2)12] was
acetate). Mp: 190°C. H NMR (400 MHz, DMSO, TMS,
d): 10.46 (s, 3H, NHCO), 7.12 (s, 3H, ArH), 6.96 (s, 3H,
ArH), 6.42 (s, 6H, ArH). FTIR (KBr, cm-1): 1110, 1340,
1560, 1615, 1710, 3265. Anal. Calcd for C27H15N9O15
(705.46): C, 45.97; H, 2.14; N, 17.87. Found: C, 45.95; H,
2.13; N, 17.89.
Synthesis of [1,3,5-tris(3,5-diamminophenyl) benzamide)]
[PAD-(NH2)6] (G1)
1
obtained in 60% yield. Mp: 275°C. H NMR (400 MHz,
PAD-(NO2)6 (3.52 g, 50 mmol in 30 ml of acetone) and Sn
(7.64 g), conc. HCl (60 ml) were mixed drop-wised in a
100 ml round-bottom flask. The flask was equipped with
condenser and the mixture was refluxed for 30 min. The
resulting mixture was cooled to room temperature and add
an aqueous sodium hydroxide to re-dissolve the initial
precipitate. The mixture was transferred into a separating
funnel and add 3 g sodium chloride powder with10 ml of
DMSO, TMS, d): 9.81 (s, 3H, NH-CO), 9.76 (s, 6H,
NH-CO), 7.27 (s, 9H, ArH), 6.84 (s, 3H, ArH), 6.62 (s, 6H,
ArH), and 6.25 (s, 12H, ArH). 13C NMR (100 MHz,
DMSO, TMS): 104.2, 112.5, 114.2, 129.8, 133.3, 135.4,
137.2, 145.2, 171.8, and 174.1 ppm. FTIR (KBr, cm-1):
1115, 1342, 1560, 1558, 1588, 1650, 1720, 3245. Anal.
Calcd for C69H39N21O33 (1690.17): C, 62.29; H, 4.77; N,
22.11. Found: C, 62.30; H, 4.78; N, 22.10.
123

Med Chem Res (2012) 21:2023–2031
2029
1331.26. Anal. Calcd for C69H39N21O9 (1330.51): C,
49.03; H, 2.33; N, 17.40. Found: C, 49.01; H, 2.24; N,
17.42.
Table 2 Minimum inhibitory concentration (MIC) of dendritic
ligand and metallodendrimers
Abbreviation
Minimum inhibitory concentration (MIC)
E. coli
S. typhi
B. subtilis
S. aureus
Synthesis of generation 1 dendritic Pt(II) complex (PtG1)
PAD-(NO2)6
PAD-(NO2)12
PdG1
160
135
90
95
70
78
72
–
175
140
118
120
102
110
105
–
110
90
90
90
84
85
82
–
112
95
88
90
82
87
80
–
0.131 g (0.5 mmol) PAD-(NH2)6] was dissolved in 10 ml
ethanol, in a round-bottom flask 0.621 g (0.15 mmol)
K2(PtCl4) was added and the reaction mixture was stirred
magnetically under reflux for 20 h forming a yellowish
brown precipitate. The precipitate was filtered off by vac-
uum filtration and washed extensively with ethanol to
afford PtG1 as a solid in 80% yield. FTIR (KBr, cm-1):
545, 1510, 1555, 1584, 1622, 1685, 3370. MALDI-TOF
MS (m/z): 1324.43 (M ? H)?. Anal. Calcd for
C27H27N9O3Pt3Cl6 (1323.57): C, 24.50; H, 2.06; N, 9.52;
Cl, 16.07; Pt, 44.22. Found: C, 24.53; H, 2.09; N, 9.50; Cl,
16.04; Pt, 44.25.
PtG1
PdG2
PtG2
Streptomycin
DMSO
Minimum inhibitory concentration in lg/ml
Streptomycin as a standard drug
DMSO solvent of positive control
Synthesis of generation 1 dendritic Pd(II) complex (PdG1)
Using the same procedure as for the synthesis of PtG1,
when G1 (0.131 g, 0.5 mmol) in ethanol (10 ml) in a
round-bottom flask was added K2(PdCl4) (0.485 g,
0.15 mmol) and the reaction mixture was stirred under
reflux for 20 h forming a brown precipitate. After filtered,
washed off and dried a brown powder was obtained in 52%
yield as a brown powder. FTIR (KBr, cm-1): 540, 1515,
1550, 1586, 1622, 1682, 3375. 1H NMR (400 MHz,
DMSO, TMS, d): 9.82 (s, 3H, NH-CO), 6.78 (s, 3H, ArH),
6.50 (s, 6H, ArH), 6.20 (s, 3H, ArH), and 4.81 (s, 12H, Ar-
NH2). 13C NMR (100 MHz, DMSO, TMS): 101.3, 128.2,
134.5, 134.8, and 172.8 ppm. MALDI-TOF MS (m/z):
1058.28 (M ? H)?. Anal. Calcd for C27H27N9O3Pd3Cl6
(1057.53): C, 30.66; H, 2.57; N, 11.92; Cl, 20.11; Pd,
30.19. Found: C, 30.64; H, 2.58; N, 11.91; Cl, 20.13; Pd,
30.14.
Fig. 5 Cytotoxicity of dendrimers and metallodendrimers
Synthesis of generation 2 dendritic Pt(II) complex (PtG2)
Synthesis of second generation dendritic ligand
[PAD-(NH2)12] (G2)
To a solution of the G2 dendritic ligand (0.665 g,
0.5 mmol) in ethanol (10 ml) was added K2(PtCl4) (1.24 g,
0.30 mmol) and the mixture stirred under reflux for 24 h.
The solvent was evaporated via rotary evaporation to give a
yellowish brown precipitate. The precipitate was then
dissolved filtered, washed off, and dried at room temper-
ature. Resulting a yellowish brown powder was found in
76% yield. FTIR (KBr, cm-1): 548, 1110, 1560, 1556,
1648, 1712, 3260. MALDI-TOF MS (m/z): 2903.04. Anal.
Calcd for C69H39N21O9Pt6Cl12 (2902.13): C, 28.56; H,
1.35; N, 10.14; Cl, 14.66; Pt, 40.33. Found: C, 28.55; H,
1.36; N, 10.12; Cl, 14.68; Pt, 40.30.
A similar procedure has been used for the synthesis of
[PAD-(NO2)12] as for the synthesis of [PAD-(NH2)6].
Resulting, a yellow brown powder was in 52% yield. Mp:
348°C. FTIR (KBr, cm-1): 1114, 1340, 1562, 1560, 1586,
1
1652, 1721, 3265. H NMR (400 MHz, DMSO, TMS, d):
4.86 (s, 48H, NH2), 10.10 (s, 3H, NH-CO), 9.82 (s, 6H,
NH-CO), 7.18 (s, 9H, ArH), 6.82 (s, 3H, ArH), 6.48 (s, 6H,
ArH), and 6.21 (s, 12H, ArH). 13C NMR (100 MHz,
DMSO, TMS): 103.2, 110.8, 115.4, 123.8, 134.2, 136.0,
138.4, 147.0, 170.6, and 173.2 ppm. MALDI-TOF MS (m/z):
123

2030
Med Chem Res (2012) 21:2023–2031
Synthesis of generation 2 dendritic Pd(II) complex (PdG2)
population doubling time (PDT) 33 h, later they were
exposed to compound at different concentration. The IC50
values were then estimated after an exposure time of 72 h.
One hundred microliters of a 5 mg/ml MTT solution in
PBS were added to each well for 4 h. After removal of the
medium, DMSO was added to each well to dissolve the
formazan crystals, and the absorbance was determined at
540 nM.
Using the same procedure as for the synthesis of PtG2 was
obtained as a brown powder in 72% yield as. FTIR (KBr,
1
cm-1): 550, 1565, 1550, 1650, 1710, and 3265. H NMR
(400 MHz, DMSO, TMS, d): 4.80 (s, 48H, NH2), 10.08 (s,
3H, NH-CO), 9.78 (s, 6H, NH-CO), 7.14 (s, 9H, ArH), 6.81
(s, 3H, ArH), 6.48 (s, 6H, ArH), and 6.20 (s, 12H, ArH).
13C NMR (100 MHz, DMSO, TMS): 103.0, 111.0, 115.3,
123.5, 132.2, 134.0, 138.6, 146.8, 170.4, and 174.0 ppm.
MALDI-TOF MS (m/z): 2371.14. Anal. Calcd for
C69H39N21O9Pd6Cl12 (2370.13): C, 34.97; H, 1.66; N,
12.41; Cl, 17.95; Pd, 26.94. Found: C, 35.00; H, 1.62; N,
12.46; Cl, 17.93; Pd, 26.92.
Acknowledgments We would like to thank the College of Science
Research Centre, King Saud University to support this work. Finan-
cial grant CHEM/2010/10.
References
Anti-bacterial and cytotoxic assay
Awerbuch TE, Lustman L, Piret JM (1989) A numerical method to
determine minimal inhibitory concentrations (MICs) of antibi-
otics directly from disc-diffusion susceptibility tests. J Microbio
Meth 9(1):1–7
The anti-bacterial activity of dendrimers and metalloden-
drimers was determined by disk diffusion method using
DMSO as a solvent (Awerbuch et al., 1989). A lawn of
bacteria was prepared by pipetting and evenly spreading
10 ll of inoculums, adjusted turbidometrically to 105–106
CFU/cm3 (CFU = colony forming units) onto agar set in
Petri dishes, using nutrient agar (NA. Whatman No. 1 filter
paper discs of 6 mm diameter were impregnated with
dimethyl sulfoxide stock solutions of the compounds
(100 lg/ml) and dried under sterile conditions. Dried discs
were then placed on previously inoculated agar surfaces.
The plates were inverted and incubated for 24 h at 37°C.
Antimicrobial activity was indicated by the presence of
clear inhibition zones around the discs and repeated at least
for three times for each anti-microbial agent. The results
were compared with Streptomycin as a standard drug for
negative control and solvent (DMSO) for positive control.
Minimum inhibitory amounts (MIC) of dendrimers and
metallodendrimers were determined by the tube dilution
method using Mueller–Hinton broth. The bactericidal
activity of the minimum inhibitory amount was determined
by spreading 0.1 ml of the appropriately diluted broth
culture of the MIC positive tube as well as above and
below the MIC tubes, on nutrient agar plates without oil or
anti-bacterial reference drugs. The Petri plates were incu-
bated at 37°C for 18–24 h. The dilutions that showed no
growth were termed as bactericidal and vice versa. In vitro
cytotoxicity IC50 (concentration of tested agent causing
50% inhibition of cell growth) of these dendrimers and
matellodendrimers was studied by using MTT colorimetric
assay (Stevens and Olsen, 1993). This assay is based on the
cleavage of the yellow tetrazolium salt (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MTT,
Sigma) forming purple formazan crystals by mitochondrial
dehydrogenates in viable cell. The MCF-7 cells concen-
tration (3 9 104 ml-1) were adjusted according to cell
Emilio J, Prez J, Vias C, Teixidor F, Nunez (2009) Polyanionic
carbosilane and carbosiloxane metallodendrimers based on
cobaltabisdicarbollide derivatives. Organometa 28:5550–5559
Guerra W, de Andrade Azevedo E, de Souza Monteiro AR,
Bucciarelli-Rodriguez M, Chartone-Souza E, Nascimento
AMA, Fontes APS, Le Moyec L, Pereira-Maia EC (2005)
Synthesis, characterization, and antibacterial activity of three
palladium(II) complexes of tetracyclines. J Inorg Biochem
99(12):2348–2354
Hou S, Zhou C, Liu Z, Young AW, Shi Z, Ren D, Kallenbach NR
(2009) Antimicrobial dendrimer active against Escherichia coli
biofilms. Bioorg Med Chem Lett 18(19):5478–5481
Machado VG, Baxter PNW, Lehn J (2001) Self-assembly in self-
organized inorganic systems: a view of programmed metallosu-
pramolecular architectures. J Braz Chem Soc 12(4):431–462
Martinovic J, Paquim AMC, Diculescu VC, Wyk JV, Iwuoha E,
Baker P, Mapolie S, Brett AMO (2008) Metallo-functionalized
first-generation salicylaldimine poly(propylenimine) tetraamine
dendrimers: electrochemical study and atomic force microscopy
imaging. ElctroChemica acta 53:4907–4919
Moamen SR, Ibrahim MED, Ivo G, Zeinab MA, Samir G (2009)
Spectroscopic characterizations and biological studies on newly
synthesized Cu(2?) and Zn(2?) complexes of first and second
generationdendrimers. Spec ActaA–Mol BiomolSpec 72:772–782
Okazaki M, Hyakawa T, Ueda M, Takeuchi K, Asai MJ (2001)
Chemoselective polyesterification by direct polycondensation.
J Polym Sci 39:78
Okazaki M, Washio I, Shibasaki Y, Ueda M (2003) Rapid synthesis
of polyamide dendrimers from unprotected AB2 building blocks.
J Am Chem Soc 125:8120–8812
Patil SA, Naik VH, Kulkarni AD, Badami PS (2010) DNA cleavage,
antimicrobial, spectroscopic and fluorescence studies of Co(II),
Ni(II) and Cu(II) complexes with SNO donor coumarin Schiff
bases. Spectrochim Acta A 75:347–354
Pavan FR, Maia PS, Leite SRA, Deflon VM, Batista AA, Sato DN,
Franzblau SG (2010) Thiosemicarbazones, semicarbazones,
dithiocarbazates and hydrazide/hydrazones: anti-mycobacterium
tuberculosis activity and cytotoxicity. Eur
45(5):1898–1905
J Med Chem
Refat MS, El-deen IM, Grabchev I, Anwer ZM, El-Ghol Z (2009)
Spectroscopic characterizations and biological studies on newly
synthesized Cu2? and Zn2? complexes of first and second
generation dendrimers. Spectrochim Acta A 72:772–782
123

Med Chem Res (2012) 21:2023–2031
2031
Stevens MG, Olsen SC (1993) Comparative analysis of using MTT
and XTT in colorimetric assays for quantitating bovine neutro-
phil bactericidal activity. J Immunol Methods 157(1–2):225–231
Ueda M, Kameyama A, Hashimoto K (1988) Diphenyl (2,3-dihydro-
2-thioxo-3-benzoxazolyl)phosphonate: a new, reactive activating
agent for the synthesis of amides and polyamides. Macromol-
ecules 21(1):19–24
Zerzankova L, Suchankova T, Vrana O, Farrell NP, Brabec V,
Kasparkova J (2010) Conformation and recognition of DNA
modified by a new anti-tumor dinuclear PtII complex resistant to
decomposition by sulfur nucleophiles. Biochem Pharmacol
79(2):112–121
Zhou Y, Li J, Liu H, Zhao L, Jiang H (2006) An unusual de-nitro
reduction of 2-substituted-4-nitroquinolines. Tetrahedron Lett
47(48):8511–8514
Yang H, Kao WJ (2006) Dendrimers for pharmaceutical and
biomedical applications. J Biomater Sci Polym 17:3–19
123