Synthesis and evaluation of gold(III) complexes as efficient DNA binders and cytotoxic agents

Article abstract of DOI:10.1016/j.saa.2013.03.037

In recent years, great interest has been focused on gold(III) complexes as cytotoxic and antitumor drugs. Recent studies demonstrated that simple bidentate or polydentate ligands containing nitrogen donor atoms may offer sufficient redox stabilization to produce viable Au(III) anticancer drug targets under physiologic conditions. So, we have synthesized square planer Au(III) complexes of type [Au(Aⁿ)Cl_x]Cl_y and characterized them using UV-Vis absorption, C, H, N elemental analysis, FT-IR, LC-MS, ¹H and ¹³C NMR spectroscopy. These compounds manifested significant cytotoxic properties in vitro for brine shrimp lethality bioassay. The metal complexes were screened for series of DNA binding activity using UV-Vis absorption titration, hydrodynamic measurement and thermal DNA denaturation study. The nucleolytic activity was performed on plasmid pUC19 DNA. The Michaelis-Menten kinetic studies were performed to evaluate rate of enhancement in metal complexes mediated DNA cleavage over the non-catalyzed DNA cleavage.

Full text of DOI:10.1016/j.saa.2013.03.037

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 20–27
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis and evaluation of gold(III) complexes as efficient
DNA binders and cytotoxic agents
⇑
Mohan N. Patel , Bhupesh S. Bhatt, Promise A. Dosi
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, 388 120 Gujarat, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
Synthesis and characterization of
gold(III) complexes.
DNA interactions studies suggest
complexes as covalent binder.
Brine shrimp lethality bioassay was
performed to check the cytotoxicity
of complexes.
ꢀ
Michaelis–Menten kinetic
calculations for DNA cleavage
reaction.
a r t i c l e i n f o
a b s t r a c t
Article history:
In recent years, great interest has been focused on gold(III) complexes as cytotoxic and antitumor drugs.
Recent studies demonstrated that simple bidentate or polydentate ligands containing nitrogen donor
atoms may offer sufficient redox stabilization to produce viable Au(III) anticancer drug targets under
physiologic conditions. So, we have synthesized square planer Au(III) complexes of type [Au(An)Clx]ꢁCly
and characterized them using UV–Vis absorption, C, H, N elemental analysis, FT-IR, LC–MS, 1H and 13C
NMR spectroscopy. These compounds manifested significant cytotoxic properties in vitro for brine shrimp
lethality bioassay. The metal complexes were screened for series of DNA binding activity using UV–Vis
absorption titration, hydrodynamic measurement and thermal DNA denaturation study. The nucleolytic
activity was performed on plasmid pUC19 DNA. The Michaelis–Menten kinetic studies were performed to
evaluate rate of enhancement in metal complexes mediated DNA cleavage over the non-catalyzed DNA
cleavage.
Received 17 December 2012
Received in revised form 23 February 2013
Accepted 3 March 2013
Available online 14 March 2013
Keywords:
Au(III) complex
Michaelis–Menten kinetic
Covalent binding
Rate constant
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
Research into the reactivity and interactions between transition
metal complexes and biomolecules such as DNA and proteins
Interaction between DNA and drug molecules is of current
general interest and importance [1,2], especially for the designing
of new DNA-targeted drugs and the screening of these in vitro.
has resulted in significant advances in the understanding of
biochemical processes and the development of therapeutic drugs.
Since it was found that cancer could be treated with cis-
[Pt(NH3)2Cl2] [3], interest has been activized to explore the anti-
cancer activity of metal complexes [4,5]. Study on the interactions
of metal complexes with nucleic acids is just one of the basic
⇑
Corresponding author. Tel.: +91 2692 226856x218.
E-mail address: jeenen@gmail.com (M.N. Patel).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.03.037

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 20–27
21
researches to catch on the biological effect of complexes on
Synthesis of ligands
nucleic acids and select potential anti-cancer active medicines
[6]. Many metal-derived drugs [7] are believed to inhibit DNA
synthesis in rapidly growing cells, such as cancer cells, by binding
to their DNA. The formation of these adducts alters DNA structure
in such a way that replication either cannot proceed or results in
nonviable daughter cells [8,9]. Thus, advances in the design and
synthesis of new anticancer agents require exhaustive knowledge
of the different DNA-binding mechanisms with a view to
obtaining other more selective agents. There are many methods
to study the DNA binding properties [3–6]. One of the routine,
very common and convincing procedures used to study the inter-
actions of different compounds with DNA is spectrophotometric
measurements [6].
2,20-(Phenylmethylene)bis(1H-pyrrole) (A1)
2,20-(Phenylmethylene)bis(1H-pyrrole) was prepared via con-
densation of the benzaldehyde and pyrrole under TFA catalysis
[15,16]. Yield: 29%, m.p.: 128 °C, Anal. Calc. for
C15H14N2
(222.29): C, 81.05; H, 6.35; N, 12.60%. Found: C, 81.24; H, 6.52;
N, 12.47%. 1H NMR (DMSO-d6, 400 MHz): d (ppm) 7.39 (d, 2H,
00 00
00 00
00
H3 ,5 , J = 8), 7.28 (d, 2H, H2 ,6 , J = 8), 7.18 (t, 1H, H4 , J = 6.8),
0
0
6.37–6.36 (complex, 2H, H5,5 ), 5.94 (t, 2H, H4,4 , J = 7.2), 5.77 (d,
2H, H3,3 , J = 8), 5.27 (s, 2H, ANH), 5.15 (s, 1H, ACH). 13C NMR
0
00
0
(DMSO-d6, 100 MHz): d (ppm) 146.07. (C1 ), 139.35 (C2,2 ), 137.92
00 00
00 00
00
0
0
(C2 ,6 ), 136.48 (C3 ,5 ), 133.80 (C4 ), 126.36 (C5,5 ), 114.57 (C4,4 ),
112.49 (C3,3 ), 48.19 (CCH). FT-IR (cmꢂ1): 3420 (m), 3060 (w),
0
There are many reports on organic metal complexes that are
capable of binding to DNA [10]. The organic metal complexes have
some advantages, such as easy preparation and variety of design
possibilities with different metal ions and ligands, for probing
DNA structure and investigating the binding process as well as for
facilitating individual applications. Among them, ruthenium com-
plex is one of the most extensively investigated members of a class
of the DNA-binding organic metal complexes. However, only little
attention has been paid to the other metal complexes as DNA inter-
acting agent. Among them, Au(III) compounds, isoelectronic and
isostructural with Pt(II) compounds, are emerging as efficient
DNA binders and anticancer agents [11–13]. Therefore, to elucidate
biochemical aspects of gold(III) compounds under physiological
conditions and, specifically, the reactivity of gold(III) compounds
with DNA, in continuation of our earlier work [14], we have synthe-
sized Au(III) complexes of 2,20-(phenylmethylene)bis(1H-pyrrole)
(A1)/2,20-((4-chlorophenyl)methylene) bis(1H-pyrrole) (A2)/N2,N6-
bis(pyridin-2-yl)pyridine-2,6-dicarboxamide (A3)/N-(pyridin-2-yl)
picolinamide (A4)/4-chloro-N-(pyridin-2-ylmethyl)aniline (A5)
and characterized them using different analytical and spectroscopic
techniques. The compounds were checked for different DNA inter-
action, kinetic and cytotoxic studies.
1590 (s), 1530 (s), 1380 (s), 1120 (w), 760 (s), 720 (m), 660 (m).
2,20-((4-Chlorophenyl)methylene)bis(1H-pyrrole) (A2)
2,20-((4 Chlorophenyl)methylene)bis(1H-pyrrole) was prepared
via condensation of the p-chlorobenzaldehyde and pyrrole under
TFA catalysis [15,16]. Yield: 26%, m.p.: 135 °C, Anal. Calc. for C15-
H13ClN2 (256.73): C, 70.18; H, 5.10; N, 10.91%. Found: C, 70.35;
H, 5.23; N, 11.02%. 1H NMR (DMSO-d6, 400 MHz): d (ppm) 7.43
00 00
00 00
(d, 2H, H3 ,5 , J = 8), 7.28 (d, 2H, H2 ,6 , J = 8), 6.36–6.36 (complex,
0
0
0
2H, H5,5 ), 5.93 (t, 2H, H4,4 , J = 7.2), 5.77 (d, 2H, H3,3 , J = 8), 5.26
(s, 2H, ANH), 5.14 (s, 1H, ACH). 13C NMR (DMSO-d6, 100 MHz): d
00
00
0
00 00
(ppm) 145.53 (C1 ), 144.82 (C4 ), 141.16 (C2,2 ), 139.33 (C2 ,6 ),
00 00
0
0
0
138.09 (C3 ,5 ), 127.41 (C5,5 ), 114.67 (C4,4 ), 112.93 (C3,3 ), 48.26
(CCH). FT-IR (cmꢂ1): 3430 (m), 3080 (w), 1600 (s), 1530 (s), 1390
(s), 1120 (s), 1060 (s), 770 (s), 720 (s), 660 (m).
N2,N6-bis(pyridin-2-yl)pyridine-2,6-dicarboxamide (A3)
A mixture of dipicolinic acid (10 mmol) and thionylchloride
(20 mL) were refluxed for 4–5 h, till the evolution of HCl gas ceases,
under anhydrous condition. Excess thionylchloride was removed
under reduced pressure. The resulting solution was cooled in an
ice bath for 15 min. Dry toluene (25–30 mL) followed by 2-amino-
pyridine (20 mmol) was added to the above solution and further
refluxed until no more HCl was evolved. The solvent was removed
under reduced pressure and the resultant white solid was washed
with petroleum ether and neutralized with 5% NaHCO3. It was fil-
tered, washed with water and then alcohol. Recrystallization from
chloroform and ethanol yield silky needles and air-dried. Yield:
88%, m.p.: 219 °C, Anal. Calc. for C17H13N5O2 (319.32): C, 63.94;
H, 4.10; N, 21.93. Found: C, 63.80; H, 4.22; N, 21.78%. 1H NMR
(CDCl3-d, 400 MHz): d (ppm) 11.26 (s, 2H, ANH), 8.56–8.52 (com-
Experimental
Materials
All the chemicals and solvents were of reagent grade and used
as purchased. Chloroauric acid, 4-chloroaniline, 2-aminopyridine,
TFA and sodium borohydride were purchased from S.d. fine-chem
Ltd. (India.). EDTA, pyrrole, benzaldehyde and p-chloro benzalde-
hyde were purchased from Spectrochem Pvt. Ltd. (India). 2-
Picolinic acid and 2,6-dipicolinic acid was purchased from Alfa
Aesar (England). Ethidium bromide was purchased from Himedia
(India). Herring Sperm DNA was purchased from Sigma Chemical
Co. (India).
0
00
0
00
plex, 4H, H3,5,6
, ), 8.40 (dd, 2H, H3 ,3 , J = 0.8, J = 4.8), 8.17 (t, 1H, H4,
6
J = 8), 7.83 (dt, 2H, H4 ,4 , J = 1.6, J = 8), 7.14 (t, 2H, H5 ,5 , J = 6). 13C
0
00
0
00
0
00
NMR (CDCl3-d, 100 MHz): d (ppm) 164.92 (CCO), 150.40 (C2 ,2 ),
0
00
0
00
149.78 (C2,6), 148.55 (C6 ,6 ), 142.47 (C4), 140.35 (C4 ,4 ), 126.92
(C3,5), 120.06 (C5 ,5 ), 115.21 (C3 ,3 ). FT-IR (cmꢂ1): 3390 (w), 3050
(m), 1700 (s), 1580 (s), 1530 (s), 1440 (s), 1320 (s), 1240 (w),
1150 (m), 780 (m), 670 (m).
0
00
0
00
Instrumentation
N-(Pyridin-2-yl)picolinamide (A4)
It was prepared following the same procedure above by taking
2-picolinic acid as starting reactant. Yield: 83%, m.p.: 163 °C, Anal.
Calc. for C11H9N3O (199.21): C, 66.32; H, 4.55; N, 21.09. Found: C,
66.56; H, 4.74; N, 21.20%. 1H NMR (CDCl3-d, 400 MHz): d (ppm)
10.45 (s, 1H, ANH), 8.56 (d, 1H, H6, J = 5.2), 8.45 (d, 1H, H3,
Infrared spectra were recorded on ABB Bomen MB 3000, FT–IR
spectrophotometer as KBr pellets in the range 4000–400 cmꢂ1
.
The LC–MS were recorded using Thermo Scientific mass
spectrophotometer (USA). The 1H NMR and 13C NMR were
recorded on a Bruker Advance (400 MHz, Germany). The electronic
spectra were recorded on a TCC-240 A, UV–Vis spectrophotometer,
Shimadzu (Japan). Thermal DNA denaturation study was
performed using Agilent 8453 UV–Vis spectrophotometer. C, H
and N elemental analyses were performed with a model 240 Perkin
Elmer elemental analyzer.
0
J = 5.2), 8.40 (t, 1H, H4, J = 4.4), 8.32 (d, 1H, H6 , J = 6.4), 7.93 (t,
0
0
1H, H5, J = 7.6), 7.79 (t, 1H, H4 , J = 8), 7.51 (dt, 1H, H5 , J = 1.6,
J = 4.8), 7.11 (dd, 1H, H3 , J = 6.4, J = 12). 13C NMR (CDCl3-d,
0
0
100 MHz): d (ppm) 160.75 (CCO), 150.38 (C2 ), 149.78 (C2,6),
0
00
0
148.55 (C6 ,6 ), 148.24 (C6), 143.48 (C4 ), 140.01 (C4), 130.59 (C5),
128.53 (C3), 118.25 (C5 ), 115.68 (C3 ). FT-IR (cmꢂ1): 3350 (m),
0
0

22
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 20–27
3060 (w), 1690 (s), 1580 (s), 1520 (s), 1440 (s), 1300 (s), 1220 (m),
1150 (m), 780 (s), 690 (s).
236 nm
368 nm (
(
e
e
= 18,004 Mꢂ1 cmꢂ1), 295 nm
(e
= 11,582 Mꢂ1 cmꢂ1),
= 2410 Mꢂ1 cmꢂ1). LC–MS: m/z 522.98 [Au(A2)Cl2]+.
[Au(A3)Cl]ꢁCl2 (3)
4-Chloro-N-(pyridin-2-ylmethyl)aniline (A5)
Similar procedure was followed using A4 as ligand, resulted in
light orange precipitate. Yield: 91%, m.p.: 277 °C, Anal. Calc. for C17-
H13AuCl3N5O2 (622.64): C, 32.79; H, 2.10; N, 11.25%. Found: C,
4-Chloroaniline (14.8 mmol) and pyridine-2-carbaldehyde
(7.40 mmol) were dissolved in 50 mL of absolute ethanol to give
a brownish-yellow solution, which was stirred for 1 h. Sodium
borohydride in 10-fold excess (37.0 mmol) was added in portions
to the ethanolic solution at 0 °C, and stirring was continued for
20 min. The solution was then refluxed for 30 min. After cooling
of the yellow solution, the ethanol was removed by rotary evapo-
ration. Water (200 mL) was added to give a yellow solution with
some precipitate present. Concentrated HCl (ca. 2 mL) was added
to neutralize the solution (pH ca. 6–7), causing the color of the
solution to lighten and giving an off-white precipitate. The solid
mass was collected and dried. Yield: 81%, m.p.: 136 °C, Anal. Calc.
for C12H11ClN2 (218.68): C, 65.91; H, 5.07; N, 12.81. Found: C,
65.83; H, 4.89; N, 12.94%. 1H NMR (CDCl3-d, 400 MHz): d (ppm)
8.60 (d, 1H, H6, J = 4.8), 7.67 (tt, 1H, H4, J = 1.6, J = 7.6), 7.33 (d,
32.67; H, 2.22; N, 11.10%.
138 mho cm2 moleꢂ1
(s, 2H, ANH), 8.55 (d, 4H, H3,5,6
K
m
(1 ꢃ 10ꢂ3 mol Lꢂ1 in DMF):
.
1H NMR (CDCl3-d, 400 MHz): d (ppm) 12.08
0
00
0
00
,
6
, J = 5.2), 8.48 (dd, 2H, H3 ,3 ,
0
00
J = 2, J = 7.6), 8.36 (t, 1H, H4, J = 6.4), 8.12 (dt, 2H, H4 ,4 , J = 1.6,
J = 8.8), 7.40 (t, 2H, H5 ,5 , J = 6.4). 13C NMR (CDCl3-d, 100 MHz): d
0
00
0
00
(ppm) 171.25 (CCO), 152.64 (C4 ,4 ), 152.12 (C4), 149.60 (C2,6),
0
00
0
00
0
00
139.33 (C6 ,6 ), 136.17 (C2 ,2 ), 131.89 (C3,5), 129.43 (C5 ,5 ), 114.51
(C3 ,3 ). FT-IR (cmꢂ1): 3600 (w), 3080 (m), 1720 (s), 1680 (s), 1640
(s), 1550 (s), 1440 (s), 1340 (w), 1300 (m), 840 (m), 680 (m). UV–
0
00
Vis
= 18,460 Mꢂ1 cmꢂ1); (Buffer): 259 nm
290 nm (
= 25,700 Mꢂ1 cmꢂ1). LC–MS: m/z 551.03 [Au(A1)Cl]2+
(DMSO):
244 nm
(
e
= 22,453 Mꢂ1 cmꢂ1),
280 nm
(e
(e
= 25,475 Mꢂ1 cmꢂ1),
e
.
0
0
1H, H3, J = 7.6), 7.22 (t, 1H, H5, J = 6.4), 7.138 (ddd, 2H, H3 ,5 ,
[Au(A4)Cl2]ꢁCl (4)
0
0
J = 3.2, J = 4.0, J = 5.6), 6.61 (ddd, 2H, H2 ,6 , J = 3.2, J = 4.0, J = 5.6),
Similar procedure was followed using A4 as ligand, resulted in
4.86 (s, 1H, ANH), 4.45 (d, 2H, ACH2, J = 5.2). 13C NMR (CDCl3-d,
yellow precipitate. Yield: 82%, m.p.: 243 °C, Anal. Calc. for
0
100 MHz): d (ppm) 155.26 (C2), 148.31 (C6), 147.58 (C1 ), 141.60
C
11H9AuCl3N3O (502.53): C, 26.29; H, 1.81; N, 8.36%. Found:
0
0
0
(C4), 136.74 (C3 ,5 ), 130.46 (C4 ), 122.53 (C3), 121.72 (C5), 116.02
C, 26.14; H, 1.97; N, 8.51%.
K
m (1 ꢃ 10ꢂ3 mol Lꢂ1 in DMF):
(C2 ,6 ), 47.04 (CCH). FT-IR (cmꢂ1): 3330 (m), 3010 (m), 1600 (s),
1480 (s), 1440 (m), 1320 (w), 1280 (m), 1190 (s), 1140 (m), 740
(s), 650 (w).
0
0
102 mho cm2 moleꢂ1
.
1H NMR (CDCl3-d, 400 MHz):
d (ppm)
10.57 (s, 1H, ANH), 8.66 (d, 1H, H6, J = 4.4), 8.46 (d, 1H, H3,
0
J = 9.6), 8.41 (t, 1H, H4, J = 4.4), 8.33 (d, 1H, H6 , J = 9.6), 7.94
0
0
(t, 1H, H5, J = 8), 7.80 (t, 1H, H4 , J = 8), 7.52 (dt, 1H, H5 , J = 1.6,
J = 4.8), 7.12 (dd, 1H, H3 , J = 6.0, J = 10.8). 13C NMR (CDCl3-d,
Synthesis of Au(III) complexes
0
0
100 MHz): d (ppm) 168.34 (CCO), 153.60 (C4 ), 152.05 (C4), 149.89
[Au(A1)Cl2]ꢁCl (1)
0
0
(C2), 144.61 (C6), 137.42 (C6 ), 134.59 (C2 ), 132.94 (C5), 130.70
(C5 ), 128.28 (C3), 115.77 (C3 ). FT-IR (cmꢂ1): 3600 (m), 3090 (w),
1690 (s), 1670 (s), 1610 (s), 1570 (s), 1440 (s), 1340 (m),
1290 (m), 840 (s), 810 (s). UV–Vis (DMSO): 246 nm
To a solution of H[AuCl4]ꢁ3H2O (0.5 mmol) in absolute ethanol
(20 mL) was added A1 (0.5 mmol), resulted in an immediate
formation of light orange precipitate. The reaction mixture was
stirred for 2 h at 60 °C. The product was isolated by filtration,
washed with ether and dried. Yield: 82%, m.p.: 226 °C, Anal. Calc.
for C15H14AuCl3N2 (525.61): C, 34.28; H, 2.68; N, 5.33%. Found: C,
0
0
(e
= 11,740 Mꢂ1 cmꢂ1), 295 nm
(
e
= 8930 Mꢂ1 cmꢂ1); (Buffer):
= 22,761 Mꢂ1 cmꢂ1),
e
= 6028 Mꢂ1 cmꢂ1). LC–MS: m/z 465.97 [Au(A2)Cl2]+.
231 nm
315 nm (
(e (e
= 28,400 Mꢂ1 cmꢂ1), 285 nm
34.46; H, 2.81; N, 5.20%.
K
m
(1 ꢃ 10ꢂ3 mol Lꢂ1 in DMF):
91 mho cm2 moleꢂ1 1H NMR (DMSO-d6, 400 MHz): d (ppm) 7.50
.
[Au(A5)Cl2]ꢁCl (5)
00 00
00 00
00
Similar procedure was followed using A5 as ligand, resulted in
dark orange precipitate. Yield: 84%, m.p.: 238 °C, Anal. Calc. for
(d, 2H, H3 ,5 , J = 8), 7.36 (d, 2H, H2 ,6 , J = 8), 7.24 (t, 1H, H4
,
0
J = 6.8), 6.79–6.78 (complex, 2H, H5,5 ), 6.20 (s, 2H, ANH), 6.05 (t,
2H, H4,4 , J = 7.2), 5.99 (d, 2H, H3,3 , J = 8), 5.20 (s, 1H, ACH). 13C
0
0
C
12H11AuCl4N2 (522.01): C, 27.61; H, 2.12; N, 5.37. Found: C,
00
0
(1 ꢃ 10ꢂ3 mol Lꢂ1 in DMF):
NMR (DMSO-d6, 100 MHz): d (ppm) 151.24 (C1 ), 141.37 (C3,3 ),
27.77; H, 1.99; N, 5.59%.
K
m
0
00 00
00 00
00
97 mho cm2 moleꢂ1
.
1H NMR (CDCl3-d, 400 MHz): d (ppm) 8.76
136.59 (C2,2 ), 135.77 (C3 ,5 ), 135.38 (C2 ,6 ), 134.12 (C4 ), 130.32
(C5,5 ), 115.95 (C4,4 ), 52.67 (CCH). FT-IR (cmꢂ1): 3490 (m), 3070
(w), 1630 (s), 1540 (s), 1400 (s), 1180 (w), 760 (s), 730 (m), 660
0
0
(d, 1H, H6, J = 5.6), 7.87 (t, 1H, H4, J = 1.6), 8.62 (d, 1H, H3,
J = 8.0), 7.48 (t, 1H, H5, J = 4.8), 7.35 (dd, 2H, H3 ,5 , J = 4.0,
0
0
(m). UV–Vis (DMSO): 276 nm
(
e
= 21,342 Mꢂ1 cmꢂ1),ꢂ1334 ꢂn1m
0
0
J = 12), 6.84 (dd, 2H, H2 ,6 , J = 4.0, J = 12), 6.17 (s, 1H, ANH), 4.78
(
e
= 24,573 Mꢂ1 cmꢂ1); (Buffer): 256 nm
(
e
= 17,200 M cm ),
(d, 2H, ACH2, J = 4.4). 13C NMR (CDCl3-d, 100 MHz): d (ppm)
322 nm
(e (e
= 12,712 Mꢂ1 cmꢂ1), 374 nm = 2680 Mꢂ1 cmꢂ1).
0
0
154.30 (C2), 149.76 (C4), 144.48 (C6), 139.88 (C1 ), 135.97 (C4 ),
LC–MS: m/z 489.03 [Au(A1)Cl2]+.
0
0
0
0
129.76 (C3 ,5 ), 126.82 (C3), 126.05 (C5), 124.53 (C2 ,6 ), 47.63
(CCH). FT-IR (cmꢂ1): 3410 (m), 3020 (m), 1670 (s), 1530 (s),
1470 (m), 1370 (w), 1320 (m), 1280 (s), 1190 (m), 800 (s), 730
[Au(A2)Cl2]ꢁCl (2)
Similar procedure was followed using A2 as ligand, resulted in
yellow precipitate. Yield: 81%, m.p.: 226 °C, Anal. Calc. for C15H13-
AuCl4N2 (560.06): C, 32.17; H, 2.34; N, 5.00%. Found: C, 31.94; H,
(w). UV–Vis (DMSO): 277 nm (
e
= 18,046 Mꢂ1 cmꢂ1); (Buffer):
288 nm (e e
= 24,565 Mꢂ1 cmꢂ1), 319 nm ( = 8032 Mꢂ1 cmꢂ1).
LC–MS: m/z 484.97 [Au(A3)Cl2]+.
2.51; N, 5.11%.
K
m (1 ꢃ 10ꢂ3 mol Lꢂ1 in DMF): 89 mho cm2 moleꢂ1
.
1H NMR (DMSO-d6, 400 MHz): d (ppm) 7.49 (d, 2H, H3 ,5 , J = 8), 7.37
Solution study
00 00
00 00
0
(d, 2H, H2 ,6 , J = 8), 6.79–6.78 (complex, 2H, H5,5 ), 6.09 (s, 2H,
0
0
ANH), 5.95 (t, 2H, H4,4 , J = 7.2), 5.85 (d, 2H, H3,3 , J = 8), 5.31 (s,
All complexes show very low solubility in water and in aqueous
buffered solutions. The studies required dissolving the complexes
in a small amount of DMSO followed by dilution with a large
excess of buffer. So we recorded UV–Vis absorption spectra of
the solution of gold(III) compounds in DMSO and by adding small
amounts of freshly prepared, concentrated solutions of 1–2 in
dimethyl sulfoxide (DMSO) to the reference buffer (10 mM
1H, ACH). 13C NMR (DMSO-d6, 100 MHz): d (ppm) 152.33 (C1 ),
00
0
00
00 00
0
141.84 (C3,3 ), 140.66 (C4 ), 139.58 (C2 ,6 ), 138.24 (C2,2 ), 136.73
(C3 ,5 ), 130.95 (C5,5 ), 117.40 (C4,4 ), 52.88 (CCH). FT-IR (cmꢂ1):
3490 (m), 3090 (w), 1640 (s), 1540 (s), 1400 (s), 1180 (s), 1080
(s), 790 (s), 730 (s), 660 (m). UV–Vis (DMSO): 278 nm
00 00
0
0
(e (e
= 22,822 Mꢂ1 cmꢂ1), 330 nm = 26,030 Mꢂ1 cmꢂ1); (Buffer):

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 20–27
23
p, for large p, and will be designated by f(p). We are interested in
phosphate, pH 7.4). Electronic spectra of the resulting mixtures
were monitored at 24 h.
the ratios [
g]/[g
]0; from these we derive,
1=3
L=Lo ¼ ð p0=
g
g0pÞ
ð3Þ
Brine shrimp lethality bioassay
where p is the axial ratio of the rods and subscript ‘0’ indicate ab-
sence of complex. Since the hydrodynamic length of DNA molecule
L/Lo, is directly proportional to the binding ratio of the complex to
DNA, the cubic root of relative viscosity is proportional to the
Brine shrimp lethality bioassay is widely used in the cytotoxic
bioassay for the bioactive compounds. The brine shrimp, Artemia
cysts, was used as a convenient monitor for the screening. The eggs
of the brine shrimp were collected and hatched in artificial seawa-
ter for 48 h to mature shrimp called nauplii. The cytotoxicity assay
was performed on brine shrimp nauplii using Meyer method. The
test samples were prepared by dissolving in DMSO (not more than
binding ratio of the complex to DNA. Representation of data was
1/3
done in terms of (
g
/g0)
versus concentration ratio ([Complex]/
g is the viscosity of DNA solution in the
[DNA] = 0.04–0.2), where
presence of complex and
g
0 is the viscosity of solution of DNA alone.
Viscosity values were calculated using following equation, g a
(t ꢂ t0); where t0 is the flow time of buffer alone and t is the flow
time for buffer containing DNA.
50 lL in 2.5 mL solution) and sea water. A vial containing 50 lL
DMSO diluted to 2.5 mL was used as a control. Then nauplii were
applied to each of all experimental vials and control vial. After
24 h, the vials were inspected using a magnifying glass and the
number of surviving nauplii in each vial were counted. The lethal
concentrations of compounds resulting in 50% mortality of the
brine shrimp (LC50) from the 24 h counts and the dose–response
data were transformed into a straight line by means of a trend line
fit linear regression analysis (MS Excel version 7); the LC50 was
derived from the best-fit line obtained.
Gel electrophoresis
Electrophoresis through agarose is the standard method used to
separate, identify, or purify DNA fragments. When an electric field
is applied across the gel, DNA, which is negatively charged at neu-
tral pH, migrates toward the anode. The intact supercoiled (SC,
form I) DNA migrates faster than the single-nicked (OC, form II)
DNA in the gel. The linear (L. Form III) DNA have an intermediate
mobility. This technique has been employed to identify the prod-
ucts of DNA cleavage, which was carried out in this work.
Interaction between complexes and DNA
Binding of complex with Herring Sperm DNA
Cleavage of pUC19 DNA (50 lM) by complexes (200 lM) was
The binding behavior of the Au(III) complexes toward Herring
Sperm DNA was assessed by monitoring the transformation in
the absorptive nature of complex (66 lM) brought by varying the
concentration of DNA by maintaining the [DNA]/[Complex] in the
range of 0.05–0.25, effect of increasing amount of DNA was nulli-
fied by adding same aliquots of DNA to reference cell. Absorption
data were utilized to calculate intrinsic binding constant (Kb) for
the complexes using following equation,
measured by the conversion of supercoiled pUC19 DNA to open
vcircular (OC) and linear (L). Gel electrophoresis of pUC19 DNA
was carried out in TAE buffer (0.04 M Tris–Acetate, pH 8.2,
0.001 M EDTA). 15
mid DNA, and complex. Reaction mixture was incubated at 37 °C.
All reactions were quenched by addition of 3 L loading buffer
lL of reaction mixture contains 100 lg/mL plas-
l
(0.25% bromophenol blue, 40% sucrose, 0.25% xylene cyanole, and
200 mM EDTA). The aliquots were loaded directly onto 1% agarose
gel and electrophoresed at 50 V in 1X TAE buffer. Gel was stained
½DNAꢄ=ðea
ꢂ
ef Þ ¼ ½DNAꢄ=ðeb
ꢂ
ef Þ þ 1=Kbðeb
ꢂ
ef
Þ
ð1Þ
with 0.5 lg/mL of EB, and was photographed on a UV illuminator.
ea
,
ef and eb corresponds to Aobsd/[Complex], the extinction coeffi-
After electrophoresis, the proportion of DNA in each fraction was
estimated quantitatively from the intensity of the bands using
AlphaDigiDoc™ RT. Version V.4.0.0 PC–Image software.
cient for free complex and the extinction coefficient for the
complexes in the fully bound form, respectively. [DNA] is the con-
centration of DNA in terms of base pairs. A plot of [DNA]/(ea
versus [DNA] gives Kb as the ratio of slope to the intercept [17].
ꢂ
ef)
Kinetic measurements using the gel electrophoresis technique
Thermal DNA denaturation study
For kinetic measurements, DNA cleavage rates at various com-
plex concentrations were measured in a TAE buffer (pH 8.2) at
37 °C for different intervals of time (50–350 min). The decrease
in the intensities of form I with time were then plotted against
complex concentrations, and were fitted well with a single-expo-
nential decay curve (pseudo-first-order kinetics) by use of Eq. (4),
where y0 is the initial percentage of a form of DNA, y is the specific
form of DNA at time t, Kobs is the hydrolysis rate or apparent rate
constant, and Vmax is the maximal reaction velocity. Careful opti-
mization of electrophoretic and densitometric techniques led to
pseudo-first-order kinetics and allowed the determination of
Michaelis–Menten kinetic parameters.
DNA melting experiments were carried out by monitoring the
absorption intensity of Herring Sperm DNA (100 lM) at 260 nm
in the range of 25 °C to 100 °C at increments of 0.5 °C minꢂ1, both
in the absence and presence of the complexes. The melting temper-
ature (Tm) of DNA was determined as the midpoint of the optically
detected transition curves. The
ference between Tm of the free DNA and Tm of the bound DNA.
DTm value was defined as the dif-
Viscosity measurements
An Ubbelohde viscometer maintained at a constant tempera-
ture of 27 0.01 °C in a thermostatic jacket, was used to measure
the flow time of DNA in phosphate buffer (Na2HPO4/NaH2PO4, pH
7.2) with accuracy of 0.01 s. and precision of 0.1 s. DNA sample
used was having approximate average length of 200 base pairs
prepared by sonication to minimize the complexities arising from
its flexibility [18]. Flow time for each sample was measured in
triplicate and an average flow time was calculated. To evaluate
y ¼ ð100 ꢂ y0Þ½1 ꢂ expðꢂK’obstÞꢄ
ð4Þ
K0obs versus [Complex] (50–350
l
M) was plotted and fit using Eq.
(5), which allows the determination of both the rate constants
and Michaelis–Menten – ‘‘type’’ kinetic values. Similar experiments
with constant complex concentrations and varying DNA (20–
80 lM) concentrations were performed, and the intensities were
plotted against substrate concentrations by use of Eq. (6).
the molecular extension from the intrinsic viscosity, [
an equation’
g], we used
½
gꢄ ¼ ðp
NAL3=90 ꢃ 103ZÞ½3=ðln 2p ꢂ 1:5Þ þ 1=ðln 2p ꢂ 2:5Þꢄ
ð2Þ
Kobs’ ¼ Vmax’½catalystꢄ=ðKM þ ½catalystꢄÞ
ð5Þ
ð6Þ
where Z is the number of nucleotides per macromolecule. The
expression in square brackets is rather insensitive to variations in
Kobs’ ¼ Vmax’½substrateꢄ=ðKM þ ½substrateꢄÞ

24
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 20–27
the IR data. The coordination of ligand results in shifting of other
Result and discussion
Synthesis and characterization
1H NMR peaks to the downfield region.
The LC–MS spectrum of complexes validates the structure of
complex proposed by above analytical and spectroscopic tech-
niques. The LC–MS spectra and fragments correspond to peaks in
LC–MS spectra of complexes 1–5 are shown in Supply 1–10.
The complexes [Au(An)Clx]Cly, have been isolated from the eth-
anolic solution containing chloroauricacid as the starting material.
Washing with ether gave the product in sufficiently pure form to
record NMR spectra. All the complexes were obtained in good yield
and were characterized by elemental analysis, 1H and 13C NMR,
UV–Vis, FT-IR and LC–MS spectroscopic techniques. Elemental
analysis data are in good agreement with the proposed structure.
The molar conductivity data reveal that the complex 3 is 1:2 elec-
trolytes, while other complexes are 1:1 electrolyte. The structure of
various ligands and their coordination site are detailed in the
Scheme 1.
UV–Vis spectra
Au(III) with its d8 electronic configuration forms a variety of
square-planar complexes. The low energy LMCT transitions are
quite important for study of Au(III) complexes. The MLCT transi-
tions do not play any role owing to the lack of reducing properties
of Au(III). The time dependent spectrophotometric analyses of
ligand to metal charge transfer transitions clearly showed that
these complexes demonstrated no observable change in their
UV–Vis spectra in DMSO and in 10 mM phosphate buffer which
demonstrate the stability of these complexes in physiological con-
ditions. However, the spectra of the samples dissolved in the refer-
ence buffer are different than in DMSO at room temperature,
implying rapid hydrolysis of the coordinated chlorides of
complexes and conversion into their hydroxy form. Such hydroly-
sis phenomena for Au(III) complexes has been also reported by
Messori et al. [19]. The hydrolyzed metal chromophore
[Au(A)(OH)x]+ fragment was sufficiently stable for at least 24 h in
10 mM phosphate buffer (pH 7.4) (physiological conditions). The
LMCT region of metal complexes with their extinction coefficient
values are shown in the experimental part.
In IR specta, shifting of the band m
(NAH) from 3420 (A1) to 3490
(complex 1) cmꢂ1; 3430 (A2) to 3490 (complex 2) cmꢂ1; 3390 (A3)
to 3600 (complex 3) cmꢂ1; 3350 (A4) to 3600 (complex 4) cmꢂ1
and 3330 (A5) to 3410 (complex 5) cmꢂ1 withdraw the possibility
of deprotonation of ANH group and suggest the N atom of ANH
group as coordinating atom.
The shifting of
cmꢂ1 suggest N atom of central pyridyl ring as a coordinating atom.
The low
(C@O) band shifting from 1710 (A3) to 1720 (complex 3)
cmꢂ1 ruled out the possibility of participation of keto group in
m
(C@N) band from 1240 (A3) to 1340 (complex 3)
m
coordination in complex 3. The shifting of
m(C@N) band from
1220 (A4) to 1340 (complex 4) cmꢂ1 suggest N atom of pyridine
ring attached with keto group as a coordinating atom in complex
4. The shifting of m
(C@N) band from 1280 (A5) to 1320 (complex
Cytotoxicity
5) cmꢂ1 suggest N atom of pyridine ring as a coordinating atom
in complex 5.
Considering that a bioassay is the first step necessary for the
drug discovery process, the complexes were screened for in vitro
toxicity using the microwell assay. This method allows the use of
smaller quantities pure compounds, and permits a larger number
of samples and dilutions within a shorter time. Brine shrimp
In 1H NMR spectra of complexes, the shifting of ANH peak from
5.27 (A1) to 6.20 (complex 1) ppm; 5.26 (A2) to 6.09 (complex 2)
ppm; 11.26 (A3) to 12.08 (complex 3) ppm; 10.45 (A4) to 10.57
(complex 4) ppm and 4.86 (A5) to 6.17 (complex 5) ppm supports
Scheme 1. Structure of the ligands.

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 20–27
25
Fig. 3. Agarose gel (1%) of pUC19 (20 lM) at 37 °C in TE buffer (pH 8) with 150 lM
complex 1–7. Lane 1, DNA control; lanes 2, DNA + metal salt; lane 3, DNA + complex
1; lane 4, DNA + complex 2; lane 5, DNA + complex 3; lane 6, DNA + complex 4; lane
7, DNA + complex 5.
increasing concentration of HS DNA. With increasing concentration
of DNA added, significant hyperchromicity and red-shift was
observed (Fig. 1). It suggests that complexes may covalently bind
to DNA in the major or minor groove. Hyperchromism results from
breakage of secondary structure of DNA due to the fact that phos-
phate group can provide the suitable anchors for coordination with
complexes. The red shift indicates coordination of complex with
DNA through N7 position of guanine. Such type of binding ob-
served in many cis-platins, chloro-ruthenium and aqua-ruthenium
complexes [23,24]. The binding constant (Kb) values for complexes
1–5 are 2.8 ꢃ 105, 2.9 ꢃ 105, 1.3 ꢃ 105, 2.3 ꢃ 105 and 2.1 ꢃ 105,
respectively as calculated from Eq. (1).
Fig. 1. Absorption spectral changes on addition of HS DNA to the solution of
complex 1 after incubating it for 10 min at room temperature in phosphate buffer at
7.2 pH.
lethality bioassay is a recent development in the assay of bioactive
compounds [20,21]. The LC50 (half-inhibition) values obtained after
the analysis of complexes 1–5 are 10, 9, 20, 14 and 16 lM, respec-
tively. The complexes may demonstrate cytotoxicity due to their
ability to effectively interact with cellular DNA or the effect of
the ligand on cellular uptake of the Au(III) complexes. Since the ac-
tive species in the physiological conditions is [Au(A)(OH)x]+, we can
assume that the mechanism of cytotoxic action of these agents is
due to some combination of DNA interaction and redox activity
of the Au(III) center. In attempt to better understand the role of
each of these factors, DNA binding affinity studies for complexes
have been carried out.
Thermal DNA denaturation and viscosity measurement
Thermal behavior of DNA in the presence of metal complexes
can give information about the interaction strength of the com-
plexes with DNA. The double-stranded DNA tends to gradually dis-
sociate to single strands on increase in the solution temperature
and generates a hyperchromic effect on the absorption spectra of
DNA bases (kmax = 260 nm). In order to identify this transition pro-
cess, the melting temperature Tm, which is defined as the temper-
ature where half of the total base pairs gets non-bonded, is a
valuable parameter. Strong interaction of compounds to DNA gen-
erally results in a considerable increase in the melting temperature
(Tm). The DNA melting studies with the complexes 1–5 show a
moderate positive shift in the melting temperature (DTm) i.e. 6.2,
6.4, 5.2, 6.0 and 5.7 °C, respectively suggesting primarily intercala-
tion or covalent binding nature.
To understand the nature of the interaction between the com-
plexes and DNA, viscosity measurements were done. Viscosity
parameter is important as it is sensitive to the change in length
of the DNA strands and provides valuable information for any
DNA binding study
UV–Vis absorption titration analysis
It is generally accepted that DNA is the primary target in the
cis-platin mechanism [22]. Similarly, interactions between small
molecules and DNA rank among the primary action mechanisms
of cytotoxic activity. In order to compare the binding strength of
the complexes with HS DNA, the intrinsic DNA binding constants
(Kb) along with the binding site of the complexes to DNA are deter-
mined by monitoring the change of the absorption intensity of the
charge transfer spectral band of the binary complexes with
Fig. 4. Decrease in the SC form and formation of the OC and L form of pUC19 DNA in
the presence of complex 1 (150 lM) with incubation time.
Fig. 2. Effect on relative viscosity of DNA under the influence of increasing amount
of complexes at 27 0.1 °C.

26
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 20–27
Fig. 5. Michaelis–Menten Reaction kinetics for the cleavage of plasmid DNA: lane 1–7, pseudo Michaelis–Menten kinetic, at constant DNA concentration ([substrate] = 50
and varying complex 1 concentration ([catalyst] = 50–350 M); lane 8–14, true Michaelic–Menten kinetic, at constant complex concentration ([catalyst] = 150) and varying
DNA concentration ([substrate] = 20–80 M).
lM)
l
l
conformational change [25]. Plot of relative specific viscosity
2.8 ꢃ 107 for complexes 1–5, respectively) over the non-catalyzed
DNA (k = 3.6 ꢃ 10ꢂ8 hꢂ1 at 37 °C [26,27]) clearly reveals the effi-
ciency of the complexes to cleave the double-stranded DNA.
Plasmid DNA cleavage was also monitored under ‘true’
Michaelis–Menten kinetic conditions using a constant complex
1/3
(g/g0)
versus [Complex]/[DNA] (Fig. 2) ratio shows decrease
the relative viscosity of HS-DNA for all the complexes, consistent
with partial intercalation or covalent nature of DNA binding, in
which breakage or bending of DNA secondary structure is ob-
served. This is accompanied by decrease in the relative viscosity
due to shortening of length of the DNA chain. The results obtained
from viscosity studies and thermal DNA denaturation studies as
well as UV–Vis spectral titration validate the covalent binding
nature of DNA interaction for all the complexes.
concentration (150
lM) and varying substrate DNA concentration
(20–80 M) (Fig. 5). The cleavage rate constants, kobs were esti-
l
mated in a similar fashion as that shown by Eq. (4) and then plot
of kobs versus [substrate] was drawn. Under these experimental
conditions, values of KM are 76, 84, 68, 80 and 73 lM for complexes
1–5, respectively. The rate enhancements obtained here for com-
plexes (2.2 ꢃ 107, 2.3 ꢃ 107, 1.8 ꢃ 107, 2.3 ꢃ 107 and 2.3 ꢃ 107 for
complexes 1–5, respectively) over the non-catalyzed DNA reveals
the efficiency of the complexes to cleave the double-stranded DNA.
Nucleolytic activity on plasmid DNA
All the Au(III) complexes were subjected to series of time-
dependent
and
concentration-dependent
DNA
cleavage
experiments. All complex ions [Au(An)(OH)2]+ showed significant
DNA cleavage properties due to their ability to interact with
DNA, where the supercoiled DNA (form I) was cleaved to the
relaxed open circular DNA (form II) over a period of 6 h as illus-
trated in Fig. 3. During this incubation period, complexes showed
pronounced capability to further cleave form II to linear DNA (form
III). The positive-charge on active chromophore group of com-
plexes facilitate their interaction with negatively charged DNA
and accelerate the cleavage of DNA, especially, complex 2 imparts
higher cleavage activity on plasmid DNA than other complexes,
consistent with other biological parameters.
Conclusion
The structural analysis that we have performed on mononuclear
gold(III) complexes, relying on the availability of different spectro-
scopic data, has clearly revealed the existence of a common and
well-conserved structural motif comprising the gold center,
chloride atoms, and ligand within a square planar arrangement.
Solution study indicates that ligand configuration appears to im-
part suitable stability of the hydroxy species under aqueous phys-
iological condition. Also the hydroxy chromophore is stable for
24 h under the physiological condition. Then efforts were made
to correlate the cytotoxic properties of the gold(III) complexes with
their DNA interaction properties and notably, positive correlation
has emerged. The complexes show potent cytotoxic properties
against Artemia cysts and the possible observed covalent interac-
tion of complexes with DNA at N7 position of guanine base could
be the target. Furthermore, Michelis–Menten kinetic study on plas-
mid DNA, which shows rate enhancement ratio of the complexes in
the order of 107 in cleaving DNA over non-catalyzed DNA, also sup-
ported above correlation data. Such correlations might be further
exploited to design new and more-active gold-based anticancer
agents and presents the attractive possibility of activating the
cleavage chemistry of covalently bound complexes.
Kinetic measurements for DNA cleavage by compounds
As a result of the very high efficiency of the complex ion
[Au(An)(OH)2]+ in cleaving the strands of plasmid DNA, all com-
plexes were subjected for detailed kinetics studies in order to ex-
plore their activities in the catalytic cleavage of plasmid DNA.
The pUC19 DNA (50 lM) was incubated with complex (200 lM)
in TE buffer at 37 °C for 50–350 min. The increase in the amount
of OC and L forms of DNA was observed to be associated with
the increase of reaction time (Fig. 4). The amount of linear DNA
was 24%, 26%, 21%, 22%, and 24% when the reaction time was
350 min for complex 1–5, respectively. The decrease in the amount
of SC form and the formation of OC form of DNA with time shows
the exponential nature of the curves. The plot of ln(%SC DNA) ver-
sus time is linear, which confirms the process is pseudo-first-order.
The rate constant k1 (7.8 ꢃ 10ꢂ5, 8.0 ꢃ 10ꢂ5, 7.0 ꢃ 10ꢂ5, 8.0 ꢃ 10ꢂ5
and 7.8 ꢃ 10ꢂ5 sꢂ1 for complexes 1–5, respectively), the slope of
the linear plot, was obtained using a complex concentration of
Acknowledgments
Authors thank Head, Department of Chemistry Sardar Patel Uni-
versity, India for making it convenient to work in laboratory and
CSIR, India for providing financial support under ‘‘CSIR-Senior
Research Fellowship’’ scheme.
200 lM.
Appendix A. Supplementary material
Plasmid DNA cleavage was monitored under ‘pseudo’
Michaelis–Menten kinetic conditions using a constant substrate
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.03.037.
DNA concentration (50
lM) and varying complex concentration
(50–350 M) (Fig. 5). The kinetic parameters for the pseudo-
l
Michaelis–Menten conditions are derived from the plots of kobs
versus [catalyst] and fit to Eq. (5). Under these experimental
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