Design and synthesis of (S)- and (R)-enantiomers of [4-(2-hydroxy-1- phenylethylimino)pent-2-ol]dimethyltin(iv) and 2,2-dimethyl-4-phenyl-1,3,2- oxazastannolidine: In vitro antitumor activity against human tumor cell lines and in vivo assay of (S)-enantiomers

Article abstract of DOI:10.1039/c2dt32155f

New dimethyltin derived antitumor drug candidates (S)- and (R)-[4-(2-hydroxy-1-phenylethylimino)pent-2-ol]dimethyltin(iv), 1 and (S)- and (R)-[2,2-dimethyl-4-phenyl-1,3,2-oxazastannolidine], 2 derived from (R)- and (S)-enantiomers of [4-(2-hydroxy-1-phenylethylimino)pent-2-ol] and 2-amino-2-phenylethanol, respectively, were synthesized and thoroughly characterized. Preliminary complex-DNA interaction studies employing various optical methods revealed that the (S)-enantiomer displayed a higher propensity towards the drug target DNA double helix. This was quantified by K_b and K_sv values of ligands L and L′ and (S)-/(R)-1 and (S)-/(R)-2 complexes, which demonstrated a multifold increase in the case of the (S)-enantiomers in comparison to their (R)-enantiomeric forms. This clearly demonstrates the chiral preference of the (S)-enantiomer over the (R)-enantiomer, and its potency to act as a chemotherapeutic agent. Therefore, the in vitro antitumor activity of the (S)-enantiomer of 1 and 2 was evaluated by the sulforhodamine-B (SRB) assay to assess cellular proliferation against five different human cell lines viz., Hop62, DWD, K562, DU145 and MCF-7. The complex (S)-1 displayed a remarkably pronounced and specific activity for K562, while complex (S)-2 exhibited significant activity towards Hop62, DWD, DU145 and MCF-7. The in vivo antitumor activity of (S)-1 and (S)-2 was carried out, which revealed significant regression in human lung tumors.

Full text of DOI:10.1039/c2dt32155f

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PAPER
Design and synthesis of (S)- and (R)-enantiomers of
[4-(2-hydroxy-1-phenylethylimino)pent-2-ol]-
dimethyltin(IV) and 2,2-dimethyl-4-phenyl-
1,3,2-oxazastannolidine: in vitro antitumor activity
against human tumor cell lines and in vivo assay of
(S)-enantiomers†
Cite this: Dalton Trans., 2013, 42, 3390
Farukh Arjmand,*a Fatima Sayeed,a Shazia Parveen,a Sartaj Tabassum,a
Aarti S. Juvekarb and Surekha M. Zingdeb
New dimethyltin derived antitumor drug candidates (S)- and (R)-[4-(2-hydroxy-1-phenylethylimino)pent-
2-ol]dimethyltin(IV), 1 and (S)- and (R)-[2,2-dimethyl-4-phenyl-1,3,2-oxazastannolidine], 2 derived from
(R)- and (S)-enantiomers of [4-(2-hydroxy-1-phenylethylimino)pent-2-ol] and 2-amino-2-phenylethanol,
respectively, were synthesized and thoroughly characterized. Preliminary complex–DNA interaction
studies employing various optical methods revealed that the (S)-enantiomer displayed a higher propen-
sity towards the drug target DNA double helix. This was quantified by Kb and Ksv values of ligands L and
L’ and (S)-/(R)-1 and (S)-/(R)-2 complexes, which demonstrated a multifold increase in the case of the
(S)-enantiomers in comparison to their (R)-enantiomeric forms. This clearly demonstrates the chiral pre-
ference of the (S)-enantiomer over the (R)-enantiomer, and its potency to act as a chemotherapeutic
agent. Therefore, the in vitro antitumor activity of the (S)-enantiomer of 1 and 2 was evaluated by the
sulforhodamine-B (SRB) assay to assess cellular proliferation against five different human cell lines viz.,
Hop62, DWD, K562, DU145 and MCF-7. The complex (S)-1 displayed a remarkably pronounced and
specific activity for K562, while complex (S)-2 exhibited significant activity towards Hop62, DWD, DU145
and MCF-7. The in vivo antitumor activity of (S)-1 and (S)-2 was carried out, which revealed significant
regression in human lung tumors.
Received 18th September 2012,
Accepted 21st November 2012
DOI: 10.1039/c2dt32155f
www.rsc.org/dalton
radiotherapy.2 Nevertheless, the curative effects of the existing
chemotherapeutic drugs are not good enough owing to severe
Introduction
Cancer or malignant neoplasms is a broad group of various health side effects, acquisition of resistance by tumor cells and
diseases, all involving unregulated cell growth, invasion and cost factors. Within the world of clinical oncology, the chemo-
metastasis. Cancer is a deadly global menace (with total prevention of cancer is perceived to be a failure. One major
number of global cancer cases in 2008 being 12 662 554; in obstacle is that cancers evolve from numerous tissues
2030 this number is expected to increase by +69%).1 The (different phenotypes) with multiple etiologies and endless
current treatment regime for cancer primarily includes surgery combinations of genetic or epigenetic alterations and there-
and chemotherapy, however, chemotherapy is used as a main- fore, the one-size-fits-all therapy approach cannot be under-
stay due to its ability to cure widespread malignancies or taken. There is thus a need to develop new strategies for the
metastatic cancers either alone or in combination with optimization of present drug regimes or to have a new outlook
in the design of multifunctional drugs considering their mech-
anisms on the molecular level of actions of the drugs used to
treat different tumors.
aDepartment of Chemistry, Aligarh Muslim University, Aligarh-202002, India.
E-mail: farukh_arjmand@yahoo.co.in
Most promising chemotherapeutics in oncology are metal-
bAdvanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata
based organometallic compounds, such as organotins.3 Cispla-
Memorial Centre, Kharghar, Navi, Mumbai-410210, India
tin and its analogues are DNA alkylating agents which are effec-
†Electronic supplementary information (ESI)
available. See DOI:
tively curing at least one phenotype of cancer (testicular cancer).
10.1039/c2dt32155f
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Their major mode of action is direct DNA damage.4 On
the other hand, organotin compounds display another
spectrum of antitumor activity; the cytotoxicity induced by the
Experimental
Materials and instruments
different kinds of organotins has been related to several mech- All reagents and solvents employed were of analytical grade
anisms. In tin derivatives, the metal center acts as a strong purchased from Sigma-Aldrich and Merck Chemical Co. and
Lewis acid which allows the expansion of the coordination were used without further purification. The elemental analyses
number by the addition of ligands or solvent molecule, were performed on a Carlo Erba analyzer model 1108. IR
thereby enhancing the coordination and altering the geometry spectra were recorded on an Interspec 2020 FTIR spectrometer
from the usual trigonal bipyramidal to distorted octahedral.5 in KBr pellets from 4000–400 cm−1. The UV-vis spectra of the
The phosphate group of the DNA sugar backbone acts as an compounds were recorded on a UV-1700 PharmaSpec UV-vis
anchoring site and the binding of the nitrogen of the DNA spectrophotometer in MeOH and the data were reported as
base are extremely effective; this often results in the stabiliz- λmax nm−1. The electrospray mass spectra of the complexes
ation of the tin center as an octahedral species.6
were recorded on a Micromass Quattro II triple quadrupol
Earlier, organotin(IV) compounds received considerable mass spectrometer. 1H, 13C and 119Sn NMR were recorded on a
attention owing to their potent biocidal activities, industrial Bruker Avance DRX-400 spectrometer in DMSO-d6 respectively.
and agricultural applications.7,8 Recently, in the context of bio- Molar conductances were measured at room temperature on a
chemical properties, a large number of organotin derivatives Digisun electronic conductivity bridge. Fluorescence measure-
have been prepared and were tested in vitro and in vivo, first ments were made on a Hitachi F-2500 fluorescence spectro-
against murine leukemia cell lines (P388 and L1210) and later photometer at room temperature.
against different panels of human cell lines.9
While the organotin moiety is crucial for cytotoxicity, the
ligand design also plays a key role in transporting and addres-
Synthesis of ligands L and L′ and their corresponding metal
complexes
sing the molecule to the target, resisting untimely exchanges
with biomolecules.3 Organotin(IV) antitumor agents with new
modulated ligand scaffolds hold more promise and prove the
state-of-the-art design for chemotherapy. Schiff bases are con-
sidered “privileged ligands” and are the most widely used due
to their versatile synthesis, good solubility in common solvents
and their novel structural features.10 Besides this, stereogenic
centers or other elements of chirality (planes, axes) can be
introduced in such a synthetic design. The overall handedness
of the DNA molecule plays a major role in the recognition of
DNA by chiral molecules due to the two-pole complementary
principle.11 Furthermore, the use of stereochemistry can give
clear insight into the mechanism of action, allowing the dis-
crimination between unspecific interactions, which are
common to both enantiomers and specific contacts that give
rise to enantioselectivity. This phenomenon is important as
many small molecules that interact with DNA in vitro do not
behave in a similar manner in vivo, and this is one of the
major obstacles in the development of drugs based on DNA
binding (usually, it is difficult to quantify the differential
binding of two enantiomers with DNA as there are subtle
differences in the binding or repulsive forces).12
The aim of the present study was to investigate new chiral
organotins for the development of robust chemotherapeutic
agents for treating cancers. The DNA binding profile revealed
that optically pure (S)-enantiomers showed higher binding
affinities than the (R)-enantiomers, and furthermore, the
Schiff base complex, 1 exhibited avid DNA binding propensity
in comparison to the 2-amino-2-phenylethanol complex 2. The
in vitro antitumor activities of complexes (S)-1 and (S)-2
have been evaluated using the SRB assay13 against different
human carcinoma cell lines, while the in vivo activity
was carried out in the human lung xenograft tumor model
(Hop62).
Synthesis of Schiff base ligands (L and L′). The Schiff base
ligand was prepared by adopting the reported procedure.14 To
a methanolic solution (20 mL) of acetylacetone (1.03 mL) was
added a stirred solution of (S)-/(R)-2-amino-2-phenylethanol
(1.37 g) in 1 : 1 molar ratio. The reaction mixture was refluxed
for 4–8 h with continuous stirring during which the color of
the solution turned to yellowish-orange. The completion of the
reaction was monitored by TLC. After completion of the reac-
tion, the mixture was cooled in an ice bath to give a solid
product. Ligands L and L′, obtained from the S- and R-enantio-
mers of 2-amino-2-phenylethanol, respectively, were washed
with hexane and dried in vacuo.
Synthesis of the (S)- and (R)-[4-(2-hydroxy-1-phenylethyli-
mino)pent-2-ol]dimethyltin(IV), 1. To a methanolic solution
(20 mL) of the Schiff base ligand (L) (1.08 g), two drops of tri-
ethylamine were added and the resulting yellow solution was
allowed to stir for 3–4 h. To this solution, 20 mL of a methano-
lic solution of dimethyltin dichloride (1.09 g) was added drop
wise at 20 °C in an equivalent molar ratio. The final product
obtained was washed with hexane and dried in vacuo. The
corresponding (R)-[4-(2-hydroxy-1-phenylethylimino)pent-2-ol]
dimethyltin(IV) 1 complex was synthesized in accordance with
the above procedure described for (S)-1 by using ligand L′.
(S)-Enantiomer. Yield: 72%; m.p. = 138 °C; [α]D = +12; %
anal. calc. for C15H21NO2Sn: C 49.22, H 5.78, N 3.83; found:
C 49.23, H 5.75, N 3.80; ESI-MS m/z [C15H21NO2Sn + H]+: 367;
1H NMR (DMSO-d6, 400 MHz) δ (ppm); 7.4–7.2 (ArH, 6H), 4.9
(vCH, 1H), 4.6–4.7 (CH–N, 1H), 3.5–3.7 (–CH2, 2H), 2.4 (OvC–
CH3, 3H), 1.8 (NvC–CH3, 3H), 0.6–1.1 (Sn–CH3, 6H); 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 126–128 (ArC), 193 (CvN), 162
(O–Cv), 95 (vC–), 38–40 (N–CH2), 10–14 (Sn–CH3). 119Sn
NMR (DMSO-d6, 146 MHz) δ (ppm): −241.
(R)-Enantiomer. Yield: 69%; m.p. = 138 °C; [α]D = −14; %
anal. calc. for C15H21NO2Sn: C 49.22, H 5.78, N 3.83; found:
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C 49.24, H 5.75, N 3.82; ESI-MS m/z [C15H21NO2Sn + 2H]+: 368; absorbance due to DNA at the measured wavelength. From the
1H NMR (DMSO-d6, 400 MHz) δ (ppm); 7.4–7.3 (ArH, 6H), 4.9 absorption titration data, the intrinsic binding constant (Kb)
(vCH, 1H), 4.9 (CH–N, 1H), 3.2 (–CH2, 2H), 2.4 (OvC–CH3, of the complexes with CT-DNA were determined using the
3H), 1.3 (NvC–CH3, 3H), 0.9–1.0 (Sn–CH3, 6H); 13C NMR Wolfe–Shimmer equation.17
(DMSO-d6, 100 MHz) δ (ppm): 126–128 (ArC), 196 (CvN), 162
½DNAꢀ=εa ꢁ εf ¼ ½DNAꢀ=εb ꢁ εf þ 1=Kbðεb ꢁ εf Þ
ð1Þ
(O–Cv), 95 (vC–), 38–40 (N–CH2), 10–14 (Sn–CH3). 119Sn
NMR (DMSO-d6, 146 MHz) δ (ppm): −240.
where εa, εf and εb correspond to Aobsd/[complex], the extinc-
tion coefficient for the free complex, and the extinction coeffi-
cient for the complexes in the fully bound form, respectively.
Plot of [DNA]/εa–εf vs. [DNA], where [DNA] is the concentration
of DNA in the base pairs, gives Kb as the ratio of the slope to
the intercept.
Fluorescence spectral studies. Luminescence titration
quenching experiments were conducted by adding increasing
concentration of the complexes (solution in DMSO) to a fixed
concentration EB–DNA system. Tris-HCl buffer was used as a
blank to make preliminary adjustments.
CD spectroscopy. While measuring the absorption spectra
an equal amount of DNA was added to both the solutions of
the compound and the reference solution to eliminate the
absorbance of the CT-DNA itself, and the CD contribution by
the CT-DNA and Tris buffer was subtracted through base line
correction.
Both enantiomeric metal complexes exhibited identical
molar conductance, IR and UV-vis spectra.
Λm: Ω−1 cm2 mol−1 = 29 (non-electrolyte, MeOH); UV-vis,
nm: 299, 258, 211; IR (KBr): ν(cm−1): 1518–1525, ν(CvN),
424–432, ν(Sn–N), 461–557, ν(Sn–O), 569–595, ν(Sn–C).
Synthesis of (S)- and (R)-[2,2-dimethyl-4-phenyl-1,3,2-oxaza-
stannolidine], 2. To a stirring solution of (S)-2-amino-2-pheny-
lethanol (1.37 g) in methanol, a solution of dimethyltin
dichloride (2.19 g) was added in 1 : 1 molar ratio. After 3–4 h
a white amorphous compound was isolated, washed with
hexane and dried in vacuo over anhydrous CaCl2. The corres-
ponding (R)-[2,2-dimethyl-4-phenyl-1,3,2-oxazastannolidine],
2 complex was also synthesized according to the above pro-
cedure by using (R)-2-amino-2-phenylethanol.
(S)-Enantiomer. Yield: 72%; m.p. = 240 °C; [α]D = +23; %
anal. calc. for C10H16NOSnCl: C 37.49; H 5.03; N 4.37; found:
C 37.47, H 5.01, N 4.39; ESI-MS m/z [C10H16NOSnCl + 0.5
1
CH3OH]: 336; H NMR (DMSO-d6, 400 MHz) δ (ppm); 7.4–7.3
Antitumor activity assays
(ArH, 6H), 4.8 (–NH2, 1H), 4.7–3.7 (–CH–N, 1H), 3.1–3.6 (–CH2–
O, 2H), 0.6–1.1 (Sn–CH3, 6H); 13C NMR (DMSO-d6, 100 MHz):
δ 66, –CH2–O; δ 38–40, CH–N; δ 128, ArC; δ 10–14, CH3–Sn;
119Sn NMR (DMSO-d6, 146 MHz) δ (ppm): −161.
(R)-Enantiomer. Yield: 78%; m.p. = 231 °C; [α]D = −20; %
anal. calc. for C10H16NOSnCl: C 37.49; H 5.03; N 4.37; found:
C 37.49, H 5.03, N 4.37; ESI-MS m/z [C10H16NOSnCl +
0.5CH3OH]: 336; 1H NMR (DMSO-d6, 400 MHz) δ (ppm);
7.7–7.3 (ArH, 6H), 4.67 (–NH2, 1H), 4.7–3.7 (–CH–N, 1H),
3.1–3.6 (–CH2–O, 2H), 0.8–1.2 (Sn–CH3, 6H); 13C NMR (DMSO-
d6, 100 MHz): δ 68, –CH2–O; δ 41, CH–N; δ 128–135, ArC;
δ 10–13.2, CH3–Sn; 119Sn NMR (DMSO-d6, 146 MHz) δ (ppm):
−166.
In vitro antitumor activity. The cell lines used for in vitro
antitumor screening activity were Hop62 (human lung), DWD
(human oral), K562 (human leukemia), DU145 (human pros-
trate) and MCF-7 (human breast). These human malignant cell
lines were procured and grown in RPMI-1640 medium sup-
plemented with 10% fetal bovine serum (FBS) and antibiotics
to study the growth pattern of these cells. The proliferation of
the cells upon treatment with chemotherapy was determined
using the semi automated Sulphorhodamine-B (SRB) assay.
Cells were seeded in 96-well plates at an appropriate cell
density to give optical densities in the linear range (from 0.5
to 1.8) and were incubated at 37 °C in a CO2 incubator for
24 h. Stock solutions of the complexes were prepared as
100 mg mL−1 in DMSO and four dilutions (i.e., 10 μL, 20 μL,
40 μL, 80 μL), and were tested in triplicates, each well receiving
90 μL of cell suspension and 10 μL of the drug solution. Appro-
priate positive control (Adriamycin) and controls were also
run. The plates with cells were incubated in a CO2 incubator
with 5% CO2 for 24 h followed by the addition of the drug. The
Both enantiomeric metal complexes exhibited identical
molar conductance, IR and UV-vis spectra.
Λm: Ω−1 cm2 mol−1 = 37 (non-electrolyte, MeOH); UV-vis,
nm: 252–257, 220–228; IR (KBr): ν(cm−1) = 3007–3002, ν(NH2)
461–466, ν(Sn–N), 569–575, ν(Sn–C), 549–550, ν(Sn–O).
DNA binding and cleavage experiments
DNA binding experiments, which include absorption spectral plates were incubated further for 48 h. The experiment was ter-
titrations, fluorescence and circular dichroism, conformed to minated by gently layering the cells with 50 μL of chilled 30%
the standard methods and practices previously adopted by our TCA (in the case of adherent cells) and 50% TCA (in the case
laboratory.15–18 DNA cleavage studies were performed accord- of suspension cell lines) for cell fixation and then being kept
ing to the protocol followed in our laboratory.19
at 4 °C for 1 h. Plates were stained with 50 μL of 0.4% SRB for
Absorption spectral experiments. Absorption spectral titra- 20 min. The bound SRB was eluted by adding 100 μL 10 mM
tion experiments were performed in DMSO at a constant Tris (pH 10.5) to each of the wells. The absorbance was read at
concentration of the complexes with varying CT-DNA 540 nm with 690 nm as the reference wavelength. All experi-
concentration. The absorbance (A) of the most shifted band of ments were repeated 3 times.
investigated complexes was recorded after successive addition
In vivo antitumor activity. After inoculation with Hop62
of CT-DNA. A reference cell contained DNA alone to nullify the (human lung tumor) cells (10 mg/100 g body weight) for 24 h,
3392 | Dalton Trans., 2013, 42, 3390–3401
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tumor-bearing mice were randomized into the experimental
groups. All complexes were dissolved in DMSO solution. All of
the complexes were administered at a dose of 0.2 mL/10 g
body weight for thirty three successive days. At the end of the
experiment, the animals were sacrificed and the tumors were
dissected and weighed. The tumor inhibitory rates were then
calculated.
Scheme 2 Transamination of acetylacetone.
All animal experiments were performed as per Institute’s
animal ethics guidelines (animal house is registered with Scheme 2. The complexes (S)-/(R)-2 were prepared by mixing
CTCSEA) at ACTREC, Tata Memorial Centre, Mumbai.
an equimolar ratio of the (S)- and (R)-forms of 2-amino-
2-phenylethanol with dimethyltin dichloride, respectively.
These complexes were characterized by spectral studies using
IR, NMR (1H, 13C and 119Sn), ESI-MS and elemental analysis.
The formation of the complexes (S)-/(R)-1 and (S)-/(R)-2 has
been ascertained by the comparison of the IR spectra of the
ligands and complexes. The spectra of the ligands revealed a
broad envelope in the 3028–3204 cm−1 region, assigned to the
intramolecular hydrogen bonded –OH group, which was found
absent in complexes of (S)-/(R)-1, indicating the deprotonation
of the hydroxyl group upon coordination to the metal ions.21
The bands corresponding to the azomethine group (–CvN) at
Results and discussion
Synthesis and characterization
The in situ condensation reaction of the chiral (S)- and
(R)-forms of 2-amino-2-phenylethanol and acetyl acetone in
1 : 1 molar ratio resulted in the light yellow Schiff base ligands
L and L′, respectively, and their organotin complexes (S)-/(R)-1
were synthesized in high yield by slight modification of the
procedure reported earlier (Scheme 1).14 The ligands, on com-
plexation with dimethyltin dichloride, behave as a dibasic tri-
dentate ONO donor ligand corresponding to the enol tautomer
of acetyl acetone.20 This was confirmed by the absence of a
ketonic frequency in the IR spectra of L and L′, which supports
the successful synthesis of the complexes. Since the ligand
exists in the enol tautomeric form, an electron shift from the
–OH group enhances the negative charge on the azomethine
nitrogen atom so the transamination reaction of the carbonyl
group is prohibited which would prevent the formation of the
ligands in a 1 : 2 stoichiometric ratio, as depicted in the
1551 cm−1 22
respectively in the spectra of the free ligands was
,
considerably shifted to lower frequencies ∼1518 cm−1 and
1525 cm−1 in complexes (S)-/(R)-1, indicating the involvement
of the azomethine nitrogen in the coordination with the tin
metal ion, which was further supported by the appearance of
new bands in their spectra at 424–432 cm−1, assigned to the
ν(Sn–N) stretching vibrations.21,23,24 The absence of ketonic
group vibrations (–CvO) at 1700 cm−1 in the IR spectra of the
ligands revealed that the ligands exist predominantly in the
enolic form rather than ketonic form.25 On the other hand,
the ligand 2-amino-2-phenylethanol revealed a band in the
3331–3275 cm−1 region, assigned to the ν(NH2)/ν(OH) stretch-
ing vibrations.26 A considerable lowering in these bands to
3007–3002 cm−1 was indicative of the coordination through
–NH2 and –OH groups for the formation of (S)-/(R)-2.
The electronic absorption spectra of the ligands and com-
plexes (S)-/(R)-1 and (S)-/(R)-2 were obtained in MeOH at room
temperature. The ligands L and L′ displayed two broad bands
around the 312–315 nm range, which were attributed to the
n→π* transition of the azomethine group,27 and the band
appearing at 230–228 nm, with a shoulder at 269–266 nm, was
assigned to the intraligand π–π* transition. In the spectra of
complexes (S)-/(R)-1, the bands of the azomethine chromo-
phore n→π* transition were shifted to lower frequencies
(310 nm), indicating the coordination of the imine nitrogen
atom to the tin metal ion. Other bands associated with com-
plexes (S)-/(R)-1 at 258 and 211 nm were attributed to MLCT or
intraligand transitions. The complexes (S)-/(R)-2 revealed two
bands in the 222–228 nm and 252–257 nm regions, attributed
to LMCT or intraligand transitions.28
1
The H NMR spectra of the ligands and complexes showed
identical functional group signatures, however in the spectra
of the complexes, additional signals due to methyl protons
attached to the tin metal ion were also observed at δ
0.60–0.97 ppm29 (Fig. S1†). The 1H NMR spectra of the
Scheme 1 Synthetic route of ligands and complexes.
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complexes (S)-/(R)-2 revealed a broad peak at δ 4.8 due to the respectively with a mean standard deviation of 0.15. Ligands
amine protons during the complexation with tin metal ion. L and L′ also differ significantly, which is attributed to the
The other characteristic peaks for (S)-/(R)-2 corresponding to selective binding affinity of CT-DNA for different enantiomers.
ArH, CH–N and CH2–OH protons appeared at δ 7.3–7.4, The Kb values of L (S-enantiomer) revealed a ca. 100-fold
δ 4.7–3.7, δ 3.1–3.6, respectively.30–32
higher preferential binding to the DNA helix in comparison
In the 13C NMR spectra of the ligands and complexes to L′ (R-enantiomeric form). The complexes (S)-1 and (S)-2
(S)-/(R)-1, the position of the azomethine peak at δ 193.5–196.6 showed greater propensity for DNA as compared to free
of the ligands was slightly shifted downfield, indicating the ligands due to the presence of the Sn(IV) metal center, which
coordination of azomethine nitrogen with the tin metal ion33 could be attributed to its strong tendency for phosphate
(Fig. S2†). Additional resonances observed in the 13C NMR binding. Furthermore, it has been observed that the (S)-enan-
spectra of the ligands and their complexes at 126–128, 162, 95, tiomer of both 1 and 2 exhibits a more pronounced propensity
28, 19 were attributed to the ArC, O–Cv, –Cv, CH3–CvN, for DNA binding in comparison to the (R)-enantiomer. DNA–
CH3–CvN carbons.33 Furthermore, the 13C NMR spectra of complex interactions are usually dependent on chirality and
(S)-/(R)-2 revealed various other resonances attributed to the the conformation of the isomers, involving two pole compli-
–CH2–O, CH–N, ArC and CH3–Sn carbons at δ 66, δ 38–40, mentary recognition hypothesis to the right handed domain of
δ128 and δ 10–14, respectively, of the coordinated 2-amino- the B-form of DNA. Complexes 1 and 2 show structural dissim-
2-phenylethanol (Fig. S3 and S4†).
ilarity due to the presence of the Schiff base ligand, which
The 119Sn NMR spectra recorded in DMSO-d6, displayed actually mutes the toxicity as well as tunes the metal to enforce
peaks in the range of δ −241 and −240 for complexes (S)-/(R)-1 the stereoselective binding to the DNA, leading to the specta-
and a peak at δ −161 and −166 for complexes (S)-/(R)-2 (Fig. S5 cular propensity for DNA binding of complex (S)-1. These
and S6†). The 119Sn chemical shift values obtained were very Kb values were compared to cisplatin and our observation
sensitive to the changes in the coordination number and the revealed that the Kb values of (S)-1 and (S)-2 were comparable
nature of the groups directly attached to the tin atom.34 These to that of cisplatin [5.73 ( 0.45) 104 M−1] as reported in the lit-
chemical shift values were typical of a five coordinated erature.38 These results suggest that the (S)-enantiomers are
environment around the tin metal ion.35 The chemical shift more avid DNA binders as compared to the (R)-enantiomers.
values are consistent with the observations of previously The preferential organotin phosphate binding to the oxygen
reported chiral organotin(IV) Schiff base complexes.36
atom of the sugar backbone and the nitrogen involved in the
CT-DNA base binding is extremely effective, often resulting in
the stabilization of the tin center as a stable octahedral
DNA binding studies
Electronic absorption titration. The comparative DNA species.6 Therefore, the above results indicate that all the
binding ability of chiral ligands (L and L′) and complexes studied complexes bind predominantly to the phosphate
(S)/(R)-1 and (S)/(R)-2 with the drug cisplatin were evaluated by group, neutralizing the negative charge of the CT-DNA and
electronic absorption spectroscopy by studying the metal causing contraction and conformational changes in the DNA
complex–DNA interactions. In the UV region, the ligands L by electrostatic interactions.
and L′ exhibited intense absorption bands around
211–258 nm, while the complexes (S)-/(R)-1 displayed bands in
Fluorescence spectroscopic studies
the range of 222–228 nm, attributed to intraligand transitions. To further elucidate the mode of binding of the optically active
Fig. 1 illustrates the respective absorption spectral changes of complexes (S)-/(R)-1 and (S)-/(R)-2 and ligand L and L′ with
the L and its complexes in the absence and presence DNA, fluorescence spectral methods were employed by follow-
of CT-DNA at a wavelength of ∼250 nm. Upon addition of ing the changes in the emission intensity of the complexes. In
CT-DNA (0–0.4 × 10−4 M) to a fixed concentration of complex/ the absence of DNA, the ligand L and complexes (S)-/(R)-1 and
ligand (0.06 × 10−4 M), an increase in the absorbance (hyper- (S)-/(R)-2 emit luminescence around 260–500 nm in Tris-HCl
chromic effect) of the intraligand band followed by a concomi- buffer (pH 7.2) when excited at 260 nm. A fixed volume (1.0 ×
tant red-shift of 2–4 nm, which arises due to the preferential 10−4 M) of the studied complexes and ligands L and L′ was
binding of the predissociated Sn(IV) to nucleotides via the titrated, respectively with an increasing concentration of DNA
phosphate group of DNA was observed. Since Lewis acidic Sn in the range of 0–0.40 × 10−4 M. As seen from Fig. 2, the inten-
(IV) cations in the trigonal bipyramidal geometry exhibit a ten- sity of the emission increases appreciably in the presence of
dency to expand their coordination number on addition of DNA. The observed enhancement could be due to the relatively
electron donor atoms (solvent molecules) or by the coordi- non-polar environment of the bound metal complex in the
nation to the nucleotides, the geometry changes to distorted presence of DNA, such that the complexes were less deeply
octahedral.37 Moreover, the affinity of Sn(IV) with a trinegative inserted inside the hydrophobic pockets or grooves of
phosphate group is very strong because of its hard Lewis acidic CT-DNA.39 The cationic complexes usually bind to DNA non-
property, which is also evidenced by the intrinsic binding con- covalently as the cationic core of the complexes exerts a strong
stant Kb values obtained for the complexes (S)-/(R)-1 and electrostatic attraction to the anionic phosphate backbone of
(S)-/(R)-2 and ligands L and L′, which were of the order 4.20 × 104, DNA thus precluding substantial overlap with the base pairs
1.42 × 104, 3.96 × 104, 1.27 × 104, 1.27 × 103 and 1.1 × 104 M−1
,
leading to the higher emission intensity, which is indicative of
3394 | Dalton Trans., 2013, 42, 3390–3401
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Fig. 1 Absorption spectra of (a) cisplatin, (b) L, (c) (S)-1, (d) (R)-1, (e) (S)-2 and (f ) (R)-2 in Tris-HCl buffer (pH = 7.2) in the absence and presence of increasing
amounts of CT-DNA. Arrows indicate the increase in the intensity upon increasing DNA concentration.
the electrostatic binding of the probe to DNA. The binding
The fluorescence quenching experiments, using K4[Fe(CN)6]
constants, Kb of complexes (S)-/(R)-1 and (S)-/(R)-2 and the as an anionic quencher, were performed to further probe the
ligands L and L′ were 2.90 × 107, 1.1 × 106, 1.82 × 106, 6.7 × 105, binding characteristics of both chiral complexes (S)-/(R)-1 and
2.4 × 105 and 3.2 × 105, respectively with a mean standard (S)-/(R)-2, and the ligand L. Fig. 3 shows a typical Stern–Volmer
deviation of 0.09.
plot of F0/F versus [Q] for complexes (S)-/(R)-1 and 2 and ligand
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Fig. 3 Emission quenching curves of (a) L, (b) (S)-1 and (c) (S)-2 in the absence
of CT-DNA ( ) and in the presence of CT-DNA ( ). [M] = 10−3 M.
■
●
resulting in a linear Stern–Volmer plot with a slope of 1.07 ×
105 and 8.57 × 104, respectively. In the presence of DNA, the
Fig. 2 Emission spectra of (a) L, (b) complex (S)-1 and (c) (S)-2 in Tris-HCl buffer
(pH 7.2) in the absence and presence of CT-DNA. Arrows indicate the change in slope of the plot was remarkably decreased to 6.42 × 104 and
3.50 × 104, respectively. Similarly, the slope of the linear Stern–
the intensity upon increasing DNA concentration.
Volmer plot for complexes (R)-1 and (R)-2 were also quenched
from 7.14 × 104 and 4.85 × 104 in absence of DNA to 5.0 × 104
L. In the absence of DNA, the positively charged complexes
(S)-1 and 2 were efficiently quenched by [Fe(CN)6]4− ions,
and 3.71
×
104 in the presence of DNA, respectively.
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Fig. 5 Effect of increasing concentration of K2HPO4 on the fluorescence inten-
sity of the complex (a) (S)-1 and (b) (S)-2. M = (10−3 M) with DNA (10−4 M).
Fig. 4 Effect of different concentrations of NaCl on the fluorescence spectra of
complex (a) (S)-1 and (b) (S)-2 M = (10−3 M) with DNA (10−4 M). Arrows indicate
the gradual decrease of emission intensity as a function of NaCl concentration.
positively charged molecules to the DNA phosphate backbone.
This invokes a competitive interaction for the phosphate
The greater decrease in the Ksv value for complexes (S)-1 and anions and subsequent addition of the cations weakens the
(S)-2 compared to complexes (R)-1 and (R)-2 corresponds to the surface binding interactions and hydrogen bonding between
stronger binding of these complexes with the DNA double the DNA and molecule. Therefore, the results implicate that
helix.40
complexes (S)-/(R)-1 and (S)/(R)-2 preferably binds to the DNA
Effect of ionic strength on the binding of (S)/(R)-1 and phosphate backbone by electrostatic interactions.41 In
(S)/(R)-2 with DNA. In order to ascertain the probable binding addition, it could be seen from Fig. 4a and b, that the effect of
mode between the molecule and DNA, fluorescence titrations salt concentration on the fluorescence intensity of the (S)-1–
were carried out under the conditions of increasing ionic DNA system was far greater than the other complex (S)-2, so
strength (0–0.40 × 10−4 M) through added amounts of NaCl. As complex (S)-1 has a greater potency for DNA binding.
shown in Fig. 4, the fluorescence intensity of the studied com-
Effect of phosphate group on the binding of (S)/(R)-1 and
plexes was appreciably quenched with increasing ionic (S)/(R)-2 with DNA. As a means of further exploring the selec-
strength. These results indicate that a cation, such as Na+, can tive binding site of complexes (S)-/(R)-1 and (S)/(R)-2 with DNA,
bind to the phosphate group of the DNA by electrostatic forces fluorescence titrations were performed in the presence of an
to form a cation atmosphere around the DNA. This cationic increasing amount of K2HPO4 at 25 °C (Fig. 5a and b). It is
atmosphere shields the DNA and inhibits the binding of observed from Fig. 5 that the fluorescence intensity of all
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complexes increases when K2HPO4 is added to the system. (S)-enantiomeric form, there is more predominant pertur-
These results demonstrate a competitive binding behavior bations in comparison to the (R)-enantiomeric form, indicative
between the phosphate group of K2HPO4 and DNA for the of preferential enantiomeric binding of the complexes These
complexes.42 Therefore, the phosphate group of K2HPO4 observations have been ascribed to the conformational change
weakens the interaction between the complexes and DNA,43 in the B→A form of DNA.45 Furthermore, both (S)-/(R)-2
this observation provides supportive evidence of the electro- revealed identical CD changes of equal magnitude but oppo-
static interactions of complexes (S)/(R)-1 and (S)/(R)-2, which site signs resulting from structural perturbations. The
bind selectively to the phosphate group of the DNA double (S)-enantiomeric form of complex 1 displayed a greater affinity
helix. Furthermore, there is strong evidence in literature for than both the (S)-/(R)-enantiomers of complex 2.
Sn(IV)–phosphate binding, which is validated by the binding
studies of Sn(IV) complexes with nucleotides employing 1H and
DNA cleavage studies
31P NMR techniques.44 The complex (S)-1 binds with relatively
To ascertain the ability of the enantiomeric complexes (S)-1
higher affinity towards CT-DNA in contrast to (R)-1 and (S)-/(R)-2
and (S)-2 to serve as potential metallonucleases, the DNA clea-
complexes.
vage were performed with pBR322 DNA incubated with varying
concentrations of the complexes in 5 mM Tris-HCl/50 mM
NaCl buffer at pH 7.2, for 1 h by agarose gel electrophoresis.46
The gel electrophoretic patterns of complexes (S)-1 and (S)-2
were obtained due to their high binding ability for DNA as evi-
denced by UV, fluorescence studies, Fig. 7a and b. Initially in
the untreated pBR322 plasmid DNA (Lane 1) two bands corre-
sponding to Form I and Form II were observed. When the
supercoiled pBR322 plasmid DNA were treated with the
0.10 mM of both the complexes (S)-1 and (S)-2, the supercoiled
DNA (Form I) is cleaved to nicked circular DNA (Form II)
without the concurrent formation of Form III (Lanes 2 and 4),
respectively. With increasing of the concentration of the
complex from 0.10–0.50 mM, there is apparently very little
transformation of the plasmid DNA from Form I to Form II
(Lanes 3 and 5). These changes in the intensity of Form I and
Form II with an increase in the concentration of the complexes
Circular dichroic studies
Circular dichroic spectral studies have been performed to diag-
nose the changes in DNA morphology during complex–DNA
interactions. The CT-DNA exhibits two consecutive bands, a
positive band at 275 nm due to base stacking, and a negative
band at 245 nm due to helicity in the UV region. The changes
in CD signals of DNA observed on interaction of both enantio-
mers of the (S)-/(R)-1 and (S)/(R)-2 complexes are depicted in
Fig. 6a and b. On addition of complex 1 to CT-DNA there is a
pronounced red- shift in both the positive and negative bands,
attributed to the helicity and base stacking of DNA and the
absorption intensities show a decrease in the intensity in both
the enantiomers to the extent that there is inversion in the
base stacking CD signal at 275 nm. However, in case of the
Fig. 7 Agarose gel electrophoresis patterns for the cleavage of pBR322
plasmid DNA as a function of increasing concentration of complex (a) (S)-1 and
(b) (S)-2. (a) Lane 1: DNA control, Lane 2: DNA + 50 μM of (S)-1, Lane 3: DNA +
100 μM of (S)-1, Lane 4: DNA + 150 μM of (S)-1, Lane 5: DNA + 200 μM of (S)-1,
Lane 6: DNA + 250 μM of (S)-1. (b) Lane 1: DNA control, Lane 2: DNA + 50 μM
of (S)-2, Lane 3: DNA + 100 μM of (S)-2, Lane 4: DNA + 150 μM of (S)-2, Lane 5:
DNA + 200 μM of (S)-2, Lane 6: DNA + 250 μM of (S)-2.
Fig. 6 CD spectra of CT-DNA alone and in the presence of complex (a) (S)-1
and (b) (S)-2 in Tris-HCl buffer at 25 °C. [Complex] = 10−4 M, [DNA] = 10−4 M.
3398 | Dalton Trans., 2013, 42, 3390–3401
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Table 1 In vitro antitumor activity of complexes (S)-1 and (S)-2a
Complex (S)-1
Complex (S)-2
ADR
LC50
Cell lines
LC50
TGI
GI50
LC50
TGI
GI50
TGI
GI50
Hop62
DWD
DU145
K562
52.6
49.5
>80
<10
63.4
29.9
32.5
57.7
<10
43.3
<10
44.0
35.1
71.4
>80
15.7
<10
43.2
>80
29.6
<10
<10
15.0
>80
<10
35
28
74
76
52
<10
<10
32
41
14
<10
<10
<10
<10
<10
15.5
30.8
<10
23.3
MCF-7
51.1
a GI50 = growth inhibition of 50% (GI50) calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, drug concentration result in a 50% reduction in the net
protein increase. GI50 value <10 μg mL−1 is considered to demonstrate activity. Tz = absorbance measurements at time zero. Ti = test growth in
the presence of a drug at the different concentration levels. TGI = tumor growth inhibition, LC50 = lethal concentration of 50% (LC50). ADR =
adriamycin (taken as positive control compound).
is due to the unwinding of the supercoiled Form I. The relaxed
state of the plasmid is generated due to the cleavage of the
DNA phosphodiester backbone. Thus, complexes (S)-1 and
(S)-2 do not cleave the plasmid DNA efficiently.
Antitumor activity
In vitro antitumor activity. The in vitro antitumor activity of
complexes (S)-1 and (S)-2 has been evaluated in terms of GI50,
TGI and LC50 values by the semi-automated sulforhodamine-
B (SRB) assay against different human carcinoma cell lines of
different histological origin, viz., Hop62 (human lung), DWD
(human oral), K562 (human leukemia), DU145 (human pros-
trate) and MCF-7 (human breast). The antitumor screening
data (Table 1) revealed that complex (S)-1 acts as a potent selec-
tive antitumor agent with pronounced GI50 values <10 μg
mL−1, specifically towards Hop62, K562 and DWD tumor cell
lines, while complex (S)-2 exhibited good in vitro activity
towards Hop62, DWD, MCF-7 and DU145 tumor cell lines.
In vivo antitumor activity. Since both complexes (S)-1 and
(S)-2 displayed a consistent potency in the antitumor activity
Fig. 8 Effect of complexes (S)-1 and (S)-2 on human lung cancer xenograft
against the Hop62 cancer cell line, with GI50 values <10 μg
Hop62 in mice.
mL−1, hence, it was of interest to evaluate the in vivo antitumor
activity of complexes (S)-1 and (S)-2 in the human lung cancer
xenograft model Hop62 in mice. The results revealed that (S)-1
showed significant regression in human lung tumors as com-
pared to (S)-2 (Fig. 8). The weight of tumor was also significantly
reduced in mice treated with complexes (S)-1 and (S)-2 (Fig. 9).
the drug cisplatin (clinically used for the treatment of cancer).
All mice displayed normal activities, and no significant body
(R)-enantiomers, which attenuates the importance of stereospeci-
fic interactions with inherently chiral DNA molecules. Interest-
ingly, (S)-1 and (S)-2 exhibited higher Kb values compared to
The observations of the DNA binding profile dictates the
potential of these chemical entities to act as good chemothera-
peutics. Therefore, it was thought worthwhile to investigate
weight loss was observed during these experiments (Fig. S8†).
the antitumor activity of (S)-1 and (S)-2 in vitro and in vivo. The
(S)-enantiomer of 1 exhibited effective cytotoxic activity against
Conclusions
Hop62 (human lung), K562 (human leukemia), DWD (human
oral) with pronounced GI50 values (<10 μg mL−1). Since com-
plexes (S)-1 and (S)-2 showed potent in vitro cytotoxicity but
did not exhibit any DNA cleavage activity, the results indicate
that these complexes exert cytotoxicity by some other mechan-
ism which may not involve the direct cleavage of DNA. The
results of the in vivo antitumor activity experiments revealed
that complex (S)-1 showed significant regression in human
lung tumors as compared to (S)-2. Hence, our experimental
Chiral organotin chemical entities 1 and 2 of the (R)- and
(S)-enantiomers of [4-(2-hydroxy-1-phenylethylimino)pent-2-ol]
and 2-amino-2-phenylethanol, respectively were synthesized,
thoroughly characterized and their in vitro DNA binding
profile (UV-vis, fluorescence, circular dichroism) was studied.
All complexes (R)-/(S)-1 and (R)-/(S)-2 bind strongly to DNA
via an electrostatic interaction mode that preferentially
involves the phosphodiester backbone; nevertheless, the
(S)-enantiomers bind more strongly in comparison to the
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Fig. 9 Growth of Hop-32 tumors treated with (a) control group, (b) ADR, (c) complex (S)-1 and (d) complex (S)-2.
results suggest that complex (S)-1 holds forth promise as a drug
candidate with potent in vitro and in vivo antitumor activity.
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Abbreviations
SRB
DMSO
Sulforhodamine-B assays
Dimethylsulphoxide
CT-DNA Calf thymus DNA
UV-vis
TLC
Ultraviolet visible
Thin layer chromatography
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