Synthesis, DNA binding, and cytotoxic evaluation of new analogs of diallyldisulfide, an active principle of garlic

Article abstract of DOI:10.1016/j.bmc.2008.06.029

Diallyldisulfide (DADS), an active principle of garlic (Allium sativum) is known for its antihyperlipidemic properties. However, its use is limited due to its extreme volatility. In the present study, we have synthesized and characterized a series of six

Full text of DOI:10.1016/j.bmc.2008.06.029

Bioorganic & Medicinal Chemistry 16 (2008) 7301–7309
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, DNA binding, and cytotoxic evaluation of new analogs
of diallyldisulfide, an active principle of garlic
Santosh Kumar Rai a, Meenakshi Sharma b, Manisha Tiwari a,
*
a Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
b Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, RI 02881, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Diallyldisulfide (DADS), an active principle of garlic (Allium sativum) is known for its antihyperlipidemic
properties. However, its use is limited due to its extreme volatility. In the present study, we have synthe-
sized and characterized a series of six new DADS analogs and investigated their interactions with differ-
ent DNA duplexes. The spectroscopic and circular dichroism (CD) analyses revealed that DADS analogs
bind preferentially with GC rich sequences. Thermodynamic parameters suggest that DADS analogs sta-
bilize the calf thymus (CT) DNA and GC rich duplex by favorable enthalpic gains and follow the hierarchy,
d(GC)7 > CT DNA > d(AT)10. Further, DADS analogs are less toxic and equally effective as the statins. The
analogs therefore have a good potential to provide a new therapeutic approach for the treatment of car-
diovascular and related diseases.
Received 9 May 2008
Revised 16 June 2008
Accepted 17 June 2008
Available online 20 June 2008
Keywords:
Diallyldisulfide
Drug–DNA interaction
Circular dichroism
Differential scanning calorimetry
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
hepatic 3-hydroxy-3-methylglutaryl-CoA reductase11 (HMGR)
activity, principally by inhibiting the transcription of the HMGR
Controlling gene expression with small DNA-binding molecules
has been a challenge at the interface of medicinal chemistry and
biology.1–3 To achieve this goal, a number of chemical approaches
have been investigated in search of small molecules (drugs) that
can selectively bind to DNA and either activate or inhibit gene
expression. This includes the examination of specific and non-
covalent interactions, of small organic molecules, with DNA and
RNA. The objective is to synthesize drugs that will act as specific
modulators of gene activity.4–6
Chemical modification of bioactive components of medicinal
plants is one of the most common approaches for drug discovery.
These bioactive components can be modified by organic synthesis
to prepare more stable and effective drugs (therapeutics).7 Dially-
ldisulfide (DADS) is one of the major constituents of Garlic and has
been shown to depress cholesterol synthesis by 10–25% at low
concentration (<0.5 mmol/L).8 The use of DADS is, however, limited
due to its extreme volatility.9 To overcome this limitation, our lab-
oratory synthesized various derivatives of DADS that display great-
er hypolipidemic activity and enhanced stability, as compared to
DADS. We have previously reported that the analogs with electron
withdrawing substituents on the phenyl ring (compound 5)
showed antihyperlipidemic activity better than parent DADS and
are comparable to that of Lovastatin.10 These compounds modulate
gene, thereby reducing cholesterol levels, in an animal model
(communicated elsewhere).
In this study, the synthesis and DNA-binding activity of a series
of new DADS analogs are reported (Fig. 1). The DADS structure has
been altered by chemical synthesis, and the analogs have been
studied with respect to their affinity to DNA, their influence on
DNA structure, and their cytotoxic effect on rat hepatocytes cul-
ture. In this work, we have employed various spectroscopic tech-
niques such as UV spectroscopy, thermal denaturation, and
circular dichroism (CD) to characterize DNA-binding activity of
the DADS analogs and to analyze their sequence specificity. The
thermodynamic parameters of DADS analogs/DNA complexes were
determined by differential scanning calorimetry (DSC). The results
conclusively prove the relationship between sequence selectivity,
* Corresponding author. Tel.: +91 11 27666272; fax: +91 11 27666248.
E-mail addresses: skrai7@gmail.com (S.K. Rai), manisha_07@hotmail.com (M.
Tiwari).
Figure 1. Structures of DADS derivatives 5–11 from the synthetic steps shown in
Scheme 1.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.029

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S. K. Rai et al. / Bioorg. Med. Chem. 16 (2008) 7301–7309
mode of binding, and the antihyperlipidemic activity of the DADS
derivatives.
binding.12 In order to compare the DNA interactions of the DADS
analogs, absorption titrations were conducted with calf thymus
DNA (CT DNA) and with DNA duplexes of either alternating AT
(d(AT)10) or GC (d(GC)7) sequences.
2. Results and discussion
2.1. Synthesis
The spectral shifts and hypochromicities on complex formation
of all analogs of DADS at drug/DNA ratio (r) of 5 are summarized in
Table 1, and some important patterns can be observed for these
complexes. There is insignificant red shift for the DADS analogs-
d(AT)10 and DADS analogs-CT DNA complexes, whereas a signifi-
cant red shift (2–4 nm) is observed with DADS analogs-d(GC)7
complexes. All the DADS analogs with CT DNA and d(GC)7 duplex
show hypochromicity of 18–22% and 28–34%, respectively,
whereas d(AT)10 duplex complexes have comparatively smaller
hypochromicity (10–16%). Compound 6 shows the largest hypo-
chromic shift with d(GC)7. Large hypochromism and the red shift
in the absorption spectra was an indication of a strong electronic
interaction between the DADS analogs and the DNA bases (CT
DNA and d(GC)7). Our result also suggests that d(AT)10 and
d(GC)7 duplexes behave differently on complex formation with
DADS analogs.
The synthetic steps of the target compounds are shown in
Scheme 1. The starting compound 2-mercaptoethanol (1) was oxi-
dized with Br2 to yield bis-(2-hydroxyethyl)disulfide(2) in the
presence of KHCO3. The compound (2) was brominated to the cor-
responding bis-(2-bromoethyl) disulfide (3) in the presence of 48%
HBr and conc H2SO4with an approximate yield of 60%. Compound 3
was then converted to the corresponding phosphonium salt (4) by
refluxing equimolar amounts of (3) and triphenylphosphine in dry
DMF. The phosphonium salt thus obtained was refluxed for 24 h
with appropriate aldehydes in the presence of sodium ethoxide
to afford the DADS analogs (5–11) with an approximate yield of
ꢀ75% (Fig. 1). In our earlier publication, we have reported the
use of lithium ethoxide for synthesizing the DADS analogs.10 In
the present study, we have modified the reaction by using sodium
ethoxide, as lithium ethoxide tends to break the disulfide linkage,
resulting in a lower yield. The product was purified by silica gel
chromatography using ethyl acetate as the eluent to afford com-
pounds 5–11. The compounds (5–11) were fully characterized by
spectroscopic analysis (mass, IR, and 1H NMR).
2.2.2. Thermal melting analysis
UV-melting profiles of double-stranded DNAs in the presence of
DADS analogs were performed at varying drug/DNA ratios. It is
known that thermal denaturation profiles provide the simplest
means for detecting binding and asserting relative binding
strength.12
2.2. DNA-binding properties
Natural genomic calf thymus DNA and two synthetic oligonu-
cleotides with alternating AT (d(AT)10) and GC (d(GC)7) base pairs
were chosen with the purpose of determining the possible se-
quence preferences of the tested compounds.
2.2.1. Absorption spectral analysis
It is a general observation that a hypochromic and red shift in
the absorption spectra accompanies the binding of molecules to
DNA. The extent of spectral change is related to the strength of
Table 2 summarizes the change in melting temperature (
DTm)
of compounds (5–11) with CT DNA, d(AT)10, and d(GC)7 duplexes
Scheme 1. Reagents and conditions: (i) Br2, KHCO3, DCM (49%); (ii) HBr, H2SO4 (59%); (iii) PPh3, DMF, reflux (72%); (iv) NaOEt, substituted benzaldehyde (70–79%).
Table 1
Photometric properties of DADS analogs (5–11) with various DNA duplexes
DADS analogs
Calf thymus DNA
% Hypochromicitya
Red shift (nm) from 258
d(GC)7
Red shift(nm) from 257
d(AT)10
Red shift (nm) from 262
% Hypochromicitya
% Hypochromicitya
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
21
20
22
19
18
19
20
1
1
0
0
1
0
0
30
34
29
31
28
32
29
3
4
3
2
4
3
3
16
14
15
13
12
14
10
0
0
0
0
0
0
0
a
Titrations were conducted at constant duplex concentration (2.5
l
M) with increasing drug concentration in 20 mM NaH2PO4/Na2HPO4 buffer (pH 7.4) containing 100 mM
M).
NaCl at room temperature. Values at saturating concentration of DADS analogs (12.5
l

S. K. Rai et al. / Bioorg. Med. Chem. 16 (2008) 7301–7309
7303
Table 2
Effect of DADS analogs on thermodynamic parameters for drug/DNA complexes
a
b
b
DDHb (kcal/mol)
D
S (cal/mol Kꢁ1
)
DDSb (cal/mol Kꢁ1
D
G25 kcal/mol
DDG25
ꢂ
ꢂ
Compound
Tm (°C)
D
Tm (°C)
D
H (kcal/mol)
C
C
AT duplex
5
6
7
8
9
10
11
52
52
49
51
52
50
51
52
—
ꢁ 43.821
ꢁ 40.121
ꢁ 32.809
ꢁ 38.899
ꢁ 36.201
ꢁ 35.138
ꢁ 36.188
ꢁ 37.301
—
ꢁ141.61
ꢁ136.38
ꢁ125.14
ꢁ136.98
ꢁ129.11
ꢁ129.57
ꢁ131.93
ꢁ132.56
—
ꢁ1.865
+0.521
+4.401
+1.921
+2.273
+3.473
+3.127
+2.202
—
+2.3
0
+3.6
+10.9
+4.9
+7.6
+8.6
+7.6
+6.5
+5.2
+16.5
+4.6
+12.5
+12.0
+9.7
+9.1
ꢁ 3
ꢁ 1
0
ꢁ 2
ꢁ 1
0
+6.2
+3.7
+4.0
+5.3
+4.9
+4.0
GC duplex
5
6
7
8
9
10
11
87
94
93
93
92
93
92
91
—
ꢁ59.637
ꢁ 63.754
ꢁ 68.795
ꢁ 62.758
ꢁ 64.694
ꢁ 61.567
ꢁ 62.080
ꢁ 60.746
—
ꢁ162.757
ꢁ 169.028
ꢁ 186.52
ꢁ 167.69
ꢁ 175.602
ꢁ 166.178
ꢁ 167.41
ꢁ 165.892
—
ꢁ11.141
ꢁ13.384
ꢁ13.212
ꢁ12.786
ꢁ12.364
ꢁ12.045
ꢁ11.893
ꢁ11.310
—
+7
+6
+6
+5
+6
+5
+4
ꢁ4.15
ꢁ9.19
ꢁ3.15
ꢁ5.09
ꢁ1.9
ꢁ6.2
ꢁ23.7
ꢁ4.9
ꢁ12.8
ꢁ3.4
ꢁ4.6
ꢁ3.1
ꢁ2.2
ꢁ2.1
ꢁ1.6
ꢁ1.2
ꢁ0.9
ꢁ0.7
ꢁ0.2
ꢁ2.48
ꢁ1.14
CT DNA
5
6
7
8
9
10
11
73
80
81
81
80
81
79
78
—
ꢁ7.501
ꢁ14.735
ꢁ15.656
ꢁ15.361
ꢁ13.234
ꢁ14.995
ꢁ10.694
ꢁ 9.963
—
ꢁ20.824
ꢁ 41.980
ꢁ 43.336
ꢁ 44.175
ꢁ 38.07
—
ꢁ1.408
ꢁ 2.224
ꢁ 2.271
ꢁ 2.196
ꢁ 1.889
ꢁ 2.286
ꢁ 1.753
ꢁ1.694
—
+7
+8
+8
+7
+8
+6
+5
ꢁ7.2
ꢁ8.1
ꢁ7.8
ꢁ5.7
ꢁ7.5
ꢁ3.2
ꢁ2.4
ꢁ21.2
ꢁ22.5
ꢁ23.3
ꢁ17.2
ꢁ21.8
ꢁ 9.2
ꢁ 6.9
ꢁ0.8
ꢁ1.3
ꢁ0.8
ꢁ0.5
ꢁ0.8
ꢁ0.3
ꢁ0.2
ꢁ 42.647
ꢁ 30.001
ꢁ 27.748
a
Experiments were carried out in PBS buffer, pH 7.4, containing 100 mM NaCl as described in Section 4.
b
Changes due to drug–DNA complexes. The changes are calculated using non-rounded values of the thermodynamic parameters. A quantitative estimate of the binding
parameters was obtained by subtracting the values describing the thermal transition of pure DNA from those derived for the drug–DNA. Complexes: DDH =
H ꢁ Ho,
DDS = So, DDG = G ꢁ Go (the zero index relates to pure DNA).
S ꢁ
D
D
D
D
D
at a saturating concentration of DADS analogs (r = 5, ‘r’ refers to
DADS analogs/ DNA ratio). As evident from Table 2, binding of
any of the compounds with CT DNA resulted in Tm being raised,
indicative of stabilization of CT DNA duplex. As the ‘r’ increases
from 0 to 5, the thermal stabilities of CT DNA increase concomi-
tantly. Inspection of Table 2 reveals that compounds 5, 8, and 11
have no effect on the melting temperature, whereas compounds
6 (Fig. 2A), 7, 9, and 10 decrease the Tm of the d(AT)10 by 3 °C,
1 °C, 2 °C, and 1 °C, respectively. Further inspection of Table 2
and Figure 2B reveals that all the compounds show an increase
in the melting temperature of the GC duplex. As the drug/DNA ra-
tio increases from 0 to 5, the thermal stabilities of GC duplex in-
crease by 4–7 °C. Compound 5 has the most stabilizing effect on
the d(GC)7. These results suggest that DADS analogs preferentially
bind to GC base pairs rich regions of double-stranded DNA. These
results are also comparable to those obtained for Daunomycin–
DNA13 and Crytolepin-DNA interaction,14 which are known
intercalators.
2.2.3. Circular dichroism (CD)
The melting experiments have provided us with useful informa-
tion on the stability of drug–DNA duplexes; however, little can be
discerned about the physical structure and mode of drug–DNA
interactions from these experiments. CD spectropolarimetry pro-
vides a second means of detecting and characterizing ligand bind-
ing. Binding of an achiral molecule within a chiral environment,
such as that afforded by right handed double helical DNA, can lead
to induced optical activity for the bound species (ligand).15 This is
manifested in the appearance of a CD absorption band, assignable
to the ligand but observed only in the presence of DNA. Panels A–C
of Figure 3 show the representative CD spectra from 200 nm to
340 nm obtained by incremental titrations of compound 6 into
CT DNA (panel A), d(GC)7 (panel B), and d(AT)10 (panel C) dissolved
in an appropriate buffer.
Figure 2. Derivative plot (Tm) for the d(AT)10 (A) and d(GC)7 (B) duplexes (solid
lines) containing a compound 6 (dashed lines, respectively). The concentration of
the d(AT)10 and d(GC)7 duplexes was 2.5 lM and compound 6 was 12.5 lM (r = 5).
Melting temperature measurements were performed in 20 mM NaH2PO4/Na2HPO4
buffer (pH 7.4) containing 100 mM NaCl.
CD signals at 280 nm and 250 nm depend on the ratio of drug to
DNA. The induced CD signals around 280 nm increase with the in-
crease in ratio of drug to DNA. These spectral changes are an
important evidence for the interaction of the drugs with the base
pairs of CT DNA.16 Since DADS derivatives are achiral molecules,
As displayed in Figure 3A, a maximum (positive CD) at 280 nm
and a minimum (negative CD) at 250 nm are induced when
compound 6 is bound to CT DNA. The intensities of the induced

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S. K. Rai et al. / Bioorg. Med. Chem. 16 (2008) 7301–7309
Figure 3. CD spectra showing the binding of compound 6 to calf thymus DNA (A), d(GC)7 (B), and d(AT)10 (C). The drug/DNA ratios were 0, 1.0, 2.0, 3.0, 4.0, and 5.0. CD
titration plots and best-fit curves for the compound 6 binding to calf thymus DNA (D) and d(GC)7 (E). Measurements were performed in 20 mM NaH2PO4/Na2HPO4 buffer (pH
7.4) containing 100 mM NaCl.
optical activity is generated in the asymmetric environment of the
DNA double helix and this induced CD signal is considered to arise
from the interaction of the transition moment of the chromophore
(DADS analogs) with the transition moment of the chirally dis-
posed surrounding DNA bases. The CD spectrum of DADS–CT
DNA complex is similar to that of some other intercalators such
as 9 aminoacridine, Daunomycin, and Nogalomycin.17
Ligand (Induced CD) spectra of DADS derivatives with d(GC)7
duplex differ significantly from that of d(AT)10 duplex. CD analyses
of Compound 6 binding to GC duplex have shown the presence of
induced cotton effect signal at 240 nm region (Fig. 3B). The varia-
tion in molar ellipticities around 240 nm regions at increasing
drug/DNA ratio displayed first a negative signal which then be-
comes positive. These signals demonstrate that DADS analogs bind
to GC rich DNA. Inspection of Figure 3C reveals that with com-
pound 6, AT duplex does not show any induced CD signal. More-
over, the maxima (at 270 nm) intensity gradually decreases with
increasing concentration of ‘r’. At higher drug/DNA ratio (5:1),
there is large negative intensity at 250 nm. Since the CD signal
around 220–240 nm is characteristic of the DNA bound DADS ana-
logs and positive CD bands were not observed with AT rich duplex,
we can conclude that this is indicative of an intercalative mode of
binding of the analogs, in GC rich regions of DNA.18 Other analogs
showed the same spectral behavior as that of compound 6.
2.6. The obtained ‘n’ value suggested that at least two DADS mole-
cules bound to these duplexes.
2.2.4. Binding affinity analysis
We have used UV-derived Tm data to obtain a quantitative esti-
mate of binding constants, which is a direct measure of drug
apparent binding affinity.20,21 The enthalpy (dHwc) of CT DNA and
GC duplex melting was determined by DSC (Table 2). Eq. (1) was
used to obtain the binding constant of the drug at the DNA melting
temperature, in the presence of saturating concentration of the
drug (12.5 lM). A value of ‘n’ used in Eq. (1) is based on the size
of drug binding site determined by CD titration spectra.15 Our re-
sults indicate that compound 6 exhibits greater binding affinity
for
CT
DNA
(KTm = 4.027 ꢃ 105 Mꢁ1
)
and
d(GC)7
(KTm = 6.9 ꢃ 106Mꢁ1), whereas compound 11 shows the least bind-
ing affinity with d(GC)7 (ꢀ102). Specifically, we find the following
hierarchy for DADS analogs binding to various duplexes (data not
shown);
dðGCÞ7 > CT DNA > dðATÞ10
This result is also consistent with our findings of thermal denatur-
ation and CD titration data that all tested DADS analogs showed
remarkable preference for alternating GC sequence, binding more
efficiently to these than to natural CT DNA or AT rich sequences.
Single wavelength titration curve extracted from CD titration
spectra of compound 6 binding to CT DNA and d(GC)7 is depicted
in Figure 3D and E, respectively. Here, ‘r’ (DADS/duplex ratio) val-
ues provide estimate for apparent number of base pairs influenced
by each event.19 The ellipticity values at 280 nm (CT DNA) and
275 nm (d(GC)7) were plotted against the compound 6/duplex ra-
tios. The slopes for d(GC)7 were steep, and reached a plateau within
the tested range of drug concentrations. On the other hand, the
slopes obtained for CT DNA were gentle, and the increase in the
signal intensity persisted at the high drug concentrations in these
cases. The DADS analogs binding to each duplex were analyzed by
curve fitting of the CD titration data. Analysis of the results shows
that CT DNA shows apparent binding site size (‘n’) of 2.3–2.5 with
all the DADS analogs, whereas d(GC)7 has a binding site size of 2.4–
2.2.5. Thermodynamic profile
Differential scanning calorimetry (DSC), which is an informative
method of obtaining direct data on thermal stability of drug–DNA
complexes, was employed to characterize the influence of the
DADS analogs on the thermal stability and energetics of CT DNA,
d(AT)10, and d(GC)7 duplexes.22 The melting enthalpy,
D
Hmelt, of
nucleic acid complexes with either groove binding or intercalating
ligands is higher than Hmelt of pure nucleic acids, whereas the en-
tropy of ligand binding ( Sbind) can have both positive and nega-
D
D
tive values, which mainly results from changes in the
environment of the hydrated structure of the ligand–nucleic acid
complex relative to the free nucleic acid.23 The thermodynamic
parameters of the DNA duplexes and its complexes with DADS ana-

S. K. Rai et al. / Bioorg. Med. Chem. 16 (2008) 7301–7309
7305
logs, calculated using equations, described in section materials and
methods, are summarized in Table 2.
Hence, from the results of thermodynamic parameters, it is evi-
dent that compounds 5 forms the most stable complexes with
d(GC)7 duplex. This result also shows that DADS analogs have more
specificity toward GC rich DNA.
The thermodynamic origin of stabilization of DADS analogs–
DNA complex is both enthalpic and entropic driven. All the DADS
analogs showed favorable free energy of binding with CT DNA
and GC duplexes (CT DNA, DDG = ꢁ0.2 to ꢁ1.3 kcal/mol and
d(GC)7, DDG = ꢁ0.2 to ꢁ2.2 kcal/mol). This favorable free energy
of binding was derived from a large negative enthalpy contribution
(CT DNA, DDH = ꢁ2.4 to ꢁ8.1 kcal/mol and d(GC)7, DDH = ꢁ1.14 to
ꢁ9.19 kcal/mol, Figure 4B) but was opposed by the entropic contri-
bution (CT DNA, DDS = ꢁ6.9 to ꢁ23.3 cal/mol Kꢁ1 and d(GC)7,
DDS = ꢁ3.1 to 23.7 cal/mol Kꢁ1). Hence, the enthalpy term is favor-
able for interaction whereas entropy term is unfavorable, which is
probably due to the more ordered structure of the hydration envi-
ronment of drug–DNA complexes compared to that of pure DNA.23
Hence, DADS analogs stabilize the duplexes by favorable enthalpic
gains.
2.3. Cytotoxicity of DADS analogs against primary rat
hepatocytes
The in vitro cytotoxicity of DADS derivatives was evaluated
using MTT method.25 The IC50 values (cytotoxicity potency indices)
of DADS analogs (5–11) against primary rat hepatocytes in culture
are summarized in Table 3. After 24 h incubation, increasing con-
centrations of DADS analogs (1–1000 lM) led up to a gradual de-
crease of the fraction of surviving cells in a dose dependant
manner (20–98%). The DADS analogs exhibited low or insignificant
cytotoxicity with high IC50 values. Among the derivatives, com-
pound 11 was found to be most potent in inhibiting the cell prolif-
Closer examination of Table 2 reveals that all the d(AT)10–DADS
analogs complexes exhibit positive DDH value, that is, enthalpy ef-
fects destabilize the duplex (Fig. 4A). It may be possible that DADS
analogs disrupt the stacking and H-bonding interaction of neigh-
boring AT bases or may be an effect of hydrophobic DADS analogs
being forced in juxtaposition to highly charged DNA.24 These
destabilizing effects are counter balanced by positive DDS values
eration with the lowest IC50 value of 140 lM suggesting that the
substitution on phenyl ring with fluoro or trifluoro methyl groups
leads to considerable increase in cytotoxicity. On comparison with
different statins, we have found the DADS analogs to be less cyto-
toxic than the statins, such as Lovastatin (IC50 = 90
statin (125 M), Simvastatin (>100 M) in primary rat
hepatocyte culture.26
lM), Atorva-
l
l
(d(AT)10
duplex.
,
DDS = +4.6 to +16.5 cal/mol Kꢁ1), which stabilize the
2.4. Effect of DADS analogs on serum and hepatic lipid content
Our results suggest that DADS analogs, when interact with CT
DNA and d(GC)7, stabilize the duplex by favorable enthalpic gains
and may interact in similar fashion but differently with AT duplex.
Thus, the order of thermodynamic stabilization for these com-
pounds is
Hypercholesterolemia was induced in rats by feeding them a
cholesterol rich diet for 2 weeks (5% cholesterol) (Table 4). An ath-
erogenic diet induced a marked increase in serum as well as tri-
glycerides levels (p < 0.001). At the end of the study, animals
treated with DADS analogs (Group V–XI) showed significantly low-
er levels of serum and hepatic cholesterol with respect to hyper-
cholesterolemic rats. DADS analogs also reduced the triglycerides
levels significantly (p < 0.001) with respect to hypercholesterol-
emic rats (Group II). Compounds 5 and 6 showed the maximum
diminution in serum and hepatic cholesterol as well as in triglycer-
ides levels with respect to hypercholesterolemic rats, and these
antihyperlipidemic properties were found to be comparable to that
of Lovastatin treated hypercholesterolemic rats.
dðGCÞ7ð5 > 6 > 7 > 8 > 9 > 10 > 11Þ > CT DNAð6 > 5 ꢄ 7 ꢄ 9
> 8 > 10 > 11Þ > dðATÞ10
3. Conclusion
In this paper, we report the synthesis and characterization of six
new analogs of DADS, by 1H NMR and mass spectroscopy. To fur-
ther evaluate their potential pharmaceutical properties, the DNA-
binding activity has been investigated by absorption, circular
dichroism, and DSC measurements. The experimental results indi-
cate that DADS analogs bind to GC rich regions of DNA duplexes.
Spectrophotometric titrations suggest that there is large hypo-
chromic shift, moderate red shift and substantial increase in the
thermal denaturation temperature, when DADS analogs bind to
CT DNA and d(GC)7 duplexes. DADS analogs which do not possess
Table 3
IC50 cytotoxicity values (lM) of DADS derivatives against primary rat heptocytes
(data derived from the mean of three independent assays)
Compound
Rat hepatocyte culture (IC50, lM)
5
6
7
8
9
10
11
335
425
350
365
275
155
140
Figure 4. DSC thermograms for the d(AT)10 (A) and d(GC)7 (B) duplexes(solid lines)
containing a compound 6 (dashed lines, respectively). The concentrations of the
d(AT)10 and d(GC)7 duplexes were 100
lM, and the buffer conditions were 20 mM
sodium phosphate, pH 7.4 and 100 mM NaCl.

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S. K. Rai et al. / Bioorg. Med. Chem. 16 (2008) 7301–7309
intrinsic optical activity become optically active when bound to
DNA (CT DNA and d(GC)7 duplex) and exhibit a CD spectrum sim-
ilar to known intercalators. The thermodynamic profile demon-
strates that compounds 5 and 6 have the most stabilizing effect
on GC rich DNA which is predominantly enthalpy driven. Our
MTT assay revealed that compounds 5 and 6 showed minimal toxic
effect in primary rat heptocytes culture, whereas compound11
shows the least IC50 value as well as the most destabilizing effect
on DNA duplexes.
All the results discussed above are also consistent with the anti-
hyperlipidemic properties of these DADS compounds evaluated by
biochemical determinations. The compounds 5 and 6, thus, seem to
be well suited for further work aiming at the development of new
antihyperlipidemic agents, which can act as specific modulators of
gene activity (HMGR gene).
4. Experimental
4.1. General
All organic solvents and common reagents were procured from
the Merck India Ltd. All the derivatives of benzaldehyde and So-
dium metal were procured from Aldrich Chemical Company, Inc.
USA. TLC was carried out on commercially available flexible TLC
silica gel (silica gel 60 F254) plates (E Merck, Germany). The purity
of all organic compounds was confirmed by TLC, 1H NMR, IR, and
Mass. 1H NMR spectra were recorded in Bruker Spectrospin Avance
300 instrument operating at 300 MHz in CDCl3 or DMSO using TMS
as an internal standard. IR spectra (KBr) were recorded on a Perkin-
Elmer BX FT-IR instrument. Melting points were determined on a
Buchi melting point B-450 instrument. Mass spectra were recorded
on a Qstar (Appllied biosystem) ESI-MS mass spectrometer.
Absorption spectra were determined using Cary varian-300
spectrophotometer. Oligodeoxynucleotides and calf thymus DNA
(CT DNA) were purchased from Sigma–Aldrich Chemical Pvt Ltd
(USA). CT DNA solution was prepared by swelling it (1 mg/ml)
overnight at 4 °C in phosphate buffer, then filtering and adjusting
the concentration to the desired value with buffer. An extinction
coefficient of 6600 Mꢁ1 cmꢁ1 has been used. The alternating AT
(20 bp, d(AT)10) and GC (14 bp, d(GC)7) self-complementary oligo-
mer duplexes were used for the determination of sequence speci-
ficity. Their concentration was determined using
a molar
extinction coefficient of 217.8 mMꢁ1 cmꢁ1 and 136.8 mMꢁ1 cmꢁ1
for d(AT)10 and d(GC)7 oligomer duplex, respectively. For experi-
ments involving UV spectroscopy and circular dichroism, titrations
of the drug with DNA, at different drug/DNA ratios (‘r’), were per-
formed with fixed DNA concentration (2.5
centrations of the drug substrates from 0
l
M) and increasing con-
lM to 12.5 M, until no
l
further changes in drug–DNA spectra were observed. All the mea-
surements were performed in 20 mM sodium phosphate (NaH2-
PO4/Na2HPO4) buffer containing 100 mM of NaCl, pH 7.4. The
absence of precipitates or changes in the absorption spectra of
the stock solutions was regularly verified.
4.2. Synthesis of bis-(2-hydroxy ethyl) disulfide (2)
To a solution of dichloromethane (40 ml) 3 ml (40 mM) of 2-
mercaptoethanol (1) was added. To this, 40 ml of 10% aqueous
potassium hydrogen carbonate (KHCO3) and a solution of 2 ml Bro-
mine (40 mM) in 10 ml dichloromethane was added to the reaction
vessel whereupon the color of bromine disappeared during addi-
tion. The mixture was stirred and cooled in ice water bath. The or-
ganic phase was separated, and the aqueous phase was extracted
with (3ꢃ 10 ml) dichloromethane. The organic phases were com-
bined and dried with anhydrous Na2SO4. The solvent was evapo-

S. K. Rai et al. / Bioorg. Med. Chem. 16 (2008) 7301–7309
7307
rated, and the compound was further purified by column chroma-
tography. (1 g, 48.8%); IR (KBr, cmꢁ1): 3330 (O–H str.), 2925 (C–H
2 ꢃ 1H(c)]; 7.21–7.49 (m, 2 ꢃ 4H, aromatic protons): MS
(C18H16Cl2S2): m/e 367 (M+).
str.), 696 (C–S str.); 1H NMR (300 MHz; CDCl3):
d 2.88 [t,
2 ꢃ 2H(a), J = 1.05 Hz]; 2.99[s, 2 ꢃ 1H (OH)]; 3.89 [t, 2ꢃ 1H(b),
4.5.3. Conversion of 4 to bis[3-(2,40-dichlorophenyl)prop-2-
ene]disulfide (7)
J = 1.05 Hz]; MS(C4H10O2S2): m/e 154 (M+), 92(100).
An orange solid compound. Yield; 77.8%: Mp 155–157 °C: IR
(KBr, cmꢁ1): 3048 (-Csp2–H str.), 2930(-Csp3–H str.), 1623 (–C@C
str.), 477 (–S–S str.), 691(–C–S str.), 670 (–C–Cl str.):1H NMR
(300 MHz, CDCl3); d 1.18 [s,2 ꢃ 2H(a)]; 4.86–4.89 [m, 2 ꢃ 1H(b)];
5.22–5.29[m, 2 ꢃ 1H(c)]; 7.35–7.61(m, 3H, aromatic protons):
MS(C18H14Cl4S2): m/e 435 (M+).
4.3. Synthesis of bis-(2-bromoethyl)disulfide (3)
To a solution of 48% HBr (70 ml) 46 ml of concd H2SO4 was
added with continuous stirring. The flask was pre-cooled in an
ice bath, and to the resulting ice-cold solution 2 g of compound 2
was added dropwise. The reaction mixture was left to stir for
24 h at room temperature. After this the reaction mixture was
heated for 3 h on a steam bath. Dichloromethane (10 ml) was
added to the reaction mixture. The upper layer was taken, washed
with water, 10% Na2CO3 solution, and then dried over anhydrous
Na2SO4. The dichloromethane was evaporated, and the desired
compound 3 was obtained. (2 g, 59%): IR (KBr, cmꢁ1); 617 (C–S
str.), 563 (C–Br str.), 445 (–S–S str.): 1H NMR (300 MHz; CDCl3);
d 2.95 (t, 2 ꢃ 2H(a), J = 1.0 Hz); 3.67(t, 2 ꢃ 2H(b), J = 1.0 Hz): MS
(C4H8Br2S2): m/e 280 (M+).
4.5.4. Conversion of 4 to bis[3-(20-bromophenyl)prop-2-ene]-
disulfide (8)
Yield: 190 mg (72.3%): Mp ꢁ220 to 224 °C: IR (KBr, cmꢁ1); 3055
(–Csp2–H str.); 2927 (–Csp3–H str.), 1685 (–C@C str.); 427(–S–S
str.); 691(–C–S str.), 560 (–C–Br str.): 1H NMR (CDCl3): d1.66[s,
2 ꢃ 2H(a)]; 4.00–4.23 [m, 2 ꢃ 1H(b)]; 4.77[s, 2 ꢃ 1H(c)]; 7.26–
7.34 (m, 2 ꢃ 4H, aromatic protons): MS(C18H16Br2S2); m/e 455
(M+).
4.5.5. Conversion of 4 to bis[3-(2,40-dibromophenyl)prop-2-
ene]disulfide (9)
4.4. Conversion of (3) to the analogous phosphonium bromide
(4)
Yield: 190 mg (79.2%): Mp ꢁ100 to 130 °C: IR (KBr, cmꢁ1); 3046
(Csp2–H str.); 3005 (broad band); 2936 (–Csp3–H str.), 1685 (–C@O
str.); 690(–C–S str.), 560 (–C–Br str.): 1H NMR (300 MHz, CDCl3);
d1.85 [d, 2 ꢃ 2H(a), J = 1.60 Hz]; 6.08 [m, 2 ꢃ 1H(b)]; 6.38
[2 ꢃ 1H(c)]; 7.02–7.48 (m, 8H, aromatic protons): MS
(C18H14Br4S2); m/e 615 (M+).
To a solution of compound 3 (2.8 g, 0.01 mmol) in dry dimeth-
ylformamide (15 ml) triphenyl phosphine (5.5 g, 0.02 mmol) was
added under nitrogen, and the mixture was refluxed for 5 h. The
mixture was allowed to cool to room temperature, and 15 ml of
hexane was added. The desired phosphonium salt precipitated
out. The solution was filtered, washed with hexane, and dried.
White crystals of (4) were obtained (5.6 g, 71.7%): Mp, 130–
132 °C: IR (KBr, cmꢁ1); 3053 (C–H str.), 691 (C–S str.): 1H NMR
4.5.6. Conversion of 4 to bis[3-(40-fluorophenyl)prop-2-ene]-
disulfide (10)
Yield: 76.5%: Mp ꢁ145.67 °C: IR (KBr, cmꢁ1): 3435 (O–H str.);
3054(–Csp2–H str.), 2918(–Csp3–H str.); 691(–C–S str.), 1100(–C–F
str.): 1H NMR (300 MHz, CDCl3); d1.41 [t, 2 ꢃ 2H(a), J = 1.77 Hz];
4.00–4.09 [m, 2 ꢃ 1H(b)]; 4.61–4.67 [m, 2 ꢃ 1H(c)]; 6.89 [d,
2 ꢃ 2H(d), J = 8.49 Hz]; 7.29 [d, 2 ꢃ 2H(e), J = 7.95]: MS
(C18H16F2S2): m/e 335 (M+).
(300 MHz, CDCl3);
d
3.00 (t, 2 ꢃ 2H(a), J = 2.82 Hz); 3.75 (t,
2 ꢃ 2H(b), J = 3.66 Hz); 7.05–7.45(s, 6 ꢃ 5H). MS(C40H38Br2P2S2):
m/e 804 (M+).
4.5. General procedure for the preparation of 5–11
4.5.7. Conversion of 4 to bis[3-{(40-trifluoromethyl)phenyl}-
prop-2 ene]disulfide (11)
To a stirred solution of compound 4 in super-dry ethanol appro-
priate substituted benzaldehyde (2 mM) was added in the pres-
ence of sodium ethoxide (formed by the addition of sodium,
25 mM, in super-dry ethanol). The reaction was set up in absolute
dry condition by flushing Nitrogen gas. The mixture was stirred at
the reflux temperature for 24 h. The reaction mixture was then
poured into ice. The product precipitated out and was filtered.
The residue was purified by column chromatography and elution
with hexane/ethyl acetate (80:20).
Yield: 70%: Mp ꢁ184 to 186 °C: IR (KBr, cmꢁ1); 3435(O–H str.);
3054(–Csp2–H str.), 2918(–Csp3–H str.); 691(–C–S str.), 610 (–C–F
str.): 1H NMR (300 MHz, CDCl3); d1.66 [s, 2 ꢃ 2H(a)]; 4.71–4.79
[m, 2 ꢃ 1H(b); 5.06–5.30 [m, 2 ꢃ 1H(c)]; 7.21–7.68 (m, 2 ꢃ 4H,
aromatic protons): MS (C20H16F6S2); m/e 435 (M+).
4.6. Spectrophotometric titrations
Titration of the drugs with DNA covering a large range of DNA–
phosphate/drug ratios was performed by keeping the DNA concen-
tration constant and varying the concentration of the analogs of
DADS. Initial concentrations of DNAs and DADS analogs were kept
4.5.1. Conversion of 4 to bis[3-(40-nitrophenyl)prop-2-ene]-
disulfide (5)
An organic solid. Yield; 78.6%: Mp 140–142 °C: IR (KBr, cmꢁ1);
3055 (Csp2–H str.), 2948 (Csp3–H str.), 1583 [–N@O str. (anti)],
1388 cmꢁ1 [–N@O str. (sym)], 690 (C–S str.): 1H NMR (CDCl3); d
1.83 [d, 2 ꢃ 2H(a), J = 1.87 Hz]; 6.00[m, 2 ꢃ 1H(b)]; 6.30 [d,
2 ꢃ 1H(c) J = 1.83]; 6.92–7.35(m, 8H, aromatic protons):
MS(C18H16N2O4S2): m/e 380 (M+). Compound 5 is the same as that
reported previously, as the spectral data are all identical.6
as 2.5 lM and 0 lM, respectively. The experiments were con-
ducted at 25 °C in phosphate buffer (pH 7.4) containing 100 mM
NaCl. After mixing, the solution was allowed to stand for 15 min,
the corresponding absorption spectra in the region from 200 nm
to 340 nm were measured at room temperature.
4.7. Tm measurements
4.5.2. Conversion of 4 to bis[3-(20-chlorophenyl)prop-2-ene]-
disulfide (6)
The thermal denaturation experiments were performed on a
Varian make CARY-100 Spectrophotometer equipped with a peltier
thermo-programmer and interfaced with a Pentium III computer
for data collection and analysis. The temperature dependence on
the absorption value of the DNA was monitored at 260 nm. The
Yellow solid. Yield: 77.0%: Mp ꢁ115 to 117 °C: IR (KBr, cmꢁ1);
3048 (–Csp2–H str.), 2930 (–Csp3–H str.), 1623 (–C@C str.), 477
(–S–S str.), 691(–C–S–str.), 670 (–C–Cl–str.): 1H NMR
(300 MHz,CDCl3); d 1.25[s, 2 ꢃ 2H(a)]; 4.78[s, 2 ꢃ 1H(b)]; 5.29 [s,

7308
S. K. Rai et al. / Bioorg. Med. Chem. 16 (2008) 7301–7309
temperature of the cell holder was increased from 25 °C to 100 °C
at a rate of 1 °C/min. A teflon-coated temperature probe, immersed
directly in a control cuvette, measured the sample temperature.
The sample solutions were overlaid with paraffin-oil to prevent
evaporation. The melting temperature (Tm) was taken to be the
midpoint of the hyperchromic transition determined from first
software (Microcal). The free energy of duplex formation at 25 °C
G25) was calculated using the standard thermodynamic relation-
(D
ship given in Eq. (2) and the corresponding
DHcal and
DScal values:
D
Gꢂ
¼
D
Hꢁcalð298:15Þ
DScal:
ð2Þ
25
The duplexes were dissolved in the buffer containing 20 mM so-
dium phosphate (NaH2PO4/Na2HPO4), pH 7.4 and 100 mM NaCl.
derivatives plots. All
DTm values are reported as the means from
at least three determinations. The accuracy of the reported Tm val-
ues is 0.5 °C. The duplexes were dissolved in the buffer containing
20 mM sodium phosphate (NaH2PO4/Na2HPO4), pH 7.4 and
100 mM NaCl.
4.11. Cell culture and cytotoxicity assay
Hepatocytes were isolated from rat liver as previously de-
scribed.28 Hepatocytes were collected and cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine ser-
um in the Thermo Forma Direct Heat CO2 incubator with 5% CO2–
95% O2 at 37 °C. The effect of DADS analogs on the viability of the
hepatocytes was determined by the MTT assay, which is based on
the reduction of a tetrazolium salt by mitochondrial dehydroge-
nase in the viable cells.25 The cells were seeded in a 96-well plate
4.8. Circular dichroism (CD)
CD Spectra were recorded on JASCO-715 Spectropolarimeter
interfaced with an IBM PC compatible computer, calibrated with
D-Camphor sulphonic acid. Five scans of the spectrum were col-
lected over the wavelength range of 220–340 nm at a scanning rate
of 100 nm/min. The desired ratios of compound to DNA were ob-
tained by adding compound to the cell containing a constant
amount of DNA. The duplexes were dissolved in the buffer contain-
ing 20 mM sodium phosphate (NaH2PO4/Na2HPO4), pH 7.4 and
100 mM NaCl. The average of multiple scans was used for analysis.
The scan of the buffer alone recorded at room temperature was
subtracted from the average scans for each DNA duplex. Data were
collected in units of millidegrees versus wavelength and normal-
ized to total DNA concentration.
at a density of 1 ꢃ 105 cells/ml and treated with 1–1000
l
M of
DADS analogs. After incubation for 24 h at 37 °C, 50
ll of the
MTT (Bio-Gene, USA) stock solution (2 mg/ml) was added to each
well to attain a total reaction volume of 200 l. After incubation
for 4 h, the plate was centrifuged at 800g for 5 min and the super-
natants were aspirated. The formazan crystals in each well were
l
dissolved in 150 ll DMSO, and the A540 was read on a scanning
multi-well spectrophotometer. All drug doses were tested in tripli-
cate and the IC50 values were derived from the mean OD values of
the triplicate tests versus drug concentration curves.
4.9. Determination of binding constants by thermal melting
study
4.12. Animals and biochemical estimations
Adult male Wistar rats, weighing 100–120 g each, were selected
from the stock colony maintained in our animal facility with daily
light/darkness cycle 14–10 h, with free access to food and water.
Experiments were performed according to the guidelines of the
Institutional Animal Ethics committee. They were given standard
sterilized commercial diet purchased from Brooke Bond India Ltd.
The animals were then divided into eleven groups of six animals
each. The control group received only rat chow and saline solution
(Group I): Control hypercholesterolemic group were fed with 5%
cholesterol in their diet for 2 weeks (Group II): The experimental
groups (Group III–XI) were fed with 5% cholesterol for 1 week
and then fed with cholesterol rich diet supplemented with Allicin,
Lovastatin and different DADS analogs (Compounds 5–11) in the
second week (20 mg/Kgwt, suspension in water, po), respec-
tively.10 The animals were immediately dissected to remove their
tissues, which were washed in ice-cold saline (0.85% NaCl). The
extraneous material was removed. Approximately 1 g of tissue
was kept for estimation of lipids and the remaining was used for
biochemical assays. The levels of cholesterol (hepatic and serum)
and triglycerides (hepatic and serum) were measured by standard
enzymatic assays (Sigma Chemical Co., St. Louis, MO, USA).
Profiles of absorbance at 260 nm versus temperature were mea-
sured between 25 °C and 100 °C, at a heating rate of 1 °C minꢁ1, in
the presenceof 100 mM of NaCl, in 20 mM sodium phosphate (NaH2-
PO4/Na2HPO4) buffer (pH 7.4) and saturating concentrations of
DADS derivatives (2.5 lM DNA (bp); 12.5 lM DADS compounds).
DNA melting temperatures (Tm) were used to calculate the binding
constant to different DNA duplexes at the DNA melting temperature
(KTm), using the equation derived by Crothers20 and McGhee21
:
1=To ꢁ 1=Tm ¼ ðR=ndHwcÞ lnð1 þ KTmaÞ
ð1Þ
m
where To is the UV-melting temperature of DNA duplex alone, Tm
m
is the melting temperature in the presence of saturating amounts of
the drug, dHwc is the enthalpy of DNA melting obtained by DSC, R is
the gas constant, KTm is the drug binding constant at Tm, ‘a’ is the
free drug activity, which is estimated by one half of the total drug
concentration, and ‘n’ is the size of the drug binding site.
4.10. Differential scanning calorimetry
Excess heat capacity (DCp) versus temperature profiles for the
thermally induced transitions of CT DNA, AT, and GC duplexes of
DADS analogs were measured using a Pyris 6-DSC Calorimeter
(Perkin-Elmer, USA). In the DSC experiments, the concentrations
Acknowledgments
of the CT DNA, d(AT)10, and d(GC)7 duplexes were 100 lM, the
The authors express their sincere gratitude to Prof. Vani Brah-
machari and Dr. Shrikant Kukreti, for their valuable suggestions.
The author S.K.R. thanks the Council of Scientific and Industrial Re-
search (CSIR), India, for the award of JRF and SRF. The author M.T.
thanks the Department of Science and Technology, Government of
India, for financial support.
heating rate was 60 °C/h and the maximum temperature was
100 °C. After reaching the maximum temperature the samples
were cooled at the same rate to the starting temperature of
25 °C. Here,
line subtracted and concentration normalized.27 The reference
scans were subtracted from the sample scans to obtain Cp versus
temperature profiles. Enthalpies ( Hcal) and entropies ( Scal) of
DCp is defined as excess heat capacity, which is base-
D
D
D
References and notes
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