Synthesis of novel 9-O-N-aryl/aryl-alkyl amino carbonyl methyl substituted berberine analogs and evaluation of DNA binding aspects

Article abstract of DOI:10.1016/j.bmc.2012.03.006

This manuscript describes the design and synthesis of three 9-O substituted analogs of plant alkaloid berberine to enhance the DNA binding affinity. Three analogs of berberine with aryl/aryl-alkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore were synthesized and characterized by NMR (¹HNMR and ¹³C NMR) and mass spectroscopy. The products were evaluated for their binding to calf thymus DNA by a wide variety of techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry. The results revealed that these analogs showed more than six times higher binding affinity to DNA compared to berberine. From fluorescence and absorbance studies it was inferred that all the analogs bound to DNA non-cooperatively in contrast to the cooperative binding of the parent alkaloid berberine. The viscosity and ferrocyanide quenching experiments confirmed that the analogs are stronger intercalative binders to DNA useful for potential biological applications. Stronger binding of the analogs was also inferred from circular dichroism studies and thermal melting experiments. Thermodynamics of the binding from isothermal titration calorimetry experiments revealed an entropy driven binding for these analogs compared to the enthalpy driven binding of berberine. The small but negative heat capacity change of the analogs along with the significant enthalpy-entropy compensation phenomenon observed established the involvement of multiple weak noncovalent interactions in the binding process. A comparative study also revealed that the spacer length is also significant in modulating the DNA binding affinities.

Full text of DOI:10.1016/j.bmc.2012.03.006

Bioorganic & Medicinal Chemistry 20 (2012) 2498–2505
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel 9-O-N-aryl/aryl–alkyl amino carbonyl methyl substituted
berberine analogs and evaluation of DNA binding aspects
Anirban Basu a,b, Parasuraman Jaisankar b, Gopinatha Suresh Kumar a,b,
⇑
a Biophysical Chemistry Laboratory, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India
b Chemistry Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
This manuscript describes the design and synthesis of three 9-O substituted analogs of plant alkaloid ber-
berine to enhance the DNA binding affinity. Three analogs of berberine with aryl/aryl–alkyl amino car-
bonyl methyl substituent at the 9-position of the isoquinoline chromophore were synthesized and
characterized by NMR (1HNMR and 13C NMR) and mass spectroscopy. The products were evaluated for
their binding to calf thymus DNA by a wide variety of techniques like spectrophotometry, spectrofluori-
metry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry. The results
revealed that these analogs showed more than six times higher binding affinity to DNA compared to ber-
berine. From fluorescence and absorbance studies it was inferred that all the analogs bound to DNA non-
cooperatively in contrast to the cooperative binding of the parent alkaloid berberine. The viscosity and
ferrocyanide quenching experiments confirmed that the analogs are stronger intercalative binders to
DNA useful for potential biological applications. Stronger binding of the analogs was also inferred from
circular dichroism studies and thermal melting experiments. Thermodynamics of the binding from iso-
thermal titration calorimetry experiments revealed an entropy driven binding for these analogs com-
pared to the enthalpy driven binding of berberine. The small but negative heat capacity change of the
analogs along with the significant enthalpy–entropy compensation phenomenon observed established
the involvement of multiple weak noncovalent interactions in the binding process. A comparative study
also revealed that the spacer length is also significant in modulating the DNA binding affinities.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 2 February 2012
Revised 29 February 2012
Accepted 1 March 2012
Available online 8 March 2012
Keywords:
9-O-N-Aryl/aryl–alkyl amino carbonyl
methyl substituted berberines
Synthesis
DNA binding
Intercalation
1. Introduction
ine binding to different DNA sequences, triplexes and quadruplex
structures.24–30 In respect of topoisomerase inhibition, the current
Berberine, the most extensively studied isoquinoline alkaloid,
has been characterized to possess multiple pharmacological ef-
fects.1–6 In recent years pharmacological activities of this alkaloid
as antihypertensive, antiinflammatory, antioxidant, antidepressant,
antidiarrhoeal, cholagogue, hepatoprotective and as antimicrobial
agent have been demonstrated.7–14 The strong antineoplastic ef-
fects of berberine appears to suppress the growth of a wide variety
of tumor cells.15,16 Nucleic acid binding properties in terms of
duplex and quadruplex structures and topoisomerase inhibition
activities were tested for berberine and several derivatives17–33
and the anticancer activity1,3 may be interpreted in relation to the
strong DNA binding affinity and enzyme inhibition property. An
intercalation model of DNA binding has been established for berber-
ine from the recent X-ray data.34 In several recent articles were also
described the selectivity, specificity and thermodynamics of berber-
model suggests that an intercalative as well as a minor groove bind-
ing component of berberine may be equally important.35 Substitu-
tions on the isoquinoline ring have been an important strategy to
enhance the efficacy of berberine. Enhanced effect of 9-substituted
analogs of berberine on drug-topoisomerase II interactions is well
documented from an early investigation based from a comparative
study of 9-ethoxycarbonyl berberine, 9-N,N-dimethylcarbamoyl
berberine and 12-bromo berberine (methoxy group at 9-position).36
The 12-bromo analogue was a stronger DNA binding agent but a
much weaker enzyme poison both in vitro and in cell-based assays
compared to the others. The results suggested that the proposed
drug domain for DNA intercalation is not a major determinant of en-
zyme inhibition for simple berberine analogs. Rather, the 9-substi-
tuent within the domain has a major influence, presumably by
facilitating drug interaction with enzyme and/or enzyme-DNA com-
plexes. Various derivatives of berberine including those with substi-
tutions at the 9-position were synthesized or prepared as
chemically modified compounds in order to enhance the pharmaco-
logical activities19,21,22 as well as DNA binding affinities.19,20,31,32
The binding affinities of berberine with duplex DNA, usually in
the range of 1.0–2.0 ꢀ 105 Mꢁ1, are modest and necessitate the
⇑
Corresponding author at: Biophysical Chemistry Laboratory, CSIR-Indian Insti-
tute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India.
Tel.: +91 33 2472 4049/2499 5723; fax: +91 33 2472 3967.
E-mail addresses: gskumar@iicb.res.in, gsk.iicb@gmail.com (G. Suresh Kumar).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.006

A. Basu et al. / Bioorg. Med. Chem. 20 (2012) 2498–2505
2499
appropriate structural modification as to serve as
a
novel
2.2. Spectroscopic experiments: Elucidation of the binding
affinity parameters
DNA-binding agent with enhanced binding affinities. In this paper,
we describe the synthesis of three new 9-O-substituted berberine
derivatives having N-aryl/aryl–alkyl amino carbonyl methyl sub-
stituent at the 9-position, their enhanced DNA-binding affinities
and the detailed thermodynamic aspects of the interaction. These
analogs have been synthesized from the linkage of berberrubine
with the intermediates 4a–b, with the aim to assess the effect of
the modification at this position on the mode, affinity and energet-
ics of DNA binding. These functional substituents used in the de-
sign of DNA-binders, we believe, are expected to increase binding
affinities through hydrogen bonding and/or electrostatic interac-
tion with DNA. Thus, the interaction of these berberine derivatives
with structural alteration at the 9-position with DNA may provide
new insights into the different aspects of association process of
berberine with DNA.
The DNA binding studies of these analogs in comparison with 1
were performed initially using absorption and fluorescence spec-
troscopy with calf thymus DNA in 10 mM citrate-phosphate buffer
of pH 7.0 at 20 °C.38–42 Hypochromic and bathochromic effects
were observed in the visible absorption bands of berberine analogs
with three sharp isosbestic points (Fig. 1A) providing unambiguous
evidence for the formation of stable complexes with equilibrium
between free and bound alkaloid molecules. The bathochromic
and hypochromic effects of analogue-DNA complexes were similar
to those observed for intercalative ligand–DNA complexation43 and
are suggestive of strong intermolecular interactions involving the
overlap of the
P electron cloud of the alkaloid with that of the base
pairs. Berberine analogs have a weak intrinsic fluorescence with
maximum around 525 nm and a hump around 445 nm when ex-
cited at 345 nm that enhanced on binding with deoxyribonucleic
acid. Figure 1B shows the results from spectrofluorimetric titration
of 6 with DNA. Pronounced enhancement of the fluorescence
intensity on binding to CT DNA was observed for the analogs 6,
5a and 5b. Saturation was obtained at P/D (DNA base pair/alkaloid
molar ratio) 18, 27 and 34 for 6, 5a and 5b which is much lower
than that for 1 where saturation was achieved at a P/D of 38. The
binding affinities of the alkaloid analogs were estimated from Scat-
chard plots fitted to McGhee and von Hippel analysis.40 The bind-
ing of 1 was cooperative as evidenced by the positive slope in the
Scatchard plot at low r values while the binding of the analogs
studied was found to be non-cooperative (negative slope) (Fig. 1C
and D). The substitution at the 9-position of BC therefore switched
the binding from a cooperative to non-cooperative mode. The
intrinsic binding affinity (Ki) values obtained from absorption
and fluorescence spectral analysis are presented in Table 1. It can
be seen that the Ki values are significantly higher for the analogs
compared to 1 and the magnitude varied as 6 > 5a > 5b > 1. The
number of excluded sites (n) was found to be around 2–3 for each
of the analogs. The binding stoichiometry of these molecules to
DNA was estimated independently by continuous variation analy-
sis procedure (Job’s plot) from fluorescence.44
2. Results and discussion
2.1. Synthesis of berberine analogs
The synthetic route of 9-O-substituted berberine analogs is
shown in Scheme 1. Commercially available berberine chloride
(1, 1.0 g) when heated at 190 °C under vacuum (10–15 mm) for
45 min underwent selective demethylation to afford berberrubine
(2) in 65% yield (625 mg).37 To the solution of either 3a or 3b
(10 mmol) in dry dichloromethane was added chloroacetyl chlo-
ride (11 mmol) dropwise under ice-cold condition. The reaction
mixture was stirred at room temperature for 40 min and then it
was poured into ice-cold water to decompose the unreacted acid
chloride. The resulting solution was neutralized with saturated
NaHCO3 solution and the organic layer was extracted with chloro-
form (3 ꢀ 50 ml), dried over Na2SO4 and the solvent was removed
under reduced pressure. The resulting solid of 4a or 4b was utilized
to prepare the desired analogs 5a–b. To a solution of berberrubine
2 (1 mmol) in dry CH3CN (10 ml) was added compounds 4a or 4b
(1.2 mmol) and the reaction mixture was refluxed for 12 h. The
progress of the reaction was monitored by TLC. Then the reaction
mixture was cooled to room temperature and the solvent was re-
moved under reduced pressure. The crude product was chromato-
graphed on a neutral Al2O3 column and eluted with increasing
concentration of MeOH in CHCl3 (CHCl3/MeOH, 99:1–98:2) to af-
ford the desired berberine analogs 5a–b. Similarly, the berberine
analogue 6 was prepared from the reaction between berberrubine
(2) and 2-chloro-N-phenyl acetamide (Scheme 1).
2.3. Binding stoichiometry (Job plot)
For determining the stoichiometry, the alkaloid: DNA molar
ratio was varied keeping the total molar concentration constant.
The stoichiometry of binding was determined from the molar ratio
where maximal binding was observed. The plot of difference
Scheme 1. Reagents and conditions: (i) 190 °C, vacuum (10–15 mm Hg), 45 min; (ii) ClCH2COCl (1.1 equiv), DCM, rt, 40 min; (iii) CH3CN, reflux, 12 h.

2500
A. Basu et al. / Bioorg. Med. Chem. 20 (2012) 2498–2505
Figure 1. (A) Absorption spectra of 6 (curve 1) with increasing concentration of CT DNA (curves 2–8) in 10 mM CP buffer, pH 7.0. (B) Fluorescence spectra of 6 (curve 1) with
increasing concentration of CT DNA (curves 2–9) in 10 mM CP buffer, pH 7.0. (C) Scatchard plot for 6-CT DNA complexation from absorbance and (D) Scatchard plot for 6-CT
DNA complexation from fluorescence.
Table 1
Association constants and thermodynamic parameters of the binding of berberine and its analogs to CT DNAa
Compound
Spectrophotometry
Spectrofluorimetry
Isothermal titration calorimetry
G0 (kcal/mol) H0 (kcal/mol)
Ki ꢀ 10ꢁ5 (Mꢁ1
)
Ki ꢀ 10ꢁ5 (Mꢁ1
)
Ka ꢀ 10ꢁ5 (Mꢁ1
)
n
D
D
TD
S0 (kcal/mol)
b
1
6
5a
5b
3.64 0.014
8.93 0.044
6.85 0.023
5.92 0.020
3.64 0.012
9.14 0.041
6.72 0.025
5.98 0.017
1.28 0.05
7.96 0.04
6.33 0.07
5.18 0.02
1.85
2.00
2.44
2.86
ꢁ6.84
ꢁ7.91
ꢁ7.79
ꢁ7.65
ꢁ3.03
ꢁ1.29
ꢁ1.49
ꢁ1.79
3.81
6.62
6.30
5.86
a
Measured in 10 mM CP buffer, pH 7.0 at 20 °C.
b
For 1, Ki = Kcoop
ꢀ x where Kcoop is the cooperative of binding affinity and x is the cooperativity factor. n (1/N) is the stoichiometry that may be equated to the number of
base pairs spanned by the binding of an alkaloid molecule.
fluorescence intensity (
mol fraction of the alkaloids revealed a single binding mode in each
case. From the inflection point, ligand was found to be 0.334, 0.32,
DF) at the wavelength maxima versus the
v
0.29 and 0.26 for 1, 6, 5a and 5b, respectively. Thus, the number of
DNA base pairs bound per berberine analog can be estimated to be
around 1.99, 2.12, 2.45 and 2.85, respectively, for 1, 6, 5a and 5b.
The values of stoichiometry are in good agreement with the num-
ber of excluded sites obtained from the McGhee-von Hippel anal-
ysis of the spectroscopic data.
2.4. Optical melting results
The binding was further tested from optical thermal melting
studies.45 Double stranded calf thymus DNA under the conditions
of the experiment had a melting temperature (Tm) value of 65 °C
(Fig. 2). The melting temperature of the DNA enhanced in the pres-
ence of the analogs and at saturation
12.0 °C, respectively, were observed for 6, 5a and 5b against a
D
Tm values of 15.5, 14.0 and
Tm
D
of 9.0 °C for 1 under identical conditions (Fig. 2). Such high stabil-
ization of the DNA helix is essentially due to the strong binding of
Figure 2. Optical thermal melting profiles of CT DNA (j), CT DNA-1 (d), CT DNA-5b
(s), CT DNA-5a (N) and CT DNA-6 complex (h).

A. Basu et al. / Bioorg. Med. Chem. 20 (2012) 2498–2505
2501
these analogs and reflected better interaction probability of the
analogs compared to 1.
and 5b. From these results it can be inferred that the bound alka-
loid analogs are sequestered away from the solvent confirming
strong intercalative binding. The trend in the values suggests that
the Ksv of 1 is higher than that of 6 and for the others it varied as
1 > 5b > 5a > 6. Thus, this data suggests that 5b is better interca-
lated than 1, and 6 has a much better intercalation capability com-
pared to 5a and 5b. These conclusions were also reaffirmed by
viscosity studies where length enhancement was maximum with
6.
2.5. Intrinsic circular dichroism studies
The association between the berberine analogs and CT DNA was
studied by means of circular dichroism spectroscopy.32 CD spec-
trum of calf thymus DNA displayed a canonical B-form conforma-
tion with a large positive band at 270–280 nm and a negative band
at 248 nm. A small positive band at 210 nm was also apparent for
the B-form structure. These CD bands of the duplex DNA are caused
due to stacking interactions between the base pairs and the helical
structure that provide asymmetric environment for the bases.
Enhancement of the long wavelength band ellipticity of the DNA
was observed in presence of the analogs (Fig. 3). The extent of
enhancement was higher for the analogs when compared with
the parent alkaloid. This indicates a stronger intercalative binding
compared to 1 because of a larger lengthening and consequent
weakening of the base stacking interactions. Furthermore, this also
suggests closer interaction of the transition moments of the bound
analogs with that of the base pairs of DNA. The intercalative mode
of binding of these analogs has been independently verified by
fluorescence quenching and hydrodynamic methods.
2.7. Verification of intercalation by viscosity measurements
The intercalative binding of the analogs was tested by viscosity
measurements. Viscometric technique is a well established, and
reliable hydrodynamic method47,48 and the most stringent test
for establishing the extension of double stranded DNA helix on
intercalation. The original hypothesis of Lerman49 proposed that
the viscosity of a rod like nucleic acid would increase upon com-
plexation on intercalation to accommodate the stacked molecules
between the base pairs. This process would result in an increase
in the contour length of the DNA. Hence, to further probe the bind-
ing mode, the viscosity of the DNA solution was measured in pres-
ence of increasing concentrations of the alkaloid analogs and the
change in relative viscosities with varying D/P (alkaoid/DNA base
pair molar ratio) value were estimated (Fig. 4). The change was
found to be more rapidly pronounced for the analogs compared
to 1. Viscosity results are expressed as length enhancement esti-
mated with respect to a standard value (b) of 1.0 for a true interca-
lator corresponding to a length enhancement of 0.34 nm per bound
drug. The b values for 1, 6, 5a and 5b binding to DNA were found to
be 0.70, 0.84, 0.79 and 0.77, respectively. Thus, a much better DNA
intercalation scenario may be envisaged for the analogs compared
to the parent alkaloid berberine. Therefore, in the confirming vis-
cosity experiment we observed greater length enhancements for
6, 5a and 5b compared to 1 on intercalation to rod like DNA sug-
gesting better intercalation geometry compared to berberine sug-
gesting that the side chain contributes to the stronger binding.
2.6. Fluorescence quenching studies and elucidation of the
mode of binding
The intercalative DNA binding mode of berberine has been
unequivocally established from the recent X-ray studies.34 So it
may be presumed that, like berberine, these analogs also may
intercalate to DNA, but what is the effect of the 9-substituted side
chain on the intercalation mode? We probed the DNA binding
mode of the analogs in comparison with berberine at first using
ferrocyanide quenching experiments.26,46 This anionic quencher
would not be able to penetrate the negatively charged double helix
and if these analogs are bound inside the DNA helix strongly by
intercalation then little or no change in fluorescence is expected.
Stern–Volmer plots for the quenching of the fluorescence of ber-
berine analogs by the DNA clearly indicated that free molecules
were quenched efficiently. More quenching was observed in case
of 1 and less quenching for the bound analogs 6, 5a and 5b indicat-
ing the bound analogs are indeed located in a relatively more pro-
2.8. Energetics of the binding from isothermal titration
calorimetry studies
Isothermal titration calorimetry (ITC) was utilized to study the
interaction in more details.43 ITC enables the direct and accurate
estimation of the binding affinity and complete thermodynamic
parameters of the complexation. Compound 1 and its three analogs
tected environment than that of 1. The quenching constants (Ksv
)
calculated were in the range 200–230 Mꢁ1 for the unbound alka-
loid analogs and 90, 57, 48 and 37 Mꢁ1, respectively, for 1, 6, 5a
Figure 4. A plot of increase in helix contour length (L/Lo) versus
r for the
Figure 3. Circular dichroic spectra of CT DNA (curve 1) with increasing concentra-
tion of 6 in 10 mM CP buffer, pH 7.0, as represented by curves 2–10.
complexation of CT DNA with 6 (h), 5a (N), 5b (s) and 1 (d) in 10 mM CP buffer, pH
7.0, at 20 0.5 °C.

2502
A. Basu et al. / Bioorg. Med. Chem. 20 (2012) 2498–2505
bound to CT DNA exhibiting monophasic binding events that were
exothermic, resulting in negative peaks in a plot of power versus
time. First of all, exothermic binding was observed in each case
and the heat evolved with 1 was higher than that with its analogs.
Secondly, the binding of the analogs with double stranded DNA
was highly entropy dominated. Since the integrated heat data in
the ITC profiles showed only one binding event, they were fitted
to a single set of identical sites model. This was also based on
the results from single binding mode revealed from the Job plot
analysis. A representative ITC thermogram of the titration of 6 with
DNA is depicted in Figure 5. The thermodynamic parameters of the
interaction elucidated from a single site fitting protocol are pre-
sented in Table 1. It can be seen that the binding affinity values
energy (
D
G0) was ꢁ6.84 kcal/mol for 1 that enhanced for all the
analogs indicating enhanced binding preference. With the intro-
duction of the side chain the enthalpy change (
duced while the entropy change (T
D
H0) was greatly re-
D
S0) was remarkably increased
indicating the entropy domination of the binding for the analogs
compared to 1. The site size (n), which is the reciprocal of stoichi-
ometry (N) values, in the range 1.85–2.86, also enhanced going
from 1 to 5b and are in excellent agreement with the stoichiometry
values obtained from Job plot analysis. Comparison of the thermo-
dynamic parameters helps to elucidate the forces that govern the
complexation. First of all, with the introduction of the substituent
at 9-position a remarkable enhancement in binding affinity was
observed. Secondly, the addition of the side chain at the 9-position
enhanced the interaction free energy (in absolute values) by an
additional 1.07 kcal/mol for 6 compared to 1, indicating more
favorable contacts by the side chain on the DNA. It is also notewor-
thy that the n values have enhanced because of the involvement of
the side chain in the binding process. It is pertinent to observe that
the entropic contribution to the binding Gibb’s energy enhanced
significantly on going from 1 to 6, indicating clearly the entropy-
driven interaction and the strong positive entropy terms is sugges-
tive of the role of the substituent in the disruption and release of
water molecules from the grooves on intercalation of 6, 5a and
5b into the DNA helix. The overall binding affinity and the binding
site size values obtained from ITC analysis are in excellent agree-
ment with the affinity values and the number of excluded site val-
ues from spectroscopy.
for 6, 5a and 5b were 7.96 ꢀ 105 Mꢁ1
,
6.33 ꢀ 105 Mꢁ1 and
5.18 ꢀ 105 Mꢁ1, respectively, against the value of 1.28 ꢀ 105 Mꢁ1
for 1. A comparative bar chart of the Ka values is presented in the
Figure 6. The binding affinity values from ITC clearly suggested
an increase of affinity from 1 to 6 and a gradual decrease from
5a to 5b compared to 6. The stoichiometry (N) values in the range
0.54–0.35 also decreased going from 1 to 5b. The binding Gibb’s
2.9. Temperature dependence of the calorimetric data: Heat
capacity changes
The constant pressure heat capacity changes (D
Cp0) of berberine
and its analogs-CT DNA interactions were determined from the
temperature dependent enthalpy results employing the standard
relationship:
D
C0p ¼ d
D
H0=dT
ð1Þ
Heat capacity change data can provide valuable insights into the
type and magnitude of forces involved in the complexation. The
thermodynamic parameters were evaluated from ITC studies per-
formed at three temperatures, viz. 10, 20 and 30 °C. As the temper-
ature increased, the association constant (Ka) of the complexation
decreased significantly in each case. For the analog 6, the Ka value
decreased from 9.4 ꢀ 105 Mꢁ1 to 6.0 ꢀ 105 Mꢁ1 on increasing the
temperature from 10 to 30 °C. Furthermore, there were remarkable
changes in the enthalpy and entropy contributions with increasing
Figure 5. Representative ITC profile for the sequential titration of successive
aliquots of 6 to CT DNA in 10 mM CP buffer, pH 7.0 (curve at the bottom), along with
the dilution profiles (curves on the top offset for clarity). The top panel represents
the raw data and the bottom panel shows the integrated heat data after correction
of the heat of dilution. The symbols (j) represent the data points that were fitted to
a one-site model and the solid line represents the best-fit data.
temperature; while the binding
D
H0 values increased, the favorable
TD
S0 values decreased. Nevertheless, only small changes occurred
in the Gibb’s energy values. The reaction enthalpy and entropy,
both of which were strong functions of temperature, compensate
each other to make the reaction free energy more or less indepen-
dent of temperature. Such compensation is observed for many bio-
molecular interactions50–52 and suggests the involvement of
significant hydrophobic interactions in the binding. The variation
H0 with temperature (not shown) afforded the
D
Cp values.
0
of
D
The slopes of the straight lines gave values of ꢁ110, ꢁ82, ꢁ91.5
and ꢁ105.5 cal/mol K, respectively, for the binding of 1, 6, 5a and
5b, respectively. Negative heat capacity values have been observed
for a large variety of small molecules binding to DNA and
RNA.43,50,53,54 Furthermore, a negative
D
Cp value is usually associ-
0
ated with changes in hydrophobic or polar group hydration and is
considered as an indicator of dominant hydrophobic interactions
in the binding process. The heat capacity change values are lower
for the three analogs compared to the parent alkaloid berberine
clearly indicating the influence of the bulky 9-O-substitution on
0
the complexation process. The differences in the
D
Cp values may
Figure 6. Comparative bar chart depicting the Ka values of 1, 6, 5a and 5b.

A. Basu et al. / Bioorg. Med. Chem. 20 (2012) 2498–2505
2503
indicate differences in the release of structured water consequent
for the design and development of berberine based therapeutic
agents with greater efficacy.
to the transfer of nonpolar groups into the interior of the helix. First
0
of all, the values of
D
Cp are non-zero and negative indicating tem-
perature dependence of the enthalpy change and significant hydro-
4. Materials and methods
4.1. Materials
phobic contribution in the binding process. Secondly, the values of
0
D
Cp in almost all cases fall within the range 100–500 cal/mol K or
are very close to the lower limit that is frequently observed for
many ligand–nucleic acid interaction.53,54 Slightly higher
D
Cp val-
0
Calf thymus (CT) DNA was obtained from Sigma–Aldrich Corpo-
ration (St. Louis, MO, USA). The concentration of the CT DNA was
determined by absorption measurements using the molar extinc-
tion coefficient value of 13,200 Mꢁ1 cmꢁ1 at 260 nm expressed in
base pairs. Berberine chloride was purchased from Sigma–Aldrich
and was used without further purification. The concentrations of
1, 6, 5a and 5b were determined by applying the molar extinction
ues for 1 compared to the analogs for binding to the DNA may sug-
gest conformational differences in its structure and also differences
in the disruption of the water structure around the base pairs of the
DNA on complexation. Four types or modes of DNA recognition and
binding by small molecules viz. sequence specific, non-specific,
minimal sequence specific and structure specific have been dis-
cussed by Murphy and Churchill.55 Small negative
D
Cp values are
0
coefficient (e ,
) values of 22,500, 25,000, 26,000, 26,500 Mꢁ1 cmꢁ1
considered to be the hallmark of a minimal sequence specific bind-
respectively, at the wavelength maximum of 345 nm. All buffer
salts and other reagents were of analytical grade or better. Solu-
tions were freshly prepared in the buffer and kept protected in
the dark. All experiments were conducted in filtered 10 mM cit-
rate-phosphate (CP) buffer, pH 7.0, prepared in deionised and triple
distilled water.
0
ing and hence the slightly negative non-zero
D
Cp value that is ob-
served here for the 9-O-berberine analogs-DNA complexation
appears to denote structure specific binding. It is known that for
DNA intercalators a large hydrophobic contribution to the binding
free energy is expected from their aromatic ring system and the
binding should be energetically favourable.53 From the records56
relationship,
from the hydrophobic transfer step of binding of these molecules
were calculated. The Ghyd values for 1, 6, 5a, 5b binding to CT
D
Ghyd = 80( 10) ꢀ
D
Cp0, the free energy contribution
4.2. General procedure for synthesis of berberine analogs
D
To the solution of N-aryl/aryl–alkyl amine (10 mmol) in dry
dichloromethane was added chloroacetyl chloride (11 mmol)
dropwise under ice-cold condition. Then the reaction mixture
was stirred at room temperature for 40 min and then it was poured
into ice-cold water to decompose the unreacted acid chloride. The
resulting solution was neutralized with saturated NaHCO3 solution
and the organic layer was extracted with chloroform (3 ꢀ 50 ml),
dried over Na2SO4 and the solvent was removed under reduced
pressure. The resulting solid (1.2 mmol) was added to a solution
of berberrubine 2 (1 mmol) in dry CH3CN (10 ml) and the reaction
mixture was refluxed for 12 h. The progress of the reaction was
monitored by TLC. Then the reaction mixture was cooled to room
temperature and the solvent was removed under reduced pressure.
The crude product was chromatographed on a neutral Al2O3 col-
umn, eluted with increasing concentration of MeOH in CHCl3
(CHCl3/MeOH, 99:1–98:2) to afford the desired berberine analogs.
DNA were deduced to be ꢁ8.80. ꢁ6.56, ꢁ7.32 and ꢁ8.44 kcal/mol,
respectively, which are all well within the range that were observed
for DNA and RNA intercalating molecules.50,53,57,58
3. Conclusion
In summary, we have designed and synthesized three new 9-O-
substituted berberine analogs. With respect to DNA binding, these
analogs have retained the DNA binding mode, viz. intercalation,
but a remarkable amplification of the DNA binding affinity and
thermal stabilization of DNA against strand separation was ob-
served. Ferrocyanide quenching and viscosity studies confirmed
that all these compounds bound to DNA by intercalation geometry
exhibiting stronger intercalative property. The binding affinity of
the analogs was dependent on the length of the side chain. The
highest binding affinity was for 6 which was six times more than
that of 1 under identical conditions. However, further chain elon-
gation gradually reduced the binding affinity. The 9-O-substitution
enhanced the thermal stabilization of DNA remarkably. Analog 6
enhanced the stability of the DNA by almost 7 °C higher than that
of 1 clearly suggesting strong additional binding effects from the
side chain. This indicates that the side chain can effectively partic-
ipate in H-bonding with either the base pairs or the phosphates of
the DNA. The Scatchard binding isotherms showed pronounced
cooperative binding for 1 in agreement with the literature data
whereas the binding of the analogs was found to be completely
non-cooperative. This might suggest significant differences in the
mechanism of binding. Circular dichroism studies confirmed that
a higher perturbation of the DNA conformation within the B-form
was also achieved for the analogs compared with the parent ber-
berine. Thermodynamics of the interaction revealed a larger entro-
pic and a smaller but favorable enthalpic contribution, that
increased significantly with temperature, to the binding free en-
ergy with the introduction of the N-aryl/aryl–alkyl amino carbonyl
methyl substituent suggesting the role of release of DNA bound
water molecules by the side chain. The small but negative heat
capacity change of the analogs along with the significant enthal-
py–entropy compensation phenomenon observed confirmed the
involvement of multiple weak noncovalent interactions in the
binding process. These results may be of potential significance
4.2.1. Synthesis of compound 5a
Berberrubine was treated with compound 4a according to the
general procedure to obtain the desired product as a yellow solid.
(24% yield). IR (KBr) mmax 3437, 1686, 1555, 1266 cmꢁ1. UV–Vis
(kmax) 228, 265, 344, 421 nm. 1H NMR (300 MHz, CD3OD) d 10.04
(s, 1H), 8.74 (s, 1H), 8.16 (d, J = 9.3 Hz, 1H), 8.06 (d, J = 9.0 Hz,
1H), 7.69 (s, 1H), 7.60 (d, J = 7.8 Hz, 2H), 7.35 (t, J = 7.8 Hz, 2H),
7.15 (t, J = 7.3 Hz, 1H), 6.99 (s, 1H), 6.13 (s, 2H), 5.13 (s, 2H),
4.96–4.90 (m, 2H), 4.62 (s, 2H), 4.13 (s, 3H), 3.32–3.27 (m, 2H).
13C NMR (150 MHz, CD3OD) 171.07, 152.42, 151.69, 150.13,
146.97, 143.73, 139.98, 139.94, 135.21, 132.06, 129.76 (1 ꢀ 2),
128.82 (1 ꢀ 2), 127.71, 125.37, 124.64, 123.43, 121.97, 121.61,
109.56, 106.72, 103.87, 73.09, 57.76, 57.46, 43.96, 28.32. ESIMS
m/z: [MꢁCl]+ 469.21. Anal. Calcd. for C28H25ClN2O5: C, 66.60; H,
4.99; N, 5.55. Found: C, 66.53; H, 5.13; N, 5.73.
4.2.2. Synthesis of compound 5b
Berberrubine was treated with compound 4b according to the
general procedure to obtain the desired product as a yellow solid
(57% yield). IR (KBr) mmax 3342, 1652, 1548, 1271 cmꢁ1. UV–Vis
(kmax) 228,264,345,423 nm. 1H NMR (600 MHz, CD3OD) d 9.89 (s,
1H), 8.71 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 9.0 Hz, 1H),
7.66 (s, 1H), 7.27–7.16 (m, 5H), 6.97 (s, 1H), 6.25 (s, 2H), 4.92 (t,
J = 6.3 Hz, 2H), 4.88 (s, 2H), 4.06 (s, 3H), 3.53 (t, J = 7.5 Hz, 2H),

2504
A. Basu et al. / Bioorg. Med. Chem. 20 (2012) 2498–2505
3.27 (t, J = 6.3 Hz, 2H), 2.86 (t, J = 7.2 Hz,2H). 13C NMR (150 MHz,
CD3OD) 171.08, 152.39, 151.65, 150.10, 146.99, 143.66, 140.47,
139.90, 135.21, 132.06, 129.95 (1 ꢀ 2), 129.67 (1 ꢀ 2), 127.76,
127.58, 125.36, 123.36, 121.95, 121.58, 109.56, 106.72, 103.85,
72.84, 57.81, 57.49, 41.88, 36.65, 28.32. ESIMS m/z: [MꢁCl]+
483.18. Anal. Calcd for C29H27ClN2O5: C, 67.11; H, 5.24; N, 5.40.
Found: C, 67.03; H, 5.35; N, 5.56.
at constant temperature of 20 1 °C under conditions of continuous
stirring. Uncorrected fluorescence spectra were recorded. The fluo-
rescence signal was recorded for solutions where the concentrations
of both DNA and the alkaloid were varied while the sum of their con-
centrations was kept constant. The difference in fluorescence inten-
sity (DF) of the alkaloids in the absence and presence of DNA was
plotted as a function of the input mol fraction of each alkaloid. Break
point in the resulting plot corresponds to the mol fraction of the
bound alkaloid in the complex. The stoichiometry was obtained in
4.2.3. Synthesis of compound 6
Berberrubine was treated with 2-chloro-N-phenyl acetamide
according to the general procedure to obtain the desired product
as a yellow solid (35% yield). IR (KBr) mmax 3267, 1692, 1537,
terms of DNA-alkaloid [(1-valkaloid)/valkaloid] where valkaloid denotes
the mole fraction of the respective alkaloid. The results reported are
averages of at least three experiments.
1262 cmꢁ1
. UV–Vis (kmax)
230, 263, 346, 423 nm. 1H NMR
(300 MHz, CD3OD) d 10.03 (s, 1H), 8.72 (s, 1H), 8.15 (d, J = 9.0 Hz,
1H), 8.05 (d, J = 9.0 Hz, 1H), 7.64 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H),
7.35 (t, J = 7.8 Hz, 2H), 7.15 (t, J = 7.5 Hz, 1H), 6.98 (s,1H), 6.10 (s,
2H), 5.06 (s, 2H), 4.96–4.89 (m, 2H), 4.13 (s, 3H), 3.33–3.32 (m,
2H). 13C NMR (150 MHz, CD3OD) 169.52, 152.42, 151.61, 150.14,
147.29, 143.99, 139.97, 139.16, 135.30, 132.10, 130.13 (1 ꢀ 2),
127.80, 125.93, 125.26, 123.54, 122.02, 121.72 (1 ꢀ 2), 121.58,
109.57, 106.75, 103.87, 72.85, 57.90, 57.54, 28.35. ESIMS m/z:
[MꢁCl]+ 455.30. Anal. Calcd for C27H23ClN2O5: C, 66.06; H, 4.72;
N, 5.71. Found: C, 66.01; H, 4.84; N, 5.86.
4.5. Thermal melting studies
Absorbance versus temperature profiles (melting curves) of
DNA and alkaloid-DNA complexes were measured on the Shima-
dzu Pharmaspec 1700 unit equipped with the peltier controlled
TMSPC-8 model accessory in eight chambered quartz cuvette of
1cm path length.45 The temperature was ramped from 20 to
100 °C at a scan rate of 0.5°C/min. monitoring the absorbance
changes at 260 nm.
4.3. Elucidation of DNA binding by spectroscopic measurements
4.6. Circular dichroism studies
Measurements were carried out at 20 °C. Binding was moni-
tored spectrophotometrically and fluorimetrically in the alkaloid
absorption and emission regions, respectively, after addition of
scalar amounts of the CT DNA into freshly prepared alkaloid solu-
tions as described previously.39,41,42 Spectrophotometric and fluo-
rimetric measurements were made with a Jasco V660 unit (Jasco
International Co. Ltd, Hachioji, Japan) and a Shimadzu RF5301 PC
fluorimeter (Shimadzu Corporation, Kyoto, Japan), respectively, in
thermostated quartz cuvettes of 1 cm path length. Binding data ob-
tained from spectrophotometric and spectrofluorimetric titrations
were converted into Scatchard plot of r/Cf versus r as described
previously.25
The circular dichroism (CD) spectra were recorded on a JASCO
J815 spectropolarimeter (Jasco International Co. Ltd, Hachioji, Ja-
pan) equipped with a Jasco temperature controller (model PFD
425L/15) interfaced with a HP PC at 20 0.5 °C using instrument
parameters reported previously.32,50 The molar ellipticity values
[h] (deg. cm2 dmolꢁ1) were calculated from the equation:
½hꢂ ¼ ½hꢂobs=10lC
ð4Þ
where [h]obs is the observed ellipticity (milli degree), C is the molar
concentration and l is the optical path length of the cuvette in cm.
The expressed molar ellipticity is in terms of DNA base pairs.
The Scatchard isotherms with positive slope at low r values
were analyzed using the following McGhee-von Hippel equation
for cooperative binding.40
4.7. Fluorescence quenching studies
Quenching studies were carried out with the anionic quencher
[Fe(CN)6]4ꢁ. Quenching experiments were performed by mixing,
in different ratios, two solutions, one containing KCl and the other
containing K4[Fe(CN)6], in addition to the normal buffer compo-
nents, at a fixed total ionic strength. Experiments were performed
at a constant P/D (DNA base pair/alkaloid molar ratio) monitoring
fluorescence intensity as a function of increasing the concentration
of the ferrocyanide ions as described in details previously.26,46 At
least four measurements were taken for each set and averaged
out. The data were plotted as Stern–Volmer plots of relative fluo-
r
Cf
¼ Kið1 ꢁ nrÞ
ꢀ
ꢁðnꢁ1Þ
rð1 ꢁ nrÞg ,
ꢀ
ꢁ
2
ð2
x
þ 1Þð1 ꢁ nrÞ þ ðr ꢁ RÞ
1 ꢁ ðn þ 1Þr þ R
2ð1 ꢁ nrÞ
ꢀ
ð2Þ
2ð
x
ꢁ 1Þð1 ꢁ nrÞ
1
2
2
where, R ¼ f½1 ꢁ ðn þ 1Þrꢂ þ 4
x
Scatchard plots with negative slopes at low r values were ana-
lyzed by the non-cooperative binding model of McGhee and von
Hippel as per the following equation:
rescence intensity (Fo/F) versus [Fe(CN)6]4ꢁ
.
r=Cf ¼ Kið1 ꢁ nrÞ½ð1 ꢁ nrÞ=f1 ꢁ ðn ꢁ 1Þrgꢂðnꢁ1Þ
ð3Þ
4.8. Hydrodynamic studies
Here, Ki is the intrinsic binding constant to an isolated binding
site, n is the number of base pairs excluded by the binding of a sin-
The viscosity of the DNA-alkaloid complexes was determined
by measuring the time needed to flow through a Cannon-Manning
semi micro size 75 capillary viscometer (Cannon Instruments Com-
pany, State College, PA, USA) that was submerged in a thermo-
stated water bath (20 1 °C) as reported previously.47,48 Small
volumes of the alkaloid solution were added to sonicated CT DNA
(280 40 base pairs) solution placed in the viscometer. Mixing
was effected by slowly bubbling nitrogen gas. Flow times were
measured in triplicate to an accuracy of 0.01 s with an electronic
stopwatch Casio Model HS-30W (Casio Computer Co. Ltd, Tokyo,
Japan). Relative viscosities for DNA either in the presence or ab-
sence of the alkaloids were calculated from the relation:
gle alkaloid molecule and
x is the cooperativity factor. All the
binding data were analyzed using the Origin 7.0 software (Origin
Labs, Northampton, MA, USA) that determines the best-fit param-
eters to Ki, n and
x to Eq. 2 and Ki and n to Eq. 3.
4.4. Stoichiometry of binding: Job plot
Continuous variation method of Job44 was employed to deter-
mine the binding stoichiometry in each case from fluorescence
spectroscopy. Steady state fluorescence measurements were per-
formed in fluorescence free quartz cuvettes of 1 cm path length
as described previously.48,59 All the measurements were performed

A. Basu et al. / Bioorg. Med. Chem. 20 (2012) 2498–2505
2505
g0sp=gsp ¼ fðtcomplex ꢁ t0Þ=t0g=fðtcomplex ꢁ t0Þ=t0g
ð5Þ
2. Kulkari, S. K.; Dhir, A. Eur. J. Pharmacol. 2008, 589, 163.
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where, gsp and gsp are specific viscosities of the alkaloid-DNA com-
plex and the DNA respectively; tcomplex, tcontrol, and to are the aver-
age flow times for the DNA-alkaloid complex, free DNA and buffer,
respectively. The relative increase in length of DNA, L/Lo, is obtained
from a corresponding increase in relative viscosity using the follow-
ing equation:
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1=3
L=L0 ¼ ð
g=
g0
Þ
¼ 1 þ br
ð6Þ
where L and L0 are the contour lengths of DNA in the presence and
in the absence of the alkaloid and and g0 are the corresponding
values of intrinsic viscosity (approximated by the reduced viscosity
sp/C where C is the DNA concentration) and b is the slope of the
g
g = g
plot of L/L0 versus r.
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4.9. Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) experiments were carried
out on a VP-ITC microcalorimeter (ITC; MicroCal LLC). Instrument
control, data acquisition and analysis were performed using the
dedicated Origin 7.0 software following methods described previ-
ously.43,54 Briefly, aliquots of degassed alkaloid solutions were ti-
trated from the rotating syringe (stirring speed 290 rpm) into the
isothermal sample chamber containing DNA (1.4235 mL) solution.
The reference cell was filled with deionized water. The heat liber-
ated or absorbed with each injection of the alkaloid is observed as a
peak that corresponds to the power required to keep the sample
and reference solutions at identical temperature. The peaks pro-
duced over the entire course of the titration were converted to heat
output per injection by integration. The area under each heat burst
curve was determined by integration using the Origin software.
Control experiments were carried out to determine the heat contri-
bution from dilution arising from buffer into DNA and alkaloids
into buffer. The resulting corrected injection heats were plotted
as a function of the molar ratio. Experimental data were fitted
using a non linear least square minimization algorithm to one site
binding theoretical curves to provide the binding affinity (Ka), the
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binding stoichiometry (N), and the enthalpy of binding (D
H0).
The binding site size has been restricted during the fitting process
to values close to that obtained from the spectroscopic data. The
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binding Gibb’s energy (D D
G0) and the entropic contribution (T S0)
were deduced from standard relationships,
D
G0 = ꢁRTlnKa
(R = 1.9872 cal molꢁ1 Kꢁ1, T = 298 K) and
D
G0 =
D
H0ꢁT
D
S0.
Acknowledgment
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This work was supported by grants from the CSIR NWP0036.
A. Basu is a NET-Senior Research Fellow of the University Grants
Commission, Government of India. The authors thank all the col-
leagues of the Chemistry and Biophysical Chemistry Laboratories
for cooperation and help during the course of this work. The critical
comments of the reviewers that enabled us to improve the manu-
script are also appreciated.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.03.006.
References and notes
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