Synthesis, antioxidant and anticancer screenings of berberine-indole conjugates

Article abstract of DOI:10.1007/s11164-015-2208-x

A variety of heterocyclic nitrogen cores in the form of indole moieties were linked to the natural isoquinoline alkaloid molecule berberine to achieve anticipated antioxidant and anticancer properties. An efficient synthetic pathway afforded final compounds 5a-j, which were tested in vitro for antioxidant potency using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical (ABTS) bioassays, and for anticancer activity using sulforhodamine B (SRB) assay against HeLa and Caski cancer cell lines. Moreover, the toxic nature of the resultant molecules was investigated using Madin-Darby canine kidney cells. The therapeutic indices of 5a-j were more appreciable against the Caski than HeLa cell line, in which compounds with electron-releasing alkyl or alkoxy functional group on indole entity as well as azaindole derivative performed well. In addition, these compounds were well endowed with antioxidant properties, in addition to the equal antioxidant effect of the compound with electron-withdrawing chlorine atom within indole entity. Adequate confirmation of the structure of the final analogues was achieved using Fourier-transform infrared (FT-IR), ¹H nuclear magnetic resonance (NMR), and mass spectroscopy and elemental (CHN) analysis.

Full text of DOI:10.1007/s11164-015-2208-x

Res Chem Intermed
DOI 10.1007/s11164-015-2208-x
Synthesis, antioxidant and anticancer screenings
of berberine–indole conjugates
Bhupendra Mistry1 Young-Soo Keum1
•
•
Doo Hwan Kim1
Received: 31 May 2015 / Accepted: 3 August 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract A variety of heterocyclic nitrogen cores in the form of indole moieties
were linked to the natural isoquinoline alkaloid molecule berberine to achieve
anticipated antioxidant and anticancer properties. An efficient synthetic pathway
afforded final compounds 5a–j, which were tested in vitro for antioxidant potency
using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,20-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) diammonium salt radical (ABTS) bioassays, and
for anticancer activity using sulforhodamine B (SRB) assay against HeLa and Caski
cancer cell lines. Moreover, the toxic nature of the resultant molecules was inves-
tigated using Madin–Darby canine kidney cells. The therapeutic indices of 5a–-
j were more appreciable against the Caski than HeLa cell line, in which compounds
with electron-releasing alkyl or alkoxy functional group on indole entity as well as
azaindole derivative performed well. In addition, these compounds were well
endowed with antioxidant properties, in addition to the equal antioxidant effect of
the compound with electron-withdrawing chlorine atom within indole entity. Ade-
quate confirmation of the structure of the final analogues was achieved using
1
Fourier-transform infrared (FT-IR), H nuclear magnetic resonance (NMR), and
mass spectroscopy and elemental (CHN) analysis.
Keywords Berberine Á Indole Á Antioxidant Á Anticancer Á Cervical cancer Á
Natural product derivatives Á Alkaloids
&
&
1
Bhupendra Mistry
bhupendra.mistry84@gmail.com
Doo Hwan Kim
kimdh@konkuk.ac.kr
Organic Research Laboratory, Department of Bioresources and Food Sciences, College of Life
and Environmental Sciences, Konkuk University, Seoul, South Korea
123

B. Mistry et al.
Introduction
The metabolic activities of the human body demand energy generation for growth
and development, with oxygen playing an essential role in the performance of these
biological functions, for example, catabolism of carbohydrates, proteins, and fats.
However, oxygen has a disadvantageous effect too, because it has some toxic effects
on living cells due to its involvement in production of reactive oxygen species
(ROS) [1–3]. When performing normal metabolism, the human body can generate
ROS such as hydroxyl, alkoxyl, and lipid peroxyl radicals, nitric oxide, and
peroxynitrite [4]. On the other hand, these can also be created via the explosion of
biomolecules produced by chemicals and ionizing radiation, thus making it more
likely for the body to suffer from diseases such as cancers, inflammation,
cardiovascular diseases (CVDs), and other illnesses [5]. The functions of essential
cell components such as proteins, nucleic acids, lipids, and carbohydrates can be
altered by ROS, which are also the factor responsible for exposing the body to a
variety of diseases. Moreover, damage to cells caused by ROS is believed to play a
central role in the aging process and disease progression. Production of ROS in
larger amounts can be considered as oxidative stress [6], which can be controlled by
the action of antioxidant molecules. Compounds that can scavenge free radicals play
an important role in the progress of these infective conditions.
Nowadays, the most severe disease to which humans are exposed is cancer, being
one of the leading causes of mortality worldwide [7]. In fact, the global burden of
cancer continues to increase, largely because of aging, which is caused by oxidative
stress. In 2012, according to GLOBOCAN estimates, there were 14.1 million new
cancer cases, 8.2 million cancer deaths, and 32.6 million people living with cancer.
Also, 65 % of cancer deaths and 57 % of newly diagnosed cancer cases occur in
developing regions of the world [8]. The cell damage exerted by ROS depends on the
equilibrium between ROS and endogenous antioxidant species. In the absence of an
antioxidant defense system, oxidative stress causes nicks in the DNA repair system,
causing DNA oxidation that generates 8-hydroxy-20-deoxyguanosine, a product that
can generate mutations in DNA, thereby causing cancer [9]. Hence, cancer and
oxidative stress can be correlated, and accordingly, compounds that reduce oxidative
stress, i.e., antioxidants, can be regarded as important molecules that also help to treat
cancer [10]. It is worthwhile to assume that compounds bearing functional groups
demonstrating potential antioxidant effects could be studied further to investigate
their efficacy as chemopreventive and chemotherapeutic agents. To further support
this statement, researchers have reported several molecules with antioxidant and
chemopreventive properties, including compounds bearing phenolic hydroxyl
group(s) [11, 12, 13] and compounds containing heterocyclic systems such as
imidazole [14, 15], thiazole [16], and thiadiazole [17, 18].
Natural products have played a key role in medicine throughout our evolution. In
fact, natural products are still used today in various regions of the world to relieve
pain, or are wrapped (for example, in leaves) to heal wounds. Over the past decade,
natural products have been the most studied for drug design and development
through the invention of molecular biology and combinatorial chemistry, which
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Synthesis, antioxidant and anticancer screenings of…
assist with the rationalization of drugs. More recently, natural products have been
investigated in clinical trials or provided leads for compounds that have entered
clinical trials, particularly for anticancer and antimicrobial agents [19–21]. Between
1981 and 2010, 34 % of the medicines approved by the Food and Drug
Administration (FDA) were based on natural product small scaffolds or their
derivatives [22–25].
Berberine is an isoquinoline quaternary alkaloid present in many therapeutic
plants such as Hydrastis canadensis, Berberis aristata, Coptis chinensis, C. rhi-
zome, C. japonica, Phellodendron amurense, P. chinense Schneid, and other local
varieties used all over the world in conventional therapeutic practice. Plants that
contain berberine have been used in protection against and therapy for many
illnesses, such as gastrointestinal infections, abdominal pain and diarrhea,
hyperglycemia, hyperlipidemia, metabolic syndrome, polycystic ovary syndrome,
obesity, fatty liver, and coronary artery disorders [26]. Berberine has been
associated with many medicinal properties, for example, anticancer [27] and
antioxidant actions [28]. Derivatization of berberine has generated molecules
exhibiting potential cytotoxic properties against HeLa, SVKO3, and Hep-2 [29].
The anticancer efficiency of berberine seems to result from its powerful interaction
with inhibition of topoisomerase enzymes, and nucleic acids and telomerases, and
binding to quadruplex structures [30–32]. Its connection with biomacromolecules,
especially DNA and RNA, at the molecular level and structural information relating
to such complexes are key issues to determine its biological features. Research on
the DNA and RNA executed factors of berberine, its analogues, and relevant
alkaloids has been substantially examined by many research groups [33–38].
Analysis of the anticancer action of berberine, in particular, has attracted extensive
interest, with quite excellent results being obtained [39]. The berberine framework
represents a naturally exciting molecule as well as an eye-catching natural lead
substance for development of various derivatized variations exhibiting appropriate
actions with other pharmacologically important heterocyclic cores. The indole core
has been studied in therapeutic chemistry and is considered to be a potent scaffold
[40–43]. Therefore, linkage of indoles to an intended biocore has been a subject of
concentrated analysis over the decades [44–50]. Indole derivatives represent an
important class of healing compounds in the therapeutic era, demonstrating e.g.
anticancer [51], antioxidant [52], and anti-human immunodeficiency virus (HIV)
[53, 54] actions; in fact, many indole derivatives are considered to be the most
potent scavengers of free radicals [55]. Prompted by the above considerations, and
given the need for new antitumor agents, we considered it of interest to prepare
combinations of the berberine moiety with the indole nucleus.
Results and discussion
Chemistry
Scheme 1 summarizes the chemical strategies adopted to obtain 5a–j efficiently.
Berberrubine (2) was furnished in good yield upon treatment of berberine
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B. Mistry et al.
Scheme 1 Synthesis of indole-linked berberine derivatives
hydrochloride in a vacuum oven at temperature of 190 °C and pressure of
20–30 mmHg [56]. Berberrubine was further alkylated using dibromoalkanes in dry
acetonitrile [57] to construct the final intermediate compound 3. The intended indole
entities were introduced into the berberine core via reaction with intermediate 3 in
dimethylformamide (DMF) for 6–8 h to produce 5a–j in reasonable yields.
Analytical data in terms of FT-IR, 1H NMR, and mass spectrometry and
determination of elemental proportions (CHN analysis) confirmed adequate
production of the title compounds. Within the aromatics, C–H stretching exhibited
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Synthesis, antioxidant and anticancer screenings of…
its characteristic bands at around 3037–3061 cm-1 in the FT-IR spectra of 5a–j,
including C=C characteristic bands near 1622–1531 cm-1. A sharp peak corre-
sponding to C–O–C linkage was found at around 1121–1077 cm-1, and similarly
for halogen linkage at 757–781 cm-1. The structure of berberine was confirmed
through assignment of its proton atoms through 1H NMR spectra (5b) as
corresponding to the H-8, H-13, H-1, H-12, H-4, and H-11 positions, resonating
in the form of singlet peaks at 9.61, 8.43, 7.62, 7.44, 7.40, and 6.51 ppm,
respectively, as well as a singlet peak observed at 6.05 ppm due to the presence of
–OCH2O of berberine ring. H-6 and H-5 proton atoms of the berberine ring
1
exhibited peaks in the form of triplets at 4.94 ppm and 2.47 ppm in the H NMR
spectra of 5b as well as a triplet corresponding to the characteristic alkyl chain
proton (H-15) observed at 4.22 ppm. Furthermore, a triplet signal at 3.20 ppm,
broad singlet at 2.40 ppm, as well as multiplet signals at 2.19 ppm were due to alkyl
chain protons H-19, H-17, and H-18 as well as H-16, respectively, while proton
atoms present on aromatic moieties attached to the indole or in the indoline or
azaindole entity exhibited corresponding signals as multiplets in the range of
7.32–6.61 ppm. Mass spectrometric data were in accurate accordance, as observed
from the M? ion values for the final compounds 5a–j. All of the novel compounds
gave C, H, N analyses within 0.4 percent points of the theoretical value, i.e., within
an acceptable range.
Evaluation of biological activities
Antioxidant activity
The free radical scavenging capability of the new berberine–indole conjugates
5a–j was determined using multiple antioxidant bioassays based on 1,1-diphenyl-2-
picrylhydrazyl radical (DPPH) and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt radical (ABTS). Overall, the title analogues presented
positively enhanced radical scavenging effects when compared with the parent
berberine, with 50 % inhibition concentration (IC50) values ranging from
17.09 0.29 to 26.38 0.16 lg/mL and 9.09 0.11 to 19.50 1.16 lg/mL in
DPPH and ABTS bioassay, respectively. In addition, it was found that neat indole
entity failed to present significant antioxidant efficacy; however, the presence of
different electron-withdrawing and electron-donating substituents on it significantly
enhanced the potency of the resultant indoles connected to the berberine core. The
title compounds served as beneficial antioxidant drug-like molecules when
comparing their potencies with that of the control drug ascorbic acid with IC50
values of 15.59 0.32 and 6.77 0.02 lg/mL in DPPH and ABTS bioassay,
respectively. An analogue with electron-withdrawing chlorine atom (5e) appeared to
be the most potent radical scavenger in DPPH assay, with IC50 level of
17.09 0.29 lg/mL, slightly lower than the control drug ascorbic acid but twice
as potent as berberine. Another analogue with indoline ring (5h) demonstrated
almost equal antioxidant power to 5e with IC50 of 19.48 0.39 lg/mL. Title
analogues with electron-releasing alkyl functionality, e.g., compound 5b with
methyl and 5g with dimethyl functional groups, exerted equal antioxidant efficacies
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B. Mistry et al.
with IC50 values of 19 lg/mL. Azaindole-based compound 5j presented an IC50
level around 19.79 0.84 lg/mL, being equal in potency to compounds with alkyl
functional groups. Final analogues with alkoxy (5d) and fluorine (5c) functional
groups as well as one with oxindole moiety (5i) exhibited IC50 values around
20 lg/mL, being considered as moderate radical scavenging agents in the present
study according to the DPPH assay. The results of the ABTS bioassay confirmed
that compound 5b with methyl indole functional group demonstrated the greatest
antioxidant power with an IC50 level of 9.09 0.11 lg/mL, being ten times more
potent than the parent molecule berberine with antioxidant effects at the IC50 level
of 90.71 1.77 lg/mL according to the ABTS assay. This compound demonstrated
activity close to that of the control drug ascorbic acid with antioxidant power at the
IC50 level of 6.77 0.02 lg/mL in the ABTS assay. Another three compounds with
electron-releasing alkoxy (5d) and electron-withdrawing chlorine atom (5e) as well
as one with indoline entity (5i) showed radical scavenging potential similar to 5b
with IC50 values of 9.40 0.33, 9.64 0.36, and 9.56 0.53 lg/mL, respec-
tively, in the ABTS assay. The remaining analogues can be regarded as antioxidant
agents with good to moderate power against ABTS radical when comparing their
potency with the control drug ascorbic acid; however, they can be regarded as very
potent when compared with berberine in the same bioassay. Overall, all the
presented analogues demonstrated enhanced potency as DPPH and ABTS radical
inhibitors compared with the parent molecule berberine (Table 1).
Anticancer activities
The in vitro action of the berberine–indole conjugates against two cervical cancer
cell lines, namely HeLa and Caski, was determined and the results are presented in
Tables 2 and 3. The sulforhodamine B (SRB) colorimetric bioassay was adopted to
investigate the anticancer action of the title compounds. In general, the compounds
Table 1 Screening results of
Compound
IC50 (lg/mL) SDa
DPPH and ABTS radical
scavenging activity of berberine
derivatives 5a–j
DPPHb
ABTSc
5a
26.38 0.16
19.90 0.34
20.55 0.89
22.27 0.62
17.09 0.29
20.37 0.12
19.48 0.39
17.25 0.47
20.58 0.53
19.79 0.84
41.87 1.63
15.59 0.32
12.09 0.62
9.09 0.11
14.02 0.87
9.40 0.33
9.64 0.36
19.50 1.16
10.71 0.38
9.56 0.53
17.34 0.69
16.46 0.57
90.71 1.77
6.77 0.02
5b
5c
5d
a
Antioxidant activities are
5e
shown as IC50 values in lg/mL.
All assays were carried out in
triplicate, and the results are
expressed as
5f
5g
5h
average standard deviation
b
DPPH = 2,2-diphenyl-1-
picrylhydrazyl
5i
5j
c
ABTS = 2,20-azino-bis(3-
Berberine
Ascorbic acid
ethylbenzothiazoline-6-sulfonic
acid)
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Synthesis, antioxidant and anticancer screenings of…
Table 2 Anticancer activity of
Compound IC50 (lg/mL) SDa CC50 (lg/mL) SDb TIc
synthesized compounds against
HeLa cancer cells and their
toxicity
HeLa
MDCK
5a
7.461 0.12
7.145 0.06
7.275 0.07
7.215 0.11
7.189 0.18
7.358 0.13
7.555 0.11
7.582 0.06
7.443 0.11
7.279 0.02
5.575 0.18
303.1 2.02
300.9 1.25
310.3 0.89
316.7 1.05
302.4 0.77
302.7 0.66
310.5 1.59
306.4 0.09
302.5 0.13
315.3 1.41
144.1 1.12
40.62
42.11
42.65
43.89
42.06
41.14
41.10
40.41
40.64
43.32
25.85
5b
5c
5d
5e
a
Anticancer activities shown as
5f
IC50 values in lg/mL. All assays
were carried out in triplicate,
and the results are expressed as
average standard deviation
5g
5h
5i
b
CC50, 50 % cytotoxicity
concentration
5j
Berberine
c
TI, therapeutic index
Table 3 Anticancer activity of
synthesized compounds against
Caski cancer cells and their
toxicity
Compound IC50 (lg/mL) SDa CC50 (lg/mL) SDb TIc
Caski
MDCK
5a
6.639 0.33
6.324 0.58
7.667 0.53
7.725 0.32
7.893 0.22
8.340 0.26
7.886 0.41
6.971 0.29
7.277 0.44
6.827 0.26
5.870 0.39
303.1 2.02
300.9 1.25
310.3 0.89
316.7 1.05
302.4 0.77
302.7 0.66
310.5 1.59
306.4 0.09
302.5 0.13
315.3 1.41
144.1 1.12
45.65
47.58
40.47
41.00
38.31
36.29
39.37
43.95
41.57
46.18
24.55
5b
5c
5d
a
Anticancer activities are
5e
shown as IC50 values in lg/mL.
All assays were carried out in
triplicate, and the results are
expressed as
5f
5g
5h
average standard deviation
b
CC50, 50 % cytotoxicity
concentration
5i
5j
Berberine
c
TI, therapeutic index
exhibited reasonable levels of cancer cell inhibitory effects against the HeLa and
Caski cell lines in terms of IC50 levels when compared with the parent molecule
berberine. However, the title compounds exhibited a better level of cytotoxic nature
when compared with berberine, furnishing therapeutic indices almost twice as
potent as those of the parent molecule berberine. The title analogues exhibited IC50
values of 7.145 0.06 to 7.582 0.06 lg/mL and 6.324 0.58 to
8.340 0.26 lg/mL against the HeLa and Caski cell lines, respectively. In
addition, the cytotoxicity level against Madin–Darby canine kidney (MDCK) cell
lines was observed to lie in the range of 300.9 1.25–316.7 1.05 lg/mL for the
title analogues 5a–j, resulting in therapeutic indices of 40.41–43.89 and
36.29–47.58 against the HeLa and Caski cervical cancer cell lines, respectively.
Overall, it can be stated that changing the nature of the substituent or functional
group present on the indole ring led to significant changes or variation in the
anticancer potency of the resultant molecules.
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B. Mistry et al.
The final analogue 5d with electron-releasing alkoxy functional group present on
the 5th position of indole ring attached to the berberine core demonstrated the most
potent anticancer effects with IC50 level of 7.215 0.11 lg/mL and low
cytotoxicity towards healthy MDCK cells (CC50 of 316.7 1.05 lg/mL), thereby
furnished a potent chemotherapeutic index of 43.89. This compound was one of the
most potent anti-HeLa cell line inhibitory agents among all those tested and was
much more potent than the parent molecule berberine with a therapeutic index of
25.85. Moreover, another compound 5j with 7-azaindole moiety connected to the
berberine core with a pentane aliphatic chain demonstrated anti-HeLa cell line
inhibitory effects almost equal to those of compound 5d, with IC50 level of
7.279 0.02 lg/mL, and low cytotoxicity towards healthy MDCK cells (CC50 of
315.3 1.41 lg/mL), thereby furnishing a chemotherapeutic index of 43.32. These
two molecules were identified as the most potent inhibitors of the HeLa cervical
cancer cell line. However, another three title derivatives, namely 5b, 5c, and 5e,
with either halo or alkyl substituent exhibited IC50 values around 7 lg/mL and
therapeutic indices of around 42, close to those of the two potent derivatives
mentioned above. Hence, based on these data, it is worth stating that more than half
of the total number of studied molecules presented powerful anticancer effects,
while the remaining analogues exhibited TIs of 40.41–41.14, still being much higher
than that of berberine. Furthermore, once again compound with electron-releasing
alkyl functional group exhibited the most potent cancer cell growth inhibitory
effects against the Caski cell line, with IC50 level of 6.324 0.58 lg/mL, whereas
the cytotoxicity was very reasonable at 300.9 1.25 lg/mL, leading to a
therapeutic index of 47.58. The potency of this analogue against the Caski cell
line was almost twice that of berberine at 24.55 in terms of the therapeutic index.
Similar to the activity of the title analogues against the HeLa cell line, in the case of
the Caski cell line, compound 5j with azaindole functionality displayed a significant
level of anticancer potential with IC50 level of 6.827 0.26 lg/mL and cytotox-
icity of 315.3 1.41 lg/mL, thereby presenting a TI value of 46.18. The final
compound with neat indole moiety was found to be inactive in other bioassay
targets, whereas in the case of activity against the Caski cell line, compound 5a with
indole moiety connected to the berberine core exhibited a therapeutic index of 45.65
and was considered to be excellently active compared with berberine itself. The
remaining analogues exhibited therapeutic indices of 36–41 and can be regarded as
good to moderate anticancer agents, suggesting that further structural refinements
are required to obtain more potent cancer therapeutics.
Experimental
Materials and methods
Highest quality chemicals and reagents were used in this study without prior
purification. A Veego VMP-D open capillary electronic apparatus was utilized to
obtain the melting points of the synthesized compounds, which are uncorrected.
A Shimadzu 8400-S FT-IR spectrophotometer (KBr pellets) and Varian 500 MHz
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Synthesis, antioxidant and anticancer screenings of…
model spectrometer [with dimethyl sulfoxide (DMSO) as solvent and tetramethyl-
1
silane (TMS) as internal standard] were used to obtain the FT-IR and H NMR
spectra of the title compounds. Thin-layer chromatography (TLC) was carried out
using appropriate mobile-phase systems of silica gel-G coated microscope glass
slides (2 9 7.5 cm), with TLC spots being observed in an ultraviolet (UV) light
1
chamber. FT-IR bands are presented in cm-1, while H NMR spectral results are
furnished in ppm downfield from TMS with s (singlet), d (doublet), m (multiplet),
and br s (broad singlet) patterns. Elemental analyses (C, H, N) were done using a
Heraeus Carlo Erba 1180 CHN analyzer.
General procedure for synthesis of berberrubine (2)
Berberine hydrochloride (10 g, 0.01 mol) was placed in a 50-mL round-bottomed
flask. The reaction system was maintained at reduced pressure (20–30 mmHg) using
an oil pump and warmed to 190 °C, followed by reaction for 40 min. The vacuum
pump was turned off after the temperature had decreased to room temperature. The
reaction product was purified using silica gel column chromatography (CHCl3:-
CH3OH 15:1 and 10:1, eluting until no compound was seen in the eluent) to acquire
compound 2 as a brownish-red amorphous powder (6.6 g, 85 %).
Synthesis of bromopentylberberrubine (3)
A solution of 2 (5 g, 0.01 mol) and 1,5-dibromopentane (0.01 mol) in dry
acetonitrile was heated at reflux temperature for 6 h, then diethyl ether was added.
The resulting solid was filtered and then subject to anion exchange to chloride form
to give compound 3.
General procedure for preparation of derivatives 5a–j
The substituent indole derivatives (0.01 mol) were added to a magnetically stirred
solution of compound 3 and anhydrous K2CO3 in dry DMF (25 mL). The reaction
mixture was heated at 80 °C for 6–8 h, and the reaction was monitored by TLC. The
resulting solid was filtered at room temperature and subjected to anion exchange to
chloride form. The crude product was chromatographed on an Al2O3 column, and
eluted with CHCl3/CH3OH (9:1, v/v) to give the proposed compound.
9-O-3-(1-(1H-Indole)pentylberberine (5a)
Light-yellow solid. Yield: 67 %. M.p. 273–275 °C. IR (KBr) cm-1: 3011 (C–H,
1
Ar), 1605–1540 (C=C, Ar), 1251–1060 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.75 (s, 1H, H-8), 8.62 (s, 1H, H-13), 7.73 (s, 1H, H-1), 7.58 (s, 1H, H-12), 7.34 (s,
1H, H-4), 7.25–6.76 (m, 6H, indole), 6.66 (s, 1H, H-11), 6.11 (s, 2H, –OCH2O),
5.05 (t, 2H, J = 6.3, H-6), 4.39 (t, 2H, J = 6.5, H-15), 4.08 (s, 3H, OCH3), 3.12 (t,
2H, J = 6.4, H-19), 2.61 (t, 2H, J = 7.4, H-5), 2.31 (br s, 4H, H-17, H-18), 2.11 (m,
2H, H-16). Anal. Calcd. for C32H31ClN2O4: C, 70.77; H, 5.75; N, 5.16. Found: C,
70.86; H, 5.64; N, 5.33.
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B. Mistry et al.
9-O-3-(1-(2-Methyl-1H-indole)pentylberberine (5b)
Light-yellow solid. Yield: 61 %. M.p. 256–258 °C. IR (KBr) cm-1: 3092 (C–H,
1
Ar), 1613–1580 (C=C, Ar), 1180–1048 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.61 (s, 1H, H-8), 8.43 (s, 1H, H-13), 7.62 (s, 1H, H-1), 7.44 (s, 1H, H-12), 7.40 (s,
1H, H-4), 7.21–6.61 (m, 5H, indole), 6.51 (s, 1H, H-11), 6.05 (s, 2H, –OCH2O),
4.94 (t, 2H, J = 6.5, H-6), 4.22 (t, 2H, J = 6.3, H-15), 4.12 (s, 3H, OCH3), 3.20 (t,
2H, J = 6.6, H-19), 2.47 (t, 2H, J = 7.1, H-5), 2.40 (br s, 4H, H-17, H-18), 2.19 (m,
2H, H-16), 2.05 (s, 3H, Ar-CH3). Anal. Calcd. for C33H33ClN2O4: C, 71.15; H, 5.97;
N, 5.03. Found: C, 71.02; H, 6.02; N, 5.14.
9-O-3-(1-(5-Fluoro-1H-indole)pentylberberine (5c)
Light-yellow solid. Yield: 56 %. M.p. 263–265 °C. IR (KBr) cm-1: 3028 (C–H,
1
Ar), 1609–1517 (C=C, Ar), 1233–1035 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.84 (s, 1H, H-8), 8.54 (s, 1H, H-13), 7.84 (s, 1H, H-1), 7.56 (s, 1H, H-12), 7.29 (s,
1H, H-4), 7.19–6.81 (m, 5H, indole), 6.70 (s, 1H, H-11), 6.09 (s, 2H, –OCH2O),
5.07 (t, 2H, J = 6.4, H-6), 4.35 (t, 2H, J = 6.3, H-15), 4.03 (s, 3H, OCH3), 3.15 (t,
2H, J = 6.5, H-19), 2.55 (t, 2H, J = 7.3, H-5), 2.37 (br s, 4H, H-17, H-18), 2.21 (m,
2H, H-16). Anal. Calcd. for C32H30ClFN2O4: C, 68.50; H, 5.39; N, 4.99. Found: C,
68.35; H, 5.27; N, 5.09.
9-O-3-(1-(5-Methoxy-1H-indole)pentylberberine (5d)
Light-yellow solid. Yield: 58 %. M.p. 244–246 °C. IR (KBr) cm-1: 3085 (C–H,
1
Ar), 1621–1556 (C=C, Ar), 1244–1022 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.65 (s, 1H, H-8), 8.63 (s, 1H, H-13), 7.75 (s, 1H, H-1), 7.41 (s, 1H, H-12), 7.38 (s,
1H, H-4), 7.28–6.74 (m, 5H, indole), 6.68 (s, 1H, H-11), 6.12 (s, 2H, –OCH2O),
4.96 (t, 2H, J = 6.5, H-6), 4.26 (t, 2H, J = 6.4, H-15), 4.04 (s, 3H, OCH3), 3.91 (s,
3H, Ar-OCH3), 3.23 (t, 2H, J = 6.3, H-19), 2.62 (t, 2H, J = 7.5, H-5), 2.44 (br s,
4H, H-17, H-18), 2.18 (m, 2H, H-16). Anal. Calcd. for C33H33ClN2O5: C, 69.16; H,
5.80; N, 4.89. Found: C, 69.31; H, 5.92; N, 4.96.
9-O-3-(1-(7-Chloro-1H-indole)pentylberberine (5e)
Light-yellow solid. Yield: 63 %. M.p. 277–279 °C. IR (KBr) cm-1: 3034 (C–H,
Ar), 1619–1561 (C=C, Ar), 1192–1019 (C–O–C), 786 (C–Cl). H NMR (CDCl3,
1
500 MHz): d 9.76 (s, 1H, H-8), 8.45 (s, 1H, H-13), 7.65 (s, 1H, H-1), 7.59 (s, 1H,
H-12), 7.27 (s, 1H, H-4), 7.15-6.82 (m, 5H, indole), 6.55 (s, 1H, H-11), 6.01 (s, 2H,
–OCH2O), 5.01 (t, 2H, J = 6.3, H-6), 4.37 (t, 2H, J = 6.5, H-15), 4.05 (s, 3H,
OCH3), 3.15 (t, 2H, J = 6.4, H-19), 2.45 (t, 2H, J = 7.4, H-5), 2.34 (br s, 4H, H-17,
H-18), 2.25 (m, 2H, H-16). Anal. Calcd. for C32H30Cl2N2O4: C, 66.55; H, 5.24; N,
4.85. Found: C, 66.42; H, 5.11; N, 4.72.
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Synthesis, antioxidant and anticancer screenings of…
9-O-3-(1-(Methyl 1H-indole-3-carboxylate)pentylberberine (5f)
Light-yellow solid. Yield: 54 %. M.p. 269–271 °C. IR (KBr) cm-1: 3071 (C–H,
Ar), 1715 (C=O), 1612–1566 (C=C, Ar), 1184–1041 (C–O–C). H NMR (CDCl3,
1
500 MHz): d 9.87 (s, 1H, H-8), 8.58 (s, 1H, H-13), 7.77 (s, 1H, H-1), 7.47 (s, 1H,
H-12), 7.35 (s, 1H, H-4), 7.31–7.05 (m, 5H, indole), 6.73 (s, 1H, H-11), 6.08 (s, 2H,
–OCH2O), 5.09 (t, 2H, J = 6.4, H-6), 4.28 (t, 2H, J = 6.5, H-15), 4.07 (s, 3H,
OCH3), 3.97 (s, 3H, Ar-OCH3), 3.21 (t, 2H, J = 6.3, H-19), 2.58 (t, 2H, J = 7.3,
H-5), 2.41 (br s, 4H, H-17, H-18), 2.16 (m, 2H, H-16). Anal. Calcd. for
C34H33ClN2O6: C, 67.94; H, 5.53; N, 4.66. Found: C, 67.82; H, 5.68; N, 4.77.
9-O-3-(1-(1-(1H-Indol-3-yl)-N,N-dimethylmethanamine)pentylberberine (5g)
Light-yellow solid. Yield: 57 %. M.p. 253–255 °C. IR (KBr) cm-1: 3046 (C–H,
1
Ar), 1625–1574 (C=C, Ar), 1254–1049 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.63 (s, 1H, H-8), 8.66 (s, 1H, H-13), 7.81 (s, 1H, H-1), 7.54 (s, 1H, H-12), 7.24 (s,
1H, H-4), 7.21–7.01 (m, 5H, indole), 6.67 (s, 1H, H-11), 5.96 (s, 2H, –OCH2O),
4.98 (t, 2H, J = 6.4, H-6), 4.36 (t, 2H, J = 6.3, H-15), 4.11 (s, 3H, OCH3), 3.55 (m,
2H, –CH2–N–), 3.19 (t, 2H, J = 6.5, H-19), 2.64 (t, 2H, J = 7.2, H-5), 2.35 (br s,
4H, H-17, H-18), 2.24 (m, 2H, H-16), 2.12 (s, 3H, Ar-CH3),1.90 (s, 3H, Ar-CH3).
Anal. Calcd. for C35H38ClN3O4: C, 70.05; H, 6.38; N, 7.00. Found: C, 70.16; H,
6.49; N, 6.93.
9-O-3-(1-(Indoline)pentylberberine (5h)
Light-yellow solid. Yield: 51 %. M.p. 278–280 °C. IR (KBr) cm-1: 3067 (C–H,
1
Ar), 1607–1552 (C=C, Ar), 1177–1037 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.78 (s, 1H, H-8), 8.49 (s, 1H, H-13), 7.69 (s, 1H, H-1), 7.45 (s, 1H, H-12), 7.37 (s,
1H, H-4), 6.95–6.65 (m, 4H, indoline), 6.58 (s, 1H, H-11), 6.10 (s, 2H, –OCH2O),
5.08 (t, 2H, J = 6.3, H-6), 4.25 (t, 2H, J = 6.4, H-15), 3.98 (s, 3H, OCH3),
3.75–3.31 (m, 4H, indoline), 3.26 (t, 2H, J = 6.4, H-19), 2.43 (t, 2H, J = 7.5, H-5),
2.33 (br s, 4H, H-17, H-18), 2.15 (m, 2H, H-16). Anal. Calcd. for C32H33ClN2O4: C,
70.51; H, 6.10; N, 5.14. Found: C, 70.39; H, 6.02; N, 5.25.
9-O-3-(1-(2-Oxindole)pentylberberine (5i)
Light-yellow solid. Yield: 60 %. M.p. 249–251 °C. IR (KBr) cm-1: 3056 (C–H,
1
Ar), 1614–1563 (C=C, Ar), 1210–1044 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.67 (s, 1H, H-8), 8.56 (s, 1H, H-13), 7.85 (s, 1H, H-1), 7.53 (s, 1H, H-12), 7.22 (s,
1H, H-4), 7.29–6.63 (m, 6H, indole), 6.71 (s, 1H, H-11), 5.98 (s, 2H, –OCH2O),
4.95 (t, 2H, J = 6.5, H-6), 4.38 (t, 2H, J = 6.5, H-15), 4.06 (s, 3H, OCH3), 3.17 (t,
2H, J = 6.3, H-19), 2.57 (t, 2H, J = 7.4, H-5), 2.42 (br s, 4H, H-17, H-18), 2.22 (m,
2H, H-16). Anal. Calcd. for C32H31ClN2O5: C, 68.75; H, 5.59; N, 5.01. Found: C,
68.86; H, 5.47; N, 5.12.
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9-O-3-(1-(7-Azaindol)pentylberberine (5j)
Light-yellow solid. Yield: 55 %. M.p. 281–283 °C. IR (KBr) cm-1: 3083 (C–H,
1
Ar), 1629–1576 (C=C, Ar), 1188–1025 (C–O–C). H NMR (CDCl3, 500 MHz): d
9.82 (s, 1H, H-8), 8.60 (s, 1H, H-13), 7.68 (s, 1H, H-1), 7.49 (s, 1H, H-12), 7.36 (s,
1H, H-4), 7.32–6.67 (m, 5H, azaindol), 6.56 (s, 1H, H-11), 6.04 (s, 2H, –OCH2O),
5.02 (t, 2H, J = 6.4, H-6), 4.27 (t, 2H, J = 6.3, H-15), 3.96 (s, 3H, OCH3), 3.22 (t,
2H, J = 6.5, H-19), 2.60 (t, 2H, J = 7.3, H-5), 2.36 (br s, 4H, H-17, H-18), 2.13 (m,
2H, H-16). Anal. Calcd. for C31H30ClN3O4: C, 68.44; H, 5.56; N, 7.72. Found: C,
68.33; H, 5.68; N, 7.61.
DPPH free radical scavenging assay
Free radicals exercise deleterious effects on biological systems and foods, and hence
radical scavenging activities are very useful. Various chemical reactions that occur in
biological systems usually furnish free radicals that are responsible for damage to
biological building blocks such as DNA, lipids, etc. Reduction of the stable free
radical 2,2-diphenyl-1-picrylhydrazyl is the basis of the DPPH antioxidant bioassay.
It has an odd electron which exhibits a maximum absorption band at 517 nm (deep-
violet color) in ethanol. The DPPH bioassay is a widely used and accepted method for
investigating the free radical scavenging efficacy of an intended compound. Such
substances donate a hydrogen atom when mixed with DPPH, thereby producing the
reduced congener diphenyl picrylhydrazine (nonradical) with loss of the violet color.
In the present study, the DPPH bioassay was adopted to screen the berberine-
based compounds for their in vitro antioxidant potency. The results of this bioassay
screening are presented in the form of the percentage radical scavenging antioxidant
activity (RSA %) for each substance. Investigation of the DPPH radical scavenging
activity was carried out according to the methodology described by Brand-Williams
et al. [58]. The stable free radical 2,2-diphenyl-1-picrylhydrazyl was allowed to
react with the berberine-based scaffolds in methanol solvent with 20 lL quantities
of the title compounds mixed with 180 lL DPPH in MeOH. The title compounds
donated hydrogen on mixing, thereby reducing the DPPH and causing a color
change from deep violet to light yellow at 517 nm after 25 min of reaction,
measured using a UV–visible spectrophotometer (PerkinElmer). Blank reading was
also performed using a mixture of methanol (20 lL) and sample (180 lL DPPH).
Ascorbic acid served as the control drug in this assay, in solution prepared by
mixing methanol (20 lL) and DPPH radical solution (180 lL). The result of this
bioassay was determined as the RSA % value (radical scavenging activity in
percentage) according to Mensor et al. [59] as described by the equation
Absorbance of blank À Absorbance of test
%Scavenging ¼
 100:
Absorbance of blank
A plot between the concentration of the test compound and the % scavenging
value was used to determine the IC50 value in the presence of ascorbic acid as
standard.
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Synthesis, antioxidant and anticancer screenings of…
ABTS radical scavenging assay
The ABTSÁ? radical cation scavenging efficacy of the test compounds was
determined according to the previously described method [60]. Mixing of an equal
amount of 7 mM 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTSÁ?)
stock solution with 2.45 mM potassium persulfate stock solution produces the
ABTSÁ? cation. The mixture was kept in the dark at temperature of 0 °C for 12 h,
and ABTS solution was diluted with MeOH to give a UV absorption value of 0.700
(
0.20) at 734 nm. The 1000 lL stock solutions of the title compounds 5a–j were
established by dissolving them in MeOH, with further dilutions furnishing 100, 10,
1, and 0.1 lL quantities of the samples. We mixed 180 lL solutions of compounds
to be evaluated and 20 lL of ABTS solution in 96-well plates in the dark and then
incubated for 10 min to measure the UV absorption at 734 nm. A mixture of
180 lL ABTS and 20 lL methanol was used for control determination, whereas
ascorbic acid was used as a reference drug. The UV absorption data represent the
radical scavenging rates, giving the corresponding IC50 values for the test
compounds.
The scavenging capability for ABTSÁ? radical was calculated using the equation
Absorbance of blank À Absorbance of test
%Scavenging ¼
 100:
Absorbance of blank
In vitro anticancer bioassay
Cell cultures The test compounds 5a–j were checked for their in vitro anticancer
potential against cervical cancer cell lines HeLa and Caski, and Madin–Darby
canine kidney (MDCK) cells purchased from the American Type Culture Collection
(ATCC). All cell lines were well maintained in a humidified cell culture incubator in
presence of 5 % CO2 at temperature of 32 °C. Dulbecco’s modified Eagle’s medium
(DMEM) and Roswell Park Memorial Institute (RPMI)-1640 medium supplemented
with 10 % fetal bovine serum (FBS) and 1 % antibiotic–antimycotic solution
(1009) were used for HeLa, Caski, and MDCK cell growth, respectively. DMEM,
RPMI-1640, trypsin–ethylenediaminetetraacetic acid (EDTA), antibiotic–antimy-
cotic solution 1009, and FBS were purchased from Welgene (150-Seongseo
Industrial Complex, Bukro, Dalseogu, Daegu, 704–948 Republic of Korea).
In the 96-well plates, both cancer cell lines HeLa and Caski, and MDCK were
seeded at concentration of 2 9 104 cells per well. Cancerous cells were allowed to
grow for 1 day initially, after which the 96-well plates were washed twice with
phosphate-buffered saline (PBS). DMEM and RPMI-1640 medium containing
trypsin–EDTA were used to dilute HeLa, Caski, and MDCK cells to 5 9 103 level,
which was used for infection followed by placing of 10 lL compound and 90 lL
cell solution onto 96-well plates in which HeLa, Caski, and MDCK cells were
grown the previous day. Test compounds at concentrations of 0.1, 1, 10, and 100 lL
were used in 96-well plates for analysis, with three replicates of observations.
Infected plates were incubated in a CO2 incubator for 48 h. After incubation, the
medium was removed followed by washing twice with PBS buffer. Thereafter,
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B. Mistry et al.
70 % acetone was added to fix the cells, which were incubated for 1 h at
temperature of 4 °C. After incubation, the solvent was removed, and the plates were
dried in an oven at temperature of 60 °C. The dried plates were incubated overnight
with 100 lL SRB (0.4 mg/L), followed by SRB removal and washing thrice with
1 % acetic acid, and dried again under hot-air oven at 60 °C. Microscopic
observation was carried out to determine the morphology of the cells, after which
the SRB stain was dissolved with 10 mM Tris base followed by incubation
overnight [61]. Spectrophotometric data were recorded at 510 nm to calculate the
50 % inhibition concentration (IC50), 50 % cytotoxic concentration (CC50), and
therapeutic index (TI) values.
Conclusions
Alkyl chain as an aliphatic linker was utilized to connect two pharmacologically
diverse natural products as well as a heterocyclic core in a compact system in the
form of berberine and indole, respectively. This design was expected to present
enhanced antioxidant and anticancer potential in DPPH, ABTS, and SRB bioassays.
Berberine–indole conjugation was successfully achieved via efficient organic
transformation, and the compounds were adequately characterized. DPPH and
ABTS bioassays suggested that all the title analogues successfully demonstrated
increased radical scavenging efficacies compared with the parent berberine
molecule, and reasonable antioxidant power when compared with the control drug
ascorbic acid. A compound with alkyl and halo functional group in the form of
methyl and chlorine atoms exhibited a significant level of antioxidant effects in
scavenging DPPH and ABTS radicals. However, the aza functional group was found
to be essential in delivering anticancer effects against both cervical cancer cell lines
studied in the present work. Furthermore, the presence of electron-releasing alkyl or
alkoxy functional group was found to be beneficial to exert anticancer potential
against HeLa and Caski cell lines. These compounds exhibited a higher level of
therapeutic index as anticancer agents when compared with berberine. Hence, in the
present study, four different molecules were identified as potent scaffolds
combining natural and heterocyclic cores for future drug discovery studies.
Acknowledgments This article was supported by the KU Research Professor Program of Konkuk
University, Seoul, South Korea.
Compliance with ethical standards
Conflict of interest Authors report no conflict of interest.
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