Synthesis and biological evaluation of a novel betulinic acid derivative as an inducer of apoptosis in human colon carcinoma cells (HT-29)

Article abstract of DOI:10.1016/j.ejmech.2015.07.035

Abstract A novel family of betulinic acid analogues, carrying a triazole unit at C-3 attached through a linker, was synthesized by the application of azide-alkyne "Click reaction". These were screened for their anticancer activity against different cancer cells and normal human PBMC by MTT assay. Compound 2c [(3S)-3-{2-(4-(hydroxymethyl-1H-1,2,3-triazol-1-yl)acetyloxy}-lup-20(29)-en-28-oic acid] was found as the most potent inhibitor of cell line HT-29 with IC₅₀ value 14.9 μM. Its role as an inducer of apoptosis was investigated in this cell line by Annexin-V/PI binding assay and by following its capability for ROS generation, depolarization of mitochondrial transmembrane potential, activation of caspases, PARP cleavage, nuclear degradation and expression of pro- and anti-apoptotic proteins. It exhibited much higher cytotoxicity than the standard drug 5-fluorouracil but showed negligible cytotoxicity towards normal PBMC. Elevated level of ROS generation, activation of caspase 3 and caspase 9, DNA fragmentation, higher expression of Bax and Bad, lower expression of Bcl2 and Bcl-xl, and increased level of Bax/Bcl-xl ratio identified 2c as a promising inducer of apoptosis that follows a mitochondria dependent pathway. Bio-physical studies indicate that compound 2c acts as a minor groove binder to the DNA.

Full text of DOI:10.1016/j.ejmech.2015.07.035

Accepted Manuscript
Synthesis and biological evaluation of a novel betulinic acid derivative as inducer of
apoptosis in human colon carcinoma cells (HT-29)
Biswajit Chakraborty, Debasmita Dutta, Sanjukta Mukherjee, Supriya Das, Nakul C.
Maiti, Padma Das, Chinmay Chowdhury
PII:
S0223-5234(15)30167-7
10.1016/j.ejmech.2015.07.035
EJMECH 8019
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 January 2015
Revised Date: 16 July 2015
Accepted Date: 17 July 2015
Please cite this article as: B. Chakraborty, D. Dutta, S. Mukherjee, S. Das, N.C. Maiti, P. Das, C.
Chowdhury, Synthesis and biological evaluation of a novel betulinic acid derivative as inducer of
apoptosis in human colon carcinoma cells (HT-29), European Journal of Medicinal Chemistry (2015),
doi: 10.1016/j.ejmech.2015.07.035.
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Graphical Abstract
Synthesis and biological evaluation of a novel betulinic acid derivative as
inducer of apoptosis in human colon carcinoma cells (HT-29)
Biswajit Chakrabortya,‡, Debasmita Duttaa,†, Sanjukta Mukherjee‡, Supriya Das§, Nakul C. Maiti§,
Padma Das*,† and Chinmay Chowdhury*,‡
†
‡Organic and Medicinal Chemistry Division, Cancer Biology and Inflammatory Disorder Division,
§Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4 Raja
S. C. Mullick Road, Kolkata 700032, India.
aContributed equally.

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Synthesis and biological evaluation of a novel betulinic acid derivative as inducer of
apoptosis in human colon carcinoma cells (HT-29)
Biswajit Chakrabortya,‡, Debasmita Duttaa,†, Sanjukta Mukherjee‡, Supriya Das§, Nakul C. Maiti§, Padma
Das*,† and Chinmay Chowdhury*,‡
†
‡Organic and Medicinal Chemistry Division, Cancer Biology and Inflammatory Disorder Division,
§Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C.
Mullick Road, Kolkata 700032, India.
aContributed equally.
Abstract: A novel family of betulinic acid analogues, carrying a triazole unit at C-3 attached through a
linker, was synthesized by the application of azide-alkyne “Click reaction”. These were screened for their
anticancer activity against different cancer cells and normal human PBMC by MTT assay. Compound 2c
[(3S)-3-{2-(4-(hydroxymethyl-1H-1,2,3-triazol-1-yl)acetyloxy}-lup-20(29)-en-28-oic acid] was found as
the most potent inhibitor of cell line HT-29 with IC50 value 14.9 µM. Its role as an inducer of apoptosis
was investigated in this cell line by Annexin-V/PI binding assay and by following its capability for ROS
generation, depolarization of mitochondrial transmembrane potential, activation of caspases, PARP
cleavage, nuclear degradation and expression of pro- and anti-apoptotic proteins. It exhibited much higher
cytotoxicity than the standard drug 5-fluorouracil but showed negligible cytotoxicity towards normal
PBMC. Elevated level of ROS generation, activation of caspase 3 and caspase 9, DNA fragmentation,
higher expression of Bax and Bad, lower expression of Bcl2 and Bcl-xl, and increased level of Bax/Bcl-xl
ratio identified 2c as a promising inducer of apoptosis that follows a mitochondria dependent pathway.
Bio-physical studies indicate that compound 2c acts as a minor groove binder to the DNA.
Abbreviations: BA, Betulinic Acid; DIPEA,
N,N-diisopropylethylamine;
DMAP, 4-
dimethylaminopyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMC,
peripheral blood mononuclear cells; ROS, Reactive oxygen species; FACS, Fluorescence-activated cell
sorting; PARP, Poly(ADP-ribose)polymerase; CD, Circular dichroism.
Keywords: Betulinic acid, Triazole derivative, Cytotoxic activities, Mitochondrial dysfunction, Caspase
activation, DNA binding.
Corresponding authors:E-mail: chinmay@iicb.res.in (CC), Tel: 91-33-2499 5862 (CC).
padmadas@iicb.res.in (PD), Tel: 91-33-2499 5899

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1. Introduction
The synthetic anticancer drugs used along with conventional radiotherapy and chemotherapy frequently
evoke adverse side effects, severe immunosuppression and alarming increase in the incidence of drug
resistance. In contrast, natural products are considered to be a better option in the development of new
drugs because of their potent therapeutic efficacy and minimum side effects. Indeed, some of the natural
products have been used directly as a drug, while others have served as a source of structural or functional
template in drug design. In recent years there has been a tremendous interest worldwide in the design and
development of analogue libraries based on natural product templates. Notably, roughly around 50% of
the currently used anti-cancer drugs were discovered from studies on either natural products or natural
product based molecules [1].
Triterpenes represent an important class of natural products. Among these, pentacyclic lupane-
type triterpenes are one of the most significant subclasses and some of them do exhibit several medicinal
properties including anticancer [2]. Betulinic acid (BA) 1 (Fig. 1), a member of this class of pentacyclic
triterpenes, has been shown to possess a range of biological activities such as anti-HIV [3a], anti-bacterial
[3b], anti-malarial [3c], anti-inflammatory [3d] etc. Additionally, BA is also found to be cytotoxic
towards a variety of cancer cell lines such as lung, ovarian, cervical, neuroblastoma, and gliobastoma
including head and neck carcinomas [4]. Betulinic acid was also reported as a selective inhibitor of
human melanoma [11a]; it induces apoptosis in human neuroblastoma [11b] as well. It has low toxicity
and high safety profile in normal cell lines [5], and is safe for use even at a dose of 500 mg/Kg body
weight [4a]. This feature calls for its development as anticancer drug in preference to other natural
products like taxol, camptothecin, ellipticine, etoposide, vinblastin and vincristin etc. Previous reports
reveal that the apoptotic properties of BA are due to modulation of Bcl-2 family and cell cycle regulatory
protein [6], downregulation of NF-kB [7], inhibition of amino peptidase N [8], and growth factor induced
angiogenesis [9]. Besides, BA acts as potent inhibitor of eukaryotic topoisomerase I and II [10]. The
cytotoxic effects of BA have been studied in xenograft mouse models and the compound is currently
undergoing clinical trials at NCI, USA [12]. From mechanistic view point, betulinic acid induces
apoptosis by direct effects on mitochondria leading to cytochrome-c release, which in turn regulates the
"downstream" caspase activation resulting in apoptosis ultimately [13]. This study indicates that
mitochondria could be an attractive target in the development of anti-cancer therapeutics based on
betulinic acid.
Despite such encouraging results, the poor solubility of BA preventing blood serum
solubilizations and its low bioavailability have limited its potential to be used as a drug candidate. In
order to overcome this lacuna, efforts are being made for structural modifications through judicious

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manipulation of the functional groups present in the molecule [14]. In continuation of our studies [15] on
structural modulations of bioactive natural products for the development of lead(s) having better efficacy
and less toxicity than the parent compound, we chose betulinic acid to prepare an analogue library. We
realised that a 1,2,3-triazole moiety can be attached at C3 hydroxy of BA through a linker, leading to the
formation of analogues 2 (Fig. 1). The rationale for selecting 1,2,3-triazole was its remarkable binding
affinity with biological receptors/enzymes through non-covalent interactions resulting in the discovery of
various potent bioactive agents [16]. Besides, this class of compounds possess exceptional metabolic
stability [17]. These favourable therapeutic indexes have led to the development of several 1,2,3-triazole
containing drugs available in the market [18]. For our present study, we synthesized a number of 1,2,3-
triazole derivatives 2 based on betulinic acid and assessed the potency of these compounds and the
parent compound for the induction of cell death in five different cancer cell lines (derived from four
different types of cancers) along with normal epithelial cell lines. 5-Fluorouracil, a well known agent in
cancer chemotherapy, was used as a standard compound. The findings show that one of the developed
molecules bears better anticancer potency against human colon carcinoma cells (HT-29) than the parent
compound (BA) and 5-fluorouracil. The results obtained so far are described herein.
Figure 1
2. Results and discussions
2.1. Chemistry:
Betulinic acid used in this study was isolated in bulk quantity (~ 50 g) from methanolic extracts of the
Dillenia indica fruits following the literature procedure [19]. Purification (>98%) was achieved by silica
gel chromatography followed by fractional crystallizations. Thereafter, we initiated the study of
derivatizations; we chose the C3 hydroxyl group of BA as functionalizations at this site are found to be
more suited for showing anticancer activities compared to other functional groups (at C28 and C20) [4a].
Thus, (3S)-3-(2-chloroacetyloxy)-lup-20(29)-en-28-oic acid (3) was prepared (Scheme 1) by treating BA
(1) with chloroacetyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) and 4-
dimethylaminopyridine (DMAP, catalytic amount). Azidation of 3 using sodium azide led to the isolation
of the intermediate (3S)-(2-azidoacetyloxy)-lup-20(29)-en-28-oic acid (4) which served as a useful
substrate for “click chemistry” employed in this work for the synthesis of targeted derivatives 2 (Scheme
1). On the other hand, some of the requisite acetylenic substrates 5 were prepared by the well-known
“Sonogashira coupling”[20] between trimethylsilyl acetylene and aryl iodides followed by base
Scheme-1

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mediated desilylation of the resulting products; other acetylenes were procured from commercial source.
Thereafter, 1,3-dipolar cycloadditions of azide 4 with acetylenes 5 were carried out at room temperature
by employing “Click Chemistry”. The crude products 2 obtained after standard work-up of the reactions
were purified (>96%) by silica gel (100-200 mesh) column chromatography; the yields (Table 1) and
spectral data (see experimental section) usually refer to these compounds. However, for anti-cancer
screening studies, these products were further purified (>99%) by HPLC using reverse phase C18 column
[21]. As can be seen from Table 1 (entries 2-10), alkyl/aryl/heteroaryl acetylenes 5b-j underwent
cycloadditions successfully with the azide 4 at room temperature within few hours affording 1,2,3-triazole
derivatives 2. Besides, even gaseous acetylene (balloon pressure) can be used as substrate in this reaction
to afford the desired product 2a with 60% yield (entry 1, Table 1). Interestingly, irrespective of the nature
of the substituents (i.e., OMe, NO2, CO2Me, CO2Et) in aryl/heteroaryl moiety of the acetylenic substrates
5, all (except entry 1 of Table 1) produced their corresponding 1,2,3-triazole derivatives 2 with very good
1
yields (80-90%). In H-NMR, signal for the proton of 1,2,3-triazole ring of compounds 2 appeared
around at δ 7.5 as singlet, while that for the methylenic protons (-COCH2N-) at the C-3 side chain
appeared in the region δ 4.6-5.6 as two separate doublets with coupling constant 12-17 Hz. Besides, the
peaks for vinylic protons (H-29) of products 2 appeared as singlets around at δ 4.6 and 4.7, respectively,
while that for H-3 was found at around δ 4.5 as multiplet (m). The remaining signals appeared at their
appropriate positions.
Table 1
In a further study, we exposed the 1,4-dioxane solution of product 2d to moist acetic acid at rt for 1h to
achieve chemoselective deprotection of one of the isopropylidene moieties; this afforded the novel sugar
derivative 2k as shown in Scheme 2.
Scheme 2
In a recent study, few important betulinic acid derivatives conjugated with a well-known anti-viral agent
AZT (3΄-azido-3´-deoxythymidine) were synthesized and evaluated [22]. In view of the immense
importance of uracil derivatives in cancer chemotherapy[23] along with our own interest[24] on these
compounds, we became interested to convert the product 2e to corresponding uracil derivative which may
serve as uracil-betulinic acid conjugate via a 1,2,3-triazole linker. Indeed, treatment of compound 2e with
trimethylsilyl chloride and sodium iodide at rt led to the formation of desired uracil derivative 2l with
good yields (Scheme 3).
Scheme 3

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2.2. Biology:
2.2.1. Cytotoxic activity of Betulinic Acid analogues:
The cytotoxic activity of betulinic acid analogues 2a-l was studied using MTT assay on cultured cancer
cells such as U937 (Human leukemic monocyte lymphoma cell line), HT-29 (Human colorectal
adenocarcinoma), Jurkat (human T cell lymphoblast-like cell line), HepG2 (human hepatocellular
carcinoma), MCF-7 (human breast adenocarcinoma) as well as normal human PBMC (peripheral blood
mononuclear cells) cells (Table 2). 5-Fluorouracil was used as the reference compound. We assessed the
effects of different concentrations (0-50 µM) of the analogues for 48 h. Considering the IC50 values of
Table 2, we conclude that compound 2c was the most potent inhibitor of HT-29 cells (IC50 14.9 µM); it
also exhibited negligible cytotoxicity towards normal PBMC. All other IC50 values were more than 40
µM. The concentration of DMSO used to solubilize these compounds was 0.1% which had no effect on
cell viability. As 2c appeared to be the best among all the analogues tested, we compared the effects of
the compound with standard drug 5-fluorouracil and the parent compound betulinic acid (see Table S1 in
supplementary material); thereafter, we performed all apoptotic studies on HT-29 cell line using this
compound (2c) as discussed below.
Table 2
2.2.2. Compound 2c triggers ROS generation:
Reactive oxygen species (ROS) are highly harmful elements to cells as they initiate oxidative stress and
ultimately cause cellular damage. Excessive ROS generation renders cells vulnerable to apoptosis. To
determine whether 2c triggers ROS generation in HT-29 cells to induce apoptosis, the ROS level was
measured with and without (control) treatment of 2c (at IC50 dose) using CM-H2DCFDA – a lipid
soluble, membrane permeable non-fluorescent reduced derivative of 2,7-dichlorofluorescein. This was
used based on the premise that the acetate groups of CM-H2DCFDA are removed by esterase cleavage
intracellularly to produce the hydrophilic, non-fluorescent dye dichlorodihydrofluorescein (DCFH2)
which is subsequently oxidized by ROS to form the highly fluorescent product dichlorofluorescein
(DCF). The fluorescence generated is directly proportional to the quantum of ROS generated [25]. The
ROS level was measured in terms of MFI. Interestingly, compound 2c was capable of inducing ROS in
HT-29 cells within 15 min of treatment, with MFI value 1270±14.6 which is more than two times that
of control, p<0.001 (Fig. 2A). Taken together, these results indicate that 2c causes oxidative imbalance in
HT-29 cells. This induction of oxidative burst is a key factor behind the anti proliferative activity of 2c.
To confirm the elevated levels of ROS generation by 2c, cells were pre-incubated with 2.5 mM of N-
acetyl cysteine (NAC), an established anti-oxidant, for 3 h before treatment with 2c. To determine the role

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of ROS in 2c mediated action, cells preincubated with NAC (2.5 mM; 3 h) were treated with 2c and
analyzed for cell viability assay. In the pretreated cells, there was no change in percentage of cell
viability. But in cells subjected to 2c treatment after incubation with NAC, cell viability increased two
times (IC50: 30.4 µM, Fig. 2B), thereby confirming that IC50 concentration of 2c was the only contributing
factor responsible for excessive ROS generation and ultimately causing apoptosis.
Figure 2A and 2B
2.2.3. Analogue 2c induces apoptosis in HT-29 cells:
In order to determine whether the 2c induced reduction in cell viability was responsible for the induction
of apoptosis, HT-29 cells were co-stained with PI and Annexin-V FITC, and the number of apoptotic cells
was estimated by flow cytometry. Translocation of phosphatidylserine from the inner aspect to the outer
leaflet of the plasma membrane occurs during apoptosis which can be detected by utilizing the high
binding affinity of Annexin V, a Ca+2 dependent phospholipid binding protein, to phosphatidylserine [26].
In contrast, the non permeable stain propidium iodide (PI) has affinity towards nucleic acids and
selectively enters necrotic or late apoptotic cells. A time-dependent increase in the percentage of
apoptotic cells was noted after the cells were treated for 12, 24, and 48 h with analogue 2c at IC50
concentration. As shown in Fig. 3, very few (0.3%) apoptotic cells were present in the control panel,
whereas after treatment with 2c the population rose to 15.6% at 12 h. Further increase to 17.6% and
23.4% occurred after treatment for 24 and 48 h, respectively. The percentage of PI positive cells is
negligible. These indicate that 2c induces significant apoptotic cell death in HT-29 cells in comparison
with control, in a time-dependent manner.
Figure 3
2.2.4. Compound 2c triggers mitochondrial pathway dependent apoptosis:
The loss of mitochondrial transmembrane potential (ꢀψm) also reflects apoptosis. This was
measured by using a cationic, lipophilic, fluorescent dye JC-1 capable of selectively entering
mitochondria and acting as a dual emission probe [27]. With normal cells (high MMP), JC-1 displays a
green fluorescence (590 nm). This is caused by spontaneous and local formation of aggregates that are
associated with a large shift in the emission. In contrast, when the mitochondrial membrane is depolarized
(low MMP), JC-1 forms monomers that emit blue fluorescence (at 530 nm). Here, the percentage of green
fluorescence represents cells with polarized mitochondria whereas that of blue fluorescence serves as an
indicator of cells with loss of mitochondrial transmembrane potential. A gradual time dependent change
from green to blue fluorescence was observed in HT-29 cells after treatment with 2c. In control cells,

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percentage of cells with depolarized mitochondria was 1.75, which increased to 16.5% at 12 h, 30.5% at
24 h and 51.4 % at 48 h of treatment, suggesting the occurrence of depolarization of mitochondria by 2c
(Fig. 4).
Figure 4
2.2.5. Activation of caspases and cleavage of PARP by 2c:
Various proteolytic enzymes (like cytochrome c which activates caspases) are released from depolarized
mitochondria and are the main executors of apoptosis. Caspases are also the key regulatory proteases of
apoptotic pathways. Activation of this family of proteins provides confirmatory evidence towards
apoptosis. Depolarization of the mitochondrial transmembrane potential causes a change in the
mitochondrial membrane permeability which helps to release apoptotic factors from mitochondria and
thereby activates caspases [28]. In order to prove that 2c induces caspase activation, cell lysates were
prepared as described in materials and methods and levels of activated caspases 8, 9 and 3 were measured
using quantitative detection of colorimetric tetrapeptide substrates. As shown in Fig. 5A and 5B, activities
of caspases 3 and 9 increased significantly after 48 h treatment with 2c as compared to control. In
contrast, the level of caspase 8 (Fig. 5C) did not increase significantly even after 48 h of treatment. To
confirm the role of caspases in apoptosis induced by 2c, we studied the specific cleavage of PARP (116
kDa), a well known substrate of caspase 3 [29]. When HT-29 cells were treated with analogue 2c at IC50
concentration, specific cleavage of PARP yielded 85 kDa fragments (as shown in the results), which was
confirmed by analysis using western blotting with anti-PARP antibodies for different time durations (Fig.
5D). A well known function of PARP is its ability to repair single-strand DNA nicks. Indeed cleavage of
PARP was found to be causing abatement of DNA repairing process leading to further cellular damage.
Figure 5A, 5B, 5C and 5D
2.2.6. Compound 2c causes DNA fragmentations and morphological changes:
An essential hallmark of apoptosis is DNA fragmentation and morphological changes caused by the
involvement of caspases. We stained the HT-29 cells with Hoechst 33258 dye. The treatment of the HT-
29 cells with 14.9 µM of compound 2c resulted in the induction of chromatin condensation,
fragmentation, membrane blebbing and clear apoptotic bodies that were visualized in confocal
microscopy (Fig. 6).
Figure 6

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2.2.7. Compound 2c induced expression of pro- and anti-apoptotic proteins:
Since Bax and Bcl-2 play a significant role in apoptosis [30], we studied the effect of analogue 2c on the
expression of pro-apoptotic protein Bax and Bad, and anti-apoptotic proteins Bcl-2 and Bcl-xl in HT-29
cells. The Western blot analysis demonstrated time dependent elevations in Bax and Bad (Fig. 7A); this
was clearly reflected in the gradual reduction in Bcl-2/Bax ratio with time (**p<0.01; Fig. 7B). The
significant reduction in the ratio suggested the involvement of Bcl-2 family proteins, a mediator of the
mitochondrial apoptosis pathway, in apoptosis of HT-29 cells induced by 2c, which is mediated by the
mitochondria-dependent pathway.
Figure 7A and 7B
2.3. Bio-physical studies of compound 2c:
2.3.1. Compound 2c decreases UV absorption of DNA binding:
The binding efficacy and mode of binding to DNA of 2c were measured by absorption spectroscopic
analysis. DNA shows an intense absorption band at 260 nm (Fig. 8A). Addition of 2c caused decrease in
the absorption. The normalized absorbance vs wavelength plot clearly indicated decrease in absorbance
with increase in concentration of 2c (Fig. 8B).[31] This implied clearly that 2c interacts with DNA
effectively. The binding isotherm (Fig. 9) for DNA-2c interaction was obtained by plotting fraction (θ) of
DNA bound to 2c vs total compound concentration. The value of θ was calculated according to the
following equation:
θ = (A0 -Ai)/(A0- Aα)
where A0 is the absorbance of DNA in the absence of the compound at 260 nm. Ai is the absorbance of
DNA in the presence of the compound and Aα is the absorbance of DNA at compound concentration for
which maximum binding took place. Kb was obtained from the reciprocal of the ligand concentration at
the half saturation point of the binding isotherm.[32] The measured binding constant was 8.375 × 104 M-1.
Figure 8A, 8B and 9
2.3.2. Compound 2c caused no significant changes in the DNA conformation:
Circular dichroism (CD) analysis was carried out to measure the effect of binding to conformational
changes in DNA duplex structure. The CD spectra of the DNA solution (500 µg/mL) in the absence and
presence of 2c (50 µM) were recorded after 2 h incubation of the mixture at 37 °C (Fig. 10). DNA
solution showed a positive absorbance at 275 nm due to base stacking and a negative absorbance at 245
nm due to the B-conformation of DNA [32]. The absorbance and peak position were not perturbed
significantly in the presence of 2c (red line in Figure 10). It is known from earlier reports that the

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electrostatic interaction or groove binding of small molecules to DNA does not severely affect the
absorbance at 275 nm, whereas, changes in absorption at 245 nm implies change in B-DNA conformation
or the formation of other structures [32]. The CD spectra of the DNA in the presence of 2c was very
similar to the CD spectra of DNA itself, indicating that 2c caused no major alteration of DNA
conformation. Thus this study implied that compound 2c may act as groove binder of DNA but not as
intercalator.
Figure 10
2.3.3. Molecular Modeling: Compound 2c binds to a minor groove of DNA as determined by Molecular
Docking:
In order to find the type of groove binding (major or minor), we carried out molecular modeling studies
on compound 2c. Out of the 100 docking runs performed for DNA-2c complexes, favorable binding
poses were obtained which presented most negative binding free energies. Molecular docking results
showed that 2c had the best binding energy of -10.09 kcal/mol when it bound with the DNA as a minor
groove binder. A crystallographic DNA with no gap (PDB entry 1DNE, resolution 2.4 Å), which was the
crystal structure of a DNA complex with the known minor groove binder [33] netropsin (obtained binding
energy -7.02 kcal/mol), was chosen as the control model so that it would be capable of accommodating a
minor groove binder (Fig. 11). Water molecules and the bound netropsin were removed from the
structure. Then, the obtained DNA structure was used as receptor model for the docking studies.
Figure 11
3. Conclusion
In this endeavor, to develop promising anticancer agent(s) based on betulinic acid, a new library of
compounds having 1,2,3-triazole moiety attached to C-3 of betulinic acid were synthesized and the anti-
cancer activities of these compounds were evaluated (in vitro) against different cancer cell lines (i.e.,
breast, colon, liver and leukemic). Interestingly, one (2c) of the compounds was found to be significantly
active against HT-29 (human colon adenocarcinoma) cell lines and this activity profile was better than
that of the parent compound (BA). Induction of apoptosis by 2c is characterized by ROS generation, the
externalization of phosphotidylserine, loss in mitochondrial membrane potential, induced expression of
pro and antiapoptotic proteins, and appreciable DNA fragmentations. The structure-activity relationship
studies (SARs) indicate that the 1,2,3-triazole moiety favors the activity when substituted at C4 (of the
heterocycle) with a hydroxymethyl group, but disfavors it when the substituent is an aromatic or
heteroaromatic moiety. Further studies to refine it are currently under progress.

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4. Experimental
4.1. Chemistry
4.1.1 General methods
Betulinic acid (1) was isolated from dried and powdered fruits of Dillenia indica. All solvents were
distilled prior to use. Petroleum ether refers to fraction boiling in the range 60-80 °C. Dichloromethane
(DCM) was dried over CaH2, distilled and stored over 3Å molecular sieves. All the reactions were
performed under argon atmosphere and anhydrous conditions unless otherwise noted. Reactions were
monitored by thin-layer chromatography (TLC) on aluminium sheets 20 × 20 cm coated with silica gel 60
F254. Visualization of the developed chromatogram was performed by UV light or iodine or Liebermann
solution. Analytical (C18, 5 µm, 4.6 × 250 mm column) and semi preparative (C18, 5 µm, 10 × 250 mm
column) HPLC were performed to purify the final compounds.
1H and 13C NMR spectra were recorded in 300 or 600 MHz spectrometer. Chemical shifts (δ) are reported
in parts per million (ppm) downfield from tetramethylsilane (δ = 0.00) with the residual proton signal of
deuterated solvents used [CDCl3 : 1H NMR, δ = 7.26 ppm (s), 13C NMR, δ = 77.0 ppm (t); CD3OD : 1H
NMR, δ = 4.86 ppm, 3.32 ppm, 13C NMR, δ = 49.0 ppm (m)]. Coupling constants (J) were expressed in
Hertz (Hz) and spin multiplicities are given as s (singlet), d (doublet), dd (double doublet), t (triplet), m
(multiplet) and br (broad). Mass spectra were obtained from ESI-TOF and FAB-EI MS mass
spectrometer. Infrared spectra (IR) were obtained as neat or KBr plate. Specific rotations were measured
on JASCO P-1020 polarimeter at 589 nm.
4.1.2. Preparations of starting materials
4.1.2.1. (3S)-3-(2-chloroacetyloxy)-lup-20(29)-en-28-oic acid (3)
The compound was prepared according to the literature procedure [34] (for details see the
supplementary material).
4.1.2.2. (3S)-(2-azidoacetyloxy)-lup-20(29)-en-28-oic acid (4)
Compound 3 (500 mg, 0.94 mmol) was dissolved in dry DMF, mixed with NaN3 (123 mg, 1.9
mmol) under argon atmosphere and the reaction mixture was allowed to reflux at 70 °C for 4 h. After
completion of the reaction (confirmed by monitoring TLC) the solvent was evaporated to dryness under
reduced pressure and the residue was extracted with ethyl acetate (3× 15 mL). The combined organic
extracts were washed with water (10 mL) and brine (10 mL), dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The resulting residue was purified through silica gel (100-200

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mesh) column chromatography using 10% ethyl acetate in petroleum ether (v/v) as eluent to obtain the
desired product 4. Yield: 77%, white solid, m.p. = 245-246°C; IR (Neat) νmax : 2944, 2107, 1738, 1695,
1
1196 cm-1; H NMR (CDCl3, 300 MHz): δ 4.74 (1H, s), 4.61-4.57 (2H, m), 3.85 (2H, s), 3.04-2.97 (1H,
m), 2.29-2.11 (2H, m), 2.03-1.91 (2H, m), 1.69 (3H, s), 1.65-1.39 (12H, m), 1.34-1.16 (6H, m), 0.97 (3H,
s), 0.93 (3H, s), 0.87 (3H, s), 0.85 (3H, s), 0.82 (3H, s), 0.79 (3H, s); 13C NMR (CDCl3, 75 MHz): δ
182.7, 168.1, 150.3, 109.7, 83.2, 56.4, 55.3, 50.6, 50.3, 49.2, 46.9, 42.4, 40.6, 38.4, 38.3, 37.8, 37.1, 34.1,
32.1, 30.5, 29.6, 27.9, 25.3, 23.6, 20.8, 19.3, 18.1, 16.4, 16.1, 15.9, 14.6; MS (ESI) 562.20 [M+Na]+.
HRMS [EI] + m/z calculated for C32H49N3O4 539.37231, found 539.37207.
4.1.3. General procedure for the synthesis of 1,2,3-triazole derivatives 2 (except the compound 2a)
employing “Click Chemistry”:
Compound 4 (1.0 eqv.) dissolved in dry DMSO (2 mL) was treated with terminal acetylenes 5 (1.1 eqv.)
in the presence of sodium ascorbate (1.01 eqv.) and CuSO4.5H2O (1.01 eqv.), and the mixture was stirred
at room temperature for 2-18 h under argon atmosphere until the complete consumption of starting
materials. It was then purified by silica gel (100-200 mesh) column chromatography using 70-88% ethyl
acetate in petroleum ether (v/v) as eluent. The yield was calculated based on the product isolated through
column chromatography; however, products screened for anti-cancer assay were further purified by
HPLC.
Synthesis of compound 2a and its spectroscopic data have been provided in supplementary material.
4.1.3.1. (3S)-3-[2-(4-butyl-1H-1,2,3-triazol-1-yl)acetyloxy]-lup-20(29)-en-28-oic acid (2b)
Yield: 98%; white solid, m.p. = 133-135 °C; [α]25 + 20.2 (c 0.23, CHCl3); IR (Neat) νmax: 3144, 3072,
D
2950, 2870, 1744, 1643, 1460, 1374, 1269, 1222 cm-1; 1H NMR (CDCl3, 300 MHz): δ 7.40 (1H, s), 5.13
(1H, d, J = 17.7 Hz), 5.11 (1H, d, J = 17.7 Hz), 4.73 (1H, s), 4.60 (1H, s), 4.54 (1H, dd, J = 11.1, 4.5 Hz),
3.00-2.99 (1H, m), 2.74 (2H, t, J = 7.6 Hz), 2.29-2.25 (1H, m), 2.17-2.15 (1H, m), 1.99-1.95 (3H, m),
1.68 (3H, s), 1.66-1.56 (9H, m), 1.52-1.32 (12H, m), 1.27-1.15 (3H, m), 0.96 (3H, s), 0.93 (3H, s), 0.92
(3H, s), 0.82 (3H, s), 0.81 (3H, s), 0.72 (3H s); 13C NMR (CDCl3,150 MHz): δ 180.5, 166.2, 150.3, 148.9,
121.9, 109.7, 83.7, 56.3, 55.3, 51.1, 50.3, 49.2, 46.8, 42.4, 40.6, 38.3, 38.2, 37.8, 37.1, 37.0, 34.1, 32.1,
31.5, 30.5, 29.6, 27.9, 25.4, 25.2, 23.7, 22.2, 20.8, 19.3, 18.1, 16.3, 16.1, 16.0, 14.6, 13.8. MS (ESI)
644.39 [M+Na]+. HRMS [EI]+ m/z calculated for C38H59N3O4 621.45056, found 621.45105.
4.1.3.2. (3S)-3-[2-(4-hydroxymethyl-1H-1,2,3-triazol-1-yl)acetyloxy]-lup-20(29)-en-28-oic acid (2c)
Yield: 84%; white solid, m.p. = 212-214 °C; [α]25 + 33.8 (c 0.24, CHCl3); IR (Neat) νmax: 3415, 3144,
D
1
3073, 2947, 2872, 1741, 1689, 1457, 1374, 1274, 1229, 1013 cm-1; H NMR (CDCl3, 300 MHz): δ 7.62

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(1H, s), 5.20 (1H, d, J = 17.4 Hz), 5.11 (1H, d, J = 17.4 Hz), 4.82 (1H, d, J = 13.5 Hz), 4.76 (1H, d, J =
13.5 Hz), 4.73 (1H, s), 4.60 (1H, s), 4.52 (1H, dd, J = 11.2, 4.65 Hz), 3.02-2.98 (1H, m), 2.31-2.18 (3H,
m), 2.10-1.91 (4H, m), 1.68 (3H, s), 1.63-1.49 (9H, m), 1.45-1.05 (10H, m), 0.95 (3H, s), 0.89 (3H, s),
0.78 (3H, s), 0.75 (3H, s), 0.54 (3H, s); 13C NMR (CDCl3,75 MHz): δ 181.1, 165.5, 150.5, 148.1, 123.2,
109.6, 83.8, 56.3, 55.5, 55.1, 51.4, 50.1, 49.2, 46.8, 42.3, 40.5, 38.0, 37.7, 37.0, 33.9, 32.2, 30.5, 29.6,
27.8, 25.2, 23.5, 20.7, 19.3, 18.2, 16.1, 16.0, 14.5; MS (ESI) 618.12 [M+Na]+. HRMS (FAB)+ m/z
calculated for C35H54N3O5 [M+H]+ 596.40635, found 596.40790.
4.1.3.3. (3S)-3-[2-(4-{[5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,2-
d][1,3]dioxol-6-yloxy]methyl}-1H-1,2,3-triazol-1-yl)acetyloxy]-lup-20(29)-en-28-oic acid (2d)
Yield: 98%; white solid, m.p. = 169-171 °C; 1H NMR (CDCl3, 300 MHz): δ 7.77 (1H, s), 5.87 (1H, d, J =
3.6 Hz), 5.17 (1H, d, J = 17.7 Hz), 5.14 (1H, d, J = 17.7 Hz), 4.86 (1H, d, J = 12.6 Hz), 4.81 (1H, d, J =
12.6 Hz), 4.73 (1H, s), 4.60 (d, J=3.3 Hz, 1H), 4.60-4.53 (2H, m), 4.34-4.28 (1H, m), 4.15-4.08 (2H, m),
4.07 (1H, brs), 4.02-3.97 (1H, m), 3.03- 2.96 (1H, m), 2.29-2.10 (3H, m), 2.04-1.91 (3H, m), 1.68 (3H, s),
1.64-1.56 (9H, m), 1.49 (3H, s), 1.42 (3H, s), 1.35 (3H, s), 1.31 (3H, s), 1.28-1.01 (10H, m), 0.96 (3H, s),
0.88 (3H, s), 0.83 (6H, brs), 0.75 (3H, s); 13C NMR (CDCl3, 75 MHz): 182.0, 165.9, 150.3, 145.2, 123.8,
111.8, 109.7, 109.0, 105.2, 83.8, 82.5, 81.7, 81.0, 72.3, 67.3, 63.9, 56.3, 55.2, 51.0, 50.2, 49.1, 46.8, 42.3,
40.6, 38.2, 37.8, 37.0, 34.1, 32.1, 30.5, 29.6, 27.9, 26.8, 26.1, 25.4, 23.5, 20.8, 19.2, 18.0, 16.3; MS (ESI)
860.24 [M+Na]+. HRMS (FAB)+ m/z calculated for C47H72N3O10 [M+H]+ 838.52177, found 838.51849.
4.1.3.4. (3S)-3-[2-{4-(2,4-dimethoxypyrimidin-5-yl)-1H-1,2,3-triazol-1-yl}acetyloxy]-lup-20(29)-en-
28-oic acid (2e)
Yield: 82%; white solid; [α]25 + 10.5 (c 0.27, MeOH); IR (Neat) νmax: 3444, 2948, 2871, 1744, 1617,
D
1568, 1467, 1381, 1284, 1223, 1141, 1086, 1043, 1013 cm-1; 1H NMR (CDCl3, 300 MHz): δ 9.17 (1H, s),
8.04 (1H, s), 5.23 (1H, d, J = 17.2 Hz), 5.21 (1H, d, J = 17.2 Hz), 4.73 (1H, s), 4.60-4.57 (1H, m), 4.08
(3H, s), 4.05 (3H, s), 3.00-2.99 (1H, m), 2.29-2.18 (3H, m), 2.04-1.95 (3H, m), 1.68 (3H, s), 1.64-1.56
(6H, m), 1.48-1.32 (10H, m), 1.28-1.01 (4H, m), 0.96 (3H, s), 0.92 (3H, s), 0.83 (3H, s), 0.79 (3H, s), 0.74
(3H, s); 13C NMR (CDCl3,75 MHz): δ 182.1, 166.8, 166.0, 164.5, 156.2, 150.4, 140.0, 123.7, 109.7,
106.2, 83.9, 56.3, 55.2, 55.0, 54.3, 51.1, 50.3, 49.2, 46.9, 42.4, 40.6, 38.3, 38.2, 37.8, 37.0, 34.1, 32.1,
30.5, 29.6, 27.9, 25.3, 23.5, 20.8, 19.3, 18.1, 16.3, 16.1; MS (ESI) 726.08 [M+Na]+. HRMS (FAB)+ m/z
calculated for C40H58N5O6 [M+H]+ 704.43871, found 704.43878.

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4.1.3.5. (3S)-3-{2-[4-(1H-indol-5-yl)-1H-1,2,3-triazol-1-yl]acetyloxy}-lup-20(29)-en-28-oic acid (2f)
Yield: 70%; white solid, m.p. = 207-209 °C; [α]25D + 16.7 (c 0.16, MeOH); IR (Neat) νmax : 3414, 2946,
1
2870, 1741, 1640, 1449, 1371, 1223, cm-1; H NMR (DMSO-d6, 300 MHz): δ 11.1 (1H, s), 8.44 (1H, s),
8.01 (1H, s), 7.58 (1H, dd, J = 8.4, 1.2 Hz), 7.44 (1H, d, J = 8.4 Hz), 7.36 (1H, t, J = 2.5 Hz), 6.47 (1H, s),
5.49 (1H, d, J = 17.4 Hz), 5.36 (1H, d, J = 17.7 Hz), 4.68 (1H, s), 4.55 (1H, s), 4.47 (1H, t, J = 7.8 Hz),
4.05 (1H, brs), 2.99-2.91 (3H, m), 2.25-2.08 (3H, m), 1.90-1.72 (3H, m), 1.63 (3H, s), 1.50 (6H, m), 1.47-
1.10 (10H, m), 0.93 (3H, s), 0.85 (3H, s), 0.80 (3H, s), 0.78 (3H, s), 0.68 (3H, s); 13C NMR (DMSO-d6, 75
MHz): δ 166.9, 150.3, 147.9, 135.7, 127.9, 126.1, 121.6, 121.5, 119.0, 116.8, 111.8, 101.4, 82.0, 55.4,
54.4, 50.6, 49.5, 48.5, 46.6, 42.0, 37.5, 36.5, 33.6, 31.7, 30.1, 29.2, 27.5, 25.0, 23.2, 20.4, 18.9, 17.6, 16.1,
15.8, 15.7, 14.3; MS (ESI) 703.36 [M+Na]+. HRMS (FAB)+ m/z calculated for C42H57N4O4
[M+H]+681.43798, found 681.43800.
4.1.3.6. (3S)-3-[2-(4-phenyl-1H-1,2,3-triazol-1-yl)acetyloxy]-lup-20(29)-en-28-oic acid (2g)
Yield: 98%; white solid, m.p. = 148-151 °C; [α]25 -119.8 (c 0.21, CHCl3); IR (Neat) νmax : 3442, 3138,
D
3074, 2946, 1746, 1718, 1643, 1465, 1376, 1266 cm-1; 1H NMR (CDCl3, 300 MHz): δ 7.90 (1H, s), 7.84
(2H, d, J = 7.2 Hz), 7.46-7.32 (3H, m), 5.21 (2H, s), 4.73 (1H, s), 4.60 (1H, s), 4.57-4.55 (1H, m), 3.00-
2.98 (1H, m), 2..28- 2.14 (3H, m), 2.04-1.95 (3H, m), 1.68 (3H, s), 1.64-1.56 (3H, m), 1.45-1.37 (10H,
m), 1.28-1.06 (6H, m), 0.96 (3H, s), 0.92 (3H, s), 0.84-0.83 (6H, m), 0.74 (3H, s); 13C NMR (CDCl3, 75
MHz): δ 181.4, 165.9, 150.3, 148.2, 130.3, 128.8, 128.2, 125.8, 120.9, 83.9, 56.3, 55.3, 51.2, 50.3, 49.2,
46.9, 42.4, 40.6, 38.3, 37.9, 37.1, 34.1, 32.1, 30.5, 29.6, 28.0, 25.4, 23.6, 20.8, 19.3, 18.1, 16.3, 16.1, 15.9,
14.6; MS (ESI) 664.36 [M+Na]+. HRMS (FAB)+ m/z calculated for C40H56N3O4 [M+H]+ 642.42708,
found 642.42600.
4.1.3.7. (3S)-3-{2-[4-(4'-nitrophenyl)-1H-1,2,3-triazol-1-yl]acetyloxy}-lup-20(29)-en-28-oic acid (2h)
Yield: 80%; white solid, m.p. = 233-235 °C; [α]25 + 18.1 (c 0.15, CHCl3);IR (Neat) νmax: 3442, 3139,
D
1
3074, 2948, 2871, 1743, 1688, 1606, 1517, 1459, 1341, 1231, 1108, 1044 cm-1; H NMR (CDCl3, 300
MHz): δ 8.31 (1H, d, J = 8.7 Hz), 8.07 (1H, s), 8.02 (1H, d, J = 8.7 Hz), 5.25 (2H, s), 4.73 (1H, s), 4.60-
4.57 (2H, m), 2.99-2.96 (1H, m), 2.28-2.15 (3H, m), 1.97-1.95 (3H, m), 1.68 (3H, s), 1.64-1.56 (8H, m),
1.45-1.37 (8H, m), 1.28-1.15 (5H, m), 0.96 (3H, s), 0.92 (3H, s), 0.85 (3H, s), 0.84 (3H, s), 0.76 (3H, s);
13C NMR (DMSO-d6, 75 MHz): δ 177.2, 166.6, 150.3, 146.7, 144.4, 136.9, 125.9, 124.9, 124.6, 115.6,
109.6, 82.2, 55.8, 55.4, 54.3, 50.8, 49.5, 48.5, 46.6, 42.0, 37.5, 37.4, 36.5, 36.3, 33.6, 32.1, 31.6, 30.1,
29.1, 29.0, 27.5, 25.0, 23.2, 20.4, 18.9, 17.6, 16.1, 15.8, 15.6, 14.3; MS (ESI) 709.57 [M+Na]+. HRMS
(FAB)+ m/z calculated for C40H55N4O6 [M+H]+ 687.41216, found 687.41205.

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4.1.3.8. (3S)-3-{2-[4-(4'-methoxycarbonyl-phenyl)-1H-1,2,3-triazol-1-yl]acetyloxy}-lup-20(29)-en-28-
oic acid (2i)
Yield: 91%; white solid, m.p. = 181-182 °C; [α]25 + 11.0 (c 0.13, MeOH); IR (Neat) νmax : 3427, 3143,
D
3072, 2948, 2871, 1722, 1616, 1458, 1375, 1281, 1226, 1109, 1017 cm-1; 1H NMR (CDCl3, 600 MHz): δ
8.10 (2H, d, J = 8.4 Hz), 8.00 (1H, s), 7.91 (2H, d, J = 8.4 Hz), 5.24 (1H, d, J = 17.4 Hz), 5.21 (1H, d, J =
17.4 Hz), 4.72 (1H, s), 4.60 (1H, s), 4.57 (1H, d, J = 4.8 Hz), 3.93 (3H, s), 3.01-2.97 (1H, m), 2.26 (1H, d,
J = 12.6 Hz), 2.17 (1H, t, J = 5.4 Hz), 2.02-1.94 (2H, m), 1.78-1.70 (2H, m), 1.68 (3H, s), 1.65-1.58 (3H,
m), 1.53-1.42 (4H, m), 1.41-1.34 (7H, m), 1.32-1.23 (4H, m), 1.85-1.62 (1H, m), 1.06-0.99 (2H, m), 0.96
13
(3H, s), 0.91 (3H, m), 0.83 (3H, s), 0.82 (3H, s), 0.78-0.76 (1H, m), 0.74 (3H, s); C NMR (CDCl3,
150MHz): δ 166.7, 165.8, 150.3, 147.2, 134.6, 130.2, 129.7, 125.6, 121.8, 109.7, 84.0, 56.2, 55.3, 52.1,
51.2, 50.3, 49.2, 46.9, 42.4, 40.6, 38.3, 38.2, 37.8, 37.06, 37.01, 36.5, 34.1, 32.1, 31.4, 30.5, 29. 7, 29.6,
28.0, 25.3, 23.6, 20.8, 19.3, 18.0, 16.3, 16.1, 16.0, 14.6. MS (ESI) 722.49 [M+Na]+. HRMS (FAB)+ m/z
calculated for C42H58N3O6 [M+H]+ 700.43256, found 700.43353.
4.1.3.9. (3S)-3-{2-[4-(4'-ethoxycarbonyl-phenyl)-1H-1,2,3-triazol-1-yl]acetyloxy}-lup-20(29)-en-28-
oic acid (2j)
Yield: 90%; white solid, m.p. = 144-146 °C; [α]25 + 16.9 (c 0.23, CHCl3); IR (Neat) νmax : 3443, 3144,
D
3072, 2947, 2871, 1714, 1617, 1460, 1372, 1277, 1228, 1107, 1018 cm-1; 1H NMR (CDCl3, 600 MHz): δ
8.10 (2H, d, J = 8.4 Hz), 8.00 (1H, s), 7.91 (2H, d, J = 7.8 Hz), 5.24 (1H, d, J = 18 Hz), 5.21 (1H, d, J =
18 Hz), 4.72 (1H, s), 4.60 (1H, s), 4.59-4.57 (1H, m), 4.40 (1H, d, J = 7.2 Hz), 4.37 (1H, d, J = 7.2 Hz),
3.01-2.97 (1H, m), 2.26 (1H, d, J = 12.6 Hz), 2.19-2.15 (1H, m), 2.05-2.00 (2H, m), 1.73-1.70 (1H, m),
1.68 (3H, s), 1.65-1.56 (3H, m), 1.54-1.46 (4H, m), 1.49-1.39 (7H, m), 1.36-1.32 (3H, m), 1.30-1.21 (4H,
m), 1.20-1.59 (1H, m), 1.07-0.99 (2H, m), 0.96 (3H, s), 0.91 (3H, s), 0.83 (3H, s), 0.82 (3H, s), 0.78-0.76
(1H, s), 0.74 (3H, s); 13C NMR (CDCl3, 150MHz): δ 166.2, 165.8, 150.3, 147.2, 134.5, 130.1, 125.5,
121.8, 109.7, 84.0, 61.0, 55.2, 51.2, 50.3, 49.2, 46.9, 42.4, 40.6, 38.3, 38.2, 37.8, 37.0, 34.1, 32.1, 30.5,
29.6, 27.9, 25.3, 23.6, 20.8, 19.3, 18.0, 16.3, 16.1, 16.0, 14.6, 14.3. MS (ESI) 736.50 [M+Na]+. HRMS
(FAB)+ m/z calculated for C43H60N3O6 [M+H]+ 714.44821, found 714.44507.
4.2. Biology
4.2.1. Ethics Statement
Ethical approvals of blood samples used in this study and consent procedure were obtained from Internal
Review Board (Ethical Committee on Human Subjects) of CSIR-Indian Institute of Chemical Biology.

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All clinical investigations have been conducted according to the principles expressed in the Declaration of
Helsinki.
4.2.2. Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from USB
Corporation (USA). Pen strep, RPMI 1640, High Glucose DMEM, and Heat inactivated Fetal Bovine
Serum (FBS), 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethyl benzimidazolyl carbocyanine iodide (JC-1), and 5-
(and-6)-chloromethyl-2',7'-dichloro dihydrofluorescein diacetate (CM-H2DCFDA) were obtained from
Invitrogen (Carlsbad, CA, USA). Caspase-3, Caspase-8, Caspase-9 colorimetric assay kits were procured
from Biovision (Milpitas, CA, USA). The antibodies against Bcl2, Bax, Bcl-xl, Bad, β-Actin, and PARP,
Alkaline phosphatase/Horseradish peroxidase conjugated secondary antibodies, and enhanced
chemiluminescence kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
4.2.3. Apoptosis Assay
In brief, HT-29 cells (2.5 x 105/ml) were incubated with or without compound 2c, the most potent
betulinic acid derivative (IC50: 14.9 ꢁM), for 12, 24 and 48 h at 37 °C, 5% CO2. Cells were then washed
twice in PBS and resuspended in Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM
CaCl2; pH 7.4). Annexin V-FITC was then added according to the manufacturer’s instructions and the
mixture was incubated for 15 min under dark conditions at 25 °C. PI (0.1 mg/ml) was added just prior to
acquisition. Data was acquired using a FACS Aria flow cytometer (Becton Dickinson, USA) at an
excitation wavelength of 488 nm and an emission wavelength of 530 nm, and analyzed with BD FACS
Diva software (Becton Dickinson, USA).
4.2.4. Measurement of Intracellular Reactive Oxygen Species (ROS)
The effect of IC50 concentration of compound 2c on generation of ROS was measured in HT-29 cells (2.5
× 105/ml). After treatment for 0-2 h, cells were washed with PBS (2000 × g, 5 min), resuspended in PBS
and then incubated with CM-H2DCFDA (5 ꢁM in1X PBS) for 30 min at 37 ⁰C in dark condition.
Subsequently, cells were again washed and resuspended in 500 ꢁl of PBS. DCF fluorescence was
determined by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 530
nm in FACS Calibur (Becton Dickinson, USA) using forward vs. side scatter to gate the cell population
and a FL1 histogram to quantify fluorescence of viable cells. The subsequent analyses were done using
BD CellQuest Pro software. To confirm the elevated levels of ROS induced by 2c for the inhibition of
ROS generation, cells were pre-incubated with 2.5 mM of N-Acetyl cysteine (NAC), an established anti-

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oxidant, for 3 h before treatment with 2c and analyzed for cell viability assay. Mean fluorescence
intensities (MFI) were obtained using the FACS Diva software.
4.2.5. Detection of Mitochondrial Membrane Potential
Briefly, HT 29 cells (2.5 × 105/ml) were incubated with compound 2c (14.9 µM) for 12, 24, and 48 h at
37 °C, 5% CO2. The cells were then washed with PBS and incubated with JC-1 (7.5 mM in PBS) under
dark conditions for 15 min at 20–25 °C. Cells were acquired using FACS and analyzed using FACS Diva
software. HT-29 cells treated with H2O2 (30 mM; 37⁰C; 15 min), illustrative of cells with depolarized
mitochondrial membrane potential, served to set the quadrants.
4.2.6. Western Blotting Analysis
Control and 2c treated (14.9 µM; 0–48 h) HT-29 cells were lysed in lysis buffer (50 mM Tris–HCl pH
7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mg/ml protease inhibitor cocktail, 5 mM PMSF and
1 mM DTT containing 1% Triton X-100), sonicated and centrifuged for 10 min at 4 °C at 10,000×g,
and the protein concentration was estimated. Electrophoretic separations (50 mg protein/lane) were
carried out on 10% SDS-polyacrylamide gel, and electrotransferred onto a PVDF membrane. Blots were
blocked for 1 h at 37 °C in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.02% Tween 20 (TBST) containing
5% skimmed milk, and probed using 1:2000 dilution of appropriate antibodies (Bax, Bad, Bcl-2, Bcl-xl,
PARP, and β-Actin) by incubating overnight at 4 °C. The membranes were washed thrice with TBST,
incubated with alkaline phosphatase/Horseradish peroxidase conjugated secondary antibody, and the
bands were visualized using a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate or
enhanced chemiluminescence kit.
4.2.7. Caspase activity analysis
The enzymatic activities of caspase -8, -9, -3 were assayed in cell lysates (100 µg protein in 50 µl lysis
buffer) using colorimetric assay kits as per the manufacturer’s instructions. Briefly, control and
compound 2c treated (14.9 µM; 0–48 h) HT-29 cells (2.5×105/ml) were washed with ice cold PBS, cell
lysates were prepared and subsequently protein concentration was estimated. Lysates were combined with
reaction buffer and incubated with specific colorimetric peptide substrates (Ac-IETD-pNA for Caspase-8,
Ac-LEHD-pNA for Caspase-9, Ac-DEVD-pNA for Caspase-3; 4 mM, 5 µl) at 37 °C for 6 h. The
emission of paranitroanilide (pNA) was measured at 405 nm in an ELISA reader every 30 min for 6 h.

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4.2.8. Cellular and Nuclear Morphology Analysis
HT 29 cells were seeded (2.5 × 105 cells/ml) in a 6-well tissue culture plate and the drug (2c) was added.
Plates were incubated for 48 h at 37 °C in a humidified 5% CO2 incubator. Hoechst 33258 solution was
prepared in MilliQ water at a concentration of 5 µg/ml. Cells were washed twice in PBS, fixed in 4%
paraformaldehyde, and incubated at 4 °C for 30 min. Cells were again washed twice in PBS. Hoechst
33258 was added to a final concentration of 5 µg/ml and incubated at 37 °C for 30 min under dark
conditions. Cells were again washed twice in PBS [35], and spotted on to poly-L-lysine coated slides and
mounted using 50% glycerol. Slides were then analyzed in a laser scanning confocal microscope (Leica
TCS SP2 System Leica Microsystem, Heidelberg, Germany, 100×). At least 20 randomly selected
microscopic fields were observed per sample.
4.3. Bio-physical studies
4.3.1. UV-vis absorption spectroscopic study
Unless otherwise stated, all chemicals were of analytical grade and were purchased from Sigma-Aldrich
Chemicals. The CT-DNA was purchased from Bangalore Genie.
The UV-vis absorption spectrophotometric studies were carried out with 1 cm path length quartz
cuvette using UV-vis spectrophotometer; (UV-2401PC, Shimadzu). The spectra were scanned in the
wavelength range 200-400 nm. Stock solution of each of the compounds having different concentration
was prepared in 60% ethanol in 50 mM Tris-HCl/NaCl buffer (pH 7.5) and then diluted to varied
concentrations with the buffer. Then the absorption spectra for different concentrations were taken to
obtain the molar extinction coefficient which was used to determine the actual concentration of the
compound throughout the experiment. CT DNA concentration was kept fixed at 50 µg/ml and the
concentration of the compound was varied from 0 to a higher concentration as required for individual
compounds. The DNA-compound complex was incubated at 37 °C for 2 h and the absorption spectra
were taken with proper dilution in the range 200-400 nm.
4.3.2. Circular dichroism (CD) spectroscopic study
CD spectroscopic measurements were performed at 25 °C with cuvettes having path length 1 mm using
CD spectrometer (Jasco J- 815) with the sensitivity of 100 millidegree and in continuous scanning mode
with a speed of 100 nm/min; band-width was 1 nm and the number of accumulations was three. Scans

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were taken from 190 to 250 nm. CT-DNA (1000 mg/ml) was used for recording spectra in the presence
and absence of compound 2c (100 µM).
4.3.3. Molecular Docking Study
A crystallographic DNA with no gap (PDB entry 1DNE, resolution 2.4 Å), which was a crystal structure
of a minor groove binder netropsin-DNA complex, was chosen as the receptor capable of accommodating
a minor groove binder. Water molecules and the bound netropsin were removed from the structure. The
obtained DNA structure was used as receptor.
The compound structure was optimized using Gaussian 09W with DFT method and basis set 3-21G. The
obtained structure was used as ligand model. Netropsin was used as control with DNA as receptor.
AutoDock Tools was used for docking. Polar and aromatic hydrogens were added with computing the
Gasteiger charges on ligand atoms. Torsional degree of freedom for the ligand was defined automatically
using the software. The grid box was centered on the receptor and the dimension of the grid was adjusted
to include the entire DNA fragment with the spacing between the grid points 0.403 Å. Grid potential
maps were calculated using the module AutoGrid 4.0. The Lamarckian genetic algorithm was used to
perform docking simulation, with an initial population of 150 randomly placed individuals with a
maximum number of 250000 energy evaluations, 150000 generations, mutation rate of 0.02, a crossover
rate of 0.8 and an elitism value of 1. The number of docking runs was chosen to be 100. Then least
energetical docking conformation was chosen from the output file.
Acknowledgements
This work is financially supported by CSIR network projects ORIGIN (CSC0108), miND (BSC-0115)
and TREAT (BSC 0116). Partial financial supports from Department of Biotechnology (DBT), Govt. of
West Bengal and Department of Biotechnology (DBT), New Delhi are also gratefully acknowledged.
The authors are thankful to Dr. B. Achari, Emeritus Scientist for checking the manuscript and to Dr. Sib
Sankar Roy, Cell Biology and Physiology Division of this institute for extending his laboratory facilities.
S.D. thanks UGC, Government of India for a fellowship during this study.
Appendix A. Supplementary material
Supplementary material related to this article can be found at http://dx.doi.orgXXX

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References and notes:
1. (a) F. E. Koehn, G. T. Carter, The evolving role of natural products in drug discovery, Nat. Rev. Drug
Disc. 4 (2005) 206-220. (b) J.W.-H. Li, J.C. Vederas, Drug Discovery and Natural Products: End of an
Era or an Endless Frontier?, Science. 325 (2009) 161-165.
2. F. Sandberg, H. Dutschewska, V. Christov, S. Spassov, Spondianthus preussii var. glaber Engler.
Pharmacological screening and occurrence of triterpens, Acta Pharm. Suec. 24 (1987) 253-256.
3. (a) F. Li, R. Goila-Gaur, K. Salzwedel, N. R. Kilgore. M. Reddick, C. Matallana, A. Castillo, D.
Zoumplis, D. E. Martin, J. M. Orenstein, G. P. Allaway, E. O. Freed, C. T. Wild, PA-457: A potent HIV
inhibitor that disrupts core condensation by targeting a late step in Gag processing, Proc. Natl. Acad. Sci.
U.S.A. 100 (2003) 13555-13560; (b) C. Chandramu, R. D. Manohar, D. G. Krupadanam, R. V.
Dashavantha, Isolation, Characterization and Biological Activity of Betulinic Acid and Ursolic Acid from
Vitex negundo L, Phytother. Res. 27 (2003) 129-134; (c) G. Bringmann, W. Saeb, L. A. Assi, G. Francois,
A. S. S. Narayanan, K. Peters, E. M. Petera, Betulinic Acid: Isolation from Triphyophyllum peltatum and
Ancistrocladus heyneanus, Antimalarial activity, and crystal structure of the benzyl ester, Planta Med. 63
(1997), 255-257; (d) P. K. Mukherjee, K. Saha, J. Das, M. Pal, B. P. Saha, Studies on the anti-
inflammatory activity of rhizomes of Nelumbo nucifera, Planta Med. 63 (1997) 367–369.
4. (a) R. H. Cichewicz, S. A. Kouzi, Chemistry, biological activity, and chemotherapeutic potential of
betulinic acid for the prevention and treatment of cancer and HIV infection, Med. Res. Rev. 24 (2004) 90-
114. (b) D. A. Eiznhamer, Z.-Q. Xu, Betulinic acid: a promising anticancer candidate, IDrugs. 7 (2004)
359-373. (c) E. Pisha, H. Chai, I. S. Lee, T. E. Chagwedera, N. R. Farnsworth, G. A. Cordell, C. W. W.
Beecher, H. H. S. Fong, A. D. Kinghorn, D. M. Brown, M. C. Wani, M. E. Wall, T. J. Hieken, T. K.
Dasgupta, J. M. Pezzuto, Discovery of betulinic acid as a selective inhibitor of human melanoma that
functions by induction of apoptosis, Nat. Med. 1 (1995) 1046-1051. (d) D. Thurnher, D. Turhani, M.
Elzmann, B. Wannemacher, B. Knerer, M. Formanek, V. Wacheck, E. Selzer, Betulinic acid: a new
cytotoxic compound against malignant head and neck cancer cells, Head Neck. 25 (2003) 732-740. (e) J.
H. Kessler, F. B. Mullauer, D. M. de Roo, J. P. Medema, Broad in vitro efficacy of plant-derived betulinic
acid against cell lines derived from the most prevalent human cancer types, Cancer Lett. 251 (2007) 132-
145. (f) S. Fulda, C. Friesen, M. Los, C. Scaffidi, W. Mier, M. Benedict, G. Nunes, P. H. Krammer, M.
E. Peter, K. M. Debatin, Betulinic acid triggers CD95 (APO-lIFas)- and p53-independent apoptosis via
activation of caspases in neuroectodermal tumors, Cancer Res. 57 (1997) 4956-4964.

ACCEPTED MANUSCRIPT
5. V. Zuco, R. Supino, S. C. Righetti, L. Cleris, E. Marchesi, C. G. Passerini, F. Formelli, Selective
cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells, Cancer Lett. 175 (2002) 17-25.
6. (a) W. Rzeski, A. Stepulak, M. Szymanski, M. Sifringer, J. Kaczor, K. Wejksza, B. Zdzisinska, M. N.-
S. Kandefer-Szerszen, Betulinic acid decreases expression of bcl-2 and cyclin D1, inhibits proliferation,
migration and induces apoptosis in cancer cells, Arch. Pharmacol. 374 (2006) 11-20; (b) E. Selzer, C.
Thallinger, C. Hoeller, P. Oberkleiner, V. Wacheck, H. Pehamberger, B. Jansen, Betulinic acid-induced
Mcl-1 expression in human melanoma: mode of action and functional significance, Mol. Med. 8 (2002)
877-884.
7. (a) Y. Takada, B. B. Aggarwal, Betulinic acid suppresses carcinogen-induced NF-κB activation
through inhibition of IκBα Kinase and p65 phosphorylation: Abrogation of cyclooxygenase-2 and matrix
metalloprotease-9, J. Immunol. 171 (2003) 3278-3286; (b) H. Kasperczyk, K. La Ferla-Bruhl, M. A.
Westhoff, L. Behrend, R. M. Zwacka, K. M. Debatin, S. Fulda, Betulinic acid as new activator of NF-κB:
molecular mechanisms and implications for cancer therapy, Oncogene. 24 (2005) 6945-6956.
8. M. F. Melzig, H. Bormann, Betulinic acid inhibits aminopeptidase N activity, Planta Med. 64 (1998)
655-657.
9. H. J. Kwon, J. S. Shim, J. H. Kim, H. Y. Cho, Y. N. Yum, S. H. Kim, J. Yu, Betulinic acid inhibits
growth factor-induced in vitro angiogenesis via the modulation of mitochondrial function in endothelial
cells, Jpn. J. Cancer Res. 93 (2002) 417-425.
10. A. Ganguly, B. Das, A. Roy, N. Sen, S. B. Dasgupta, S. Mukhopadhayay, H. K. Majumder, Betulinic
acid, a catalytic inhibitor of Topoisomerase I, inhibits reactive oxygen species–mediated apoptotic
Topoisomerase I–DNA cleavable complex formation in prostate cancer cells but does not affect the
process of cell death, Cancer Res. 67 (2007) 11848-11858.
11(a) Y. Tan, R. Yu, J.M. Pezzuto, Betulinic acid-induced programmed cell death in human melanoma
cells involve mitogen-activated protein kinase activation, Clin. Cancer Res. 9 (2003) 2866-2875; (b) M.
L. Schmidt, K. L. Kuzmanoff, L. Ling-Indeck, J. M. Pezzuto, Betulinic acid induces apoptosis in human
neuroblastoma cell lines, Eur. J. Cancer. 33 (1997) 2007-2010.
12. http://clinicaltrials.gov/show/NCT00346502.
13. S. Fulda, K.-M. Debatin, Betulinic acid induces apoptosis through direct effect on mitochondria in
neuroectodermal tumors, Med. Pediatr. Oncol. 35 (2000) 616-618.

ACCEPTED MANUSCRIPT
14. (a) Y. Kashiwada, F. Hashimota, L.M. Cosentina, C.H. Chen, P.E. Garren, K.H. Lee, Betulinic Acid
and Dihydrobetulinic Acid Derivatives as Potent Anti-HIV Agents, J. Med. Chem. 39 (1996) 1016-1017;
(b) Y. Kashiwada, J. Chiyo, Y. Ikeshino, T. Nagao, H. Okabe, L.M. Cosentino, K. Fowke, K.H. Lee,
3,28-Di-O-(dimethylsuccinyl)-betulin isomers as anti-HIV agents, Bioorg. Med. Chem. Lett. 11 (2001)
183-185; (c) I. D. Bori, H. Y. Hung, K. Qian, C. H. Chen, S.L. M. Natschke, K. H. Lee, Anti-AIDS
agents 88. Anti-HIV conjugates of betulin and betulinic acid with AZT prepared via click chemistry,
Tetrahedron Letters. 53 (2012) 1987-1989; (d) K. Nakagawa-Goto, K. Yamada, M. Taniguchi, H.
Tokuda, K.-H. Lee, Cancer preventive agents 9. Betulinic acid derivatives as potent cancer
chemopreventive agents, Bioorg. Med. Chem. Lett. 19 (2009) 3378-3381; (e) R. Majeed, P. L. Sangwan,
P. K. Chinthakindi, I. Khan, N. A. Dangroo, N. Thota, A. Hamid, P. R. Sharma, A. K. Saxena, S. Koul,
Synthesis of 3-O-propargylated betulinic acid and its 1,2,3-triazoles as potential apoptotic agents, Eur. J.
Med. Chem. 63 (2013) 782-792; (f) W. Zhang, X. Men, P. Lei, Review on anti-tumor effect of triterpene
acid compounds, J. Can. Res. Ther. 10 (2014) 14-19; (g) R.Csuk, Betulinic acid and its derivatives: a patent
review (2008-2013) Expert Opin. Ther. Patents. 24 (2014) 913-923; (h) D. Emmerich, K. Vanchanagiri, L.
C. Baratto, H, Schmidt, R. Paschke, Synthesis and studies of anticancer properties of lupane-type
triterpenoid derivatives containing a cisplatin fragment, Eur. J. Med. Chem. 75 (2014) 460-466; (i) S.
Schwarz, B. Siewert, N. M. Xavier, A. R. Jesus, A. P. Rauter, R. Csuk, A “natural” approach: Synthesis
and cytoxicity of monodesmosidic glycyrrhetinic acid glycosides, Eur. J. Med. Chem. 72 (2014) 78-83;
(j) H. Zhang, P. Zhu, J. Liu, X. Yang, S. Xu, H. Yao, J. Jiang, W. Ye, X. Wu, J. Xu, Synthesis and
antitumor activity of novel 3-oxo-23-hydroxybetulinic acid derivatives, Eur. J. Med. Chem. 87 (2014)
159-167.
15. (a) R. Preet, B. Chakraborty, S. Siddharth, P. Mohapatra, D. Das, S. R. Satapathy, S. Das, N. C. Maiti,
P.R. Maulik. C. N. Kundu, C. Chowdhury, Synthesis and biological evaluation of andrographolide
analogues as anti-cancer agents, Eur. J. Med. Chem. 85 (2014) 95-106; (b) B. Das, C. Chowdhury, D.
Kumar, R. Sen, R. Roy, P. Das, M. Chatterjee, Synthesis, cytotoxicity, and structure–activity relationship
(SAR) studies of andrographolide analogues as anti-cancer agent, Bioorg. Med. Chem. Lett. 20 (2010)
6947-6950; (c) V. Srivastava, M. P. Darokar, A. Fatima, J.K. Kumar, C. Chowdhury, H.O. Saxsena, G.R.
Dwivedi, K. Shrivastava, V. Gupta, S.K. Chattopadhya, S. Luqman, M.M. Gupta, A.S. Negi, P.S.
Khanuja, Synthesis of diverse analogues of Oenostacin and their antibacterial activities, Bioorg. Med.
Chem. 15 (2007) 518-525.
16. See a recent review article: S. G. Agalave, S.R. Maujan, V. S. Pore, Click Chemistry: 1,2,3-Triazoles
as Pharmacophores, Chem. Asian J. 6 (2011) 2696-2718.

ACCEPTED MANUSCRIPT
17. J. M. Holub, K. Kirshenbaum, Tricks with clicks: modification of peptidomimetic oligomers via
copper-catalyzed azide-alkyne [3 + 2] cycloaddition, Chem. Soc. Rev. 39 (2010) 1325−1337
18. (a) M. J. Soltis, H. J. Yeh, K. A. Cole, N. Whittaker, R. P. Wersto, E. C. Kohn, Identification and
characterization of human metabolites of CAI [5-amino-1-1(4'-chlorobenzoyl-3,5-dichlorobenzyl)-1,2,3-
triazole- 4-carboxamide), Drug Metab. Dispos. 24 (1996) 799-806; (b) C. Sheng, W. Zhang, New Lead
Structures in Antifungal Drug Discovery, Curr. Med. Chem. 18 (2011) 733-766 and see also reference 16.
19. D. Kumar, S. Mallick, J. R. Vedasiromoni, B. C. Pal, Anti-leukemic activity of Dillenia indica L. fruit
extract and quantification of betulinic acid by HPLC, Phytomedicine. 17 (2010) 431.
20. (a) K. Sonogashira, Y. Tohda, N. A. Haghihara, Convenient synthesis of acetylenes: catalytic
substitutions of acetylenic hydrogen with bromoalkenes, iodoarenes and bromopyridines, Tetrahedron
Lett. 16 (1975) 4467-4470; (b) K. Sonogashira, in Comprehensive Organic Synthesis, ed. B. M. Trost,; I.
Fleming; Pergamon, Oxford, 1991, vol 3, pp. 521; (c) K. Sonogashira, In Metal Catalysed Cross Coupling
Reactions; F. Diederich, A. de Meijera, , Eds; Wiley-VCH:Weinheim, 2004, vol 1, pp 319.
21. Products 2a, 2b-d, 2k and 2l were separated in HPLC using acetonitrile/water/acetic acid (65:35:1) as
mobile phase with flow rate 2.5 mL/min. The retention time (tR) of these compounds were found to be
30.2, 22.5, 15.0, 18.8, 19.2 and 26.4 min, respectively. The other products (2f, 2g-i and 2j) were separated
in HPLC using acetonitrile/water/acetic acid (90:10:1) as mobile phase with the same flow rate. The
retention times (tR) were 18.2, 17.3, 14.3, 15.6 and 17 min, respectively.
22. I. D. Bori, H.-Y. Hung, K. Qian, C.-H. Chen, S. L. Morris-Natschke, K.-H. Lee, Tetrahedron Lett. 53
(2012) 1987-1989.
23.(a) C. Heidelberger, J. F. Holand , E. Frei (Eds.), Pyrimidine and Pyrimidine Antimetabolites in
Cancer Medicine, Lea and Febiger, Philadelphia, PA, 1984, pp. 801–824; (b) D. Pizzirani, C. Pagliuca,
N. Realini, D. Branduardi, G. Bottegoni, M. Mor, F. Bertozzi, R. Scarpelli, D. Piomelli, T. Bandiera,
Discovery of a New Class of Highly Potent Inhibitors of Acid Ceramidase: Synthesis and
Structure−Activity Relationship (SAR), J. Med. Chem. 56 (2013) 3518−3530; (c) F. Focher, D. Ubiali, M.
Pregnolato, C. Zhi, J. Gambino, G. E. Wright, S. Spadari, Novel Nonsubstrate Inhibitors of Human
Thymidine Phosphorylase, a Potential Target for Tumor-Dependent Angiogenesis, J. Med. Chem. 43
(2000) 2601-2607.
24. N. G. Kundu, J. S. Mahanty, C. Chowdhury, S. K. Dasgupta, B. Das, C. P. Spears, J. Balzarini, E.
De Clercq, 5-(Acylethynyl)uracils, 5-(Acylethynyl)-2'-deoxyuridines and 5-(Acylethynyl)-1-(2-

ACCEPTED MANUSCRIPT
hydroxyethoxy)methyluracils. Their synthesis, antiviral and cytotoxic activities, Eur. J. Med. Chem. 34
(1999) 389-398.
25. L. Gibellini, M. Pinti, M. Nasi, S. D. Biasi, E. Roat, Interfering with ROS Metabolism in Cancer
Cells: The Potential Role of Quercetin, Cancers. 2 (2010) 1288–1311.
26. R. C. Widaua, A. D. Parekha, M. C. Rancka, D. W. Goldena, K. A. Kumara, R. F. Sooda, S. P.
Pitrodaa, Z. Liaoa, X. Huanga, T. E. Dargaa, D. Xua, L. Huangb, J. Andradeb, B. Roizmanc, R. R.
Weichselbauma, N. N. Khodareva, RIG-I–like receptor LGP2 protects tumor cells from ionizing
radiation, PNAS. (2014) E484-E491; see also reference 15b.
27. D. Deeb, X. Gao, H. Jiang, B. Janic, A. S. Arbab, Y. Rojanasakul, S. A. Dulchavsky, S. C. Gautam,
Oleanane triterpenoid CDDO-Me inhibits growth and induces apoptosis in prostate cancer cells through a
ROS-dependent mechanism, Biochem Pharmacol. 79 (2010) 350–360.
28. M. Leist, M. Jäättelä, Four deaths and a funeral: From caspases to alternative mechanisms, Nat. Rev.
Mol. Cell. Biol. 2 (2001) 589–598.
29. C. Mandal, A. Dutta, A. Mallick, S. Chandra, L. Misra, R. S. Sangwan, C. Mandal, Withaferin A
induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells
of lymphoid and myeloid origin through mitochondrial death cascade, Apoptosis. 13 (2008) 1450–1464.
30. M.H. Harris, C.B. Thompson, The role of the Bcl-2 family in the regulation of outer mitochondrial
membrane permeability, Cell Death Differ. 7 (2000) 1182–1191.
31. S.K. Samanta, D. Dutta, S. Roy, K. Bhattacharya, S. Sarkar, A.K. Dasgupta, B.C. Pal, C. Mandal, C.
Mandal, Mahanine a DNA minor groove binding agent exerts cellular cytotoxicity with involvement of
C-7-OH and -NH functional groups, J. Med. Chem. 56 (2013) 5709-5721.
32. S. Panja, B. Jana, P. Aich, T. Basu, In vitro interaction between calf thymus DNA and Escherichia
coli LPS in the presence of divalent cation Ca2+, Biopolymers. 89 (2008) 606-613; see also reference 15a.
33. M. Coll , J. Aymami, G.A. Van der Marel, J.H. Van Boom, A. Rich, A.H. J. Wang, Molecular
structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT
segment, Biochemistry. 28 (1989) 310-320.

ACCEPTED MANUSCRIPT
34. R. D. Goff, J. S. Thorson, Enhancing the Divergent Activities of Betulinic Acid via Neoglycosylation,
Org. Lett. 11(2) (2009) 461-464.
35. R. Roy, D. Kumar, B. Chakraborty, C. Chowdhury, P. Das, Apoptotic and Autophagic Effects of
Sesbania grandiflora Flowers in Human Leukemic Cells, Plos One (2013) 8(8): e71672.
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Captions for Figures, Schemes and Tables
Figure 1. Betulinic acid (1) and its designed analogs (2)
Scheme 1: Preparation of 1,2,3-triazole derivatives of betulinic acid 2. Reagents and conditions: (a)
chloro acetyl chloride, N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP, cat.),
THF, r.t., 2 h, 86%; (b) sodium azide, DMF, 70 °C, 4 h, 77%; (c) alkyl/aryl acetylene 5, sodium
ascorbate, copper sulphate (CuSO4.5H2O), DMSO, 2-18 h, 70-98%.
Table 1. Synthesis of 1,2,3-triazole derivatives 2 using “Click chemistry”
Table 2. Cytotoxic activity of betulinic acid derivatives 2 on different cancer cell lines
Figure 2: Analogue 2c triggers redox imbalance in HT-29 cells. (A) Increase in ROS levels. HT29
cells (2.5×105/mL) were treated with IC50 concentration of 2c for 0–2 h (time-dependence). After
treatment, cells were washed and resuspended in PBS, incubated with CM-H2DCFDA (5 µM) for 30 min,
and the fluorescence was measured using flow cytometry as described in materials and methods. (B) For
the inhibition of ROS generation, cells were pre-treated with NAC (2.5 mM) for 3 h before treatment with

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2c (14.9 µM; 48 h) and ROS was similarly quantified. Values are expressed as Mean Fluorescence
Intensity ± SEM of three independent experiments ( ***p<0.001).
Figure 3: Induction of apoptosis by 2c. Control and 2c (at IC50 concentration) treated (for 12, 24, and 48
h) HT-29 cells (2.5×105/mL). HT-29 cells were co-stained with PI and Annexin-V FITC and analysed
using flow cytometry as described in materials and methods. The figure is a representative profile of at
least three experiments.
Figure 4: Analogue 2c causes depolarization of mitochondrial transmembrane potential. Estimation
of involvement of mitochondrial pathway in apoptosis induced by compound 2c and loss of mitochondrial
membrane potential. Cells were incubated with 2c (14.9 µM, 0-48 h) and loaded with JC-1 for flow
cytometric analysis of mitochondria transmembrane potential. Data is a representative of three different
experiments.
Figure 5: Caspase activation by 2c. Activities of caspase 3 (A), 9 (B), 8 (C) were measured in control
and 2c treated (14.9 µM, 0-48 h) HT-29 cells using colorimetric tetrapeptide substrates. Results are
expressed as mean±SEM from three independent experiments. (D) PARP cleavage was evaluated by
Western blotting analysis in extracts of control and 2c (14.9 µM, 0-48 h) treated cells. The compound
enhances PARP cleavage. Data was confirmed with three different sets of experiments.
.
Figure 6: Nuclear degradation induced by 2c. Control and 2c treated (14.9 µM; 0–48 h) HT-29 cells
(2.5×105/mL) were stained with Hoechst 33258 and observed under a Leica confocal microscope (100×).
The figure is a representative profile of at least three experiments.
Figure 7: Expression of pro- and anti-apoptotic proteins in control and 2c treated HT-29 cells. (A)
Expression levels of pro- and anti-apoptotic proteins. Whole cell lysates were prepared from control and
HT-29 cells (2.5×105/mL) treated with IC50 concentration of betulinic acid analogue 2c. Then equal
amounts of cell lysates were loaded in each lane for SDS-PAGE and probed with specific antibodies
against Bax, Bad, Bcl-2, Bcl-xl and β-actine. Analysis was confirmed with three different sets of extracts.

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β-Actin served as a loading control. (B) Histogram shows time dependent decrease in Bcl-2/Bax ratio
(**p<0.01, as compared with control).
Figure 8: UV-VIS absorption spectra of DNA (100 µg/mL) in 50 mM Tris-HCl/NaCl, pH 7.5, after
incubation with different concentrations of 2c, at 37 °C for 2 h. (A) original plot, (B) normalized plot.
Figure 9: Binding isotherm of DNA in presence of compound 2c.
Figure 10: CD spectra of DNA in the presence of 2c in 50 mM Tris-HCl, NaCl buffer: Blue and red lines
represent the spectra of DNA itself (500 µg/mL) and DNA in the presence of compound 2c (50 mM)
respectively.
Figure 11: (A) Energy minimized docked structure of compound 2c and DNA (PDB ID: 1DNE);
DNA is shown in surface representation (with colour code green for C, blue for N, red for O, white for H,
orange for P), whereas 2c is represented as sticks (whole compound was coloured in white) model. (B)
Energy minimized docked structure of netropsin and DNA (PDB ID: 1DNE); DNA is shown in surface
representation (with colour code green for C, blue for N, red for O, white for H, orange for P), whereas
netropsin is represented as sticks (whole compound was coloured in white) model.

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Table 1
29
20
30
O
R
N
28
1
H
OH
O
10
3
O
5
N
N
6
2
R
CH, (5)
R=
% Yieldb
Time in
hours
Products 2
Entry
1c
2
2a
2b
(5a)
H
18
10
60
98
(5b)
(5c)
4
2
84
98
2c
2d
3
4
OH
O
O
O
H
O
(5d)
O
O
N
N
OMe
3
5
(5e)
82
70
2e
2f
5
MeO
(5f)
6
7
N
H
2g
2h
1.5
2
(5g)
98
80
(5h)
NO2
8
2
COOMe (5i)
2i
2j
91
90
9
COOEt
2
(5j)
10
aReagents and conditions: Acetylene 5 (1.1 eqv.), azide 4 (1.0 eqv.), sodium ascorbate (1.01 eqv.)
b
and CuSO4.5H2O (1.01 eqv.) in dry DMSO (2 mL) were stirred at r.t. for 2-18 h. Yield was
calculated based on the pure product obtained through silica gel (100-200 mesh) column
chromatography. cFor product 2a, acetylene gas (balloon pressure) was used in lieu of any acetylenic
reagent.

ACCEPTED MANUSCRIPT
Table 2
Compounds
Cell growth inhibition in terms of IC50 (µM)
U937
HT29
Jurkat
HepG2
MCF-7
PBMC
2a
2b
2c
2d
2e
2f
>50
>50
>50
>50
>50
>50
>50
49.8±0.9
>50
>50
>50
>50
N.D
>50
N.D
>50
N.D
>50
>50
N.D
N.D
N.D
N.D
>50
14.9±1.3
>50
41.41±1.9
>50
>50
>50
>50
>50
41.93±2.1
>50
39.80±2.4
>50
>50
>50
44.69±2.9
>50
>50
>50
2g
2h
2i
>50
>50
>50
>50
>50
>50
>50
44.8±1.6
>50
42.7±2.2
>50
45.7±1.7
>50
>50
>50
2j
40.1±2.8
>50
>50
>50
>50
>50
2k
2l
>50
>50
>50
>50
>50
>50
>50
>50
>50
Log phase cells (1.25-2.5×104/100 µL of RPMI 1640 medium/well) were treated with betulinic acid
analogs (0–50 µM) for 48 h, and cell viability was measured by MTT assay as described in materials
and methods. Each IC50 (Mean ± SEM) has been derived from at least three experiments in duplicate.