A convergent synthesis of novel alkyne–azide cycloaddition congeners of betulinic acid as potent cytotoxic agent

Article abstract of DOI:10.1016/j.steroids.2017.04.002

In an endeavour to develop potent anti-tumor agents from betulinic acid (BA), a series of C-28 derived 1,2,3-triazolyl derivatives were designed and synthesized by employing Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction. All the derivatives were evaluated for cytotoxic activity by MTT assay against five different human cancer cell lines: lung (A549), colon (HCT116), prostate (PC3), pancreatic (MIA PaCa-2) and breast (T47D). The data revealed that compounds 11c, 11d, 11g, 11h and 13a possess most promising cytotoxic potential. The compound 11h was one of the most active compounds, with IC₅₀ values in the range of 4–6?μM against all the five cancer cell lines. The results of this study suggested that derivatives with free –OH (11c, 11d and 11g) and free –COOH (11h and 13a) substitutions in the triazole moiety introduced at the C-28 position significantly improved the anti-tumor activity and may be the favourable position to synthesize potent anticancer leads from BA. Introduction of a non polar alkyl groups at C-28 position (10, 12 and 14) resulted in the significant loss of the activity. Further, DAPI staining, ROS generation and wound healing experiments revealed that compound 11h induces apoptosis in HCT-116 cells.

Full text of DOI:10.1016/j.steroids.2017.04.002

Accepted Manuscript
A convergent synthesis of novel alkyne–azide cycloaddition congeners of be-
tulinic acid as potent cytotoxic agent
Nisar A. Dangroo, Jasvinder Singh, Santosh K. Rath, Nidhi Gupta, Arem
Qayum, Shashank Singh, Payare L. Sangwan
PII:
S0039-128X(17)30055-7
http://dx.doi.org/10.1016/j.steroids.2017.04.002
STE 8092
DOI:
Reference:
Steroids
To appear in:
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Revised Date:
Accepted Date:
10 December 2016
11 April 2017
12 April 2017
Please cite this article as: Dangroo, N.A., Singh, J., Rath, S.K., Gupta, N., Qayum, A., Singh, S., Sangwan, P.L., A
convergent synthesis of novel alkyne–azide cycloaddition congeners of betulinic acid as potent cytotoxic agent,
Steroids (2017), doi: http://dx.doi.org/10.1016/j.steroids.2017.04.002
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A convergent synthesis of novel alkyne–azide cycloaddition congeners of betulinic acid as
potent cytotoxic agent #
Nisar A. Dangrooa, Jasvinder Singhb,c, Santosh K. Ratha,c, Nidhi Guptaa, Arem Qayum b,c, Shashank
Singhb,c,* and Payare L. Sangwana,c,*
aBioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu-180001,
India
bCancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu-180001,
India
cAcademy of Scientific and Innovative Research (AcSIR), CSIR-IIIM Campus, Jammu, India
#IIIM manuscript no. IIIM/2004/2017
*Corresponding author. E-mails; sksingh@iiim.ac.in (S. Singh); plsangwan@iiim.ac.in (P.L.
Sangwan)
Keywords: Betulinic acid; Apoptosis; click chemistry; Triazole derivative; Colon
1

ABSTRACT
In an endeavour to develop potent anti-tumor agents from betulinic acid (BA), a series of C-28
derived 1,2,3-triazolyl derivatives were designed and synthesized by employing Cu(I) catalyzed
Huisgen 1,3-dipolar cycloaddition reaction. All the derivatives were evaluated for cytotoxic activity
by MTT assay against five different human cancer cell lines: lung (A549), colon (HCT116),
prostate (PC3), pancreatic (MIA PaCa-2) and breast (T47D). The data revealed that compounds
11c, 11d, 11g, 11h and 13a possess most promising cytotoxic potential. The compound 11h was
one of the most active compounds, with IC50 values in the range of 4-6 µM against all the five
cancer cell lines. The results of this study suggested that derivatives with free –OH (11c, 11d and
11g) and free -COOH (11h and 13a) substitutions in the triazole moiety introduced at the C-28
position significantly improved the anti-tumor activity and may be the favourable position to
synthesize potent anticancer leads from BA. Introduction of a non polar alkyl groups at C-28
position (10, 12 and 14) resulted in the significant loss of the activity. Further, DAPI staining, ROS
generation and wound healing experiments revealed that compound 11h induces apoptosis in HCT-
116 cells.
1. Introduction
Since last few decades, natural products (NPs) have been extensively explored for the
development of anti-cancer drugs and currently about 80% of clinically important anti-
cancer agents originate from natural products [1]. In recent years bioactive plant derived
triterpenoid scaffolds such as betulinic acid 1, oleanolic acid 1a and ursolic acid 1b (Fig. 1)
have renewed much interest globally due to their diverse biological activities, and hundreds
of new derivatives are being reported every year [2,3]. Among these, the pentacyclic lupane-
type triterpenes represent a diverse assemblage of bioactive natural products [4]. Betulinic
2

acid (BA) 1, a lupane-type pentacyclic triterpene possesses diverse biological properties
including antitumor, anti-HIV, anti-malarial, immunomodulatory, anti-inflammatory and
hepatoprotective [5,6]. However, BA is well known and most highly regarded for its
anticancer properties because it inhibits a broad range of tumours, exhibits high cancer cell
selectivity with favourable safety profile [7-10]. The anticancer properties of BA and its
analogs have been extensively investigated using a variety of human cancer cell lines,
primary tumour samples and xenograft mice models [11-14].
Figure 1.
BA, a natural product (NP) is under clinical evaluation (Phase I and Phase II, NCT00701987
and Phase II, NCT00346502) for the treatment of dysplastic nevi [9]. Many mechanisms,
including induction of apoptosis, cell cycle arrest, multidrug resistance (MDR) reversal,
immune regulation, as well as autophagy induction have been proposed for its activity [10].
Being the biologically attractive scaffold and high safety profile of BA in the area of cancer
therapy, numerous structural modifications have been carried out to improve its potency and
efficacy as well as to investigate structure activity relationships. Despite of the outstanding
in vitro biological activity, only a few BA derivatives e.g. B10 (
), DRF 4012 ( ) pyrazine betulinic acid ( ), 3-acetoxybetulinic acid glucoside (
acetylbetulinic (2-(2-aminoethyl)aminoethyl)amide betulonic acid ( ) etc. (Fig. 2) have
2
), NVX-207 (
3
), ALS 862
(
4
5
6
7
), 3-O-
9
shown excellent and selective in vivo cytotoxicity and might have some clinical utility [9,12-
17]. One of the major drawbacks of BA and its derivatives for cancer therapy is its poor
hydrophilicity and one of the simple strategies to overcome such limitation is to introduce a
heterocyclic moiety like 1,2,3-triazoles. Due to extra stability of triazoles in typical
3

physiological conditions, they act as ideal linkers and could improve drug likeness and
hydrophilic property of the parent NP via hydrogen bonding [15,16].
Figure 2.
Betulonic acid/ketobetulonic acid (KBA), oxidised C-3-OH of BA has been reported to be
more cytotoxic than BA [17]. The literature survey as well as based on our previous
communications, it was observed that polar substituent at C-3 and/or C-28 enhanced
antitumor activity of BA [18,19]. In context with literature and our research interest for the
chemical modification of NPs for better potency and lesser toxicity [18-25], herein, a series
of C-28 derived 1, 2, 3 triazole analogs of KBA were synthesised via the cupper (I)
catalyzed azide alkyne cycloaddition (CuAAC) and screened against a panel of human
cancer cell lines. It was demonstrated that many analogs exhibited better anti-cancer efficacy
than BA.
2. Experimental
2.1. Chemistry
Unless stated otherwise, all the reagents and solvents for carry out the present work were
obtained from Sigma Aldrich and used without further purification. All the chemical
reactions were monitored by TLC on 0.25 mm silica gel 60 F254 plates (E. Merck) using 2%
ceric ammonium sulphate solution and/or dragendorff as a spraying reagent for detection of
the spots on the TLC. Purification of all the compounds was carried out by column
1
chromatography using Silica gel 60-120 mesh stationary phase or by crystallization. H
13
NMR and C NMR spectra were recorded on Bruker DPX 400 and DPX 500 instruments
using CDCl3 or CD3OD as the solvents with TMS as internal standard. The chemical shifts
4

were expressed in
δ ppm and coupling constants in Hertz. High resolution mass spectra
(HRESIMS) were recorded on Agilent Technologies 6540 instrument.
2.1.1. Isolation of betulinic acid (1)
Betulinic acid (1) was isolated in bulk quantity from DCM- MeOH extract of the stem bark of
Platanus orientalis by column chromatography over silica gel 60-120 mesh. The compound was
further purified by crystallization and characterized using various spectroscopic techniques [18,26].
2.1.2. Preparation of betulonic acid (9)
The compound was prepared by oxidation of compound
1 (2.2 mmol) in DCM using
pyridinium chlorochromate (PCC) (2.4 mmol) as a catalyst [27]. Upon completion, the
reaction mixture was passed through celite, the filtrate was concentrated on rota evaporator
and subjected to column chromatography with elution in 10% ethyl acetate in hexane to
furnish betulonic acid 9
. yield: 70%; mp: 250°C; HRESIMS m/z calcd for C30H47O3 [M+H]+
455.3525, found 455.3523. 1H NMR and 13C NMR (supporting information).
2.1.3. Preparation of C28-propargylbetulonate (10)
The propargylation of compound
9 (1.0 g, 2.2 mmol) by propargyl bromide (0.31 ml, 2.6
mmol) and anhydrous K2CO3 (0.45 g, 3.30 mmol) in acetone (30 ml) at 50°C for 2-3 h
yielded C-28 propargylated betulonic ester 10 (yield 96%). HRESIMS m/z calcd for
1
C33H49O3 [M+H]+ 493.3681, found 493.3680. H NMR and 13C NMR (supporting
information).
2.1.4. General experimental procedure for preparation of aryl azides
The detail procedure for the preparation of organic azides is described in earlier
communication [28].
2.1.5. General experimental procedure for synthesis of triazolyl derivatives (11a-p)
5

Compound 10 (0.2 mmol) and appropriate organic azides (0.24 mmol) were suspended in 20
ml of 1:1 water: -butanol mixture followed by the addition of 20 mg sodium ascorbate and
t
25 mg copper (II) sulphate pentahydrate (10 mol% each). The heterogeneous reaction
mixture was stirred vigorously at room temperature till the completion of reaction. The
reaction mixture was extracted using ethyl acetate and water. The combined organic layer
was dried over anhydrous sodium sulphate and purified through column chromatography
and/or by crystallization to furnish different triazolyl derivatives in excellent yields (70-
1
89%). The H NMR assignments was carried by the literature based on 2D NMR technique
reported by us earlier [18,19,25,26].
2.1.5.1. Experimental data of (1-(2-cyanophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester (11a)
1H NMR (CDCl3, 400 MHz): δ 8.81 (1H, s, N-C
Ar- ), 7.64 (1H, m, Ar- ), 5.34 (2H, d, = 4.0 Hz -OCH2C), 4.74 and 4.60 (1H, each, s,
H), 7.88 (2H, m, 2x Ar-H), 7.83 (1H, m,
H
H
J
H-29), 3.01 (1H, m, H-19), 2.49 and 1.89 (1H, each, m, H-22), 2.44 and 1.84 (1H each, m,
H-16), 2.39 (2H, m, H-2), 2.23 (1H, m, H-13), 1.92 (1H, m, H-18), 1.89 and 1.43 (1H each,
m, H-6), 1.85 and 1.59 (1H each, m, H-7), 1.69 (3H, s, H-30), 1.61 and 1.25 (1H each, m, H-
12), 1.55 and 1.29 (1H each, m, H-15), 1.43 and 1.35 (1H each, m, H-21), 1.41 (1H, m, H-
9), 1.37 and 1.29 (1H each, m, H-11), 1.31 and 1.22 (1H each, m, H-1), 1.05 (3H, s, H-26),
0.97 and 0.95 (3H, each, s, H-27 and H-23), 0.90 (1H, m, H-5), 0.85 (3H, s, H-24), 0.75
(3H, s, H-25); 13C NMR (125 MHz, CDCl3) δ 218.36, 175.97, 150.36, 144.10, 138.36,
134.44, 134.33 129.73, 125.40, 124.53, 115.55, 109.75, 106.67, 56.83, 56.57, 54.83, 49.81,
49.39, 47.32, 46.89, 42.39, 40.59, 39.59, 38.39, 36.86, 34.12, 33.45, 31.92, 30.54, 29.71,
29.61, 26.64, 25.46, 21.36, 21.01, 19.60, 19.38, 15.95, 15.51, 14.60. HRESIMS m/z calcd
for C40H53N4O3 [M+H]+ 637.4117, found 637.4112.
6

2.1.5.2. Experimental data of 1-(3-(hydroxymethyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic
ester (11b)
1H NMR (CDCl3, 400 MHz): δ 8.10 (1H, s, N-C
Hz Ar- ), 7.52 (1H, dd, = 7.6, 7.8 Hz, Ar- ), 7.45 (1H, d,
H
), 7.78 (1H, s, Ar-
H
), 7.65 (1H, d,
J = 7.8
H
J
H
J
= 7.6 Hz Ar-H
), 5.34 (2H, m,
-OCH2C), 4.81 (2H, s, CH2O), 4.74 and 4.61 (1H each, s, H-29), 3.01 (1H, m, H-19), 2.44
and 1.88 (1H, each, m, H-22), 2.40 and 1.85 (1H each, m, H-16), 2.15 (2H, m, H-2), 1.92
(1H, m, H-13), 1.83 (1H, m, H-18), 1.80 and 1.41 (1H each, m, H-6), 1.85 and 1.59 (1H
each, m, H-7), 1.71 (3H, s, H-30), 1.65 and 1.29 (1H each, m, H- 12), 1.45 and 1.23 (1H
each, m, H-15), 1.35 and 1.11 (1H each, m, H-21), 1.30 (1H, m, H-9), 1.27 and 1.21 (1H
each, m, H-11), 1.32 and 1.13 (1H each, m, H-1), 1.04 (3H, s, H-26), 0.98 and 0.94 (3H,
13
each, s, H-27 and H-23), 0.89 (1H, m, H-5), 0.84 (3H, s, H-24), 0.67 (3H, s, H-25); C
NMR (100 MHz, CDCl3) δ 218.40, 176.14, 150.29, 143.82, 143.41, 137.03, 129.80, 127.04,
122.50, 119.43, 118.78, 109.74, 64.19, 56.97, 56.54, 54.84, 49.80, 49.44, 47.28, 46.95,
42.37, 40.56, 39.57, 38.45, 36.83, 34.07, 33.44, 31.92, 30.57, 29.68, 26.64, 25.49, 22.67,
21.38, 20.95, 19.46, 15.83, 15.44, 14.57, 14.10. HRESIMS m/z calcd for C40H56N3O4
[M+H]+ 642.4271, found 642.4256.
2.1.5.3. Experimental data of 1-(3,4-methylenedioxyphenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic
ester (11c)
1H NMR (CDCl3, 400 MHz): δ 7.97 (1H, s, N-C
Hz Ar- ), 6.90 (1H, d, = 8.0 Hz Ar- ), 6.07 (2H, s, -OCH2O-), 5.34 (2H, m, -OCH2C),
H), 7.14 (1H, s, Ar-H), 7.13 (1H, d, J = 8.0
H
J
H
4.74 and 4.61 (1H each, s, H-29), 3.01 (1H, m, H-19), 2.45 and 1.86 (1H, each, m, H-
22),2.41 and 1.89 (1H each, m, H-16), 2.29 (2H, m, H-2), 1.91 (1H, m, H-13), 1.85 (1H, m,
H-18), 1.81 and 1.42 (1H each, m, H-6), 1.83 and 1.54 (1H each, m, H-7), 1.70 (3H, s, H-
7

30), 1.64 and 1.27 (1H each, m, H- 12), 1.61 and 1.20 (1H each, m, H-15), 1.37 and 1.12
(1H each, m, H-21), 1.30 (1H, m, H-9), 1.26 and 1.22 (1H each, m, H-11), 1.32 and 1.13
(1H each, m, H-1), 1.05 (3H, s, H-26), 0.96 and 0.94 (3H, each, s, H-27 and H-23), 0.90
(1H, m, H-5), 0.84 (3H, s, H-24), 0.68 (3H, s, H-25); 13C NMR (100 MHz, CDCl3) δ 218.12,
176.13, 150.31, 148.68, 148.15, 143.69, 131.41, 122.66, 114.24, 109.73, 108.46, 102.81,
102.16, 57.04, 56.55, 54.87, 49.83, 49.46, 47.27, 46.97, 42.38, 40.58, 39.59, 38.40, 36.86,
34.07, 33.47, 31.93, 30.59, 29.69, 26.66, 25.50, 22.67, 21.40, 20.95, 19.58, 15.85, 15.50,
14.58, 14.09. HRESIMS m/z calcd for C40H53N3O5 [M+H]+ 656.1463, found 656.1468.
2.1.5.4. Experimental data of 1-(4-hydroxy-3-methylphenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic
ester (11d)
1H NMR (CDCl3, 400 MHz): δ 7.78 (1H, s, N-C
), 7.15 (1H, d,
d, = 3.8 Hz Ar- ), 6.80 (1H, d, = 7.9 Hz Ar-H), 5.32 (2H, m, -OCH2C), 4.72 and 4.60
H
J = 7.9 Hz Ar-H), 6.85 (1H,
J
H
J
(1H each, s, H-29), 3.00 (1H, m, H-19), 2.47 and 1.87 (1H, each, m, H-22), 2.39 and 1.83
(1H each, m, H-16), 2.30 (2H, m, H-2), 2.11 (3H, s, Ar-CH3), 1.91 (1H, m, H-13), 1.81 (1H,
m, H-18), 1.79 and 1.41 (1H each, m, H-6), 1.70 and 1.52 (1H each, m, H-7), 1.69 (3H, s,
H-30), 1.63 and 1.25 (1H each, m, H- 12), 1.59 and 1.23 (1H each, m, H-15), 1.42 and 1.12
(1H each, m, H-21), 1.35 (1H, m, H-9), 1.31 and 1.17 (1H each, m, H-1), 1.29 and 1.21(1H
each, m, H-11), 1.09 (3H, s, H-26), 1.01 and 0.95 (3H, each, s, H-27 and H-23), 0.91 (1H,
13
m, H-5), 0.87 (3H, s, H-24), 0.79 (3H, s, H-25); C NMR (125 MHz, CDCl3) δ 219.39,
176.17, 157.92, 150.28, 142.80, 135.25, 127.25, 126.08, 117.93, 113.73, 109.82, 57.00,
56.57, 54.87, 50.19, 49.80, 49.30, 47.38, 46.92, 42.40, 40.58, 39.59, 38.40, 36.86, 34.15,
33.46, 31.88, 30.50, 29.72, 26.70, 25.56, 22.71, 21.38, 21.01, 19.60, 19.34, 17.82, 15.98,
8

15.74, 14.59, 14.29. HRESIMS m/z calcd for C40H56N3O4 [M+H]+ 642.4271, found
642.4269.
2.1.5.5. Experimental data of 1-(2-(chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11e)
1H NMR (CDCl3, 400 MHz): δ 8.10 (1H, s, N-C
Ar- ), 5.33 (2H, m, -OCH2C), 4.72 and 4.61 (1H each, s, H-29), 2.99 (1H, m, H-19), 2.46
H), 7.62 (1H, m, 2x Ar-H), 7.49 (1H, m, 2x
H
and 1.89 (1H, each, m, H-22), 2.31 and 1.85 (1H each, m, H-16), 2.21and 1.71 (1H each, m,
H-2), 1.91 (1H, m, H-13), 1.69 (1H, m, H-18), 1.79 and 1.41 (1H each, m, H-6), 1.70 (3H,
s, H-30), 1.67 and 1.49 (1H each, m, H-7), 1.63 and 1.27 (1H each, m, H- 12), 1.59 and 1.29
(1H each, m, H-15), 1.42 and 1.12 (1H each, m, H-21), 1.37 (1H, m, H-9), 1.33 and 1.21
(1H each, m, H-1), 1.31 and 1.21(1H each, m, H-11), 1.09 (3H, s, H-26), 1.03 and 0.95 (3H,
13
each, s, H-27 and H-23), 0.91 (1H, m, H-5), 0.86 (3H, s, H-24), 0.78 (3H, s, H-25); C
NMR (125 MHz, CDCl3) δ 218.29, 176.07, 150.34, 143.01, 134.72, 130.88, 130.85 128.49,
127.99, 127.83, 126.16, 109.76, 56.97, 56.55, 54.86, 49.82, 49.34, 47.31, 46.92, 42.40,
40.58, 39.59, 38.3, 36.88, 34.12, 33.48, 31.91, 30.97, 30.51, 29.57, 26.63, 25.47, 21.36,
21.01, 19.58 19.35, 15.94, 15.61, 14.59. HRESIMS m/z calcd for C39H53ClN3O3 [M+H]+
646.3775, found 646.3733.
2.1.5.6. Experimental data of 1-(3-(nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester (11f)
1H NMR (CDCl3, 400 MHz): δ 8.60 (1H, s, N-C
), 8.33 (1H, d,
m, 2x Ar- ), 7.68 (1H, dd, = 8.4 Hz each, Ar-H) 5.31 (2H, m, -OCH2C), 4.74 and 4.61
H
J = 8.4 Hz, Ar-H), 8.2 (2H,
H
J
(1H each, s, H-29), 3.02 (1H, m, H-19), 2.46 and 1.85 (1H, each, m, H-22), 2.39 and 1.89
(1H each, m, H-16), 2.29 and 1.72 (1H each, m, H-2), 1.91 (1H, m, H-13), 1.85 and 1.38
(1H each, m, H-6), 1.73 (1H, m, H-18), 1.68 (3H, s, H-30), 1.63 and 1.44 (1H each, m, H-7),
9

1.59 and 1.39 (1H each, m, H- 12), 1.49 and 1.23 (1H each, m, H-15), 1.39 and 1.11 (1H
each, m, H-21), 1.33 (1H, m, H-9), 1.31 and 1.27 (1H each, m, H-1), 1.29 and 1.21(1H each,
m, H-11), 1.03 (3H, s, H-26), 0.97 and 0.94 (3H, each, s, H-27 and H-23), 0.89 (1H, m, H-
13
5), 0.80 (3H, s, H-24), 0.68 (3H, s, H-25); C NMR (125 MHz, CDCl3) δ 218.14, 176.19,
150.22, 148.92, 144.65, 137.59, 131.07, 126.03, 123.40, 122.48, 115.36, 109.84, 56.85,
56.55, 54.86, 49.78, 49.40, 47.30, 46.95, 42.37, 40.58, 39.56, 38.46, 36.83, 34.10, 33.45,
31.88, 30.53, 29.70, 29.59, 26.54, 25.45, 21.34, 20.98, 19.50, 19.35, 15.79, 15.45, 14.60.
HRESIMS m/z calcd for C39H53N4O5 [M+H]+ 657.4016, found 657.4049.
2.1.5.7. Experimental data of 1-(4-(hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11g)
1H NMR (CDCl3, 400 MHz): δ 8.39 (1H, s, N-C
7.04 (2H, m, 2x Ar- ) 5.31 (2H, m, -OCH2C), 4.74 and 4.60 (1H each, s, H-29), 3.03 (1H,
H), 8.17 (1H, s, Ar-H), 7.37 (1H, m, Ar-H),
H
m, H-19), 2.41 and 1.82 (1H, each, m, H-22), 2.36 and 1.87 (1H each, m, H-16), 2.28 and
1.60 (1H each, m, H-2), 2.01 (1H, m, H-13), 1.88 and 1.37 (1H each, m, H-6), 1.77(1H, m,
H-18), 1.66 (3H, s, H-30), 1.59 and 1.21 (1H each, m, H-7), 1.43 and 1.19 (1H each, m, H-
12), 1.41 and 1.17 (1H each, m, H-15), 1.34 and 1.02 (1H each, m, H-21), 1.33 (1H, m, H-
9), 1.31 and 1.07 (1H each, m, H-1), 1.28 and 0.86 (1H each, m, H-11), 0.99 (3H, s, H-26),
0.91 and 0.88 (3H, each, s, H-27 and H-23), 0.95 (1H, m, H-5), 0.74 (3H, s, H-24), 0.58
(3H, s, H-25); 13C NMR (100 MHz, CDCl3) δ 218.50, 176.13, 158.71, 150.26, 143.60,
137.40, 130.55, 122.57, 116.94, 109.80, 109.75, 108.90, 56.57, 56.41, 54.80, 49.75, 49.49,
47.23, 46.96, 42.31, 40.47, 39.54, 38.50, 36.78, 34.09, 33.30, 31.96, 30.60, 29.69, 29.52,
26.68, 25.46, 21.30, 20.89, 19.44, 19.39, 15.72, 15.24, 14.55. HRESIMS m/z calcd for
C39H54N3O4 [M+H]+, 628.4114 found 628.4106.
10

2.1.5.8. Experimental data of 1-(3-(carboxyphenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11h)
1H NMR (CDCl3, 400 MHz): δ 8.43 (1H, s, N-C
Hz, Ar- ), 8.09 (1H, d, = 8.4 Hz, Ar- ), 7.68 (1H, dd,
H
), 8.24 (1H, s, Ar-
H
), 8.20 (1H, d,
J = 7.6
H
J
H
J
= 8.0 Hz each, Ar-H
), 5.33 (2H,
m,-OCH2C), 4.74 and 4.61 (1H each, s, H-29), 3.03 (1H, m, H-19), 2.44 and 1.86 (1H, each,
m, H-22), 2.40 and 1.90 (1H each, m, H-16), 2.21 and 1.71 (1H each, m, H-2), 1.91 (1H, m,
H-13), 1.88 and 1.40 (1H each, m, H-6), 1.81 (1H, m, H-18), 1.68 (3H, s, H-30), 1.63 and
1.44 (1H each, m, H-7), 1.59 and 1.39 (1H each, m, H- 12), 1.49 and 1.23 (1H each, m, H-
15), 1.39 and 1.11 (1H each, m, H-21), 1.33 (1H, m, H-9), 1.31 and 1.27 (1H each, m, H-1),
1.29 and 1.21(1H each, m, H-11), 1.03 (3H, s, H-26), 0.97 and 0.93 (3H, each, s, H-27 and
13
H-23), 0.88 (1H, m, H-5), 0.79 (3H, s, H-24), 0.65 (3H, s, H-25); C NMR (125 MHz,
CDCl3) δ 218.73, 176.32, 169.39, 157.15, 144.13, 142.42, 137.11, 130.42, 130.14, 125.42,
122.74, 121.69, 109.80, 56.93, 56.57, 54.80, 49.78, 49.42, 47.30, 46.95, 42.37, 40.55, 39.57,
38.48, 36.81, 34.09, 33.41, 31.94, 30.56, 29.37, 26.67, 25.48, 22.71, 21.37, 20.95, 19.47,
15.85, 15.39, 14.59, 14.16. HRESIMS m/z calcd for C40H54N3O5 [M+H]+ 656.4063, found
656.4057.
2.1.5.9. Experimental data of 1-(4-(methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11i)
1H NMR (CDCl3, 400 MHz): δ 8.00 (1H, s, N-C
(2H, d, = 8.8 Hz, 2x Ar- ), 5.30 (2H, m, -OCH2C), 4.74 and 4.60 (1H each, s, H-29), 3.86
H), 7.62 (2H, d, J = 8.8 Hz, 2x Ar-H), 7.02
J
H
(3H, s, -OCH3), 3.03 (1H, m, H-19), 2.45 and 1.89 (1H, each, m, H-22), 2.41 and 1.87 (1H
each, m, H-16), 2.28 and 1.60 (1H each, m, H-2), 2.11 (1H, m, H-13), 1.88 and 1.37 (1H
each, m, H-6), 1.77 (1H, m, H-18), 1.67 (3H, s, H-30), 1.59 and 1.21 (1H each, m, H-7),
11

1.43 and 1.19 (1H each, m, H- 12), 1.41 and 1.17 (1H each, m, H-15), 1.34 and 1.02 (1H
each, m, H-21), 1.33 (1H, m, H-9), 1.31 and 1.07 (1H each, m, H-1), 1.28 and 0.86 (1H
13
each, m, H- (3H, s, H-24), 0.67 (3H, s, H-25); C NMR (100 MHz, CDCl3) δ 218.14,
176.16, 159.96, 150.35 (2x
CH), 143.67 (2x CH), 130.37, 122.52, 122.22, 114.74, 109.73,
57.10, 56.54, 55.71, 54.87, 49.82, 49.45, 47.28, 46.97, 42.39, 40.57, 39.59, 38.46, 36.85,
34.09, 33.47, 31.91, 30.59, 29.57, 26.67, 25.50, 21.39, 20.94, 19.56, 19.37, 15.90, 15.89,
15.42, 14.61. HRESIMS m/z calcd for C40H56N3O4 [M+H]+ 642.4271, found 642.4275.
2.1.5.10. Experimental data of 1-(3-(cyanophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11j)
1H NMR (CDCl3, 400 MHz): δ 8.13 (1H, s, N-C
Ar- ), 7.70 (1H, m, Ar- ), 5.30 (2H, m, -OCH2C), 4.74 and 4.60 (1H each, s, H-29), 3.03
H), 8.04 (2H, m, 2x Ar-H), 7.75 (1H, m,
H
H
(1H, ddd, J = 4.8, 4.4, 3.6 Hz, H-19), 2.45 and 1.89 (1H, each, m, H-22), 2.41 and 1.87 (1H
each, m, H-16), 2.28 and 1.60 (1H each, m, H-2), 2.11 (1H, m, H-13), 1.88 and 1.37 (1H
each, m, H-6), 1.77 (1H, m, H-18), 1.67 (3H, s, H-30), 1.59 and 1.21 (1H each, m, H-7),
1.43 and 1.19 (1H each, m, H- 12), 1.41 and 1.17 (1H each, m, H-15), 1.34 and 1.02 (1H
each, m, H-21), 1.33 (1H, m, H-9), 1.31 and 1.07 (1H each, m, H-1), 1.28 and 0.86 (1H
each, m, H-11), 1.04 (3H, s, H-26), 0.99 and 0.94 (3H, each, s, H-27 and H-23), 0.88 (1H,
13
m, H-5), 0.83 (3H, s, H-24), 0.67 (3H, s, H-25); C NMR (126 MHz, CDCl3) δ 218.20,
176.17, 150.22, 144.59, 137.40, 132.22, 130.97, 124.45, 123.65, 122.29, 117.27, 114.23,
109.85, 56.85, 56.54, 54.88, 49.77, 49.39, 47.32, 46.95, 42.37, 40.58, 39.57, 38.45, 36.84,
34.11, 33.46, 31.88, 30.52, 29.58, 26.55, 25.45, 21.36, 21.01, 19.52, 19.35, 15.87, 15.49,
14.60. HRESIMS m/z calcd for C40H53N4O3 [M+H]+, 637.4117, found 637.4113.
12

2.1.5.11. Experimental data of 1-(4-(acetamidophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11k)
1H NMR (CDCl3, 400 MHz): δ 8.04 (1H, s, N-C
Ar1-
H), 7.44 (1H, s, N-H), 7.66-7.71 (4H, m, 4x
H
), 5.32 (2H, m, -OCH2C), 4.73 and 4.60 (1H each, s, H-29), 3.01 (1H, m , H-19), 2.44
and 1.87 (1H, each, m, H-22), 2.40 and 1.85 (1H each, m, H-16), 2.28 and 1.59 (1H each, m,
H-2), 2.22 (3H, s, CH3CO), 2.09 (1H, m, H-13), 1.89 and 1.37 (1H each, m, H-6), 1.70
(1H, m, H-18), 1.67 (3H, s, H-30), 1.60 and 1.21 (1H each, m, H-7), 1.41 and 1.19 (1H each,
m, H- 12), 1.38 and 1.17 (1H each, m, H-15), 1.36 and 1.18 (1H each, m, H-21), 1.33 (1H,
m, H-9), 1.31 and 1.21 (1H each, m, H-1), 1.28 and 0.86 (1H each, m, H-11), 1.04 (3H, s, H-
26), 0.98 and 0.93 (3H, each, s, H-27 and H-23), 0.88 (1H, m, H-5), 0.81 (3H, s, H-24),
13
0.67 (3H, s, H-25); C NMR (125 MHz, CDCl3) δ 218.82, 176.12, 169.06, 150.26, 143.79,
139.02, 132.55, 129.54, 122.41, 121.22, 120.60, 115.43, 109.79, 56.96, 56.53, 54.79, 49.78,
49.40, 47.31, 46.95, 42.37, 40.53, 39.57, 38.44, 36.83, 34.13, 33.42, 31.92, 30.55, 29.56,
26.70, 25.47, 24.52, 22.69, 21.38, 20.98, 19.57, 19.36, 15.92, 15.40, 14.58. HRESIMS m/z
calcd for C41H57N4O4 [M+H]+ 669.4380, found 669.4381.
2.1.5.12. Experimental data of 1-(4-(benzamidophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11l)
1H NMR (CDCl3, 400 MHz): δ 8.11 (1H, s, N-C
), 7.89 (3H, m, 3x
Ar1- ), 7.73 (2H, d, J = 8.8 Hz, 2x Ar1-
H), 7.59 (1H, d, J = 8.4 Hz, Ar-H), 7.51 (2H, t, J =
H
), 8.07 (1H, s, Ar-
H
H
7.2 Hz, 2x Ar-H), 5.30 (2H, m, -OCH2C), 4.74 and 4.60 (1H each, s, H-29), 3.01 (1H, ddd, J
= 4.8, 4.0, 1.6 Hz H-19), 2.44 and 1.87 (1H, each, m, H-22), 2.39 and 1.85 (1H each, m, H-
16), 2.28 and 1.59 (1H each, m, H-2), 2.09 (1H, m, H-13), 1.89 and 1.37 (1H each, m, H-6),
1.70 (1H, m, H-18), 1.68 (3H, s, H-30), 1.60 and 1.21 (1H each, m, H-7), 1.41 and 1.19 (1H
13

each, m, H- 12), 1.38 and 1.17 (1H each, m, H-15), 1.36 and 1.18 (1H each, m, H-21), 1.33
(1H, m, H-9), 1.31 and 1.21 (1H each, m, H-1), 1.28 and 0.86 (1H each, m, H-11), 1.03 (3H,
s, H-26), 0.97 and 0.94 (3H, each, s, H-27 and H-23), 0.89 (1H, m, H-5), 0.83 (3H, s, H-24),
13
0.69 (3H, s, H-25); C NMR (100 MHz, CDCl3) δ 218.29, 176.08, 166.03, 150.26, 143.87,
138.90, 134.49, 132.94, 132.11, 128.78 (2x
CH), 127.21 (2x CH), 122.21 (2x CH), 121.19
(2x CH), 121.14, 109.73, 57.02, 56.56, 54.92, 49.84, 49.48, 47.28, 46.96, 42.42, 40.60,
39.58, 38.47, 36.85, 34.07, 33.50, 31.95, 30.61, 29.58, 29.65, 26.64, 25.53, 21.41, 20.98,
19.58, 19.38, 15.86, 15.47, 14.58. HRESIMS m/z calcd for C46H59N4O4 [M+H]+ 731.4536,
found 731.4541.
2.1.5.13. Experimental data of 1-(4-(cyanophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11m)
1H NMR (CDCl3, 400 MHz): δ 8.16 (1H, s, N-C
( 2H, d, J = 8.4 Hz, 2x Ar- ), 5.32 (2H, m, -OCH2C), 4.73 and 4.61 (1H each, s, H-29),
H), 7.93 (2H, d, J = 8.4 Hz, 2x Ar-H), 7.86
H
3.01 (1H, m, H-19), 2.46 and 1.87 (1H, each, m, H-22), 2.39 and 1.85 (1H each, m, H-16),
2.27 and 1.59 (1H each, m, H-2), 2.17 (1H, m, H-13), 1.89 and 1.37 (1H each, m, H-6),
1.72 (1H, m, H-18), 1.67 (3H, s, H-30), 1.63 and 1.19 (1H each, m, H-7), 1.58 and 1.21 (1H
each, m, H- 12), 1.41 and 1.17 (1H each, m, H-15), 1.39 and 1.18 (1H each, m, H-21), 1.33
(1H, m, H-9), 1.31 and 1.21 (1H each, m, H-1), 1.28 and 0.86 (1H each, m, H-11), 1.04 (3H,
s, H-26), 0.99 and 0.94 (3H, each, s, H-27 and H-23), 0.85 (1H, m, H-5), 0.83(3H, s, H-24),
13
0.67 (3H, s, H-25); C NMR (100 MHz, CDCl3) δ 217.90, 176.16, 150.19, 144.66, 139.6,
133.94 (2x
CH), 122.05, 120.59 (2x CH), 117.56, 112.66, 109.83, 56.82, 56.57, 54.94,
50.79, 49.81, 49.42, 47.31, 46.96, 42.41, 40.59, 39.58, 38.48, 36.84, 34.09, 33.51, 31.90,
14

30.55, 29.63, 26.52, 25.48, 21.36, 21.03, 19.52, 19.32, 15.82, 15.44, 14.60. HRESIMS m/z
calcd for C40H53N4O3 [M+H]+ 637.4117, found 637.4120.
2.1.5.14. Experimental data of 1-(2-(chloropyridin-3-yl)-1H-1,2,3-triazol-4-yl)methyl betulonic
ester (11n)
1H NMR (CDCl3, 400 MHz): δ 8.57 (1H, d, J = 3.2 Hz, Ar-
(1H, 1H, d, J = 6.0 Hz, Ar- ), 7.50 (1H, m, Ar- ), 5.34 (2H, m, -OCH2C), 4.73 and 4.60
H), 8.22 (1H, s, N-CH), 8.07
H
H
(1H each, s, H-29), 3.01 (1H, m, H-19), 2.47 and 1.87 (1H, each, m, H-22), 2.42 and 1.85
(1H each, m, H-16), 2.29 and 1.59 (1H each, m, H-2), 2.17 (1H, m, H-13), 1.89 and 1.37
(1H each, m, H-6), 1.72 (1H, m, H-18), 1.67 (3H, s, H-30), 1.63 and 1.19 (1H each, m, H-7),
1.58 and 1.21 (1H each, m, H- 12), 1.41 and 1.17 (1H each, m, H-15), 1.39 and 1.18 (1H
each, m, H-21), 1.35 (1H, m, H-9), 1.31 and 1.21 (1H each, m, H-1), 1.28 and 0.86 (1H
each, m, H-11), 1.05 (3H, s, H-26), 1.00 and 0.95 (3H, each, s, H-27 and H-23), 0.89 (1H,
13
m, H-5), 0.86 (3H, s, H-24), 0.77 (3H, s, H-25); C NMR (100 MHz, CDCl3) δ 218.04,
176.01, 150.34, 150.29, 144.86, 143.61, 135.86, 131.96, 125.76, 123.36, 109.78, 56.83,
56.58, 54.93, 49.85, 49.38, 47.30, 46.91, 42.44, 40.63, 39.59, 38.40, 36.88, 34.09, 33.54,
31.91, 30.54, 29.58, 29.65, 26.61, 25.50, 21.38, 21.01, 19.60, 19.36, 15.91, 15.64, 14.59.
HRESIMS m/z calcd for C38H52ClN4O3 [M+H]+ 647.3728, found 647.3723.
2.1.5.15. Experimental
data
of
1-(2-carboxy-4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl
betulonic ester (11o)
1H NMR (CDCl3, 400 MHz): δ 8.07 (1H, s, N-C
H
), 7.92 (1H, s, Ar-
H), 7.86 (1H, d, J = 8.4
Hz, Ar-H), 7.42 (1H, d, J = 8.4 Hz, Ar-H), 5.30 (2H, m, -OCH2C), 4.72 and 4.59 (1H each,
s, H-29), 2.99 (1H, m, H-19), 2.44 and 1.85 (1H, each, m, H-22), 2.37 and 1.83 (1H each, m,
H-16), 2.24 and 1.59 (1H each, m, H-2), 2.02 (1H, m, H-13), 1.89 and 1.37 (1H each, m, H-
15

6), 1.72 (1H, m, H-18), 1.67 (3H, s, H-30), 1.62 and 1.19 (1H each, m, H-7), 1.57 and 1.21
(1H each, m, H- 12), 1.41 and 1.18 (1H each, m, H-15), 1.39 and 1.16 (1H each, m, H-21),
1.33 (1H, m, H-9), 1.31 and 1.21 (1H each, m, H-1), 1.28 and 0.86 (1H each, m, H-11), 1.05
(3H, s, H-26), 1.01 and 0.94 (3H, each, s, H-27 and H-23), 0.90 (1H, m, H-5), 0.87 (3H, s,
13
H-24), 0.78 (3H, s, H-25); C NMR (100 MHz, CDCl3) δ 218.46, 178.13, 176.02, 150.29,
139.25, 136.20, 136.03, 134.52, 132.47, 131.70, 128.05, 114.06, 109.77, 56.54, 54.93,
49.85, 49.37, 47.32, 46.67, 42.41, 40.63, 39.60, 38.36, 36.87, 34.09, 33.81, 31.92, 31.46,
29.68, 26.60, 25.49, 22.68, 21.34, 20.85, 19.59, 19.32, 15.82, 1568, 14.58, 14.10. HRESIMS
m/z calcd for C40H53ClN3O5 [M+H]+ 690.3673, found 690.4049.
2.1.5.16. Experimental data of 1-(3-(hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(11p)
1H NMR (CDCl3, 400 MHz): δ 9.06 (1H, bs, O
7.37 (1H, dd, J = 8.0, 7.6 Hz, Ar- ), 7.06 (1H, d, J = 8.4 Hz, Ar-
Hz, Ar- ), 5.30 (2H, m, -OCH2C), 4.74 and 4.60 (1H each, s, H-29), 3.03 (1H, m, H-19),
H
), 8.34 (1H, s, N-C
H
), 8.16 (1H, s, Ar-
H),
H
H), 6.99 (1H, d, J = 8.4
H
2.41 and 1.84 (1H, each, m, H-22), 2.37 and 1.83 (1H each, m, H-16), 2.27 and 1.53 (1H
each, m, H-2), 2.02 (1H, m, H-13), 1.89 and 1.37 (1H each, m, H-6), 1.72 (1H, m, H-18),
1.66 (3H, s, H-30), 1.62 and 1.19 (1H each, m, H-7), 1.57 and 1.21 (1H each, m, H- 12),
1.41 and 1.18 (1H each, m, H-15), 1.39 and 1.16 (1H each, m, H-21), 1.33 (1H, m, H-9),
1.31 and 1.21 (1H each, m, H-1), 1.28 and 0.86 (1H each, m, H-11), 0.99 (3H, s, H-26), 0.90
and 0.89 (3H, each, s, H-27 and H-23), 0.86 (1H, m, H-5), 0.74 (3H, s, H-24), 0.58 (3H, s,
H-25); 13C NMR (100 MHz, CDCl3) δ 218.66, 176.12, 158.75, 150.23, 143.58, 137.39,
130.54, 122.58, 116.93, 109.80, 108.88, 56.55, 56.48, 54.78, 49.74, 49.48, 47.24, 46.95,
42.30, 40.45, 39.53, 38.49, 36.77, 34.09, 33.28, 31.95, 30.59, 29.67, 29.48, 26.68, 25.45,
16

21.30, 20.89, 19.44, 19.39, 15.72, 15.23, 14.54, 14.13. HRESIMS m/z calcd for C39H54N3O4
[M+H]+, 628.4114 found 628.4121.
2.1.6. Synthesis of C-28 propargylated betulinic ester (12).
Propargylation of compound
1 (0.250 g, 0.55 mmol) was carried out using propargyl
bromide (0.08 ml, 0.65 mmol) and K2CO3 (0.11 g, 0.82 mmol) in acetone (20 ml) at 50°C
1
13
for 2-3 h to furnish C-28 propargylated betulinic ester 12 (96% yield). H NMR and C
NMR [18]. HRESIMS m/z calcd for C33H51O3 [M+H]+, 495.3838 found 495.3822.
2.1.6.1. Synthesis of 1-(3-(carboxyyphenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester (13a)
The compound was prepared by the procedure mentioned in section 2.1.5 from compound
1
12. H NMR (CDCl3, 400 MHz): δ 8.41 (1H, s, Ar-
H
), 8.36 (1H, s, N-C
H), 8.16 (1H, s, Ar-
H
), 8.03 (1H, d, J = 8.0 Hz, Ar-
H
), 7.70 (1H, s, Ar-H), 5.30 (2H, m, -OCH2C), 4.74 and
4.61 (1H each, s, H-29), 3.12 (1H, t, J = 8.4 Hz,, H-3), 3.04 (1H, m, H-19), 2.29 and 1.69
(1H, each, m, H-22), 2.20 and 1.63 (1H each, m, H-16), 2.05 and 1.53 (1H each, m, H-2),
1.92 (1H, m, H-13), 1.89 and 1.37 (1H each, m, H-6), 1.68 (3H, s, H-30), 1.65 (1H, m, H-
18), 1.62 and 1.19 (1H each, m, H-7), 1.57 and 1.21 (1H each, m, H- 12), 1.41 and 1.18 (1H
each, m, H-15), 1.39 and 1.16 (1H each, m, H-21), 1.33 (1H, m, H-9), 1.31 and 1.21 (1H
each, m, H-1), 1.28 and 0.86 (1H each, m, H-11), 0.94 (3H, s, H-26), 0.72 and 0.71 (3H,
each, s, H-27 and H-23), 0.63 (1H, m, H-5), 0.69 (3H, s, H-24), 0.57(3H, s, H-25); 13C NMR
(125 MHz, CDCl3) 176.32, 169.39, 157.15, 144.13, 142.42, 137.11, 130.42, 130.14, 125.42,
122.74, 121.69, 113.34, 82.39, 64.43, 60.47, 59.29, 54.37, 53.43, 50.98, 46.11, 44.46, 42.67,
42.34, 40.90, 40.55, 38.04, 35.77, 34.36, 33.39, 31.48, 30.63, 29.35, 24.65, 24.27, 22.80,
21.99, 19.29, 18.25, 17.56, 17.54. HRESIMS m/z calcd for C40H56N3O5 [M+H]+, 658.4220
found 658.4198.
17

2.1.6.2. Synthesis of 1-(2-carboxy-4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl betulonic ester
(13b)
The compound was prepared by the procedure mentioned in section 2.1.5 from compound
1
12. H NMR (CDCl3, 400 MHz): δ 8.07 (1H, s, N-C
H
), 7.92 (1H, s, Ar-H), 7.63 (1H, d, J =
8.4 Hz, Ar-
H
), 7.41 (1H, d, J = 8.4 Hz, Ar-
H
), 5.29 (2H, m, -OCH2C), 4.72 and 4.59 (1H
each, s, H-29), 3.18 (1H, t, J = 8.4 Hz,, H-3), 2.98 (1H, m, H-19), 2.31 and 1.69 (1H, each,
m, H-22), 2.25 and 1.63 (1H each, m, H-16), 2.12 and 1.53 (1H each, m, H-2), 1.79 (1H, m,
H-13), 1.73 and 1.37 (1H each, m, H-6), 1.68 (3H, s, H-30), 1.65 (1H, m, H-18), 1.62 and
1.19 (1H each, m, H-7), 1.57 and 1.21 (1H each, m, H- 12), 1.43 and 1.18 (1H each, m, H-
15), 1.39 and 1.16 (1H each, m, H-21), 1.37 (1H, m, H-9), 1.32 and 1.21 (1H each, m, H-1),
1.16 and 0.84 (1H each, m, H-11), 0.94 (3H, s, H-26), 0.93 and 0.87 (3H, each, s, H-27 and
13
H-23), 0.65 (1H, m, H-5), 0.77 (3H, s, H-24), 0.74 (3H, s, H-25); C NMR (125 MHz,
CDCl3) δ 176.22, 166.83 150.39, 139.31, 136.27, 134.35, 132.31, 131.62, 127.92, 126.07,
114.10, 109.76, 79.13, 56.55, 55.26, 53.48, 49.40, 46.91, 42.33, 40.65, 38.89, 38.26, 37.12,
36.88, 34.19, 33.85, 31.94, 30.51, 28.96, 27.95, 27.15, 25.45, 22.72, 20.84, 19.33, 16.11,
15.82, 15.41, 14.67, 14.16. HRESIMS m/z calcd for C40H55ClN3O5 [M+H]+, 692.3830 found
692.3823.
2.1.7. Preparation of methyl betulonate (14)
To a solution of
9 (0.24 mmol) in 20 ml of acetone was added K2CO3 (0.24 mmol), CH3I
(0.3 mmol) and stirred at room temperature. After completion, the reaction mixture was
dried under vacuum and product 14 was crystallized in methanol as white solid (yield 98%).
HRESIMS m/z calcd for C31H49O3 [M+H]+ 469.3681, found 469.3686. See supporting
information for 1H NMR and 13C NMR.
18

2.2. Pharmacology
2.2.1. Cell culture and treatment
The human cancer cell lines including breast cancer cell MCF-7, lung cancer A549, colon
cancer HCT-116, leukemia MOLT-4, prostate carcinoma cell PC-3 and pancreatic cancer
cell MIA PaCa-2 were purchased from ECACC, England. Cancer cells were grown in
RPMI/DMEM growth media containing 10% FCS, 100U penicillin G, and 100 µg
streptomycin per ml. Cells were grown in a CO2 incubator (Thermocon Electron
Corporation, Texas, USA) at 37°C with 95% humidity. Non tumor human breast epithelial
cell line fR2 (ECACC 98031102) were procured from European Collection of Authenticated
Cell Cultures (ECACC), UK. BA and its analogues were dissolved in DMSO and delivered
to cell culture in complete medium.
2.2.2. Cell Proliferation Assay
The cell viability was determined by the standard MTT method. Cells A-549, HCT-116, PC-
3, MIA PaCa-2 and T47D were seeded (as per their doubling time) 7000, 5000, 7000, 10000
and 12000 cells/well/100µL of media into 96 well plates respectively. Beside this, all the
necessary controls i.e untreated cells, RPMI/DMEM as medium, DMSO as solvent and
positive controls were also kept. The concentration of DMSO in wells was >1%. After 4 h,
cells were treated with different concentrations of given compounds and placed in incubator
for 48 h. MTT dye (20 µL) was added to the plates 4 h prior to experiment termination at
concentration of 2.5 mg/mL, dissolved in media. MTT formazon crystals formed were
dissolved in 150 µL DMSO and absorbance at 570 nm was recorded.
2.2.3. Wound Healing Assay
19

The HCT-116 cells were seeded in six well plates and on the next day a wound was created
by 20–200 µL pipette tip and the cells washed with PBS to remove the debris [29]. 5h was
added at desired concentrations and positive control Paclitaxel (1 µM) was also added and
plates were incubated for 48 h in CO2 incubator. After 48 h the photographs were taken (x10
objective) at zero h and 48 h and length of the wound is measured image J (version 1.46)
software.
2.2.4. Nuclear morphology studies by DAPI staining
The cells were seeded in 6-well tissue culture plates at the density of 2×105 cells per ml/well
in complete medium supplemented with 10% FCS for 24 h [30]. The stock solution was
prepared in DMSO and added to the medium to achieve the desired final conc. of 1, 4, 8 and
12 µM. After 24 h, cells were incubated with different concentrations of 5h and paclitaxel
(positive control) for 48 h. To assess the mechanism of cell death, after incubation for
specified time at 37°C, the cells were processed for fluorescent microscopic studies. For
fluorescent DAPI staining, air dried smears of HCT 116 cells were fixed in methanol at -
20°C for 20 min, air dried and stained with DAPI at 1 µg/ml in PBS at room temperature for
20 min in the dark and the slides were mounted in glycerol–PBS (1:1) and examined in an
inverted fluorescence microscope (Olympus, 1X81).
2.2.5. Detection of intracellular reactive oxygen species (ROS) accumulation
Human colon cancer (HCT-116) cells (2x105/mL/well) were seeded in six well plates [31].
After 24 h, cells were incubated with different concentrations of 5h for 48 h. The
intracellular ROS levels were monitored by fluorescence microscopy after staining with
DCFH-DA (dichlorodihydro-fluorescein diacetate) and observed under fluorescence
microscope using 20X lens (Olympus, 1X81). H2O2 (0.05%) was used as positive control.
20

2.2.6. DNA cell cycle analysis
HCT 116 cells (1 × 106 cells/2 ml/well) were treated with compound 5h at different
concentration i.e. 1, 4, 8 and 12 µM for 24 h. After 24 h, cells were gathered, washed twice
in PBS, and fixed in 70% cold ethanol overnight. Subsequently, cells were again washed
with PBS in order to remove the ethanol completely and digested with RNase (0.1 mg/ml) at
370C for 90 min and finally cells were incubated with PI (10 mg/ml). Cells are then
analyzed immediately on FACS Calibur Flow Cytometer (Becton Dickinson, Franklin
Lakes, NJ).The results were analyzed by Mod fit (Verity Software House Inc., Topsham).
Results and discussion
2.
3.1. Chemistry
Betulinic acid (1) was isolated from the stem bark of Platanus orientalis and used as a
starting material for chemical modification. Three sites C-3, C-20 and C-28 are readily
available position for carrying out chemical modification of BA (Fig. 1) to yield derivatives
for structure activity relationship (SAR) study. In the present study, we selected C-3 and C-
28 positions and demonstrated that chemical modification at these sites can produce
potentially important analogs. The present procedure involves oxidation of C-3-OH of BA
to give betulonic acid (
9
). Compound
9
on reaction with propargyl bromide furnished C-28
propargylated betulonic ester 10
.
Scheme 1
Compound 10 was subjected to copper (I)-catalysed alkyne-azide cycloaddition (click
chemistry approach) with various substituted aromatic azides to produce desired products
11a-p in excellent yields (70-95%) as shown in Scheme 1. Compound
1 upon reaction with
propargyl bromide furnished 12 which under click chemistry conditions with appropriate
21

aromatic azides furnished desired products 13a and
behind the synthesis such analogs was to retain the C-3 hydroxyl and insert a triazole moiety
at C-28 with a free –COOH group. Compound 14 was synthesised by treating with methyl
b, as shown in Scheme 2. The rationale
9
iodide in presence K2CO3, as shown in Scheme 3. All the derivatives were fully
1
characterized by various spectroscopic methods including H NMR, 13C NMR and
HRESIMS.
Scheme 2
Scheme 3
3.2. Biological evaluation
3.2.1. In vitro anti-proliferative activity
The in vitro antitumor activity of all the derivatives were evaluated by MTT assay on five different
human cancer cell lines: lung (A549), colon (HCT116), prostate (PC3), pancreatic (MIA PaCa-2)
and breast (T47D) cell lines along with parent compound 1 taken as reference standard. Preliminary
cytotoxicity screening of the all the analogs was carried out at 30 µM concentration for 48 h and the
percentage of growth inhibition was determined.
The results are summarized in Table 1, and the values given are the average of the triplicate
analysis. Betulinic acid
variable growth inhibition effects on different human cancer cell lines. Conversion of
10 and 14 resulted in the loss of activity. In agreement with the literature [32], conversion of
into increased the cytotoxicity against A-549, HCT-116 and PC-3 and displayed the
1
showed 68-90% of growth inhibition and its derivatives displayed
1
into
1
9
growth inhibition of 71, 93 and 87% respectively.
Table 1
22

Among the triazolyl derivatives, 11a
significant inhibition against any of the cell lines tested. Compound 11f and 12 were active
only against HCT-116 cell line. Compound 11c 11d 11g 11h 11m 11p and 11a were
11g and 11h were the
, 11b, 11e, 11i-l, 11n, 11o and 13b did not display any
,
,
,
,
,
active against all the tested human cancer cell lines. Among them 11c
potent analogs displayed 63-91% growth inhibition.
3.2.2. IC50 and structure activity relationship
,
Compounds that exhibited significant percentage growth inhibition (≥70%) at 30 µM
concentration against various selected human cancer cell lines were further evaluated at
different concentrations to calculate their IC50 (concentration for 50% growth inhibition)
values. The IC50 of the potent molecules are shown in Table 2. Conversion of
(betulonic acid) resulted in slight improvement in the activity against all the cell lines tested
except T47D. Conversion of into 12 (C 28 betulinic ester) and into 14 (C 28 betulonic
ester) significantly reduced the activity. Among the triazolyl analogs 11c 11g 11h and 13a
were active against all the cell lines tested, amongst them 11c and 11h were four fold more
active than parent against HCT-116 and displayed the IC50 values of 4 and 4.5 µM
respectively. Compound 11h also displayed a low IC50 value of 5 µM against breast cancer
cell line T47D which is five times more active than . Compound 11d was the most potent
1 into 9
1
-
9
-
,
,
1
1
compound among all the analogs against HCT-116 with IC50 value of 3.5 µM. Compound
11p was active only against colon carcinoma cell line and did not displayed significant
antitumor activity against other cell lines. Compound 13a was most potent against PC-3 cell
line had the IC50 of 6 µM.
From percentage inhibition data and IC50 values, a general structure activity relationship
SAR could be drawn as follow: Conversion of 28-COOH into simple ester reduces the
23

antitumor activity (compound 10
polar substituent can produce potentially important derivatives. Thus among triazoles having
electron rich substituents in the aromatic moiety particularly at the para position (11c 11d
and 11g) were strongly cytotoxic. The loss of toxic effect of 11p meta–OH) in the triazole
, 12 and 14). However, modification of 1 at 28-COOH by
,
(
moiety against A-549, PC3, MIA PaCa-2 and T47D suggested that position of the
substituent (–OH) is also important for retaining the activity. Analogs with electron
withdrawing substituents particularly at ortho and meta positions in the triazole moiety (11a
11e 11f, and 11j) did not show significant antitumor activity. However, 11m with para
cyano was active against lung and colon cancer cells. Derivatives with a free meta –COOH
11h and 11a) were strongly cytotoxic than analogs with ortho –COOH 11o and 13b).
,
,
(
(
Compound 11h was the most active derivative and subsequently selected for the further
studies. It seems that all the factors including size, position of appropriate substituent,
electrostatic sensitivity and strength of hydrogen bond formation are collectively responsible
for the antitumor effects of BA derivatives.
Table 2
3.2.3. Nuclear morphology studies by DAPI staining
To gain further insight into the anti-proliferative activity, compound 11h was subjected to
apoptosis assay on HCT-116 [33]. To examine the formation of apoptotic bodies, the HCT-
116 cells were treated with test compound 11h at 1, 4, 8 and 12 µM for 48 h and stained
with 6-diamidino-2-phenylindole (DAPI) to examine the nuclear morphological changes.
Increased the formation of apoptotic bodies and chromatin condensation were observed in
concentration dependent manner in nuclei of HCT-116 cells treated with 11h (Fig. 3). These
results suggest that 11h inhibits growth of HCT-116 cells by inducing apoptosis.
24

Figure 3.
3.2.4. Reactive Oxygen Species (ROS) generation study
Reactive Oxygen Species (ROS) are the common mediators of apoptotic pathway [34].
Many anticancer agents display their cytotoxic effects by the ROS generation. Thus, the role
of compound 11h in stimulating the production of ROS in the HCT-116 cells was evaluated.
In treated cells, after 48 h exposure with compound 11h, the fluorescence intensity of
dichlorofluorescein (DCF) was increased in a dose dependent manner when evaluated at 1,
4, 8 and 12 µM, (Fig. 4).
Figure 4.
3.2.5. Wound Healing Assay
Cell motility and migration correlates with the invasiveness and metastatic properties of the
cancer cells [35]. To examine this, scratch wound healing assay was performed to evaluate
the effect of 11h on HCT-116 cell migration. After creating the wound over the cell’s
confluent monolayer, cells were treated compound 11h for 24 h while DMSO and Paclitaxel
were taken as negative and positive controls respectively. Images were captured for the 0 h
and 24 h, and compared with untreated and positive control. In comparison to
DMSO/vehicle (control) healing was strongly abrogated in the cells treated with 11h in a
dose dependent manner, (Fig. 5). These findings demonstrated that 11h effectively blocked
the motility and migration capabilities in colon carcinoma (HCT-116) cell lines.
Figure 5.
3.2.6. DNA cell cycle analysis
HCT 116 cells were treated with the different concentrations (1, 4, 8 and 12 µM of 5 h for
24 h in which increased percentage arrest of cells in G1 phase (47.97, 52.31, 63.68 and
25

78.12%) compared with the control (G1 19.62%, S phase 80.38%, G0 0%). (B) Bar diagram
represents percentage of cells population in different phases (G2, S, and G1) of cell cycle
Fig. 6. The flow cytometry analysis of different phases of cell cycle showed dose dependent
changes in the distribution cell cycle population in which 5h showed enhanced G1 arrest as
the concentrations advances and confers the ability to induce cytostatic effect.
Figure 6.
4.
Conclusion
To sum up, a series of betulinic acid derived 1,2,3-triazolyl derivatives were designed and
synthesized by employing Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction and
characterized by various spectroscopic techniques. All the derivatives were evaluated for
anti-cancer activity by MTT assay on five different human cancer cell lines: lung (A549),
colon (HCT116), prostate (PC3), pancreatic (MIA PaCa-2) and breast (T47D) along with
parent compound
many compounds exhibited better anti-tumor activity against the tested cancer cell lines.
Especially compounds 11c 11d 11g 11h and 13a were found to be the most promising
anti-tumor derivatives. The results also indicated that derivatives with free –OH 11c 11d
and 11g) and free -COOH 11h and 11a) substitutions in the triazole moiety introduced at
the C-28 position significantly improved the anti-tumor activity. Introduction of a non polar
alkyl group at C-28 position (10 12 and 14) resulted in the significant loss of activity. The
1 taken as reference. From the pharmacological data, it was evident that
,
,
,
(
,
(
,
compound 11h was one of the most active compounds, having the IC50 values of 6.0, 4.0,
5.5, 6.3, and 5.0 µM on all the five cancer cell lines respectively. Further, DAPI staining,
ROS generation and wound healing experiments revealed that compound 11h has the
potential to induce apoptosis in HCT-116 cells which confers its anti-invasive and anti-
26

metastatic behaviour towards cancer cells. Further compound 11h portray as cytostatic agent
arresting G1 phase of cell cycle in HCT-116 cells. The results suggest that these compounds
can be transformed into promising lead molecule and justify detailed investigations.
Acknowledgements
Authors NAD and SKR thank to Council of Scientific and Industrial Research (CSIR-UGC), New
Delhi and ICMR, New Delhi, respectively, for the award of senior research fellowship (SRF). Staff
of instrumentation division (NMR) of our institute is well acknowledged for recording the
spectroscopic data. This research was supported in part by a grant from the CSIR 12th five-year
project (BSC0108, and BSC-0106).
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