A novel triazole derivative of betulinic acid induces extrinsic and intrinsic apoptosis in human leukemia HL-60 cells

Article abstract of DOI:10.1016/j.ejmech.2015.11.018

In an attempt to arrive at more potent cytotoxic agent than the bioactive natural product betulinic acid, influence of small structural modifications of its 1, 2, 3 triazole derivatives tethered at C-28 and both C3, C-28 using click chemistry approach has been studied. The chemically characterized triazoles have been screened for in vitro cytotoxicity against four human cancer cell lines HL-60, MiaPaCa-2, PC-3 and A549 which has allowed to identify triazole derivative 28{1N (4-fluoro phenyl)-1H-1, 2, 3-triazol-4-yl} methyloxy betulinic ester having better potency profile than the parent compound with IC₅₀ values in the range of 5-7 μM. It caused disruption of mitochondrial membrane potential, rendered Bcl-2 cleavage, Bax translocation and decrease Bcl-2/Bax ratio. These events are accompanied by activation of caspases -9, -3, which cleave the PARP-1. It also induces caspase-8, which is involved in extrinsic apoptotic pathway. Therefore, it induces apoptosis through both intrinsic and extrinsic pathways in human leukemia HL-60 cells.

Full text of DOI:10.1016/j.ejmech.2015.11.018

Accepted Manuscript
A novel triazole derivative of betulinic acid induces extrinsic and intrinsic apoptosis in
human leukemia HL-60 cells
Imran Khan, Santosh K. Guru, Santosh K. Rath, Praveen K. Chinthakindi, Buddh
Singh, Surrinder Koul, Shashi Bhushan, Payare L. Sangwan
PII:
S0223-5234(15)30354-8
10.1016/j.ejmech.2015.11.018
EJMECH 8208
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 August 2015
Revised Date: 9 November 2015
Accepted Date: 12 November 2015
Please cite this article as: I. Khan, S.K. Guru, S.K. Rath, P.K. Chinthakindi, B. Singh, S. Koul, S.
Bhushan, P.L. Sangwan, A novel triazole derivative of betulinic acid induces extrinsic and intrinsic
apoptosis in human leukemia HL-60 cells, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.11.018.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Novel tethered triazole derivatives of betulinic acid
induce G1 arrest and apoptosis via extrinsic and
Leave this area blank for abstract info.
intrinsic apoptotic pathways in human leukemia HL-60 cells
I. Khan, S.K. Guru, S.K. Rath, P.K. Chinthakindi, S. Bhushan*, B. Singh, S. Koul and P.L. Sangwan*
Cytotoxic study of new betulinic acid analogs showed the anti-leukemic potential of C28-aryl
substituted 1,2,3 triazoles derivatives of betulinic acid that induce G1 arrest and apoptosis.
N
N
N
OH
O
COOH
O
HO
HO

ACCEPTED MANUSCRIPT
A novel triazole derivative of betulinic acid induces extrinsic and intrinsic apoptosis in human
#
leukemia HL-60 cells
a
b
a,c,$
a,$
a
Imran Khan , Santosh K. Guru , Santosh K. Rath , Praveen K. Chinthakindi , Buddh Singh ,
a
b,
a,c,
Surrinder Koul , Shashi Bhushan * and Payare L. Sangwan *
a
b
Bioorganic Chemistry Division, Cancer Pharmacology Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road Jammu-180001, India
c
Academy of Scientific and Innovative Research (AcSIR), CSIR-IIIM Campus, Jammu, India
#
IIIM manuscript no. IIIM/1859/2015
$
authors equally contributed
*
Corresponding authors. Tel.: +91 191 2585006-13 Extn. 371; fax +91 191 2586333. E-mails;
plsangwan@iiim.ac.in (P.L. Sangwan), sbhushan@iiim.ac.in (S. Bhushan)
Keywords: Betulinic acid; Apoptosis; click chemistry; Triazole derivative; human leukemia
1

ACCEPTED MANUSCRIPT
ABSTRACT
In an attempt to arrive at more potent cytotoxic agent than the bioactive natural product betulinic
acid, influence of small structural modifications of its 1, 2, 3 triazole derivatives tethered at C-28
and both C3, C-28 using click chemistry approach has been studied. The chemically characterized
triazoles have been screened for in vitro cytotoxicity against four human cancer cell lines HL-60,
MiaPaCa-2, PC-3 and A549 which has allowed to identify triazole derivative 28{1N (4-fluoro
phenyl)-1H-1, 2, 3-triazol- 4-yl} methyloxy betulinic ester having better potency profile than the
parent compound with IC50 values in the range of 5-7 µM. It caused disruption of mitochondrial
membrane potential, rendered Bcl-2 cleavage, Bax translocation and decrease Bcl-2/Bax ratio.
These events are accompanied by activation of caspases -9, -3, which cleave the PARP-1. It also
induces caspase-8, which is involved in extrinsic apoptotic pathway. Therefore, it induces apoptosis
through both intrinsic and extrinsic pathways in human leukemia HL-60 cells.
1
. Introduction
Betulinic acid (1) is a bioactive pentacyclic lupane type triterpenoid (Fig. 1) which can be
directly isolated from Platanus orientalis stem bark as well as from many other plants such as birch
tree Betula spp. (Betulaceae), Ziziphus spp. (Rhamnaceae), Syzygium spp. (Myrteceae), Diospyros
spp. (Ebenaceae) and Paeonia spp. (Paeoniaceae) [1, 2]. The plant extracts bearing triterpenoids of
lupane class have been found in the past few decades to exhibit anticancer properties on various in
vivo cancer model systems [3-7], with betulinic acid of this class shown to possess various
important pharmacological properties that include antitumor, anti-HIV [8], anti-inflammatory,
antibacterial, antitrypanosomal and analgesic activities [9]. The broad spectrum of betulinic acid
against a tumour cell lines (derived from lung, colorectal, breast, prostrate and cervical cancers
which are most prevalent with highest human mortalities) has attracted considerable awareness,
2

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being active in micro-molar concentrations with high selectivity [10-13]. The mechanism of action
involves mitochondrial membrane permeabilization with release of factors like cytochrome c, Smac
or AIF in a permeability transition pore-dependent manner, activating caspases and nuclear
fragmentation [14, 15]. The natural product (NP) is reported to induce apoptosis in a p-53 and CD-
9
5 independent manner [16-21] and has been also identified as a potential activator of NF-kB in a
number of cancer cell lines [22]. In continuation of our curiosity for the modification of potential
plant-derived natural products for better activity and lesser toxicity than the parent molecule/s,
betulinic acid has been chosen for chemical modification to yield analogue/s with significantly
improved potency.
In the present paper we report the synthesis and chemical characterization of C28-aryl substituted 1,
2
, 3 triazoles derivatives of betulinic acid (1), study of the influence of minor structural changes on
the anticancer properties, and in vitro anticancer activity cum structure activity relationship study.
Therefore, our study not only describes the anti-leukemic potential of C28-aryl substituted 1, 2, 3
triazoles derivatives of betulinic acid but also insights the role of natural product in the discovery
and development of novel anticancer therapeutics.
2
. Results and discussion
The structure of betulinic acid consists of 30-carbon skeleton which has three available sites for
chemical modification at C-3, C-20 and C-28 position (Fig. 1). A series of betulinic acid derivatives
were synthesized and the strategy envisaged involved the prologue of nitrogen atom to generate
molecules bearing heterocyclic moiety. This was achieved by preparation of C-28 propargylated
betulinic ester (2), C-3 propragyl derivative (2a), and C-3, C-28-di-propargylated derivative (2b)
and subsequent reaction of 2 and 2b to copper (I)-catalysed one pot cycloaddition reaction (click
chemistry strategy) with various substituted aromatic azides generated in situ by aryl boronic acids
3

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in methanol and water (1:1) in the presence of reducing agent sodium ascorbate [23] to give very
good chemical yields. A library of 32 triazoles derivatives bearing structural diversity could thus be
prepared and chemically identified by spectral analysis (Scheme1, 2).
N
N
N R
O
O
COOH
Br
RꢀB(OH) , NaN , MeOH:H O (1:1)
2
3
2
O
O
CuSO , Sod. Ascorbate
K CO , Dry Acetone,
reflux, 14 hrs, 98% HO
4
2
3
HO
HO
2
3-31
OCH3
1
O
O
N
Cl
I
OCH3
OCH3
3
CN
11
F
NH
Ph
9
10
7
4
5
6
8
12
13
22
O
O
F
O
Br
CH3
3-31 R=
O
NO2
F
Br
Cl
3
H C
Br
F
15
O
17
21
1
9
20
1
4
16
18
O
O
O
O
CN
F
O
25
CHO
30
CH3
31
OH
28
24
26
29
2
3
27
Scheme 1. Synthesis of C-28 propargylated betulinic ester and their triazole derivatives
Scheme 2. Synthesis of C-3 and C-28 dipropargylated betulinic ester and their 1,2,3-triazole
derivatives
4

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2
.1. Cell growth inhibition
The anti-proliferative activity of synthesized compounds 2-34 along with compound 1 as standard
was determined by MTT assay in four different cell lines viz. HL-60 (leukemia), MiaPaca-2
(pancreatic), PC-3 (prostrate) and A549 (lungs). The results are summarized in Table-1. In the
primary screening betulinic acid (1) showed 68-70% of growth inhibition at 10 ꢁM concentration,
and many of its derivatives displayed different growth inhibition effects on different cancer cell
lines. Compounds 3, 6, 8, 9, 15-21, 24-30, and 32-34 showed no significant activity against any of
the cell lines. Compounds 2, 4, 10, 11, 22 and 23 showed significant inhibition against three cancer
cells lines HL-60, PC-3 and A549 and weak inhibition on MiaPaca-2 cell line. Compound 13 and
1
1
5 show significant cytotoxicity against A549 and HL-60 cells, respectively. Compound 5, 7, 12,
4 and 31 displayed inhibition against all the four experimental cancer cell lines. Maximum growth
inhibition effect as well as inhibitory effect against the entire cell lines were displayed by 12 which
was more than the effect observed for the parent molecule as can be seen from the Table-1. While
1
2 displayed 75% cell growth inhibition against MiaPaCa-2 (Pancreas) cell line to that of 70 %
displayed by 1, both were found equipotent against HL-60 and was found with marginally lower
potency against PC-3 and A549 cell lines.
The structure activity relationship of structural modifications of (1) described above can be
summarized as follow: among para- and ortho-cyano derivatives of betulinic ester, p-cyanophenyl
methyloxy betulinic ester (5) showed better cytotoxicity compared to o-cyanophenyl methyloxy
betulinic ester (27). The more electronegative p-fluoro (12) exhibited much better cytotoxicity in all
the cancer cell lines compared to p-bromo derivative (14) which showed only 61 and 54% of
growth inhibition against MiaPaCa-2 and A549 cancer cell lines. The probable reason for the same
may be the high lipophilicity, by molecule can penetrate easily to the cells and exerted its activity.
5

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Generally, compounds containing electron withdrawing functional groups (-F, -Cl) exhibited more
potent cytotoxic effects against the cancer cells compared to the electron donor functional groups (-
CH , -OCH ). In a similar trend for compound (9), m-methoxy aryl betulinic ester did not exhibit
3
3
any significant inhibition against all the cancer cell lines while o-methoxy derivative (11) showed
0% of growth inhibition against MiaPaCa-2 cell line. In case of compound 2,5-dimethyl aryl
5
betulinic ester (22) showed only 51% of growth inhibition against HL-60 while p-tolyl betulinic
ester (31) showed 56, 67 and 52% of growth inhibition against HL-60, PC-43 and A549
respectively.
The IC50 (50% growth inhibition) of the selected analogues of betulinic acid against different cancer
cells is shown in Table-2. Among all the tested compounds the compound 12 displayed promising
antiproliferative activity in all four cell lines (HL-60, MIAPaCa2, PC-3, A549) showing IC values
5
0
of 7, 5, 7 and 7 µM, respectively. The cytotoxic profile of compound 12 was similar to betulinic
acid. Therefore, we have chosen it for further cell death mechanistic studies in HL-60 cells.
2
.2. Compound 12 induced G1 arrest in HL-60 cells
The effect of compound 12 on the DNA damage and apoptosis was investigated using cell cycle
analysis. It was observed that compound 12 up to 10µM showed mild induction of apoptosis (7%)
(
Fig. 2 A, C). Further, the cell cycle G1 phase was significantly blocked by compound 12 at 20 µM
concentration indicating that it caused delay in cell cycle.
.3. Compound 12 triggers mitochondrial potential loss
2
Mitochondria play an important role in activating apoptotic induction in cancer cells. Damaged
mitochondria release a variety of proteins that activates the procaspase cascade inside the cells and
triggers apoptosis. Mitochondrial potential loss were analysed by using Rh-123, a cationic
fluorescence which intra cellular fluorescence after metabolic reduction in mitochondria.
6

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Compound 12 induces loss of mitochondrial membrane potential in concentration dependent
manner (Fig. 2B). Compound 12 at 5 µM concentration induces 7% loss of mitochondrial
membrane potential, which increases to 18% and 27% at 10 and 20 µM concentrations,
respectively.
2
.4. Compound 12 triggers extrinsic and intrinsic apoptosis in human leukemia HL-60 cells
Cancer cells avoid apoptosis and continue to proliferate uncontrollably, which is the key process in
cancer development and progression. Molecules that induce apoptosis in cancer cells are always
preferred in cancer drug discovery. Mitochondria and Bcl-2 family of proteins play a pivotal role in
the induction of apoptosis [24]. Bcl-2 associated proteins have both pro-apoptotic and anti-
apoptotic effects in cancer cells. These proteins regulate mitochondrial outer membrane potential
and control the release of many apoptotic factors originating in the mitochondria. The compound 12
decreased the expression of mitochondrial associated anti-apoptotic proteins Bcl-2 in a
concentration-dependent manner (Fig. 2D, E). The down regulation of antiapoptotic proteins
changes the symmetry of mitochondria and activates the mitochondrial permeability transition
pores (PTP) and induces the loss of mitochondrial membrane potential (Ψmt). Loss of mitochondrial
membrane potential is an early apoptotic event and damaged mitochondria relay many signals to
downstream elements that initiate intrinsic apoptotic death-signalling in cells. Compound 12 treated
HL-60 cells show significant loss of mitochondrial membrane potential (Fig. 2A). The loss of
mitochondrial membrane potential further triggers the apoptotic machinery via release of pro-
apoptotic factors such as Bax. During apoptosis, Bax is translocated to the mitochondria and creates
pores in the mitochondrial outer membrane which results in an increase in mitochondrial
permeability [25]. Compound 12 significantly decrease the level of mitochondrial antiapoptotic
protein Bcl-2 and increase the expression of pro-apoptotic proteins Bax with concurrent fall in Bcl-
7

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2
/Bax ratio (Fig. 2D, E). Bcl-2 (apoptotic inhibitior) and Bax (apoptotic inducer) ratio determine
the cell susceptibility to apoptosis. The role of caspase in apoptosis is well established and active
caspase-8 or caspase-9 activate various effector caspases including caspase-3, -6, and -7, which
results in the enhanced expression of protease activity in the cell and cleavage of various proteins
like PARP-1 [26]. Compound 12 activates these caspases and finally PARP-1 cleavage (Fig.2D).
Compound 12 inhibits both Procaspase-9 and Procaspase-8 that indicates that it induced apoptosis
via both extrinsic and intrinsic pathway. Therefore, we can conclude that it induce apoptosis via
both intrinsic and extrinsic activation pathways in HL-60 cells (Fig. 3).
3
. Conclusion
The chemically synthesized analogous of betulinic acid for anticancer activity displayed adverse
effects and toxicity. The present study demonstrate that introduction with proper substituent at C-
2
8, positions of compound 12 displayed promising antiproliferative activity can produce substantial
evidence for their apoptotic mode of action against all the cancer cell lines with improved
antiproliferative activity for the development of novel anticancer agent. We are reporting here for
the first time that compound 12 induces apoptosis via both extrinsic and intrinsic apoptotic
pathways in human leukaemia HL-60 cells. In summary, the present studies highlight the
importance of tethered triazole derivatives of betulinic acid in the discovery and development of
novel anti-leukemic agents. This class of agents emerge to merit auxiliary preclinical testing in a
broad array of lymphoid malignancies for its development as a future anticancer agent.
4
. Experimental
4
.1. Chemistry
All reagents used for chemical synthesis were purchased from Sigma Aldrich. Solvents used
were distilled before use. The progress of all reactions were monitored by TLC on silica gel 60 F254
8

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plates (E. Merck) using 2% ceric ammonium sulfate reagent for detection of the spots. Purification
1
13
of the products was carried out by column chromatography. All NMR spectra ( H and C NMR)
were recorded on Bruker DPX 200, DPX 400 and DPX 500 instruments using CDCl , pyridin-d or
3
5
CD OD as solvent with TMS as internal standard. Chemical shifts are expressed in δ and coupling
3
constant in Hertz. High resolution mass spectra (HRMS) were recorded on Agilent Technologies
6
540 instrument and mass spectra were recorded on ESI-esquire 3000 Bruker Daltonics instrument.
IR recorded on an FT-IR Bruker (270-30) spectrophotometer. Melting points (uncorrected) of
compounds were recorded on Buchi melting point apparatus B-542.
4
.1.1. Isolation of betulinic acid (1)
Betulinic acid was isolated in bulk quantity from DCM: MeOH (1:1) as well as from MeOH
extracts of the stem bark of Platanus orientalis. Purity was obtained >97% which was well
characterized by spectroscopic study (data were found in agreement with literature) [2,3].
4
.1.2. Preparation of C -O-propargyl betulinic ester (2)
28
The compound prepared by the propargylation of compound 1 (10.0 g, 22 mmol) with propargyl
bromide (4.4 g, 32 mmol) and K
2
CO
3
in dry acetone (reflux for 14 h) to give after usual workup
1
propargylated product 2 (Scheme-1), a colorless solid (9.8 g, 98 % yield) mp 222-224 ºC. H NMR
(
200 MHz, CDCl ): δ 0.65 (1H, m, H-5), 0.75 (3H, s, H-25), 0.81 (3H, s, H-24), 0.88 and 1.61 (1H
3
each, m, H-1), 0.92 (3H, s, H-26), 0.96 (6H, s, H-27 and H-23), 1.14 and 2.43 (1H each, m, H-11),
.21 and 1.99 (1H each, m, H-12), 1.25 and 1.90 (1H each, m, H-15), 1.36 and 1.54 (1H each, m,
1
H-6), 1.37 and 2.15 (1H each, m, H-21), 1.38 (1H, m, H-9), 1.39 and 1.43 (1H each, m, H-7), 1.43
and 2.20 (1H each, m, H-22), 1.51 and 2.25 (1H each, m, H-16), 1.62 (1H, m, H-18), 1.68 (3H, s,
H-30), 1.78 (2H, m, H-2), 2.42 (1H, m, H-13), 2.43 (1H, s, -OCH
2
CCH-), 2.97 (1H, m, H-19), 3.14
1
3
(
1H, m, H-3), 4.65 and 4.73 (1H each, s, H-29), 4.67 (2H, m, -OCH -). C NMR (100 MHz, CDCl
2
3
9

ACCEPTED MANUSCRIPT
): δ 14.71, 15.37, 16.00, 16.13, 18.29, 19.39, 20.87, 25.53, 27.37, 27.99, 29.63, 30.50, 31.94, 34.33,
3
7
1
6.78, 37.18, 38.25, 38.73, 38.84, 40.80, 42.38, 46.83, 49.50, 50.57, 51.32, 55.36, 56.57, 74.36,
8.13, 78.94, 109.67, 150.39, 175.16. IR γmax (neat): 3309, 2940, 2870, 1731, 1642, 1453, 1389,
-1
376, 1317, 1270, 1245, 1149, 1123, 1106, 1062, 1009, 983, 885, 757, 631 cm . HRMS m/z calcd.
+
for C H O [M + H] 495.3832, found 495.3821.
3
3
50
3
4
.1.3. Preparation of C -O-propargyl betulinic ester (2), 3-O-propargylbetulinic acid (2a), and
28
dipropargyl betulinic ester (2b)
The compounds prepared by the propargylation of compound 1 (1.0 g, 2.2 mmol) using the
propargyl bromide (0.44 g, 4.84 mmol) and NaH in dry THF under nitrogen atmosphere for 24 h,
after usual work with ethyl acetate and water to give the propargylated product 2 (0.4 g, 40% yield),
1
2
a, (0.2 g, 20% yield) and 2b (0.4 g, 40 % yield) respectively (Scheme-2). H NMR (200 MHz,
CDCl ) of compound 2a: δ 0.68 (1H, m, H-5), 0.75 (3H, s, H-25), 0.83 (3H, s, H-24), 0.88 and 1.61
3
(
1H each, m, H-1), 0.93 (3H, s, H-26), 0.97 (6H, s, H-27 and H-23), 1.17 and 2.43 (1H each, m, H-
1), 1.21 and 1.99 (1H each, m, H-12), 1.27 and 1.94 (1H each, m, H-15), 1.34 and 1.51 (1H each,
m, H-6), 1.36 and 1.41 (1H each, m, H-7), 1.37 and 2.14 (1H each, m, H-21), 1.38 (1H, m, H-9),
.47 and 2.20 (1H each, m, H-22), 1.46 and 2.25 (1H each, m, H-16), 1.61 (1H, m, H-18), 1.69 (3H,
s, H-30), 1.74 (2H, m, H-2), 2.31 (1H, m, H-13), 2.35 (1H, s, -OCH CCH-), 2.98 (2H, m, H-3, H-
1
1
2
1
3
1
1
3
8
1
4
9), 4.20 (2H, m, -OCH -), 4.61 and 4.74 (1H each, s, H-29). C NMR (100 MHz, CDCl ): δ
2 3
4.12, 16.04, 16.1, 16.17, 18.22, 19.4, 20.89, 22.69, 25.53, 28.0, 29.70, 30.60, 32.18, 34.34, 37.06,
7.17, 38.44, 38.57, 38.61, 40.74, 42.43, 46.93, 49.31, 50.49, 55.93, 56.42, 56.42, 73.42, 80.96,
5.88, 109.69, 150.43, 182.10. IR γmax (neat): 3434, 3310, 2942, 2868, 1713, 1453, 1317, 1149,
-1
+
50 3
123, 1043, 1009, 983, 885 cm . HRMS m/z calcd. for C33H O [M + H] 495.38327, found
1
95.38213. H NMR (400 MHz, CDCl
3
) of compound 2b: δ 0.68 (1H, m, H-5), 0.70 (3H, s, H-25),
1
0

ACCEPTED MANUSCRIPT
0
2
.76 (3H, s, H-24), 0.88 and 1.61 (1H each, m, H-1), 0.93 (3H, s, H-26), 0.99 (6H, s, H-27 and H-
3), 1.15 and 2.43 (1H each, m, H-11), 1.21 and 1.99 (1H each, m, H-12), 1.27 and 1.94 (1H each,
m, H-15), 1.34 and 1.51 (1H each, m, H-6), 1.36 and 1.41 (1H each, m, H-7), 1.37 and 2.14 (1H
each, m, H-21), 1.38 (1H, m, H-9), 1.47 and 2.20 (1H each, m, H-22), 1.46 and 2.25 (1H each, m,
H-16), 1.61 (1H, m, H-18), 1.71 (3H, s, H-30), 1.74 (2H, m, H-2), 2.35 (1H, m, H-13), 2.35 (1H, s,
-
OCH CCH-), 2.42 (1H, s,-OCH CCH-), 2.98 and 3.01 (2H, m, H-3, H-19), 4.20 (2H, m, -OCH -),
2
2
2
1
3
4
1
3
7
5
4
.64 (2H, m, -OCH
2
-), 4.68 and 4.73 (1H each, s, H-29). C NMR (125 MHz, CDCl
3
): δ 14.19,
4.69, 16.02, 16.22, 18.24, 20.91, 22.64, 25.53, 27.46, 27.99, 29.65, 31.94, 36.31, 36.80, 37.83,
8.23, 38.57, 40.11, 44.57, 46.84, 47.22, 48.27, 49.09, 49.48, 50.53, 51.35, 55.90, 56.48, 72.13,
+
8.68, 83.52, 85.78, 121.74, 148.18, 107.71, 172.90. HRMS m/z calcd. for C H O [M + H]
3
6
52
3
33.4048, found 533.4042.
.1.4. General experimental procedure for preparation of 1,2,3-triazoles of betulinic ester (3-34)
The compounds (3-34) were prepared by NaN (1.2 equiv.), CuSO (0.1 equiv), and boronic
3
4
acids (1.2 equiv) in methanol (10 mL) were allowed to react for 1-4 h, followed by addition of
water (10 mL), sodium ascorbate (0.5 equiv), and propargylated betulinic ester (1.0 equiv)
(Scheme-1). The contents were stirred vigorously at room temperature for 6-8 h (as monitored by
TLC analysis). After completion of the reaction, the contents diluted with water and extracted with
ethyl acetate three times. The combined ethyl acetate extract was washed with brine, dried over
anhydrous Na SO and evaporated under reduced pressure on a rota vapour. The crude product
2
4
obtained thus subjected was put to column chromatography (silica gel) with appropriate EtOAc :
Hexane mixture as eluent to afford the desired pure products in good yields. All the synthesized
1
13
compounds were characterized by H NMR, C NMR, IR and HRMS spectroscopic analysis.
1
1

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4
.1.4.1. Synthesis of 28{1N (4-methoxy phenyl)-1H-1,2,3-triazol- 4-yl} methyloxy betulinic ester
(
3). The title compound prepared by the reaction of propargylated betulinic ester (100 mg, 0.20
mmol) and p-methoxyphenyl boronic acid (36.85 mg, 0.24 mmol) as per the method described in
o
1
section 4.1.4. to give yellow solid (123 mg, 95 % yield) mp 152-153 C. H NMR (500 MHz,
CDCl ): δ 0.63 (1H, m, H-5), 0.64 (3H, s, H-25), 0.72 (3H, s, H-24), 0.73 (3H, s, H-26), 0.87 and
3
1
1
.42 (1H each, m, H-1), 0.93 and 0.94 (3H each, s, H-23 and H-27), 1.13 and 1.85 (1H each, m, H-
1), 1.21 and 1.59 (1H each, m, H-12), 1.16 and 1.42 (1H each, m, H-15), 1.22 and 1.56 (1H each,
m, H-6), 1.26 and 1.42 (1H each, m, H-7), 1.28 and 1.67 (1H each, m, H-21), 1.17 (1H, m, H-9),
.47 and 1.86 (1H each, m, H-22), 1.46 and 2.15 (1H each, m, H-16), 1.49 (1H, m, H-18), 1.54 (2H,
1
m, H-2), 1.67 (3H, s, H-30), 2.28 (1H, m, H-13), 2.96 (1H, m, H-19), 3.16 (1H, m, H-3), 3.87 (3H,
s, -OCH ), 4.60 and 4.73 (1H each, s, H-29), 5.25 and 5.34 (1H each, d, J = 12.56 Hz, -OCH -),
3
2
1
3
7
.02 (2H, d, J = 9.0 Hz, Ar-H), 7.63 (2H, d, J = 9.0 Hz, Ar-H), 7.99 (1H, s, NCH). C NMR (100
MHz, CDCl ): δ 14.69, 15.34, 15.72, 16.04, 18.23, 19.39, 20.88, 22.70, 25.53, 27.40, 27.97, 30.64,
3
3
5
1.94, 34.22, 36.89, 37.17, 38.40, 38.72, 38.85, 40.67, 42.36, 47.02, 49.55, 50.52, 55.33, 55.66,
6.59, 57.12, 78.97, 109.67, 114.79 x 2, 122.25 x 2, 122.54, 139.28, 143.73, 150.43, 159.1, 176.21.
IR γmax (neat): 3433, 2924, 2853, 1724, 1641, 1612, 1519, 1463, 1377, 1302, 1255, 1151, 1126,
-
1
+
1
6
4
041, 983, 909, 884, 832 cm . HRMS m/z calcd. for C H N O [M + H] 644.4415, found
40 58 3 4
44.4421.
.1.4.2. Synthesis of 28{1N (2-chloropyridine)-1H-1,2,3-triazol- 4-yl} methyloxy betulinic ester
(4). The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40
mmol) and 6-chloro pyridine-3-yl-3-boronic acid (76.33 mg, 0.48 mmol) as per the method
o
1
described in section 4.1.4 to give brown solid (244 mg, 94 % yield) mp 214-216 C. H NMR (500
MHz, CDCl ): 0.63 (3H, s, H-25), 0.64 (1H, m, H-5), 0.72 (3H, s, H-24), 0.73 (3H, s, H-26), 0.93
3
δ
1
2

ACCEPTED MANUSCRIPT
and 0.95 (3H each, s, H-23 and H-27), 0.87 and 1.42 (1H each, m, H-1), 1.13 and 1.85 (1H each, m,
H-11), 1.21 and 1.60 (1H each, m, H-12), 1.16 and 1.42 (1H each, m, H-15), 1.22 and 1.56 (1H
each, m, H-6), 1.26 and 1.42 (1H each, m, H-7), 1.28 and 1.67 (1H each, m, H-21), 1.17 (1H, m, H-
9
), 1.47 and 1.86 (1H each, m, H-22), 1.46 and 2.05 (1H each, m, H-16), 1.49 (1H, m, H-18), 1.54
2H, m, H-2), 1.67 (3H, s, H-30), 2.25 (1H, m, H-13), 3.0 (1H, m, H-19), 3.16 (1H, m, H-3), 4.60
and 4.73 (1H each, s, H-29), 5.27 and 5.32 (1H each, d, J = 12.56 Hz, -OCH -), 7.54 (1H, d, J =
(
2
1
3
8
.55 Hz, Ar-H), 8.11 and 8.13 (1H each, d, J = 8.65 Hz, Ar-H), 8.78 (1H, s, -NCH). C NMR (125
MHz, CDCl ): δ 14.66. 15.35, 15.82, 16.09, 18.22, 19.35, 20.81, 25.45, 27.35, 27.95, 29.59, 30.5,
1.95, 34.21, 36.90, 37.14, 38.30, 38.66, 38.83, 40.65, 42.34, 46.93, 49.41, 50.45, 55.26, 56.57,
6.80, 78.92, 109.72, 123.36, 125.86, 131.93, 135.88, 143.62, 144.85, 150.32, 150.34, 176.06. IR γ
3
3
5
max (neat): 3435, 2942, 2868, 1723, 1641, 1571, 1481, 1452, 1424, 1376, 1231, 1195, 1150, 1128,
-
1
+
1
6
4
097, 1042, 981, 884, 756 cm . HRMS m/z calcd. for C H ClN O [M + H] 649.3888, found
38 54 4 3
49.3878.
.1.4.3. Synthesis of 28{1N(4-cyanophenyl)-1H-1,2,3-triazol-4-yl} methyloxy betulinic ester (5).
The title compound prepared by the reaction of propargylated betulinic ester (196 mg, 0.39 mmol)
and 4-cyanophenylboronic acid (69.80 mg, 0.47 mmol) as per the method described in section 4.1.4
o
1
1
to give yellow solid (245 mg, 97 % yield) mp 176-178 C. H NMR (500 MHz, CDCl ): H NMR
3
(
500 MHz, CDCl3):
.87 and 1.48 (1H each, m, H-1), 0.92 and 0.94 (3H each, s, H-23 and H-27), 1.13 and 1.86 (1H
each, m, H-11), 1.21 and 1.59 (1H each, m, H-12), 1.16 and 1.43 (1H each, m, H-15), 1.23 and 1.57
1H each, m, H-6), 1.26 and 1.42 (1H each, m, H-7), 1.28 and 1.66 (1H each, m, H-21), 1.17 (1H,
m, H-9), 1.43 and 1.86 (1H each, m, H-22), 1.46 and 2.05 (1H each, m, H-16), 1.49 (1H, m, H-18),
.57 (2H, m, H-2), 1.67 (3H, s, H-30), 2.26 (1H, m, H-13), 3.00 (1H, m, H-19), 3.15 (1H, m, H-3),
δ 0.61 (3H, s, H-25), 0.62 (1H, m, H-5), 0.70 (3H, s, H-24), 0.73 (3H, s, H-26),
0
(
1
1
3

ACCEPTED MANUSCRIPT
4
2
.60 and 4.73 (1H each, s, H-29), 5.26 and 5.35 (1H each, d, J = 12.65 Hz, -OCH -), 7.85 (2H, d, J
1
3
=
8.42 Hz, Ar-H), 7.92 (2H, d, J = 8.63 Hz, Ar-H), 8.16 (1H, s, -NCH). C NMR (100 MHz,
CDCl ): δ 14.69, 15.36, 15.64, 16.01, 18.21, 19.34, 20.85, 25.48, 27.37, 27.96, 29.61, 30.59, 31.96,
3
3
7
1
1
4
4.20, 36.84, 37.14, 38.44, 38.69, 38.83, 40.66, 42.35, 47.01, 49.51, 50.45, 55.29, 56.59, 56.81,
8.89, 109.76, 112.63, 117.60, 120.60 x 2CH, 122.17, 133.92 x 2CH, 139.68, 144.68, 150.27,
76.21. IR γmax (neat): 3434, 2943, 2869, 2231, 1722, 1641, 1608, 1518, 1451, 1376, 1174, 1125,
-
1
+
044, 983, 883, 756 cm . HRMS m/z calcd. for C40
H
55
N
4
O
3
[M + H] 639.4251, found 639.4268.
The
.1.4.4. Synthesis of 28{1N(1-phenyl)-1H-1,2,3-triazol-4-yl} methyloxy betulinic ester (6)
.
title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol) and
phenylboronic acid (59.14 mg, 0.48 mmol) as per the method described in section 4.1.4 to give
o
1
yellow brown solid (235 mg, 96 % yield) mp 109-111 C. H NMR (500 MHz, CDCl ):
δ
0.63 (1H,
m, H-5), 0.64 (3H, s, H-25), 0.70 (3H, s, H-24), 0.72 (3H, s, H-26), 0.86 and 1.42 (1H each, m, H-
), 0.92 and 0.93 (3H each, s, H-23 and H-27), 1.13 and 1.87 (1H each, m, H-11), 1.20 and 1.59
1H each, m, H-12), 1.16 and 1.42 (1H each, m, H-15), 1.24 and 1.57 (1H each, m, H-6), 1.26 and
.42 (1H each, m, H-7), 1.28 and 1.66 (1H each, m, H-21), 1.16 (1H, m, H-9), 1.43 and 1.86 (1H
each, m, H-22), 1.45 and 2.11 (1H each, m, H-16), 1.49 (1H, m, H-18), 1.57 (2H, m, H-2), 1.67
3H, s, H-30), 2.27 (1H, m, H-13), 3.02 (1H, m, H-19), 3.16 (1H, m, H-3), 4.60 and 4.74 (1H each,
s, H-29), 5.26 and 5.35 (1H each, d, J = 12.54 Hz, -OCH -), 7.46 (1H, m, Ar-H), 7.54 (2H, dd, J =
3
1
(
1
(
2
1
3
8
.02, 8.44 Hz, Ar-H), 7.73 (2H, d, J = 8.44 Hz, Ar-H), 8.08 (1H, s, -NCH). C NMR (100 MHz,
CDCl ): δ 14.69, 15.34, 15.70, 16.00, 18.21, 19.39, 20.87, 25.53, 27.40, 27.97, 29.62, 30.63, 32.02,
3
3
7
4.22, 36.88, 37.16, 38.41, 38.72, 38.84, 40.67, 42.36, 47.02, 49.55, 50.51, 55.32, 56.59, 57.07,
8.95, 109.68, 120.59, 120.60, 122.40, 128.91 x 2CH, 129.76 x 2CH, 136.97, 150.42, 176.20. IR
1
4

ACCEPTED MANUSCRIPT
γ
7
4
max (neat): 3434, 2943, 2869, 1722, 1641, 1599, 1503, 1453, 1234, 1218, 1151, 1044, 983, 885,
-1
+
58 cm . HRMS m/z calcd. for C H N O [M + H] 614.4330, found 614.4316.
3
9
56
3
3
.1.4.5. Synthesis of 28{1N(3,4-methylenedioxyphenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic
ester (7).The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40
mmol) and 3,4-methylenedioxy phenylboronic acid (80 mg, 0.48 mmol) as per the method
o
1
described in section 4.1.4 to give yellow solid (250 mg, 95 % yield) mp 222-244 C. H NMR (500
MHz, CDCl ): 0.64 (1H, m, H-5), 0.65 (3H, s, H-25), 0.73 (6H, s, H-24 and H-26), 0.87 and 1.45
1H each, m, H-1), 0.93 (6H, s, H-23 and H-27), 1.15 and 1.87 (1H each, m, H-11), 1.21 and 1.58
1H each, m, H-12), 1.15 and 1.45 (1H each, m, H-15), 1.25 and 1.58 (1H each, m, H-6), 1.26 and
.42 (1H each, m, H-7), 1.29 and 1.67 (1H each, m, H-21), 1.18 (1H, m, H-9),1.43 and 1.86 (1H
each, m, H-22), 1.47 and 2.11 (1H each, m, H-16), 1.49 (1H, m, H-18), 1.57 (2H, m, H-2), 1.67
3H, s, H-30), 2.28 (1H, m, H-13), 3.02 (1H, m, H-19), 3.16 (1H, m, H-3), 4.60 and 4.73 (1H each,
s, H-29), 5.29 and 5.34 (1H each, d, J = 12.56 Hz, -OCH -), 6.10 (2H, s, -OCH O-), 6.91 (1H, dd, J
3
δ
(
(
1
(
2
2
=
7.75, 1.12 Hz, Ar-H), 7.12 (1H, dd, J = 8.11, 1.79 Hz, Ar-H), 7.24 (1H, dd, J = 1.29 and 1.12 Hz,
1
3
Ar-H), 7.96 (1H, s, -NCH). C NMR (125 MHz, CDCl
3
): δ 14.28, 14.97, 15.33, 15.63, 17.82,
1
4
1
1
8.99, 20.45, 25.09, 26.98, 27.58, 29.21, 30.20, 31.59, 33.79, 36.47, 36.75, 37.97, 38.30, 38.45,
0.24, 41.93, 46.60, 49.11, 50.07, 54.90, 56.16, 56.66, 78.51, 101.77, 102.45, 108.08, 109.30,
13.86, 122.35, 131.01, 143.32, 147.73, 148.26, 150.02, 175.79. IR γmax (neat): 3434, 2942, 2869,
-1
721, 1640, 1507, 1466, 1376, 1246, 1127, 1039, 936, 885, 756 cm . HRMS m/z calcd. for
+
C H N O [M + H] 658.4209, found 658.4214.
40
56
3
5
4
.1.4.6. Synthesis of 28{1N(4-benzamido phenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester
8).The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40
mmol) and 4-benzamidophenyl boronic acid (116.93 mg, 0.48 mmol) as per the method described
(
1
5

ACCEPTED MANUSCRIPT
o
1
in section 4.1.4 to give yellow solid (271 mg, 92 % yield) mp 175-177 C. H NMR (400 MHz,
CDCl3): 0.62 (1H, m, H-5), 0.65 (3H, s, H-25), 0.72 (3H, s, H-24), 0.73 (3H, s, H-26), 0.88 and
.45 (1H each, m, H-1), 0.93 and 0.94 (3H each, s, H-23 and H-27), 1.15 and 1.87 (1H each, m, H-
1), 1.21 and 1.59 (1H each, m, H-12), 1.15 and 1.45 (1H each, m, H-15), 1.25 and 1.59 (1H each,
δ
1
1
m, H-6), 1.27 and 1.42 (1H each, m, H-7), 1.28 and 1.68 (1H each, m, H-21), 1.18 (1H, m, H-9),
.43 and 1.87 (1H each, m, H-22), 1.46 and 2.10 (1H each, m, H-16), 1.51 (1H, m, H-18), 1.56 (2H,
m, H-2), 1.68 (3H, s, H-30), 2.27 (1H, m, H-13), 3.02 (1H, m, H-19), 3.15 (1H, m, H-3), 4.60 and
1
4
7
8
2
4
1
1
1
7
4
.73 (1H each, s, H-29), 5.26 and 5.34 (1H each, d, J = 12.40 Hz, -OCH
2
-), 7.54 (3H, m, Ar-H),
.73 (2H, d, J = 8.8 Hz, Ar-H), 7.85 (2H, d, J = 8.80 Hz, Ar-H), 7.90 (2H, d, J = 7.20 Hz, Ar-H),
1
3
.05 (1H, s, -NCH). C NMR (100 MHz, CDCl ): δ 14.70, 15.36, 15.70, 16.08, 18.25, 19.40,
3
0.88, 25.54, 27.39, 27.97, 29.40, 30.64, 32.03, 34.24, 36.91, 37.17, 38.41, 38.72, 38.85, 40.68,
2.38, 47.01, 49.55, 50.52, 55.33, 56.60, 57.06, 78.99, 109.69, 120.98 x 2CH, 121.39 x 2CH,
22.29, 127.08 x 2CH, 128.90 x 2CH, 132.25, 133.10, 134.48, 138.63, 143.94, 150.41, 165.80,
76.20. IR γmax (neat): 3350, 3068, 2929, 2868, 1724, 1655, 1640, 1605, 1579, 1520, 1448, 1409,
-1
+
61 4 4
317, 1263, 1151, 1073, 1044, 1029, 838, 756 cm . HRMS m/z calcd. for C46H N O [M + H]
33.4673, found 733.4687.
.1.4.7. Synthesis of 28{1N(3-methoxy phenyl)-1H-1,2,3-triazol-4-yl} methyloxy betulinic ester (9).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 3-methoxyphenyl boronic acid (73.7 mg, 0.48 mmol) as per the method described in section
o
1
4
0
.1.4 to give yellow solid (248 mg, 96 % yield) mp 166-168 C. H NMR (500 MHz, CDCl ): δ
3
.62 (1H, m, H-5), 0.64 (3H, s, H-25), 0.71 (3H, s, H-24), 0.72 (3H, s, H-26), 0.86 and 1.45 (1H
each, m, H-1), 0.92 and 0.94 (3H each, s, H-23 and H-27), 1.15 and 1.87 (1H each, m, H- 11), 1.22
and 1.59 (1H each, m, H-12), 1.15 and 1.45 (1H each, m, H- 15), 1.25 and 1.59 (1H each, m, H-6),
1
6

ACCEPTED MANUSCRIPT
1
1
1
.27 and 1.42 (1H each, m, H-7), 1.28 and 1.66 (1H each, m, H-21), 1.20 (1H, m, H-9), 1.43 and
.87 (1H each, m, H-22), 1.46 and 2.10 (1H each, m, H-16), 1.51 (1H, m, H-18), 1.56 (2H, m, H-2),
.67 (3H, s, H-30), 2.27 (1H, m, H-13), 3.02 (1H, m, H-19), 3.15 (1H, m, H-3), 3.88 (3H, s, -
OCH ), 4.60 and 4.74 (1H each, s, H-29), 5.24 and 5.35 (1H each, d, J = 12.58 Hz, -OCH -), 6.98
3
2
(1H, d, J = 8.24 Hz, Ar-H), 7.26 (1H, d, J = 7.95 Hz, Ar-H), 7.33 (1H, s, Ar-H) 7.41 (1H, dd, J =
1
3
8
1
3
1
3
.18 and 8.24 Hz, Ar-H), 8.08 (1H, s, -NCH). C NMR (125 MHz, CDCl ): δ 14.66, 15.35, 15.67,
3
5.99, 18.19, 19.38, 20.83, 25.47, 27.35, 27.96, 29.59, 30.58, 31.97, 34.15, 36.85, 37.11, 38.34,
8.67, 30.82, 40.60, 42.30, 46.98, 49.48, 50.44, 55.26, 55.63, 56.54, 57.01, 78.89, 106.43, 109.70,
12.42, 114.71, 122.58, 130.52, 137.90, 143.84, 150.40, 160.59, 176.20. IR γ max (neat): 3428,
-1
073, 2927, 2855, 1724, 1642, 1611, 1596, 1294, 1258, 1234, 1126, 1044, 882, 773 cm . HRMS
+
m/z calcd. For C H N O [M + H] 644.4409, found 644.4421.
4
0
58
3
4
4
.1.4.8. Synthesis of 28{1N(2-iodo phenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester (10).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 2-iodophenyl boronic acid (120.22 mg, 0.48 mmol) as per the method described in section
o
1
4
0
.1.4. to give yellow solid (273 mg, 92 % yield) mp 213-215 C. H NMR (400 MHz, CDCl
3
): δ
.65 (1H, m, H-5), 0.74 (3H, s, H-25), 0.78 (3H, s, H-24), 0.79 (3H, s, H-26), 0.85 and 1.43 (1H
each, m, H-1), 0.94 and 0.95 (3H each, s, H-23 and H-27), 1.13 and 1.87 (1H each, m, H-11), 1.21
and 1.59 (1H each, m, H-12), 1.15 and 1.43(1H each, m, H-15), 1.25 and 1.59 (1H each, m, H-6),
1
1
1
.27 and 1.41 (1H each, m, H-7), 1.28 and 1.67 (1H each, m, H-21), 1.20 (1H, m, H-9), 1.43 and
.87 (1H each, m, H-22), 1.48 and 2.15 (1H each, m, H-16), 1.50 (1H, m, H-18), 1.52 (2H, m, H-2),
.69 (3H, s, H-30), 2.27 (1H, m, H-13), 3.05 (1H, m, H-19), 3.15 (1H, m, H-3), 4.59 and 4.72 (1H
2
each, s, H-29), 5.30 and 5.34 (1H each, d, J = 12.56 Hz, -OCH -), 7.25 (1H, m, Ar-H), 7.43 (1H, d,
1
3
J = 8.08 Hz, Ar-H), 7.51 (1H, m, Ar-H), 7.95 (1H, s, -NCH), 8.01 (1H, d, J = 8.80 Hz, Ar-H). C
1
7

ACCEPTED MANUSCRIPT
NMR (100 MHz, CDCl
3
): δ 14.70, 15.37, 16.05, 16.41, 18.27, 19.36, 20.89, 25.53, 27.42, 27.99,
2
5
1
1
7
4
9.69, 30.67, 32.02, 34.31, 36.95, 37.21, 38.34, 38.73, 38.87, 40.76, 42.41, 46.99, 49.49, 50.55,
5.33, 56.61, 57.03, 78.97, 109.71, 125.97, 127.67, 127.84, 129.23, 131.56, 139.95, 140.35, 143.14,
50.43, 176.07. IR γmax (neat): 3434, 3063, 2940, 2869, 1724, 1604, 1507, 1454, 1376, 1253, 1124,
-1
+
053, 1045, 1023, 884, 753 cm . HRMS m/z calcd. for C H IN O [M + H] 740.3288, found
3
9
55
3
3
40.3282.
.1.4.9. Synthesis of 28{1N(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester (11).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 2-methoxyphenyl boronic acid (73.71 mg, 0.48 mmol) as per the method described in section
o
1
4
.1.4 to give yellow solid (247 mg, 96% yield) mp 163-165 C. H NMR (400 MHz, CDCl ): δ 0.64
3
(
1H, m, H-5), 0.68 (3H, s, H-25), 0.73 (6H, s, H-24 and H-26), 0.86 and 1.44 (1H each, m, H-1),
.93 and 0.94 (3H each, s, H-23 and H-27), 1.11 and 1.87 (1H each, m, H-11), 1.21 and 1.58 (1H
each, m, H-12), 1.15 and 1.44 (1H each, m, H-15), 1.25 and 1.58 (1H each, m, H-6), 1.27 and 1.40
1H each, m, H-7), 1.27 and 1.67 (1H each, m, H-21), 1.20 (1H, m, H-9), 1.44 and 1.87 (1H each,
m, H-22), 1.45 and 2.10 (1H each, m, H-16), 1.51 (1H, m, H-18), 1.55 (2H, m, H-2), 1.67 (3H, s, H-
0), 2.26 (1H, m, H-13), 3.01 (1H, m, H-19), 3.15 (1H, m, H-3), 3.88 (3H,s, -OCH ), 4.59 and 4.72
1H each, s, H-29), 5.29 and 5.32 (1H each, d, J = 12.56 Hz, -OCH -), 7.10 (2H, m, Ar-H), 7.41
0
(
3
3
(
(
(
2
1
3
1H, dd, J = 8.01, 8.08 Hz, Ar-H), 7.76 (1H d, J = 8.0 Hz, Ar-H), 8.19 (1H, s, -NCH). C NMR
125 MHz, CDCl ): δ 14.51, 15.25, 15.45, 15.91, 18.05, 19.18, 20.66, 25.31, 27.18, 27.82, 29.42,
3
3
5
1
0.36, 31.80, 34.04, 36.71, 36.95, 38.12, 38.53, 38.66, 40.43, 42.15, 46.82, 49.26, 50.29, 55.12,
5.78, 56.37, 56.96, 78.65, 109.52, 112.01, 120.97, 125.27, 125.91, 126.14, 130.03, 142.39, 150.30,
50.83, 175.93. IR γmax (neat): 3434, 3072, 2940, 2869, 1724, 1641, 1604, 1507, 1454, 1376, 1314,
1
8

ACCEPTED MANUSCRIPT
-
1
+
1
6
4
58 3 4
287, 1253, 1151, 1124, 1045, 1023, 884, 753 cm . HRMS m/z calcd. for C40H N O [M + H]
44.4409, found 644.4421.
.1.4.10. Synthesis of 28{1N(4-fluorophenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester (12).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 4-fluorophenyl boronic acid (67.43 mg, 0.48 mmol) as per the method described in section
o
1
4
.1.4. to give yellow crystalline solid (248 mg, 98 % yield) mp 205-207 C. H NMR (400 MHz,
CDCl ): δ 0.63 (1H, m, H-5), 0.64 (3H, s, H-25), 0.72 (3H, s, H-24), 0.73 (3H, s, H-26), 0.86 and
.44 (1H each, m, H-1), 0.92 and 0.94 (3H each, s, H-23 and H-27), 1.17 and 1.87 (1H each, m, H-
1), 1.21 and 1.59 (1H each, m, H-12), 1.16 and 1.44 (1H each, m, H-15), 1.25 and 1.56 (1H each,
3
1
1
m, H-6), 1.29 and 1.40 (1H each, m, H-7), 1.27 and 1.67 (1H each, m, H-21), 1.17 (1H, m, H-9),
.44 and 1.87 (1H each, m, H-22), 1.46 and 2.08 (1H each, m, H-16), 1.51 (1H, m, H-18), 1.56 (2H,
m, H-2), 1.67 (3H, s, H-30), 2.20 (1H, m, H-13), 2.98 (1H, m, H-19), 3.15 (1H, m, H-3), 4.60 and
1
4
8
.73 (1H each, s, H-29), 5.25 and 5.34 (1H each, d, J = 12.40 Hz, -OCH -), 7.24 (2H, dd, J = 8.08,
2
1
3
.01 Hz, Ar-H), 7.71 (2H, dd, J = 8.08, 8.01 Hz, Ar-H), 8.03 (1H s, -NCH). C NMR (100 MHz,
): δ 14.69, 15.35, 15.68, 16.01, 18.22, 19.37, 20.86, 25.52, 27.39, 27.97, 29.62, 30.62, 32.00,
4.22, 36.87, 37.16, 38.42, 38.72, 38.84, 40.67, 42.36, 47.02, 49.54, 50.49, 55.32, 56.78, 56.99,
8.93, 109.70, 116.62, 116.85, 122.56, 122.60, 133.21, 144.06, 150.37, 161.29, 163.77, 176.21. IR
CDCl
3
3
7
γmax (neat): 3434, 2943, 2869, 1721, 1641, 1518, 1452, 1376, 1294, 1235, 1188, 1153, 1127, 1044,
-1
+
9
93, 885, 756 cm . HRMS m/z calcd. For C H FN O [M + H] 632.4223, found 632.4221.
39 55 3 3
4
.1.4.11. Synthesis of 28{1N(2-vinylphenyl)-1H-1,2,3-triazol-4-yl methyloxy betulinic ester (13).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and trans-2-phenylvinyl boronic acid (71.78 mg, 0.48 mmol) as per the method described in section
o
1
4
3
.1.4. to give brown solid (243 mg, 95 % yield) mp 129-131 C. H NMR (400 MHz, CDCl ): δ
1
9

ACCEPTED MANUSCRIPT
0
.63 (1H, m, H-5), 0.70 (3H, s, H-25), 0.72 (3H, s, H-24), 0.73 (3H, s, H-26), 0.87 and 1.44 (1H
each, m, H-1), 0.93 (6H, s, H-23 and H-27), 1.17 and 1.87 (1H each, m, H-11), 1.22 and 1.59 (1H
each, m, H-12), 1.16 and 1.44 (1H each, m, H-15), 1.25 and 1.56 (1H each, m, H-6), 1.27 and 1.39
(1H each, m, H-7), 1.27 and 1.67 (1H each, m, H-21), 1.22 (1H, m, H-9), 1.44 and 1.87 (1H each,
m, H-22), 1.46 and 2.1 (1H each, m, H-16), 1.51 (1H, m, H-18), 1.56 (2H, m, H-2), 1.68 (3H, s, H-
3
5
0), 2.26 (1H, m, H-13), 3.02 (1H, m, H-19), 3.15 (1H, m, H-3), 4.60 and 4.74 (1H each, s, H-29),
.22 and 5.34 (1H each, d, J = 12.40 Hz, -OCH -), 7.14 (1H, d, J = 14.80 Hz, Ar-H), 7.48 (4H, m,
2
1
3
Ar-H), 7.73 (1H, d, J = 14.80 Hz, Ar-CH=CH), 7.97 (1H, s, -NCH). C NMR (100 MHz, CDCl
3
):
δ 14.67, 15.32, 15.77, 16.07, 18.23, 19.39, 20.88, 25.53, 27.37, 27.97, 29.60, 30.60, 32.00, 34.23,
3
1
1
9
4
6.85, 37.16, 38.37, 38.71, 38.82, 40.71, 42.36, 46.97, 49.52, 50.50, 55.32, 56.57, 56.95, 78.91,
09.69, 121.53, 122.24, 122.84, 126.75 x 2CH, 129.03, 130.01 x 2CH, 133.44, 143.57, 150.36,
76.18. IR γmax (neat): 3429, 2943, 2869, 1722, 1658, 1642, 1453, 1376, 1350, 1310, 1233, 1128,
-1
+
83, 884, 753 cm . HRMS m/z calcd. for C H N O [M + H] 640.4465, found 640.4472.
4
1
58
3
3
.1.4.12. Synthesis of 28{1N(4-bromophenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester (14).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 4-bromophenyl boronic acid (97.42 mg, 0.48 mmol) as per the method described in section
o
1
4
0
.1.4. to give yellow solid (262 mg, 94 % yield) mp 166-168 C. H NMR (400 MHz, CDCl ): δ
3
.61 (1H, m, H-5), 0.65 (3H, s, H-25), 0.72 (3H, s, H-24), 0.73 (3H, s, H-26), 0.87 and 1.44 (1H
each, m, H-1), 0.93 and 0.95 (3H each, s, H-23 and H-27), 1.17 and 1.87 (1H each, m, H-11), 1.22
and 1.59 (1H each, m, H-12), 1.16 and 1.48 (1H each, m, H-15), 1.25 and 1.55 (1H each, m, H-6),
1
1
1
.27 and 1.39 (1H each, m, H-7), 1.27 and 1.67 (1H each, m, H-21), 1.22 (1H, m, H-9), 1.40 and
.87 (1H each, m, H-22), 1.46 and 2.08 (1H each, m, H-16), 1.52 (1H, m, H-18), 1.59 (2H, m, H-2),
.61 (3H, s, H-30), 2.21 (1H, m, H-13), 2.98 (1H, m, H-19), 3.15 (1H, m, H-3), 4.59 and 4.73 (1H
2
0

ACCEPTED MANUSCRIPT
2
each, s, H-29), 5.25 and 5.34 (1H each, d, J = 12.80 Hz, -OCH -), 7.61 (2H, d, J = 8.4 Hz, Ar-H),
1
3
7
1
3
2
2
.67 (1H, d, J = 8.4 Hz, Ar-H), 8.05 (1H, s, -NCH). C NMR (125 MHz, CDCl ): δ 14.63, 15.32,
3
5.60, 15.97, 18.16, 19.32, 20.79, 25.43, 27.32, 27.92, 29.55, 30.54, 31.93, 34.13, 36.81, 37.08,
8.33, 38.64, 38.78, 40.58, 42.28, 46.94, 49.44, 50.40, 55.23, 56.51, 56.90, 78.88, 109.68, 121.89 x
CH, 122.27, 122.55, 132.88 x 2CH), 135.83, 144.15, 150.31, 176.18. IR γmax (neat): 3420, 2941,
-1
868, 1722, 1641, 1498, 1453, 1376, 1234, 1044, 1151, 1125, 983, 757 cm . HRMS m/z calcd. for
+
C
39
H
55BrN
3
O
3
[M + H] 694.3399, found 694.3409.
4
.1.4.13. Synthesis
betulinic ester (15). The title compound prepared by the reaction of propargylated betulinic ester
200mg, 0.40 mmol) and 2-bromo-4,5-difluorophenyl boronic acid (114.84 mg, 0.48 mmol) as per
of
28{1N(2-bromo-4,5-difluorophenyl)-1H-1,2,3-triazol-4-yl}methyloxy
(
o
1
the method described in section 4.1.4. to give brown solid (273 mg, 94 % yield) mp 125-127 C. H
NMR (400 MHz, CDCl ): δ 0.67 (1H, m, H-5), 0.73 (3H, s, H-25), 0.74 (3H, s, H-24), 0.76 (3H, s,
3
H-26), 0.87 and 1.44 (1H each, m, H-1), 0.94 and 0.95 (3H each, s, H-23 and H-27), 1.17 and 1.87
(
1H each, m, H-11), 1.23 and 1.60 (1H each, m, H-12), 1.16 and 1.49 (1H each, m, H-15), 1.26 and
.55 (1H each, m, H-6), 1.28 and 1.39 (1H each, m, H-7), 1.28 and 1.59 (1H each, m, H-21), 1.22
1H, m, H-9), 1.41 and 1.80 (1H each, m, H-22), 1.46 and 2.08 (1H each, m, H-16), 1.52 (1H, m, H-
1
(
1
3
8), 1.60 (2H, m, H-2), 1.67 (3H, s, H-30), 2.21 (1H, m, H-13), 3.01(1H, m, H-19), 3.18 (1H, m, H-
), 4.59 and 4.71 (1H each, s, H-29), 5.13 and 5.23 (1H each, d, J = 12.40 Hz, -OCH -), 7.47 (1H,
2
1
3
dd, J = 9.2, 9.4 Hz, Ar-H), 7.55 (1H, dd, J = 9.2, 9.4 Hz, Ar-H), 8.05 (1H s, -NCH). C NMR (100
MHz, CDCl ): δ 14.67, 15.31, 15.97, 16.07, 18.30, 19.33, 20.86, 25.50, 27.39, 27.96, 29.63, 30.59,
3
3
5
1
2.18, 34.28, 34.36, 36.90, 38.36, 38.72, 38.85, 40.74, 42.38, 46.88, 49.48, 50.50, 55.34, 56.61,
6.81, 78.95, 109.71, 112.72, 117.56, 122.64, 126.10, 132.64, 143.35, 148.20, 150.73, 152.09,
76.08. IR γmax (neat): 3435, 2942, 2869, 1723, 1641, 1606, 1508, 1464, 1376, 1317, 1283, 1234,
2
1

ACCEPTED MANUSCRIPT
-
1
+
1
7
4
2 3 3
219, 1152, 1042, 879, 757 cm . HRMS m/z calcd. for C39H53BrF N O [M + H] 730.3204, found
30.3221.
.1.4.14. Synthesis of 28{1N(2-methoxynaphthalene-6-yl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic
ester (16). The title compound prepared by the reaction of propargylated betulinic ester (200 mg,
.40 mmol) and 6-methoxy-2-naphthalene boronic acid (97.99 mg, 0.48 mmol) as per the method
0
o
1
described in 4.1.4. to give yellow solid (263 mg, 95 % yield) mp 140-142 C. H NMR (500 MHz,
CDCl ): δ 0.62 (1H, m, H-5), 0.63 (3H, s, H-25), 0.64 (3H, s, H-24), 0.67 (3H, s, H-26), 0.88 and
.42 (1H each, m, H-1), 0.92 (6H, s, H-23 and H-27), 1.16 and 1.87 (1H each, m, H-11), 1.23 and
.60 (1H each, m, H-12), 1.16 and 1.49 (1H each, m, H-15), 1.26 and 1.55 (1H each, m, H-6), 1.28
3
1
1
and 1.38 (1H each, m, H-7), 1.28 and 1.67 (1H each, m, H-21), 1.20 (1H, m, H-9), 1.41 and 1.80
(1H each, m, H-22), 1.46 and 1.99 (1H each, m, H-16), 1.52 (1H, m, H-18), 1.59 (2H, m, H-2), 1.68
(
3H, s, H-30), 2.29 (1H, m, H-13), 3.01(1H, m, H-19), 3.15 (1H, m, H-3), 3.95 (1H, s, -OCH ), 4.60
3
and 4.74 (1H each, s, H-29), 5.27 and 5.40 (1H each, d, J = 12.57 Hz, -OCH -), 7.21 (1H, s, Ar-H),
2
7
.26 (1H, m, Ar-H), 7.82 (2H, m, Ar-H), 7.89 (1H, d, J = 8.8 Hz, Ar-H), 8.08 (1H, s, Ar-H), 8.17
1
3
(
1H, s, -NCH). C NMR (100 MHz, CDCl
3
): δ 14.65, 15.27, 15.77, 15.96, 18.18, 19.39, 20.87,
2
4
1
3
5.52, 27.38, 27.95, 29.49, 30.64, 32.03, 34.19, 36.87, 37.14, 38.38, 38.69, 38.81, 40.66, 42.34,
7.01, 49.57, 50.48, 55.30, 55.40, 56.59, 57.11, 78.92, 105.89, 109.65, 114.05, 118.78, 119.49,
20.49, 122.65, 128.53, 129.64, 132.62, 134.37, 143.91, 150.40, 158.63, 176.21. IR γmax (neat):
-1
400, 2927, 2867, 1723, 1609, 1513, 1451, 1390, 1265, 1222, 1152, 1123, 1042, 885, 758 cm .
+
HRMS m/z calcd. for C H N O [M + H] 694.4566, found 694.4578.
4
4
60
3
4
4
.1.4.15. Synthesis of 28{1N(2-bromo-6-fluoro-3-methoxyphenyl)-1H-1,2,3-triazol- 4-yl} methyloxy
betulinic ester (17). The title compound prepared by the reaction of propargylated betulinic ester
200 mg, 0.40 mmol) and 2-bromo-6-fluoro-3-methoxyphenyl boronic acid (120.71 mg, 0.48
(
2
2

ACCEPTED MANUSCRIPT
mmol) as per the method described in section 4.1.4. to give yellow solid (275 mg, 93 % yield) mp
o
1
1
2
2
15-117 C. H NMR (500 MHz, CDCl ): δ 0.65 (1H, m, H-5), 0.74 (3H, s, H-25), 0.78(3H, s, H-
3
4), 0.80 (3H, s, H-26), 0.88 and 1.42 (1H each, m, H-1), 0.94 and 0.95 (3H each, s, H-23 and H-
7), 1.14 and 1.87 (1H each, m, H-11), 1.23 and 1.60 (1H each, m, H-12), 1.16 and 1.39 (1H each,
m, H-15), 1.26 and 1.56 (1H each, m, H-6), 1.27 and 1.38 (1H each, m, H-7), 1.28 and 1.66 (1H
each, m, H-21), 1.21 (1H, m, H-9), 1.39 and 1.80 (1H each, m, H-22), 1.46 and 2.04 (1H each, m,
H-16), 1.58 (1H, m, H-18), 1.59 (2H, m, H-2), 1.66 (3H, s, H-30), 2.28 (1H, m, H-13), 2.97 (1H, m,
H-19), 3.15 (1H, m, H-3), 3.95 (1H, s, -OCH
each, d, J = 12.57 Hz, -OCH -), 7.12 (1H, d, J = 8.6 Hz, Ar-H), 7.26 (1H, d, J = 8.7 Hz, Ar-H), 7.84
1H s, -NCH). C NMR (100 MHz, CDCl ): δ 16.44, 15.35, 15.90, 16.09, 18.26, 19.35, 20.86,
3
), 4.58 and 4.71 (1H each, s, H-29), 5.29 and 5.34 (1H
2
1
3
(
3
2
4
1
1
5.44, 27.41, 27.98, 29.62, 30.56, 31.91, 34.27, 36.96, 37.19, 38.26, 38.74, 38.85, 40.71, 42.42,
6.92, 49.41, 50.57, 55.36, 56.62, 56.90, 57.15, 78.98, 109.64, 112.25, 113.80, 115.23, 125.63,
26.95, 143.11, 150.43, 150.76, 153.40, 176.09. IR γmax (neat): 3435, 2928, 2868, 1725, 1641,
-1
504, 1463, 1438, 1376, 1258, 1103, 1042, 1003, 885, 757 cm . HRMS m/z calcd. for
+
C
40
H
56BrFN
3
O
4
[M + H] 742.3420, found 742.3421.
4
.1.4.16. Synthesis of 28{1N(3-chloro-4-propoxyphenyl)-1H-1,2,3-triazol- 4-yl}methyloxy betulinic
ester (18). The title compound prepared by the reaction of propargylated betulinic ester (200 mg,
.40 mmol) and 3-chloro-4-propoxyphenyl boronic acid (104.02 mg, 0.48 mmol) as per the method
0
o
1
described in section 4.1.4. to give yellow solid (265 mg, 94 % yield) mp 190-192 C. H NMR (500
MHz, CDCl ): δ 0.62 (1H, m, H-5), 0.63 (3H, s, H-25), 0.72 (3H, s, H-24), 0.76 (3H, s, H-26), 0.86
3
and 1.42 (1H each, m, H-1), 0.87 (3H, t, J = 6.92 Hz, -CH CH ), 0.92 and 0. 93 (3H each, s, H-23
2
3
and H-27), 1.11 and 1.87 (1H each, m, H-11), 1.22 and 1.60 (1H each, m, H-12), 1.17 and 1.38 (1H
each, m, H-15), 1.25 and 1.55 (1H each, m, H-6), 1.27 and 1.38 (1H each, m, H-7), 1.28 and 1.66
2
3

ACCEPTED MANUSCRIPT
2 3
(1H each, m, H-21), 1.21 (1H, m, H-9), 1.39 and 1.80 (1H each, m, H-22), 1.41 (2H, m, -CH CH ),
1
.46 and 2.17 (1H each, m, H-16), 1.58 (1H, m, H-18), 1.59 (2H, m, H-2), 1.66 (3H, s, H-30), 2.28
(
1H, m, H-13), 3.03 (1H, m, H-19), 3.16 (1H, m, H-3), 4.05 (2H, t, J = 6.52 Hz, -OCH CH ), 4.60
2 2
and 4.74 (1H each, s, H-29), 5.25 and 5.36 (1H each, d, J = 12.57 Hz,-OCH -), 7.03 (1H, d, J = 8.88
2
1
3
Hz, Ar-H), 7.57 (1H, d, J = 8.81 Hz, Ar-H), 7.73 (1H, s, Ar-H), 8.00 (1H s, -NCH). C NMR (100
MHz, CDCl ): δ 10.42, 14.66, 15.31, 15.77, 16.03, 18.21, 19.38, 20.87, 22.41, 25.51, 27.39, 27.95,
3
2
5
1
9.49, 30.62, 34.21, 36.85, 37.16, 38.39, 38.71, 38.84, 40.68, 42.33, 47.00, 49.55, 50.49, 53.38,
5.33, 56.57, 57.00, 71.10, 78.94, 109.66, 113.47, 120.08, 122.58, 122.96, 124.06, 130.22, 143.95,
-1
50.38, 155.11, 176.19. IR γmax (neat): 2925, 2853, 1724, 1641, 1508, 1463, 1126, 1043, 882 cm .
+
HRMS m/z calcd. for C H ClN O [M + H] 706.4363, found 706.4345.
4
2
61
3
4
4
.1.4.17. Synthesis of 28{1N(1-dibenzofuran)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester (19).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 4-dibenzofuran boronic acid (102.84 mg, 0.48 mmol) as per the method described in section
o
1
4
.1.4. to give yellow brown solid (260 mg, 92 % yield) mp 117-119 C. H NMR (500 MHz,
CDCl ): δ 0.55 (3H, s, H-25), 0.57 (1H, m, H-5), 0.60 (3H, s, H-24), 0.66 (3H, s, H-26), 0.87 and
.42 (1H each, m, H-1), 0.90 (6H, s, H-23 and H-27), 1.11 and 1.87 (1H each, m, H-11), 1.21 and
.60 (1H each, m, H-12), 1.19 and 1.38 (1H each, m, H-15), 1.25 and 1.55 (1H each, m, H-6), 1.27
3
1
1
and 1.38 (1H each, m, H-7), 1.29 and 1.66 (1H each, m, H-21), 1.21 (1H, m, H-9), 1.39 and 1.88
(
1H each, m, H-22), 1.45 and 2.04 (1H each, m, H-16), 1.58 (1H, m, H-18), 1.59 (2H, m, H-2), 1.68
3H, s, H-30), 2.31 (1H, m, H-13), 3.12 (1H, m, H-19), 3.13 (1H, m, H-3), 4.60 and 4.75 (1H each,
(
s, H-29), 5.37 and 5.46 (1H each, d, J = 12.58 Hz,-OCH -), 7.47 (1H, m, Ar-H), 7.53 (2H, m, Ar-H)
2
7
8
.66 (1H, d, J = 8.25 Hz, Ar-H), 8.01 (2H, d, J = 7.67 Hz, Ar-H), 8.16 (1H, d, J = 7.67 Hz, Ar-H),
1
3
3
.74 (1H, s, -NCH). C NMR (100 MHz, CDCl ): δ 14.66, 15.28, 15.57, 15.82, 18.14, 19.38,
2
4

ACCEPTED MANUSCRIPT
2
4
1
0.83, 25.53, 27.36, 27.94, 29.68, 30.65, 32.03, 34.21, 36.89, 37.08, 38.40, 38.67, 38.79, 40.60,
2.35, 47.05, 49.58, 50.48, 55.28, 56.62, 57.07, 78.90, 109.69, 112.08, 119.74, 120.67, 121.01,
22.24, 123.46, 123.68, 123.77, 124.95, 126.77, 128.23, 143.73, 145.54, 150.45, 156.21, 176.13. IR
-
γmax (neat): 3436, 2927, 2868, 1724, 1640, 1509, 1457, 1376, 1193, 1151, 1097, 1043, 884, 751 cm
1
+
.
HRMS m/z calcd. for C H N O [M + H] 704.4416, found 704.4421.
45 58 3 4
4
.1.4.18. Synthesis of 28{1N(3-nitrophenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester (20).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 3-nitrophenyl boronic acid (80.97 mg, 0.48 mmol) as per the method described in section 4.1.4.
o
1
to give yellow brown solid (243 mg, 92 % yield) mp 113-115 C. H NMR (500 MHz, CDCl
3
): δ
0
.62 (3H, s, H-25), 0.63 (1H, s, H-5), 0.68 (3H, s, H-24), 0.71 (3H, s, H-26), 0.87 and 1.42 (1H
each, m, H-1), 0.93 (6H, s, H-23 and H-27), 1.11 and 1.87 (1H each, m, H-11), 1.21 and 1.61 (1H
each, m, H-12), 1.17 and 1.38 (1H each, m, H-15), 1.25 and 1.57 (1H each, m, H-6), 1.27 and 1.39
(1H each, m, H-7), 1.28 and 1.66 (1H each, m, H-21), 1.21 (1H, m, H-9), 1.39 and 1.88 (1H each,
m, H-22), 1.44 and 2.07 (1H each, m, H-16), 1.58 (1H, m, H-18), 1.59 (2H, m, H-2), 1.68 (3H, s, H-
3
5
0), 2.29 (1H, m, H-13), 3.04 (1H, m, H-19), 3.16 (1H, m, H-3), 4.60 and 4.74 (1H each, s, H-29),
.27 and 5.38 (1H each, d, J = 12.65 Hz, -OCH -), 7.77 (1H, dd, J = 8.18, 8.09 Hz, Ar-H), 8.18
2
(1H, d, J = 8.07 Hz, Ar-H), 8.20 (1H, s, Ar-H), 8.33 (1H, d, J = 8.4 Hz, Ar-H), 8.60 (1H, s, -NCH).
1
3
C NMR (125 MHz, CDCl ): δ 14.64, 15.28, 15.62, 15.91, 18.14, 19.33, 20.79, 25.43, 27.31,
3
2
5
1
1
6
7.91, 29.67, 30.54, 31.92, 34.13, 36.82, 37.08, 38.38, 38.62, 38.78, 40.62, 42.29, 46.96, 49.46,
0.39, 55.21, 56.55, 56.80, 78.89, 109.73, 115.33, 122.46, 123.34, 125.96, 130.98, 137.56, 144.66,
48.90, 150.27, 176.22. IR γmax (neat): 3074, 2926, 2854, 1724, 1641, 1537, 1455, 1445, 1350,
-1
+
55 4 5
234, 1151, 1127, 1073, 1043, 885, 737 cm . HRMS m/z calcd. for C39H N O [M + H]
59.4161, found 659.4166.
2
5

ACCEPTED MANUSCRIPT
4
.1.4.19. Synthesis of 28{1N(4-tertiarybutyl phenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester
(
21). The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40
mmol) and 4-tertiary-butylphenyl boronic acid (86.36 mg, 0.48 mmol) as per the method described
o
1
in section 4.1.4. to give yellow brown solid (253 mg, 94% yield) mp 221-223 C. H NMR (500
MHz, CDCl ): δ 0.58 (3H, s, H-25), 0.61 (1H, m, H-5), 0.67 (3H, s, H-24), 0.71 (3H, s, H-26), 0.88
3
and 1.42 (1H each, m, H-1), 0.92 and 0.93 (3H each, s, H-23 and H-27), 1.15 and 1.87 (1H each, m,
H-11), 1.22 and 1.60 (1H each, m, H-12), 1.17 and 1.35 (1H each, m, H-15), 1.25 and 1.58 (1H
each, m, H-6), 1.27 and 1.39 (1H each, m, H-7), 1.29 and 1.66 (1H each, m, H-21), 1.22 (1H, m, H-
9
1
3
3 3
), 1.36 (9H, s, C(CH ) ), 1.39 and 1.88 (1H each, m, H-22), 1.43 and 2.06 (1H each, m, H-16),
.58 (1H, m, H-18), 1.59 (2H, m, H-2), 1.67 (3H, s, H-30), 2.28 (1H, m, H-13), 3.02 (1H, m, H-19),
.15 (1H, m, H-3), 4.60 and 4.73 (1H each, s, H-29), 5.25 and 5.36 (1H each, d, J = 12.55 Hz,-
1
3
OCH -), 7.53 (2H, d, J = 8.66 Hz, Ar-H), 7.64 (2H, d, J = 8.64 Hz, Ar-H), 8.05 (1H, s, -NCH). C
2
NMR (100 MHz, CDCl ): δ 14.65, 15.30, 15.71, 16.00, 18.19, 19.38, 20.86, 25.51, 27.38, 27.95,
3
2
9.68, 30.63, 31.25 x 3CH , 32.01, 34.19, 34.78, 36.83, 37.14, 38.39, 38.69, 38.82, 40.64, 42.32,
3
4
7.01, 49.56, 50.48, 55.30, 56.56, 57.06, 78.92, 109.64, 120.21 x 2CH, 122.38, 126.56 x 2CH,
1
34.50, 143.75, 150.42, 152.26, 176.17. IR γmax (neat): 3400, 2928, 2868, 1723, 1641, 1521, 1462,
-1
+
1
233, 1151, 1125, 1044, 837, 758 cm . HRMS m/z calcd. for C H N O [M + H] 670.4943,
43 64 3 3
found 670.4942.
4
.1.4.20. Synthesis of 28{1N(2,5-dimethylphenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester
22). The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40
mmol) and 2,5-dimethylphenyl boronic acid (72.74 mg, 0.48 mmol) as per the method described in
(
o
1
section 4.1.4. to give yellow brown solid (237 mg, 92 % yield) mp 132-134 C. H NMR (500
MHz, CDCl ): δ 0.63 (1H, m, H-5), 0.72 (3H, s, H-25), 0.73 (3H, s, H-24), 0.76 (3H, s, H-26), 0.88
3
2
6

ACCEPTED MANUSCRIPT
and 1.42 (1H each, m, H-1), 0.94 and 0.95 (3H each, s, H-23 and H-27), 1.15 and 1.86 (1H each, m,
H-11), 1.23 and 1.61 (1H each, m, H-12), 1.17 and 1.36 (1H each, m, H-15), 1.25 and 1.58 (1H
each, m, H-6), 1.27 and 1.39 (1H each, m, H-7), 1.28 and 1.65 (1H each, m, H-21), 1.23 (1H, m, H-
9
), 1.39 and 1.89 (1H each, m, H-22), 1.42 and 2.14 (1H each, m, H-16), 1.58 (1H, m, H-18), 1.59
(
2H, m, H-2), 1.67 (3H, s, H-30), 2.16 and 2.37 (3H each, s, 2 x Ar-CH ), 2.28 (1H, m, H-13), 3.0
3
(1H, m, H-19), 3.17 (1H, m, H-3), 4.59 and 4.72 (1H each, s, H-29), 5.28 and 5.37 (1H each, d, J =
1
3
1
2.55 Hz, -OCH
): δ 14.62, 15.30, 15.91, 16.05, 17.33, 18.20, 19.32, 20.62, 20.81, 25.46, 27.34, 27.93,
0.54, 31.59, 31.90, 34.21, 36.87, 37.13, 38.27, 38.67, 38.80, 40.66, 42.32, 46.93, 49.44, 50.45,
2
-), 7.14 (1H, s, Ar-H), 7.24 (2H, m, Ar-H), 7.83 (1H, s, -NCH). C NMR (100
MHz, CDCl
3
3
5
1
1
4
5.28, 56.54, 57.03, 78.85, 109.61, 125.55, 126.38, 130.13, 130.57, 131.24, 136.08, 136.76, 142.93,
50.34, 176.07. IR γmax (neat): 3429, 2940, 2868, 1723, 1641, 1513, 1453, 1377, 1232, 1151, 1129,
-1
+
043, 882, 757 cm . HRMS m/z calcd. for C H N O [M + H] 642.4628, found 642.4629.
4
1
60
3
3
.1.4.21. Synthesis of 28{1N(2,3-dimethoxy phenyl)-1H-1,2,3-triazol-4-yl}methyloxy betulinic ester
23). The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40
mmol) and 2,3-dimethoxyphenyl boronic acid (88.28 mg, 0.48mmol) as per the method described
(
o
1
in section 4.1.4. to give yellow brown solid (248 mg, 92 % yield) mp 132-134 C. H NMR (500
MHz, CDCl ): δ 0.71 (1H, m, H-5), 0.74 (3H, s, H-25), 0.75 (3H, s, H-24), 0.93 (3H, s, H-26), 0.88
3
and 1.42 (1H each, m, H-1), 0.93 and 0.95 (3H each, s, H-23 and H-27), 1.15 and 1.86 (1H each, m,
H-11), 1.23 and 1.61 (1H each, m, H-12), 1.17 and 1.36 (1H each, m, H-15), 1.25 and 1.58 (1H
each, m, H-6), 1.27 and 1.39 (1H each, m, H-7), 1.28 and 1.65 (1H each, m, H-21), 1.23 (1H, m, H-
9
), 1.39 and 1.87 (1H each, m, H-22), 1.42 and 2.16 (1H each, m, H-16), 1.58 (1H, m, H-18), 1.59
2H, m, H-2), 1.66 (3H, s, H-30), 2.27 (1H, m, H-13), 3.02 (1H, m, H-19), 3.16 (1H, m, H-3), 3.72
and 3.94 (3H each, s, 2 x -OCH ), 4.58 and 4.72 (1H each, s, H-29), 5.24 and 5.30 (1H each, d, J =
(
3
2
7

ACCEPTED MANUSCRIPT
1
2.58 Hz,-OCH
2
-), 7.02 (1H, d, J = 8.32 Hz, Ar-H), 7.21 (1H, dd, J = 8.32, 8.2 Hz, Ar-H), 7.36
1
3
(
1H, d, J = 8.2 Hz, Ar-H), 8.20 (1H, s, -NCH). C NMR (125 MHz, CDCl ): δ 14.63, 15.32, 15.72,
3
1
3
1
3
7
4
6.01, 18.20, 19.33, 20.79, 25.46, 27.34, 27.93, 29.57, 30.54, 31.96, 34.19, 36.85, 37.12, 38.27,
8.66, 38.73, 40.60, 42.31, 46.93, 49.44, 50.46, 55.27, 56.52, 56.35, 57.08, 61.10, 78.93, 109.62,
12.80, 116.94, 124.39, 125.75, 131.00, 141.30, 142.99, 150.41, 153.54, 175.92. IR γmax (neat):
429, 2926, 2854, 1725, 1641, 1598, 1492, 1462, 1377, 1237, 1271, 1151, 1106, 1107, 1043, 884,
-1
+
59 cm . HRMS m/z calcd. For C41
60 3 5
H N O [M + H] 674.4523, found 674.4527.
.1.4.22. Synthesis of 28{1N(1,4-biphenyl)-1H-1,2,3-triazol- 4-yl}methyloxy betulinic ester (24).
The title compound prepared by the reaction of propargylated betulinic ester (200 mg, 0.40 mmol)
and 4-biphenyl boronic acid (96.06 mg, 0.48 mmol) as per the method described in section 4.1.4. to
o
1
give yellow white solid (245 mg, 89 % yield) mp 152-154 C. H NMR (500 MHz, CDCl ): δ 0.63
3
(
1H, m, H-5), 0.65 (3H, s, H-25), 0.69 (6H, s, H-24 and H-26), 0.88 and 1.42 (1H each, m, H-1),
.92 and 0.93 (3H each, s, H-23 and H-27), 1.15 and 1.87 (1H each, m, H-11), 1.22 and 1.61 (1H
each, m, H-12), 1.17 and 1.37 (1H each, m, H-15), 1.25 and 1.58 (1H each, m, H-6), 1.28 and 1.39
1H each, m, H-7), 1.29 and 1.65 (1H each, m, H-21), 1.23 (1H, m, H-9), 1.39 and 1.87 (1H each,
m, H-22), 1.42 and 2.03 (1H each, m, H-16), 1.59 (1H, m, H-18), 1.60 (2H, m, H-2), 1.67 (3H, s, H-
0
(
3
5
0), 2.28 (1H, m, H-13), 3.02 (1H, m, H-19), 3.15 (1H, m, H-3), 4.60 and 4.74 (1H each, s, H-29),
.28 and 5.37 (1H each, d, J = 12.57 Hz,-OCH -), 7.40 (1H, m, Ar-H), 7.48 (2H, m, Ar-H), 7.62
2
(2H, d, J = 8.55 Hz, Ar-H), 7.74 (2H, d, J = 8.68 Hz, Ar-H), 7.80 (2H, d, J = 8.67 Hz, Ar-H), 8.12
1
3
(
1H, s, -NCH). C NMR (100 MHz, CDCl ): δ 14.67, 15.30, 15.71, 16.01, 18.22, 19.38, 20.87,
3
2
4
1
5.53, 27.38, 27.94, 29.49, 30.64, 31.91, 34.21, 36.87, 37.16, 38.42, 38.70, 38.82, 40.67, 42.36,
7.02, 49.56, 50.50, 55.32, 56.59, 57.05, 78.95, 109.67, 120.84 x 2CH, 122.35, 127.09 x 2CH,
28.35, 128.69 x 2CH, 128.99 x 2CH, 136.02, 139.60, 141.98, 143.99, 150.40, 176.21. IR γmax
2
8