Synthesis of novel benzylidene analogues of betulinic acid as potent cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2017.04.062

Different benzylidene derivatives (15a-o and 16a-o) of betulinic acid were designed and synthesized in an effort to develop potent anticancer agents. All the synthesized derivatives along with betulinic acid were evaluated for cytotoxicity against a panel of five different human cancer cell lines A-549 (Lung), PC-3 (Prostate), HCT 116 (Colon), MCF-7 (Breast) and MIA PaCa-2 (Pancreatic) using SRB assay. Pharmacological results showed that compounds 15b, 15c, 15i, 15k, 16a-c and 16l were found to have promising cytotoxic profile against various cancer cell lines tested (IC₅₀ 1–2?μM). Best results were observed for compound 16c with IC₅₀ values 1.5, 1.6, 1.36, 3.5 and 3.2?μM against A-549, PC-3, HCT 116, MCF-7 and MIA PaCa-2?cell lines, respectively. Mechanistic study of compound 16c revealed that it inhibits the colony formation and restrict the migration in HCT 116?cells in?vitro. It also induces growth arrest with characterized morphological changes and loss of mitochondrial membrane potential (MMP) in a concentration dependent manner.

Full text of DOI:10.1016/j.ejmech.2017.04.062

Accepted Manuscript
Synthesis of novel benzylidene analogues of betulinic acid as potent cytotoxic agents
Nidhi Gupta, Santosh K. Rath, Arem Qayum, Jasvinder Singh, Shashank Singh,
Payare L. Sangwan
PII:
S0223-5234(17)30336-7
10.1016/j.ejmech.2017.04.062
EJMECH 9413
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2017
Revised Date: 10 April 2017
Accepted Date: 22 April 2017
Please cite this article as: N. Gupta, S.K. Rath, A. Qayum, J. Singh, S. Singh, P.L. Sangwan, Synthesis
of novel benzylidene analogues of betulinic acid as potent cytotoxic agents, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.062.
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ACCEPTED MANUSCRIPT
Synthesis of novel benzylidene analogues of betulinic acid as potent
cytotoxic agents
Nidhi Gupta, Santosh K. Rath, Arem Qayum, Jasvinder Singh, Shashank Singh*, and Payare L.
Sangwan*
Design and synthesis of C-2 and C-3 derived benzylidene analogues of betulinic acid as potent
cytotoxic agents

ACCEPTED MANUSCRIPT
Synthesis of novel benzylidene analogues of betulinic acid as potent cytotoxic agents
Nidhi Guptaa, Santosh K. Ratha,c, Arem Qayumb,c, Jasvinder Singhb,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. IIIN/2010/2017
*Corresponding authors. E-mails sksingh@iiim.ac.in (Shashank Singh); plsangwan@iiim.ac.in
(P. L. Sangwan)
Keywords: Betulinic acid; benzylidene derivatives; cytotoxicity; colony formation
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Abstract
Different benzylidene derivatives (15a-o and 16a-o) of betulinic acid were designed and
synthesized in an effort to develop potent anticancer agents. All the synthesized derivatives along
with betulinic acid were evaluated for cytotoxicity against a panel of five different human cancer
cell lines A-549 (Lung), PC-3 (Prostate), HCT 116 (Colon), MCF-7 (Breast) and MIA PaCa-2
(Pancreatic) using SRB assay. Pharmacological results showed that compounds 15b, 15c, 15i,
15k, 16a-c and 16l were found to have promising cytotoxic profile against various cancer cell
lines tested (IC50 1-2 µM). Best results were observed for compound 16c with IC50 values 1.5,
1.6, 1.36, 3.5 and 3.2 µM against A-549, PC-3, HCT 116, MCF-7 and MIA PaCa-2 cell lines,
respectively. Mechanistic study of compound 16c revealed that it inhibits the colony formation
and restrict the migration in HCT 116 cells in vitro. It also induces growth arrest with
characterized morphological changes and loss of mitochondrial membrane potential (MMP) in a
concentration dependent manner.
1. Introduction
Triterpenoids represents one of the major classes of plant derived natural products and
some representative structural scaffolds shown in Fig. 1 such as lupane group betulinic acid (1)
and lupeol (2), oleanane group oleanolic acid (3) and maslinic acid (4), ursolic acid (5) and
ursane group β-boswellic acid (6) has gained considerable attention worldwide in recent past
owing to their various pharmacological activities and substantial number of novel derivatives
have been synthesized based on these scaffolds [1,2]. Betulinic acid (1), a pentacyclic lupane-
type triterpenoid is known to possess a broad range of biological effects, particularly anticancer
[3, 4]. Cytotoxic effects of betulinic acid has been studied on large variety of cancer cell lines [5-
7] as well as applying xenograft mice models and primary tumor samples [8] and the compound
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is currently under clinical evaluation in Phase I / II clinical trials (NCT00346502) for the
treatment of dysplastic nevi (moderate to severe dysplasia) [9]. Further, mechanistic studies
revealed that betulinic acid induces the cancer death via triggering the mitochondrial pathway of
apoptosis. It increases mitochondrial membrane permeabilisation leading to caspase activation
and nuclear fragmentation with the release of apoptogenic factors such as cytochrome c, Smac
and AIF [10]. Compound 1 is also reported to modulate the expression level of Bcl-2 family
proteins, induce apoptosis in p53 and CD-95 independent manner and inhibit the TNFα induced
activation of NFκB in human prostate cancer cells (PC-3) [11,12].
Figure 1
Betulinic acid represents a biologically active scaffold with high safety profile in cancer
therapy and is suitable to carry out chemical transformations because of the several key positions
available on the molecule i.e. C-2, C-3, C-20 and C-28 (Fig. 1). Therefore, to investigate the
structural features responsible for anticancer activity of betulinic acid and also to develop the
structure activity relationship (SAR) studies, considerable structural modification has been done
on betulinic acid for the improvement of its anticancer activity [13-15]. The oxidized product
betulonic acid (7) having carbonyl moiety at C-3 position reportedly possess better anticancer
profile than betulinic acid [16]. Also, several A-ring modified derivatives at C-2 and C-3 position
of betulinic acid have been reported to possess potent anticancer activity. You et al. synthesized
the various A-ring modified betulinic acid derivatives. Among them, compounds 8a and 8b
showed potent cytotoxicity against M2 cell line with IC50 values 0.81 and 0.13µM respectively
[17]. Grishko et al. reported the anticancer activity of ring-A fused azole derivatives of betulin
and 1,2,4-triazine analog (9) was found as the most potent against HCT-116 cell line with an IC50
value of 1.4 µM [18]. Csuk et al. reported the anticancer activity of several alkylidene branched
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lupane derivatives and some analogs including compounds 10 and 11 were found to be potent
derivatives [19]. Borkova et al. reported the cytotoxic activity of 2,2-difluoroderivative of
dihydrobetulinic acid (12a) [20]. Urban et al. reported the 2-bromo-3-oxo (12b) and 2-
hydroxymethylene-3-oxo (12c) derivatives of betulinic acid and studied their cytotoxic effects on
CEM cell line [21]. Ngoc reported the anticancer activity of 12d, a diosphenol derivative of
betulinic acid [22]. B10 (13) and NVX-207 (14) (Fig. 2.) are C-3 modified derivatives of
betulinic acid having potent clinical applications [23, 24]. Kumar et al. [25] along with the work
of Dar et al. [26] reported that introduction of benzylidene moiety into triterpene natural products
(β-boswellic acid and ursolic acid) produced analogs with improved anticancer potential.
Furthermore, benzylidene derivatives having α, β- unsaturated carbonyl moiety in biological
systems interacts with targeted proteins or enzymes via Michael addition and exhibits various
pharmacological activities [27, 28].
Based on these findings and as a part of our ongoing work on structural modification of
natural products to get the anticancer lead molecules [5, 6, 14, 29], benzylidene derivatives of
betulinic acid were designed to improve its antitumour potential. In the present paper, we report
the synthesis of benzylidene derivatives 15a-o of betulinic acid by aldol condensation reactions
which were further reduced at C-3 position to produce derivatives 16a-o. All these derivatives
were evaluated for in vitro cytotoxic activity against five human cancer cell lines (A-549, PC-3,
HCT 116, MCF-7 and MIA PaCa-2) and it was demonstrated that many analogs exhibited better
anticancer potential than betulinic acid. All the active compounds including lead (16c) were
tested on normal breast epithelial human cell line (fR2). Further, the mechanistic study of
compound 16c was carried out on colon cancer cell line (HCT 116).
Figure 2
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2. Result and Discussion
2.1. Chemistry (Design and synthesis of betulinic acid benzylidene derivatives)
In the present study, betulinic acid was isolated from DCM:MeOH (1:1) extract of stem
bark of Plantanus orientalis and was taken for structural modification studies. C-2 position was
targeted and benzylidene derivatives 15a-o were designed and synthesized. Reduction of
carbonyl group at C-3 position of these derivatives was carried out to produce derivatives 16a-o.
The strategy behind this was to study the effect of benzylidene derivatives on the anticancer
activity while retaining the C3 hydroxyl group. The procedure for the synthesis of benzylidene
derivatives 15a-o involved two steps (Scheme 1). In the first step, 1 was oxidized with
Pyridinium chlorochromate (PCC) in dichloromethane (DCM) to provide betulonic acid 7. In
second step, compound 7 was reacted with different aldehydes to carry out aldol condensation
reaction. Initially, 7 was reacted with 3-nitrobenzaldehyde and was taken as a model reaction to
optimize reaction conditions. For this, we used different bases such as K2CO3, KOEt, NaOH,
TEA, DMAP and NaH and best results were observed in case of NaH. Different solvents were
also screened taking NaH as a base and THF was found to be best solvent (Supplementary
information, Table S1). These optimized set of conditions then used for the condensation of
various aromatic aldehydes with betulonic acid 7 to provide a series of benzylidene analogues
15a-o. Further, reduction of 15a-o with sodium borohydride (NaBH4) in methanol produces
derivatives 16a-o with hydroxyl group at C-3 (Scheme 1).
Scheme 1
All the reactions were carried out at variable temperature conditions (0oC to room
temperature) and provided derivatives in good to excellent yields. Structures of all the
derivatives were confirmed by spectroscopic techniques (1H NMR, 13C NMR and HRMS). In 13C
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1
NMR, signal at δ 204, 135 & 133 and in H NMR, a singlet at δ 7.2-7.5 correspond to olefinic
proton of α, β-unsaturated carbonyl system confirm the product formation of 15a-o. For
compounds, 16a-o, 13C NMR is distinguished from the spectrum of 15a-o by the absence of
signal from carbonyl group at δ 204 and appearance of signal from C-3 atom of lupane scaffold
at δ 81 whereas in 1H NMR, singlet from the proton at C-3 atom (δ 3.7-3.9) and upfield shift of
olefinic proton signal (δ 6.4-6.7) is observed. Further confirmation for the formation of all the
derivatives was done by DEPT and HRMS data.
2.2. Biology
2.2.1. Cell growth inhibition
All the synthesized derivatives were subjected to preliminary cytotoxicity screening at 10
µM concentration against a panel of five different human cancer cell lines namely, A-549
(Lung), PC-3 (Prostate), HCT 116 (Colon), MCF-7 (Breast) and MIA PaCa-2 (Pancreatic) along
with compound 1 (parent molecule) taken as reference standard in present study to evaluate their
cytotoxic potential using SRB assay. Results are summarized in table 1 and values given are the
average of triplicate analysis. Betulinic acid 1 showed 26-49% growth inhibition against all the
cancer cell lines. Compound 7 displayed better anticancer effects than 1 which was in agreement
with the literature [16] and exhibited cytotoxicity against A-549, HCT 116, MCF-7 and MIA
PaCa-2 cell lines with 62, 49, 43 and 36% inhibition respectively. Among benzylidene analogs,
many of them exhibited ≥90% inhibition against various experimental cancer cell lines.
Compounds 15f-j, 15l-o and 16a-c displayed significant growth inhibition effects against all the
experimental cancer cell lines. Compounds 15a, 15b, 15d, 15e, 15g, 15h, 15m-o, 16a, 16b and
16d affected A-549 the most whereas compounds 15c, 15f, 16e, 16g-l, 16n and 16o showed
maximum inhibition effects against MCF-7 than other four experimental cancer cell lines.
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Compounds 15k, 15l and 16c exhibited maximum growth inhibition against colon cancer cell
line with 92, 99 and 92% respectively. Compound 15i affected MCF-7 and MIA PaCa-2 (97%
inhibition) and 15j affected A-549 and HCT 116 cell lines (89% inhibition) to same extent.
Compound 16f did not exhibit significant growth inhibition against any of the cancer cell line
examined whereas compound 16m showed fewer inhibition against MCF-7 and MIA PaCa-2 cell
lines.
Table1
2.2.2. IC50 and SAR (structure activity relationship)
Compounds showed significant inhibition effects at 10 µM concentration were further
screened at three more concentrations 1, 2.5 and 5 µM and IC50 was calculated along with the
parent molecule (Table 2). Conversion of 1 into 7 resulted in the improvement of activity with
IC50 value 5.7 µM against A-549 cell line. In benzylidene analogs, compounds 15f-j, 15l, 15n,
15o, 16a, 16c and 16n exhibit potent cytotoxic effects against all the cell lines with single digit
IC50 value (<10 µM). Compounds (15a-d, 15f, 15h-m, 15o, 16a-d, 16i-l and 16n) were found to
be most promising against A-549 cell line with IC50 <5 µM among all the cell lines tested. In
case of PC-3 and HCT 116 cell lines, compound 16c displayed most potent activity with IC50
value 1.6 and 1.36 µM respectively. Compound 16l displayed most potent activity against MCF-
7 and MIA PaCa-2 cell lines with IC50 value 1.18 and 1.21 µM respectively. Overall, best results
were observed for compound 16c with IC50 values 1.5, 1.6, 1.36, 3.5 and 3.2 µM against A-549,
PC-3, HCT 116, MCF-7 and MIA PaCa-2 cell lines respectively. All the active compounds were
tested on normal breast epithelial human cell line (fR2) Table 2. Based on the results the
selectivity index for cancer cells were determined Table 3. Moreover, IC50 value of 16c was
found to be high in normal human breast epithelial cells (fR2) which shows its selectivity for
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cancer cells (Table 2 and 3) and justify its potency to develop as an anticancer agent. On the
basis of growth inhibition and IC50 values, structure activity relationship of structural
modifications of 1 can be summarized as follows:
a)
b)
Betulonic acid (7) showed better anticancer potential than the parent (1).
In general, benzylidene derivatives having α, β-unsaturated carbonyl moiety showed
better anticancer effects than their corresponding derivatives with reduced keto group at C-3
position. However, some analogs with C3-OH (16a-c) exhibit cytotoxic effects better or
comparable to that of their corresponding keto derivatives (15a-c) on certain cell lines examined.
c)
Among the benzylidene derivatives, analogs containing ortho or para substitued electron
withdrawing groups (-NO2, -Br and -F) (15b, 15c, 15g, 15l, 16b, 16c and 16l) showed better
anticancer effects than meta- substituted analogs (15a, 15d, 16a and 16d). However, analogs
with meta- and para- disubstituted halo groups (15j and 16j) were found to be favourable for the
improvement of cytotoxic activity for all cell lines examined compared to the ortho disubstituted
analogs (15e and 16e). Analog with electron releasing group (-OCH3) (15n and 16n) showed
potent anticancer effects against all cell lines. Pyridine containing analog (15i and 16i) exhibited
better cytotoxic effects against colon, breast, lung and pancreatic cancer cells than analogs
containing other heterocyclic moieties such as furan (15f and 16f) and thiophene (15h and 16h).
Derivatives with extended conjugation (15m) and with polycyclic aromatic hydrocarbon moiety
(15o and 16o) also inhibited various cancer cell lines. It appears that many factors such as size,
position, electronic effects (resonance and inductive) of the substituent and presence of
heterocyclic groups are collectively responsible for anticancer effects of betulinic acid
benzylidene derivatives. Compound 16c was found to be most potent among synthesized series
hence taken for further cell death mechanistic study.
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Table 2
2.2.3. Compound 16c inhibited cell proliferation during colony formation assay in HCT 116
cells.
Cell proliferation inhibiting ability of compound 16c was measured using colony formation assay
or clonogenic assay (Fig. 3.). Clonogenic assay is an in vitro cell survival assay depends on cell’s
ability to grow into a colony. Initially, this assay was used for studying the effect of radiations on
cells, now it is also used for studying the effects of chemotherapeutic agents having potential
clinical applications. This assay tests each cell in the population to undergo extensive divisions
and also monitors the cells that have retained the capacity for producing colonies after treatment
with cell death causing agents (radiations or chemotherapeutic agents) [30]. The effect of
cytotoxic compounds on colony forming ability of cancer cells is measured. HCT 116 cells were
treated with different concentrations of 16c at 0.7, 1.4 and 2.8 µM in which colonies were
formed after 14 days treatment. It was found that 16c significantly decreased colony formation in
HCT 116 cells in a concentration dependent manner as compared to untreated control (A).
Figure 3
2.2.4. Compound 16c inhibited cell migration during in vitro wound healing assay in HCT 116
cells
In vitro wound healing assay or in vitro scratch assay is a method to study cell migration in
which the cells on the edge of the newly created scratch or gap will move towards the opening to
close this gap until the new cell-cell contacts are established again. Images were captured at the
beginning and after termination in order to determine the rate of cell migration [31]. Herein, the
monolayer of HCT 116 cells was scratched and treated with 16c at various concentrations (1.4,
2.0 and 2.5 µM) for 24 h. The area of the wound was measured at two different time points 0 and
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24 h and % reduction in cell migration was assessed by recovered area of the scratch as
compared with 0 h post scratch. Rate of migration was calculated as compared to the
corresponding control (A). It was observed that cell migration was inhibited in a dose dependent
manner (Fig. 4). As higher concentration of 16c correlates significantly in restricting metastasis
by decreasing cell migration as well as inhibiting cell motility as data showed that the wound-
healing rate in HCT 116 cells is slower at lower concentration in comparison to the control cells.
So, migration analyzed by wound-healing assay revealed that 16c clearly inhibited wound
closure in HCT116 cells and displayed an anti-migratory effect in HCT 116 cells.
Figure 4
2.2.5. Compound 16c induced morphological changes in HCT 116 cells
The HCT 116 cells were treated with 16c at 0.7, 1.4, 2.0 and 2.5 µM for 24 h and observed for
morphological changes by using phase contrast microscope. Cell growth arrest and characteristic
changes were observed in the morphology of treated cells in a concentration-dependent manner
(Fig. 5.).
Figure 5
2.2.6. Compound 16c triggered mitochondrial membrane potential (MMP) loss
Loss of MMP causes depolarization of mitochondrial membrane with the release of apoptogenic
factors and ultimately a cell death [32]. The changes of mitochondrial membrane were assessed
by staining with rhodamine-123 (RH-123), a fluorescent dye. The rate of fluorescence decay is
proportional to the loss in membrane potential. The loss of mitochondrial membrane integrity
causes leakage of RH-123 which cause decrease in fluorescence intensity. HCT-116 cells were
treated with different concentrations of 16c at 0.7, 1.4, 2.0 and 2.5 µM and observed loss in
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MMP in a concentration dependent manner whereas cytoplasm of untreated cells (control) were
having intact mitochondria (Fig. 6).
Figure 6
3. Conclusion
To conclude, benzylidene derivatives of betulinic acid were synthesized at C-2 position by
employing aldol condensation approach and screened against five human cancer cell lines (A-
549, PC-3, HCT 116, MCF-7 and MIA PaCa-2) along with parent (1) as reference. Results
demonstrated that C-2 position is favorable site to carry out modification as many analogs
displayed better anticancer effects than betulinic acid. Compound 16c was found as most potent
analog among the synthesized series and further its mechanistic study through colony formation,
wound healing, phase contrast microscopy and MMP loss experiments in HCT 116 cell line
showed its potential to develop as potent anticancer agent.
4. Experimental
4.1. Chemistry
All the reagents and solvents for synthesis were purchased from Sigma-Aldrich. All the
chemical reactions were monitored by TLC on silica gel 60 F254 plates (E. Merck) using 2% ceric
ammonium sulphate solution as spraying reagent for detection of spots. Purification of all
derivatives was carried out by column chromatography using silica gel 60-120 mesh as
stationary phase. All NMR spectra were recorded on Bruker DPX 400 and DPX 500 instruments
using CDCl3 as the solvent taking TMS as the internal standard. The chemical shifts are
expressed in δ and coupling constant in Hertz. High Resolution Mass Spectra (HRMS) were
recorded on Agilent Technologies 6540 instrument.
4.1.1. Isolation of betulinic acid (1)
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Betulinic acid was isolated in bulk quantity from DCM: MeOH (1: 1) extract of stem
bark of Plantanus orientalis and characterized by spectroscopic techniques as reported
previously [5].
4.1.2. Preparation of betulonic acid (7)
To a solution of compound 1 (5 g, 11 mmol) in DCM was added PCC (3.54 g, 16 mmol)
dissolved in DCM dropwise till dark colour appears and kept it at r. t. for 2 h. After completion,
reaction mixture was passed through celite and filtrate was concentrated at rotavapour.
Purification was done through column chromatography with EtoAc: Hexane (1: 13) as the eluent
1
to afford product 7 colourless solid (3.5 g, 70% yield). H NMR (400 MHz, CDCl3): δ 4.74 and
4.62 (1H each, s, H-29), 3.01 (1H, m, H-19), 2.5 and 2.39 (1H each, m, H-2), 2.29 and 1.99 (1H
each, m, H-22), 2.21 and 1.97 (1H each, m, H-16), 1.9 (1H, m, H-13), 1.7 (3H, s, H-30), 1.74 and
1.62 (1H each, m, H-1), 1.63 (1H, m, H-5), 1.56 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-6),
1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m,
H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.2 (1H each, m, H-11),
1.07 (3H, s, H-23), 1.02 (3H, s, H-24), 0.99 and 0.97 (3H each, s, H-26 and H-27), 0.93 (3H, s,
H-25). 13C NMR (125 MHz, CDCl3): δ 219.75, 183.61, 151.75, 111.22, 57.83, 56.35, 51.26,
50.60, 48.77, 48.32, 43.91, 42.05, 41.03, 39.93, 38.47, 38.34, 35.56, 35.02, 33.52, 31.97, 31.10,
28.06, 26.90, 22.79, 22.43, 21.05, 20.79, 17.39, 17.25, 16.05. HRMS m/z calcd for C30H47O3 [M
+ H]+ 455.352, found 455.3507.
4.1.3. General experimental procedure for preparation of benzylidene derivatives 15a-o
To synthesize compounds 15a-o, compound 7 (1 equiv) was dissolved in dry THF and
NaH (1.2 equiv) was added to it at 0oC. After 10 min, respective aldehyde (1.5 equiv) was added
to reaction mixture. The reaction mixture was stirred at room temperature for 1.5-2 h till the
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completion (monitored by TLC analysis) [25]. Workup of the reaction was done by diluting the
reaction mixture with ice-cold water and extracting it with ethyl acetate (3 times). The combined
organic layers were dried over sodium sulphate and concentrated on rotavapour. The crude
product obtained was purified by column chromatography on silica gel 60-120 mesh with EtoAc:
hexane (1: 10) as the eluent to afford the desired pure products 15a-o in 90-93% yield. The
spectral data of all the derivatives 15a-o are given below.
4.1.3.1. Synthesis of 2-(3-nitrobenzylidene)betulonic acid (15a). The title compound prepared by
the reaction of betulonic acid (100 mg, 0.22 mmol) and 3-nitrobenzaldehyde (50 mg, 0.33 mmol)
as per the method described in Section 4.1.3 to furnish 15a colourless solid (120 mg, 93% yield).
1H NMR (400 MHz, CDCl3): δ 8.13 (2H, m, 2×Ar-CH), 7.54 (2H, m, 2 × Ar-CH), 7.41 (1H, s, -
CH=C-CO-), 4.67 and 4.57 (1H each, s, H-29), 2.92 (1H, m, H-19), 2.89 and 2.16 (1H each, m,
H-1), 2.21 and 1.93 (1H each, m, H-22), 2.18 and 1.89 (1H each, m, H-16), 2.13 (1H, m, H-13),
1.64 (3H, s, H-30), 1.58 (1H, m, H-18), 1.56 and 1.33 (1H each, m, H-6), 1.52 and 1.29 (1H
each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and
1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.2 (1H each, m, H-11), 1.08 and 0.96 (3H
each, s, H-23 and H-24), 0.91 (3H, s, H-27), 0.81 (3H, s, H-26), 0.77 (1H, m, H-5), 0.74 (3H, s,
H-25). 13C NMR (125 MHz, CDCl3): δ 207.83, 181.59, 150.29, 148.30, 137.64, 137.03, 135.47,
134.42, 129.48, 124.60, 122.88, 109.82, 56.44, 52.95, 49.11, 48.33, 46.85, 45.41, 44.03, 42.52,
40.54, 38.39, 37.09, 36.70, 33.01, 30.04, 27.09, 25.44, 22.70, 22.37, 21.62, 20.31, 19.45, 15.84,
15.50, 14.62, 14.13. HRMS m/z calcd for C37H50NO5 [M + H]+ 588.3684, found 588.3678.
4.1.3.2. Synthesis of 2-(4-bromobenzylidene)betulonic acid (15b). The title compound prepared
by the reaction of betulonic acid (100 mg, 0.22 mmol) and 4-bromobenzaldehyde (61 mg, 0.33
mmol) as per the method described in Section 4.1.3 to furnish 15b colourless solid (125 mg, 91%
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yield). 1H NMR (400 MHz, CDCl3): δ 7.53 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 7.4 (1H, s, -CH=C-
CO), 7.27 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 4.76 and 4.66 (1H each, s, H-29), 3.02 (1H, m, H-19),
2.96 and 2.16 (1H each, m, H-1), 2.29 and 1.99 (1H each, m, H-22), 2.22 and 1.98 (1H each, m,
H-16), 1.96 (1H, m, H-13), 1.72 (3H, s, H-30), 1.68 (1H, m, H-18), 1.65 (1H, m, H-5), 1.54 and
1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7),
1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35
and 1.2 (1H each, m, H-11), 1.14 and 1.12 (3H each, s, H-23 and H-24), 1.02 (3H, s, H-27), 0.97
(3H, s, H-26), 0.78 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 207.92, 181.92, 150.44,
136.03, 134.88, 134.85, 131.70 × 2, 131.68 × 2, 122.66, 109.74, 56.43, 52.85, 49.18, 48.45,
46.84, 45.23, 44.39, 42.52, 40.54, 38.46, 37.03, 36.53, 33.06, 32.06, 30.60, 29.68, 29.43, 25.59,
22.34, 21.67, 20.33, 19.50, 15.80, 15.48, 14.62. HRMS m/z calcd for C37H50BrO3 [M + H]+
621.2938, found 621.2921.
4.1.3.3. Synthesis of 2-(4-nitrobenzylidene)betulonic acid (15c). The title compound prepared by
the reaction of betulonic acid (100 mg, 0.22 mmol) and 4-nitrobenzaldehyde (50 mg, 0.33 mmol)
as per the method described in Section 4.1.3 to furnish 15c colourless solid (120 mg, 93% yield).
1H NMR (400 MHz, CDCl3): δ 8.26 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 7.54 (2H, d, J = 8.0 Hz, 2 ×
Ar-CH), 7.48 (1H, s, -CH=C-CO), 4.75 and 4.65 (1H each, s, H-29), 3.01 (1H, m, H-19), 2.97
and 2.3 (1H each, m, H-1), 2.24 and 2.0 (1H each, m, H-22), 2.21 and 1.98 (1H each, m, H-16),
1.96 (1H, m, H-13), 1.72 (3H, s, H-30), 1.69 (1H, m, H-18), 1.64 (1H, m, H-5), 1.54 and 1.33
(1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and
1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08
(1H each, m, H-11), 1.16 and 1.14 (3H each, s, H-23 and H-24), 1.03 (3H, s, H-27), 0.97 (3H, s,
H-26), 0.79 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 209.29, 184.12, 151.79, 148.49,
14

ACCEPTED MANUSCRIPT
143.89, 139.17, 135.95, 132.10 × 2, 125.13 × 2, 111.25, 57.86, 54.24, 50.51, 49.76, 48.26, 46.81,
45.82, 43.93, 41.93, 39.84, 38.46, 38.02, 34.40, 33.45, 31.97, 31.07, 30.77, 26.92, 23.80, 23.08,
21.71, 20.89, 17.29, 16.89, 16.03. HRMS m/z calcd for C37H48NO5 [M - H]– 586.3538, found
586.3562.
4.1.3.4. Synthesis of 2-(3-bromobenzylidene)betulonic acid (15d). The title compound prepared
by the reaction of betulonic acid (100 mg, 0.22 mmol) and 3-bromobenzaldehyde (61 mg, 0.33
mmol) as per the method described in Section 4.1.3 to furnish 15d colourless solid (125 mg, 91%
yield). 1H NMR (400 MHz, CDCl3): δ 7.53 (1H, d, J = 12.0 Hz, Ar-CH), 7.45 (1H, m, Ar-CH),
7.38 (1H, s, -CH=C-CO), 7.31 (2H, m, 2 × Ar-CH), 4.76 and 4.64 (1H each, s, H-29), 3.0 (1H,
m, H-19), 2.96 and 2.16 (1H each, m, H-1), 2.29 and 2.0 (1H each, m, H-22), 2.24 and 1.97 (1H
each, m, H-16), 1.92 (1H, m, H-13), 1.72 (3H, s, H-30), 1.66 (1H, m, H-18), 1.63 (1H, m, H-5),
1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m,
H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9),
1.35 and 1.08 (1H each, m, H-11), 1.14 and 1.12 (3H each, s, H-23 and H-24), 1.03 (3H, s, H-
27), 0.97 (3H, s, H-26), 0.78 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 208.02, 181.89,
150.42, 138.10, 135.66, 133.22, 131.25, 130.66, 129.94, 128.08, 122.55, 109.74, 56.44, 52.91,
49.15, 48.36, 46.84, 45.31, 44.13, 42.52, 40.53, 38.42, 37.02, 36.62, 33.04, 32.05, 30.61, 29.68,
29.35, 25.52, 22.36, 21.63, 20.32, 19.49, 15.82, 15.50, 14.63. HRMS m/z calcd for C37H50BrO3
[M + H]+ 621.2938, found 621.2946.
4.1.3.5. Synthesis of 2-(2, 6-dichlorobenzylidene)betulonic acid (15e). The title compound
prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and 2, 6-dichlorobenzaldehyde
(57.75 mg, 0.33 mmol) as per the method described in Section 4.1.3 to furnish 15e colourless
1
solid (124 mg, 92 % yield). H NMR (400 MHz, CDCl3): δ 7.35 (2H, d, J = 8.0 Hz, 2 × Ar-CH),
15

ACCEPTED MANUSCRIPT
7.25 (1H, s, -CH=C-CO), 7.21 (1H, t, J = 8.0 Hz, Ar-CH), 4.71 and 4.58 (1H each, s, H-29), 2.97
(1H, m, H-19), 2.41 and 1.84 (1H each, m, H-1), 2.26 and 1.99 (1H each, m, H-22), 2.19 and
1.95 (1H each, m, H-16), 1.93 (1H, m, H-13), 1.67 (3H, s, H-30), 1.61 (1H, m, H-18), 1.58 (1H,
m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H
each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H,
m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.17 and 1.14 (3H each, s, H-23 and H-24), 0.97
13
(3H, s, H-27), 0.94 (3H, s, H-26), 0.81 (3H, s, H-25). C NMR (125 MHz, CDCl3): δ 208.46,
183.77, 152.0, 140.26, 135.75, 135.59 × 2, 133.96, 130.74, 129.38 × 2, 111.07, 57.83, 54.85,
50.50, 49.49, 48.27, 47.16, 44.51, 43.89, 41.88, 39.85, 38.44, 37.97, 34.58, 33.45, 31.95, 31.05,
30.14, 26.84, 23.85, 22.78, 21.62, 20.81, 17.06, 16.95, 16.01. HRMS m/z calcd for C37H47Cl2O3
[M - H]– 609.2908, found 609.293.
4.1.3.6. Synthesis of 2-{(5-bromofuran-2-yl)methylene}betulonic acid (15f). The title compound
prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and 5-bromo-2-furaldehyde
(57.75 mg, 0.33 mmol) as per the method described in Section 4.1.3 to furnish 15f colourless
solid (124 mg, 92 % yield). 1H NMR (400 MHz, CDCl3): δ 7.23 (1H, s, -CH=C-CO), 6.52 (1H,
d, J = 4.0 Hz, Ar-CH), 6.44 (1H, d, J = 4.0 Hz, Ar-CH), 4.78 and 4.66 (1H each, s, H-29), 3.0
(1H, m, H-19), 2.96 and 2.1 (1H each, m, H-1), 2.32 and 2.01 (1H each, m, H-22), 2.28 and 1.99
(1H each, m, H-16), 1.8 (1H, m, H-13), 1.74 (3H, s, H-30), 1.68 (1H, m, H-18), 1.65 (1H, m, H-
5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each,
m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-
9), 1.35 and 1.08 (1H each, m, H-11), 1.14 and 1.07 (3H each, s, H-23 and H-24), 1.04 (3H, s, H-
13
27), 0.99 (3H, s, H-26), 0.82 (3H, s, H-25). C NMR (125 MHz, CDCl3): δ 208.71, 183.57,
155.88, 151.87, 133.31, 126.17, 124.67, 118.42, 115.56, 111.15, 57.87, 53.89, 50.58, 49.91,
16

ACCEPTED MANUSCRIPT
48.28, 46.32, 43.92, 41.90, 39.89, 38.45, 37.43, 34.41, 33.48, 32.05, 31.13, 31.04, 28.34, 26.99,
23.63, 23.15, 21.79, 20.93, 17.75, 16.86, 16.07. HRMS m/z calcd for C35H48BrO4 [M + H]+
611.273, found 611.2748.
4.1.3.7. Synthesis of 2-(2-bromobenzylidene)betulonic acid (15g). The title compound prepared
by the reaction of betulonic acid (100 mg, 0.22 mmol) and 2-bromobenzaldehyde (61 mg, 0.33
mmol) as per the method described in Section 4.1.3 to furnish 15g colourless solid (125 mg, 91%
yield). 1H NMR (400 MHz, CDCl3): δ 7.61 (1H, d, J = 8.0 Hz, Ar-CH), 7.49 (1H, s, -CH=C-
CO), 7.33 (1H, t, J = 8.0 Hz, Ar-CH), 7.19 (2H, m, 2 × Ar-CH), 4.72 and 4.6 (1H each, s, H-29),
3.0 (1H, m, H-19), 2.82 and 1.97 (1H each, m, H-1), 2.28 and 1.98 (1H each, m, H-22), 2.2 and
1.96 (1H each, m, H-16), 1.93 (1H, m, H-13), 1.69 (3H, s, H-30), 1.63 (1H, m, H-18), 1.61 (1H,
m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H
each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H,
m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.17 and 1.13 (3H each, s, H-23 and H-24), 0.99
(3H, s, H-27), 0.95 (3H, s, H-26), 0.8 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 209.67,
184.18, 151.93, 138.07, 137.77, 137.39, 134.30, 131.57, 130.89, 128.49, 126.28, 111.11, 57.87,
54.66, 50.52, 49.75, 48.26, 47.14, 44.78, 43.91, 41.95, 39.83, 38.47, 38.26, 34.56, 33.47, 31.99,
31.08, 30.36, 26.90, 23.87, 22.88, 21.62, 20.86, 17.17, 16.96, 16.03. HRMS m/z calcd for
C37H50BrO3 [M + H]+ 621.2938, found 621.295.
4.1.3.8. Synthesis of 2-(thiophen-3-ylmethylene)betulonic acid (15h). The title compound
prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and 3-thiophenecarboxaldehyde
(37 mg, 0.33 mmol) as per the method described in Section 4.1.3 to furnish 15h colourless solid
(108 mg, 90 % yield). 1H NMR (400 MHz, CDCl3): δ 7.47 (2H, m, 2 × Ar-CH), 7.36 (1H, m, Ar-
CH), 7.25 (1H, s, -CH=C-CO), 4.78 and 4.67 (1H each, s, H-29), 3.04 (1H, m, H-19), 3.0 and 2.2
17

ACCEPTED MANUSCRIPT
(1H each, m, H-1), 2.32 and 2.01 (1H each, m, H-22), 2.29 and 1.99 (1H each, m, H-16), 1.97
(1H, m, H-13), 1.74 (3H, s, H-30), 1.68 (1H, m, H-18), 1.67 (1H, m, H-5), 1.54 and 1.33 (1H
each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24
(1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H
each, m, H-11), 1.15 and 1.09 (3H each, s, H-23 and H-24), 1.03 (3H, s, H-27), 0.99 (3H, s, H-
26), 0.81 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 208.14, 181.97, 150.58, 137.70, 132.71,
130.88, 129.66, 127.80, 125.63, 109.71, 56.45, 52.50, 49.16, 48.61, 46.85, 45.24, 44.96, 42.51,
40.50, 38.48, 37.04, 36.21, 33.02, 32.06, 30.62, 29.68 × 2, 25.69, 22.18, 21.81, 20.41, 19.52,
16.13, 15.43, 14.64. HRMS m/z calcd for C35H47O3S [M - H]- 547.3251, found 547.3276.
4.1.3.9. Synthesis of 2-(pyridin-3-ylmethylene)betulonic acid (15i). The title compound prepared
by the reaction of betulonic acid (100 mg, 0.22 mmol) and 3-pyridinecarboxaldehyde (35.31 mg,
0.33 mmol) as per the method described in Section 4.1.3 to furnish 15i colourless solid (108 mg,
1
90 % yield). H NMR (400 MHz, CDCl3): δ 8.67 (1H, s, Ar-CH), 8.54 (1H, d, J = 4.0 Hz, Ar-
CH), 7.73 (1H, d, J = 8.0 Hz), 7.39 (1H, s, -CH=C-CO), 7.37 (1H, m, Ar-CH), 4.73 and 4.61 (1H
each, s, H-29), 3.02 (1H, m, H-19), 2.97 and 2.21 (1H each, m, H-1), 2.3 and 2.01 (1H each, m,
H-22), 2.27 and 1.98 (1H each, m, H-16), 1.96 (1H, m, H-13), 1.7 (3H, s, H-30), 1.61 (1H, m, H-
18), 1.64 (1H, m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48
and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-
12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.13 and 1.11 (3H each, s, H-23 and
H-24), 1.0 (3H, s, H-27), 0.96 (3H, s, H-26), 0.76 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ
207.76, 180.89, 150.60, 150.47, 148.30, 137.42, 136.78, 133.09, 132.13, 123.65, 109.70, 56.31,
52.78, 49.57, 48.41, 46.87, 45.26, 44.49, 42.52, 40.51, 38.35, 37.10, 36.52, 33.03, 32.17, 30.58,
18

ACCEPTED MANUSCRIPT
29.70, 29.40, 25.57, 22.34, 21.68, 20.33, 19.42, 15.85, 15.49, 14.58. HRMS m/z calcd for
C36H48NO3 [M - H]– 542.364, found 542.3681.
4.1.3.10. Synthesis of 2-(3-bromo-4-fluorobenzylidene)betulonic acid (15j). The title compound
prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and 3-bromo-4-
fluorobenzaldehyde (67 mg, 0.33 mmol) as per the method described in Section 4.1.3 to furnish
15j colourless solid (126 mg, 90 % yield). 1H NMR (400 MHz, CDCl3): δ 7.58 (1H, m, Ar-CH),
7.36 (1H, s, -CH=C-CO), 7.32 (1H, m, Ar-CH), 7.16 (1H, m, Ar-CH), 4.76 and 4.65 (1H each, s,
H-29), 3.01 (1H, m, H-19), 2.95 and 2.15 (1H each, m, H-1), 2.28 and 2.01 (1H each, m, H-22),
2.22 and 1.98 (1H each, m, H-16), 1.96 (1H, m, H-13), 1.72 (3H, s, H-30), 1.67 (1H, m, H-18),
01.64 (1H, m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48
and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-
12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.14 and 1.12 (3H each, s, H-23 and
H-24), 1.02 (3H, s, H-27), 0.98 (3H, s, H-26), 0.78 (3H, s, H-25).13C NMR (125 MHz, CDCl3):
δ 207.78, 181.91, 159.96, 150.36, 135.45, 135.32, 134.71, 133.69, 130.14, 116.59, 109.75,
109.34, 56.44, 52.92, 49.19, 48.42, 46.84, 45.29, 44.10, 42.54, 40.56, 38.44, 37.02, 36.63, 33.05,
32.06, 30.62, 29.69, 29.36, 25.54, 22.33, 21.67, 20.32, 19.50,15.81, 15.50, 14.63. HRMS m/z
calcd for C37H48BrFO3 [M - H]- 639.2844, found 639.2838.
4.1.3.11. Synthesis of 2-(5-bromo-2-methoxybenzylidene)betulonic acid (15k). The title
compound prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and 5-bromo-2-
methoxybenzaldehyde (71 mg, 0.33 mmol) as per the method described in Section 4.1.3 to
furnish 15k colourless solid (130 mg, 92 % yield). 1H NMR (400 MHz, CDCl3): δ 7.57 (1H, s, -
CH=C-CO), 7.37 (2H, m, 2 × Ar-CH), 6.76 (1H, m, Ar-CH), 4.74 and 4.62 (1H each, s, H-29),
3.83 (3H, s, -OCH3), 3.0 (1H, m, H-19), 2.89 and 2.03 (1H each, m, H-1), 2.28 and 1.99 (1H
19

ACCEPTED MANUSCRIPT
each, m, H-22), 2.22 and 1.95 (1H each, m, H-16), 1.93 (1H, m, H-13), 1.7 (3H, s, H-30), 1.66
(1H, m, H-18), 1.62 (1H, m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m,
H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H
each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.14 and 1.12 (3H each, s,
13
H-23 and H-24), 1.01 (3H, s, H-27), 0.97 (3H, s, H-26), 0.8 (3H, s, H-25). C NMR (125 MHz,
CDCl3): δ 207.68, 182.27, 157.19, 150.24, 135.20, 132.18, 131.33, 131.23, 127.14, 112.41,
111.88, 109.84, 56.45, 55.76, 53.24, 49.14, 48.23, 46.87, 45.47, 43.43, 42.51, 40.55, 38.40,
37.03, 36.80, 33.13, 32.08, 30.55, 29.71, 29.18, 25.42, 22.59, 21.44, 20.24, 19.48, 15.69, 15.57,
14.64. HRMS m/z calcd for C38H52BrO4 [M + H]+ 651.3043, found 651.3051.
4.1.3.12. Synthesis of 2-(4-fluorobenzylidene)betulonic acid (15l). The title compound prepared
by the reaction of betulonic acid (100 mg, 0.22 mmol) and 4-fluorobenzaldehyde (41 mg, 0.33
mmol) as per the method described in Section 4.1.3 to furnish 15l colourless solid (111 mg, 90 %
1
yield). H NMR (400 MHz, CDCl3): 7.56 (1H, s, -CH=C-CO), 7.32 (2H, m, 2 × Ar-CH), 7.17
(1H, t, J = 8.0 Hz, Ar-CH), 7.09 (1H, t, J = 8.0 Hz, Ar-CH), 4.74 and 4.63 (1H each, s, H-29),
3.0 (1H, m, H-19), 2.91 and 2.1 (1H each, m, H-1), 2.29 and 1.99 (1H each, m, H-16), 2.22 and
1.97 (1H each, m, H-1), 1.90 (1H, m, H-13), 1.71 (3H, s, H-30), 1.66 (1H, m, H-18), 1.62 (1H,
m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H
each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H,
m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.15 and 1.14 (3H each, s, H-23 and H-24), 1.01
(3H, s, H-27), 0.97 (3H, s, H-26), 0.79 (3H, s, H-25). δ 13C NMR (125 MHz, CDCl3): δ 207.82,
182.16, 161.89, 150.58, 136.25, 130.10, 123.87 × 2, 122.79, 115.87, 115.69, 109.68, 56.43,
53.48, 53.01, 49.11, 48.34, 46.83, 45.41, 43.97, 42.50, 40.52, 38.41, 37.03, 33.09, 32.04, 30.57,
20

ACCEPTED MANUSCRIPT
29.66, 29.22, 25.53, 22.46, 21.53, 20.27, 19.47, 15.79, 15.52, 14.61. HRMS m/z calcd for
C37H48FO3 [M - H]– 559.3593, found 559.3637.
4.1.3.13. Synthesis of 2-((E)-3-phenylallylidene)betulonic acid (15m). The title compound
prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and cinnamaldehyde (43.5 mg,
0.33 mmol) as per the method described in Section 4.1.3 to furnish 15m colourless solid (113
mg, 90 % yield). 1H NMR (400 MHz, CDCl3): δ 7.49 (2H, m, 2 × Ar-CH ), 7.32 (3H, m, 3 × Ar-
CH), 7.23 (1H, d, J = 12.0 Hz, -CO-C=CH-), 6.93 (2H, m, 2 × Ar-CH=CH-), 4.79 and 4.67 (1H
each, s, H-29), 3.06 (1H, m, H-19), 2.98 and 2.07 (1H each, m, H-1), 2.33 and 2.01 (1H each, m,
H-22), 2.28 and 1.98 (1H each, m, H-16), 1.96 (1H, m, H-13), 1.74 (3H, s, H-30), 1.67 (1H, m,
H-18), 1.65 (1H, m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15),
1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m,
H-12), 1.38 (1H, m, H-9), 1.35 and 0.97 (1H each, m, H-11), 1.11 and 1.09 (3H each, s, H-23
13
and H-24), 1.03 (3H, s, H-26), 1.0 (3H, s, H-24), 0.82 (3H, s, H-25). C NMR (125 MHz,
CDCl3): δ 207.50, 182.52, 150.58, 140.66, 137.26, 136.70, 133.07, 128.77 × 3, 127.18 × 2,
123.35, 109.74, 56.48, 52.89, 49.17, 48.39, 46.90, 45.07, 42.72, 42.54, 40.53, 38.56, 37.08,
36.20, 33.17, 32.09, 30.58, 29.69, 29.41, 25.67, 22.35, 21.71, 20.33, 19.48, 15.91, 15.57, 14.62.
HRMS m/z calcd for C39H53O3 [M + H]+ 569.3989, found 569.3985.
4.1.3.14. Synthesis of 2-(4-methoxybenzylidene)betulonic acid (15n). The title compound
prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and 4-methoxybenzaldehyde (45
mg, 0.33 mmol) as per the method described in Section 4.1.3 to furnish 15n colourless solid (113
mg, 90 % yield). 1H NMR (400 MHz, CDCl3): δ 7.46 (1H, -CH=C-CO), 7.41 (2H, d, J = 8.0 Hz,
2 × Ar-CH), 6.95 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 4.77 and 4.66 (1H each, s, H-29), 3.85 (3H, s, -
OCH3), 3.06 (1H, m, H-19), 3.02 and 2.2 (1H each, m, H-1), 2.31 and 2.01 (1H each, m, H-22),
21

ACCEPTED MANUSCRIPT
2.28 and 1.98 (1H each, m, H-16), 1.96 (1H, m, H-13), 1.74 (3H, s, H-30), 1.68 (1H, m, H-18),
1.65 (1H, m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and
1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H each, m, H-12),
1.38 (1H, m, H-9), 1.35 and 0.97 (1H each, m, H-11), 1.14 and 1.11 (3H each, s, H-23 and H-
24), 1.03 (3H, s, H-27), 0.98 (3H, s, H-26), 0.79 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ
208.13, 181.36, 159.82, 150.67, 137.31, 132.26 × 2, 131.96, 128.66, 114.01 × 2, 109.64, 56.42,
55.34, 52.63, 49.17, 48.51, 46.83, 45.01, 44.68, 42.51, 40.52, 38.45, 37.03, 36.40, 33.06, 32.06,
30.62, 29.68, 29.63, 25.66, 22.33, 21.69, 20.39, 19.54, 15.88, 15.48, 14.63. HRMS m/z calcd for
C38H53O4 [M + H]+ 573.3938, found 573.3953.
4.1.3.15. Synthesis of 2-(naphthalene-1-ylmethylene)betulonic acid (15o). The title compound
prepared by the reaction of betulonic acid (100 mg, 0.22 mmol) and 1-naphthaldehyde (51.5 mg,
0.33 mmol) as per the method described in Section 4.1.3 to furnish 15o colourless solid (118 mg,
1
91 % yield). H NMR (400 MHz, CDCl3): δ 8.08 (1H, -CH=C-CO), 7.94 (1H, m, Ar-CH), 7.86
(2H, m, 2 × Ar-CH), 7.51 (3H, m, 3 × Ar-CH), 7.34 (1H, d, J = 8.0Hz, Ar-CH), 4.69 and 4.58
(1H each, s, H-29), 2.97 (1H, m, H-19), 2.91 and 2.04 (1H each, m, H-1), 2.26 and 1.97 (1H
each, m, H-22), 2.18 and 1.94 (1H each, m, H-16), 1.93 (1H, m, H-13), 1.66 (3H, s, H-30), 1.59
(1H, m, H-18), 1.58 (1H, m, H-5), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m,
H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H
each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 0.97 (1H each, m, H-11), 1.22 and 1.18 (3H each, s,
H-23 and H-24), 0.97 (3H, s, H-27), 0.94 (3H, s, H-26), 0.79 (3H, s, H-25). 13C NMR (125 MHz,
CDCl3): δ 207.95, 181.41, 150.41, 136.02, 135.95, 133.51, 133.13, 132.0, 128.62, 128.54,
126.48, 126.37, 126.12, 125.17, 124.68, 109.66, 56.36, 53.42, 49.13, 48.27, 46.79, 45.63, 43.52,
22

ACCEPTED MANUSCRIPT
42.49, 40.53, 38.40, 37.0, 36.76, 33.21, 32.05, 30.56, 29.65, 29.14, 25.46, 22.64, 21.39, 20.27,
19.42, 15.64, 15.56, 14.58. HRMS m/z calcd for C41H51O3 [M - H]- 591.3844, found 591.3849.
4.1.4. General experimental procedure for synthesis of benzylidene derivatives 16a-o
Compounds 16a-o were prepared by addition of NaBH4 (1.5 equiv) to stirring methanolic
solution of corresponding benzylidene derivative 15a-o (1 equiv) at 0oC. After 10 min, reaction
mixture was stirred at room temperature for 1-1.5 h till the completion (monitored by TLC
analysis) [32]. Workup of the reaction was done by diluting the reaction mixture with ice-cold
water and extracting it with ethyl acetate (3 times). The combined organic layers were dried over
sodium sulphate and concentrated on rotavapour. The crude product obtained was purified by
column chromatography on silica gel 60-120 mesh with EtoAc: Hexane (1: 10) as the eluent and
recrystallized with hot methanol to afford the desired pure products 16a-o in 70-75% yield. The
spectral data of all the derivatives are given below.
4.1.4.1. Synthesis of 2-(3-nitrobenzylidene)betulinic acid (16a). 1H NMR (400 MHz, CDCl3): δ
7.99 (2H, m, 2 × Ar-CH), 7.39 (2H, m, 2 × Ar-CH), 6.67 (1H, s, -CH=C-CHOH), 4.63 and 4.51
(1H each, s, H-29), 3.81 (1H, s, -CHOH), 2.89 (1H, m, H-19), 2.8 and 1.51 (1H each, m, H-1),
2.19 and 1.89 (1H each, m, H-22), 2.07 and 1.87 (1H each, m, H-16), 1.85 (1H, m, H-13), 1.6
(3H, s, H-30), 1.58 (1H, m, H-18), 1.56 and 1.33 (1H each, m, H-6), 1.52 and 1.29 (1H each, m,
H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H
each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.2 (1H each, m, H-11), 1.06 (3H, s, H-26), 0.91 and
13
0.8 (3H each, s, H-27 and H-23), 0.67 (3H, s, H-24), 0.77 (1H, m, H-5), 0.62 (3H, s, H-25). C
NMR (125 MHz, CDCl3): δ 182.3, 150.24, 148.2, 143.42, 139.89, 134.96, 129.01, 123.52,
120.99, 120.73, 109.77, 81.0, 56.36, 55.89, 49.78, 49.21, 46.89, 42.49, 41.88, 41.84, 40.91,
23

ACCEPTED MANUSCRIPT
40.64, 38.35, 37.04, 34.1, 32.14, 30.56, 29.69, 28.55, 25.37, 20.95, 19.37, 18.38, 16.29, 15.9,
15.53, 14.66. HRMS m/z calcd for C37H50NO5 [M - H]– 588.3694, found 588.3699.
4.1.4.2. Synthesis of 2-(4-bromobenzylidene)betulinic acid (16b). 1H NMR (400 MHz, CDCl3): δ
7.34 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 6.98 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 6.55 (1H, s, -CH=C-
CHOH), 4.66 and 4.53 (1H each, s, H-29), 3.77 (1H, s, -CHOH), 2.91 (1H, m, H-19), 2.83 and
1.47 (1H each, m, H-1), 2.19 and 1.92 (1H each, m, H-22), 2.1 and 1.88 (1H each, m, H-16),
1.87 (1H, m, H-13), 1.61 (3H, s, H-30), 1.56 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-6),
1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m,
H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.2 (1H each, m, H-11),
1.05 (3H, s, H-26), 0.92 and 0.81 (3H each, s, H-27 and H-23), 0.66 (3H, s, H-24), 0.69 (1H, m,
H-5), 0.59 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 180.42, 149.86, 140.89, 136.51, 130.73
× 2, 130.01 × 2, 121.09, 119.36, 109.29, 80.58, 55.78, 55.37, 49.30, 48.71, 46.39, 41.98, 41.46,
41.13, 40.40, 39.89, 37.86, 36.54, 33.63, 31.62, 30.01, 29.18, 28.03, 24.89, 20.54, 18.84, 17.90,
15.71, 15.37, 15.03, 14.19. HRMS m/z calcd for C37H50BrO3 [M - H]- 621.2949, found 621.2979.
4.1.4.3. Synthesis of 2-(4-nitrobenzylidene)betulinic acid (16c). 1H NMR (400 MHz, CDCl3): δ
8.12 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 7.27 (2H, d, J = 8.0 Hz, 2 × Ar-CH), 6.71 (1H, s, -CH=C-
CHOH), 4.75 and 4.65 (1H each, s, H-29), 3.83 (1H, s, -CHOH), 2.09 (1H, m, H-19), 2.84 and
1.55 (1H each, m, H-1), 2.22 and 1.91 (1H each, m, H-22), 2.09 and 1.89 (1H each, m, H-16),
1.88 (1H, m, H-13), 1.61 (3H, s, H-30), 1.54 (1H, m, H-18), 1.52 and 1.33 (1H each, m, H-6),
1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m,
H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 0.77 (1H each, m, H-11),
1.08 (3H, s, H-26), 0.93 and 0.81 (3H each, s, H-27 and H-23), 0.71 (1H, m, H-5), 0.68 (3H, s,
H-24), 0.59 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 181.85, 150.29, 145.96, 145.38,
24

ACCEPTED MANUSCRIPT
144.42, 129.51 × 2, 123.58 × 2, 121.35, 109.85, 81.01, 56.30, 55.77, 53.48, 49.79, 49.17, 46.89,
42.48, 41.93, 40.9, 40.7, 38.34, 37.04, 34.06, 32.10, 30.49, 29.71, 28.58, 25.31, 21.05, 19.35,
18.40, 16.23, 15.86, 15.58, 14.68. HRMS m/z calcd for C37H50NO5 [M - H]– 588.3694, found
588.3717.
4.1.4.4. Synthesis of 2-(3-bromobenzylidene)betulinic acid (16d). 1H NMR (400 MHz, CDCl3): δ
7.32 (2H, m, 2 × Ar-CH), 7.14 (2H, m, 2 × Ar-CH), 6.63 (1H, s, -CH=C-CHOH), 4.73 and 4.6
(1H each, s, H-29), 3.84 (1H, s, -CHOH), 2.92 (1H, m, H-19), 2.8 and 1.56 (1H each, m, H-1),
2.28 and 1.97 (1H each, m, H-22), 2.25 and 1.95 (1H each, m, H-16), 1.93 (1H, m, H-13), 1.68
(3H, s, H-30), 1.6 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H each, m,
H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and 1.25 (1H
each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.12 (3H, s, H-26), 0.99
and 0.9 (3H each, s, H-27 and H-23), 0.73 (3H, s, H-24), 0.81 (1H, m, H-5), 0.69 (3H, s, H-25).
13C NMR (125 MHz, CDCl3): δ 179.06, 150.58, 142.13, 140.67, 131.55, 129.54, 128.71, 127.32,
121.96, 121.31, 109.34, 80.56, 56.14, 55.98, 49.80, 49.06, 46.96, 42.42, 41.89, 41.5, 40.81,
40.32, 38.21, 37.02, 34.09, 32.15, 30.43, 29.58, 28.15, 25.42, 20.93, 18.94, 18.33, 16.07, 15.55,
15.43, 14.41. HRMS m/z calcd for C37H50BrO3 [M - H]- 621.2949, found 621.2974.
1
4.1.4.5. Synthesis of 2-(2, 6-dichlorobenzylidene)betulinic acid (16e). H NMR (400 MHz,
CDCl3): δ 7.28 (2H, m, 2 × Ar-CH), 7.1 (1H, t, J = 8.0 Hz, Ar-CH), 6.42 (1H, s, -CH=C-
CHOH), 4.69 and 4.59 (1H each, s, H-29), 3.92 (1H, s, -CHOH), 2.96 (1H, m, H-19), 2.25 and
1.48 (1H each, m, H-1), 2.12 and 1.95 (1H each, m, H-22), 2.06 and 1.92 (1H each, m, H-16),
1.91 (1H, m, H-13), 1.67 (3H, s, H-30), 1.56 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-6),
1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m,
H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.03 (1H each, m, H-11),
25

ACCEPTED MANUSCRIPT
1.14 (3H, s, H-26), 0.97 and 0.81 (3H each, s, H-27 and H-23), 0.88 (1H, m, H-5), 0.81 (3H, s,
H-24), 0.56 (3H, s, H-25).13C NMR (125 MHz, CDCl3): δ 182.33, 150.58, 144.25, 135.7 × 2,
135.51, 128.15 × 2, 127.95, 117.67, 109.64, 80.77, 56.40, 55.72, 49.80, 49.17, 46.93, 43.85,
42.41, 41.78, 40.79, 39.94, 38.32, 37.03, 34.06, 32.12, 30.54, 29.68, 28.36, 25.32, 20.84, 19.35,
18.47, 15.78, 15.71, 15.52, 14.67. HRMS m/z calcd for C37H49Cl2O3 [M - H]- 611.3064, found
611.3089.
4.1.4.6. Synthesis of 2-{(5-bromofuran-2-yl)methylene}betulinic acid (16f). 1H NMR (400 MHz,
CDCl3): δ 6.37 (1H, s, -CH=C-CHOH), 6.26 (1H, d, J = 4.0 Hz, Ar-CH), 6.17 (1H, d, J = 4.0 Hz,
Ar-CH), 4.76 and 4.63 (1H each, s, H-29), 3.79 (1H, s, -CHOH), 3.03 (1H, m, H-19), 3.37 and
1.73 (1H each, m, H-1), 2.3 and 1.98 (1H each, m, H-22), 2.26 and 1.96 (1H each, m, H-16),
1.94 (1H, m, H-13), 1.71 (3H, s, H-30), 1.68 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-6),
1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m,
H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11),
1.1 (3H, s, H-26), 1.02 and 0.94 (3H each, s, H-27 and H-23), 0.76 (3H, s, H-24), 0.81 (1H, m,
H-5), 0.66 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 182.79, 155.32, 150.40, 140.89,
119.94, 112.54, 110.57, 110.54, 109.77, 81.17, 56.45, 56.26, 49.75, 49.21, 46.92, 42.84, 42.51,
42.40, 41.03, 40.98, 38.46, 37.08, 34.21, 32.16, 30.59, 29.75, 28.43, 25.55, 21.0, 19.44, 18.45,
15.98, 15.89, 15.37, 14.74. HRMS m/z calcd for C35H46BrO4 [M - H]- 611.2571, found 611.2621.
4.1.4.7. Synthesis of 2-(2-bromobenzylidene)betulinic acid (16g). 1H NMR (400 MHz, CDCl3): δ
7.55 (1H, m, Ar-CH), 7.24 (1H, d, J = 8.0 Hz, Ar-CH), 7.1 (2H, m, 2 × Ar-CH), 6.62 (1H, s, -
CH=C-CHOH), 4.7 and 4.59 (1H each, s, H-29), 3.89 (1H, s, -CHOH), 2.96 (1H, m, H-19), 2.66
and 1.54 (1H each, m, H-1), 2.23 and 1.95 (1H each, m, H-22), 2.14 and 1.93 (1H each, m, H-
16), 1.92 (1H, m, H-13), 1.66 (3H, s, H-30), 1.61 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-
26

ACCEPTED MANUSCRIPT
6), 1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each,
m, H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.06 (1H each, m, H-
11), 1.13 (3H, s, H-26), 0.97 and 0.84 (3H each, s, H-27 and H-23), 0.8 (3H, s, H-24), 0.88 (1H,
13
m, H-5), 0.6 (3H, s, H-25). C NMR (125 MHz, CDCl3): δ 181.03, 149.45, 140.36, 137.35,
131.38, 129.86, 126.8, 125.81, 123.49, 121.9, 108.63, 79.99, 55.33, 54.83, 48.84, 48.19, 45.88,
41.42, 41.31, 40.71, 39.84, 39.29, 37.36, 36.0, 33.11, 31.11, 29.51, 28.65, 27.43, 24.37, 19.92,
18.32, 17.40, 14.81 × 2, 14.44, 13.65. HRMS m/z calcd for C37H50BrO3 [M - H]- 621.2949, found
621.2954.
1
4.1.4.8. Synthesis of 2-(thiophen-3-ylmethylene)betulinic acid (16h). H NMR (400 MHz,
CDCl3): δ 7.25 (1H, m, Ar-CH), 7.05 (2H, m, 2 × Ar-CH), 6.59 (1H, s, -CH=C-CHOH), 4.74
and 4.61 (1H each, s, H-29), 3.81 (1H, s, -CHOH), 3.13 and 1.64 (1H each, m, H-1), 3.0 (1H, m,
H-19), 2.29 and 1.98 (1H each, m, H-22), 2.26 and 1.96 (1H each, m, H-16), 1.94 (1H, m, H-13),
1.68 (3H, s, H-30), 1.61 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H
each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and
1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.11 (3H, s, H-
26), 1.0 and 0.91 (3H each, s, H-27 and H-23), 0.76 (3H, s, H-24), 0.83 (1H, m, H-5), 0.7 (3H, s,
H-25). 13C NMR (125 MHz, CDCl3): δ 182.44, 150.37, 140.1, 138.6, 128.76, 124.83, 121.77,
116.79, 109.76, 81.3, 56.4, 56.21, 49.76, 49.29, 46.98, 42.51, 41.81, 40.96, 40.53, 39.68, 38.48,
37.07, 34.22, 32.19, 30.59, 29.74, 28.47, 25.5, 21.14, 19.36, 18.42, 16.24, 15.92, 15.46, 14.74.
HRMS m/z calcd for C35H49O3S [M - H]– 549.3408, found 549.3409.
4.1.4.9. Synthesis of 2-(pyridin-3-ylmethylene)betulinic acid (16i). 1H NMR (400 MHz, CDCl3):
δ 8.44 (2H, m, 2 × Ar-CH), 7.52 (1H, d, J = 8.0 Hz, Ar-CH), 7.27 (1H, m, Ar-CH), 6.68 (1H, s, -
CH=C-CHOH), 4.72 and 4.58 (1H each, s, H-29), 3.88 (1H, s, -CHOH), 3.04 (1H, m, H-19), 2.8
27

ACCEPTED MANUSCRIPT
and 1.54 (1H each, m, H-1), 2.3 and 2.01 (1H each, m, H-22), 2.18 and 1.98 (1H each, m, H-16),
1.96 (1H, m, H-13), 1.68 (3H, s, H-30), 1.61 (1H, m, H-18), 1.56 and 1.33 (1H each, m, H-6),
1.51 and 1.29 (1H each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m,
H-21), 1.43 and 1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11),
1.13 (3H, s, H-26), 1.13 and 0.98 (3H each, s, H-27 and H-23), 0.85 (3H, s, H-24), 0.89 (1H, m,
H-5), 0.74 (3H, s, H-25). 13C NMR (125 MHz, CDCl3): δ 180.53, 150.71, 148.76, 146.04,
143.73, 136.94, 134.44, 123.43, 118.89, 109.57, 80.94, 56.28, 55.83, 49.81, 49.32, 47.04, 42.49,
41.93, 41.79, 40.89, 40.56, 38.41, 37.12, 34.12, 32.42, 30.71, 29.75, 28.55, 25.4, 21.02, 19.41,
18.41, 16.27, 15.92, 15.56, 14.70. HRMS m/z calcd for C36H52NO3 [M + H]+ 546.3942, found
546.3957.
4.1.4.10. Synthesis of 2-(3-bromo-4-fluorobenzylidene)betulinic acid (16j). 1H NMR (400 MHz,
CDCl3): δ 7.37 (1H, m, Ar-CH), 7.06 (2H, m, 2 × Ar-CH), 6.6 (1H, s, -CH=C-CHOH), 4.73 and
4.6 (1H each, s, H-29), 3.83 (1H, s, -CHOH), 2.99 (1H, m, H-19), 2.83 and 1.5 (1H each, m, H-
1), 2.26 and 1.97 (1H each, m, H-22), 2.15 and 1.95 (1H each, m, H-16), 1.94 (1H, m, H-13),
1.68 (3H, s, H-30), 1.65 (1H, m, H-18), 1.54 and 1.33 (1H each, m, H-6), 1.51 and 1.29 (1H
each, m, H-15), 1.48 and 1.41 (1H each, m, H-7), 1.46 and 1.24 (1H each, m, H-21), 1.43 and
1.25 (1H each, m, H-12), 1.38 (1H, m, H-9), 1.35 and 1.08 (1H each, m, H-11), 1.12 (3H, s, H-
26), 0.98 and 0.89 (3H each, s, H-27 and H-23), 0.72 (3H, s, H-24), 0.85 (1H, m, H-5), 0.67
13
(3H, s, H-25). C NMR (125 MHz, CDCl3): δ 179.23, 158.26, 150.62, 141.98, 135.85, 133.51,
129.17, 120.42, 116.02, 109.5, 108.49, 80.67, 56.14, 55.8, 49.74, 49.08, 46.89, 42.41, 41.81,
41.55, 40.81, 40.36, 38.16, 37.06, 34.06, 32.16, 30.47, 29.6, 28.34, 25.37, 20.95, 19.22, 18.34,
16.19, 15.69, 15.5, 14.56. HRMS m/z calcd for C37H49BrFO3 [M - H]- 639.2855, found 639.2883.
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