Synthesis, characterization and cytotoxic activity of betulinic acid and Sec -betulinic acid derivatives against human colorectal carcinoma

Article abstract of DOI:10.13005/ojc/330128

Different derivatives based on the betulinic acid triterpenoidal scaffold were synthesized and characterized spectroscopically. The derivatives including synthetic intermediates were evaluated for their cytotoxic activity against the human colorectal carc

Full text of DOI:10.13005/ojc/330128

ISSN: 0970-020 X
CODEN: OJCHEG
017, Vol. 33, No. (1):
Pg. 242-248
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
2
www.orientjchem.org
Synthesis, Characterization and Cytotoxic Activity of Betulinic
Acid and Sec -Betulinic Acid Derivatives against Human
Colorectal Carcinoma
1
1
1
MOHD TAJUDIN MOHD ALI* , HABSAH ZAHARI , AISYAH ALIASAk ,
SIONꢀ MENꢀ LIM , kALAꢁATHY RAMASAMY and ALLAN PATRICk ꢀ. MACABEO4
2
,3
2,3
1
Faculty of Applied Sciences, Universiti Teknologi MARA(UiTM),40450, Shah Alam, Selangor, Malaysia.
2
Faculty of Pharmacy, Universiti Teknologi MARA (UiTM),
4
2300 Bandar Punck Alam, Selangor, Malaysia.
3
Collaborative Drug Discovery Research (CDDR) Group,Pharmaceutical and Life Community
of Research, Universiti Teknologi MARA, (UiTM),40450, Shah Alam, Selangor, Malaysia.
4
Laboratory for Organic Reactivity, Discovery and Synthesis (LORDS), Research Center for the Natural
and Applied Sciences, University of Santo Tomas, Espana Blvd., Manila 1015, Philippines.
*
Corresponding author E-mail: tajudinali@salam.uitm.edu.my
http://dx.doi.org/10.13005/ojc/330128
(Received: November 24, 2016; Accepted: January 05, 2016)
ABSTRACT
Different derivatives based on the betulinic acid triterpenoidal scaffold were synthesized and
characterized spectroscopically. The derivatives including synthetic intermediates were evaluated
for their cytotoxic activity against the human colorectal carcinoma cell line (HCTT111) using a
colorimetric sulforhodamine assay (SRB).Among the synthesized compounds, compound 6 featuring
a 20-keto seco-betulinic acid conjugated with a lactone benzyl ester through an amide linkage at
C-28, exhibited the lowest IC50 value.
keywords: Betulinic acid, Seco-betulinic acid, Lactone derivatives,
Ester derivatives, cytotoxicity, cancer.
1
INTRODUCTION
from plants in 1788 .It exhibits a number of biological
activities including antitumor properties versus a
panel of cancer cell lines. While previous studies
suggest that betulinic acid is selectively cytotoxic
The pentacyclic lupane triterpenoid
betulinic acid, 1 (3b, hydroxy-lup-20(29)-en-28-oic
acid) is one of the first natural products identified
2
against melanoma cell lines , it was also reported

243
ALI et al., Orient. J. Chem., Vol. 33(1), 242-248 (2017)
to exhibit activity against other types of human (quartet), m (multiplet), and br (broad). MS spectra
cancers including neuroblastoma, glioblastoma, were recorded on a Agilent GC–7890A MS 5975 and
medulloblastoma, Ewing tumor, leukemia as well as Agilent LCMS QTof 6520.TLCs and preparative thin-
several carcinoma, i.e.head and neck, colon, breast, layer chromatography were performed on silica gel
hepatocellular, lung, prostate, renal cell, ovarian or GF/UV 254, and the chromatograms were performed
cervix carcinoma.Mechanistic studies indicate that it on silica gel (100-200 mesh) visualized under UV
3
triggers the mitochondrial path to apoptosis , inhibits light at 254 nm and 365 nm. All solvents were of
aminopeptidase N, an enzyme that is involved in reagent grade and, when necessary, were purified
the regulation of angiogenesis and overexpressed and dried by standard methods. Concentration of
4
-6
in several cancers , modulate activity of the solutions after reactions and extractions involved the
transcription factor nuclear factor-kB (NF-kB), a key use of a rotary evaporator operating at a reduced
regulator of stress-induced transcriptional activation. pressure of ca. 20 Torr. Organic solutions were dried
Because it exhibits relative selective cytotoxicity over anhydrous sodium sulfate.
against malignant over normal cells, betulinic acid
is a promising anti-cancer agent.
Chemistry
In this work, a series of the new betulinic
Several derivatization studies have been acid and C-20 seco derivatives were synthesized and
reported for betulinic acid leading to the synthesis characterized spectroscopically. Subsequently, the
of a library of betulinic acid congeners. Structurally, cytotoxicity of the derivatives including intermediates
the triterpenoidal skeleton possesses three sites against human colorectal carcinoma cell line were
amenable for modification, including the C-3 explored, and the results are described below.
hydroxyl, C-20 alkene, and C-28 carboxylic acid
positions. While it holds a great potential to be Synthesis of compounds 4 and 8
developed as anti-tumor agent, it suffers from poor
To a solution of betulinic acid 1 (30.0 mg,
solubility in aqueous solutions and thus limiting its 0.07 mmol) in dry THF (2 mL) was added HOBt
7, 8
activity in vivo
.
(11.2 mg, 1.1 equiv.), HBTU (30.0 mg, 1.0 equiv.) at
°C and trimethylsiloxycyclohexene amine TFA salt
0
In the light of the purported potential 3 or benzyl-D-alanine salt (1.1 equiv.) was added
of betulinic acid and its derivatives as cancer next. The ice bath was removed and the reaction
chemopreventive agents, there exists a niche to mixture was stirred for 24 h at room temperature.The
develop new betulinic acid-inspired natural product solvent was removed by rotary evaporator and the
analogs that may present better cytotoxic activity. In reaction mixture was redissolved in EtOAc (5 mL),
this paper we disclose the synthesis and evaluation and washed with 1M HCl (5 mL), 5% NaHCO (5 mL)
3
of cytotoxicity versus human colorectal cancer cells and brine (5 mL). The organic layer was dried over
of new betulinic acid and C-20 seco derivatives in by Na SO and the solvent was evaporated under
2
4
vitro.
reduced pressure.The crude product was purified by
column chromatography on silica gel (hexane: ethyl
acetate, 15:1) to give products 4 as white solid or 8
EꢂPERIMENTAL
(
as white solid) in 69%or 85% yields, respectively.
Material
General. Infrared (IR) spectra (KBr) were
(1R,3aS,5aR,5bR,9S,11aR)-9-hydroxy-
recordedonaThermoNicoletImpact6700instrument 5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-
KBr pellet). 1H NMR spectra were recorded with a N-((1R,6R)-6-trimethylsilyloxy)cyclohex-3-enyl)
(
Bruker Ultra Shield 300 MHz spectrometer at 300 icosahydro-1H-cyclopenta[a]chrysene-3a-
K, using TMS as an internal standard. Chemical carboxamide (4).
shifts are expressed in parts per million (ppm, units).
Coupling constants are in units of hertz (Hz).The spin
multiplicities are indicated by the symbols s (singlet), Yields: 69%; m.p. 124- 126 C. H-NMR (300 MHz
Rƒ= 0.66 (SiO , hexane/ethylacetate 4:1);
2
o
1
,
d (doublet), dd (doublet of doublets), t (triplet), q CDCL ); d= 5.23-5.35 (m, 2H, CH-Olefin), 4.73
3

ALI et al., Orient. J. Chem., Vol. 33(1), 242-248 (2017)
244
-
1
(
1
(
2
brs,1H, H-29), 4.61 (brs,1H, H-29), 3.66-3.75 (m,
H, CHO), 3.53-3.65 ( m, 1H, CHN), 3.31-3.37
m,1H, C-OH), 3.17-3.20 (m, 1H, CH, H-19)), 2.26-
.55 (m, 2H, CH ), 1.96-2.11(m, 2H, CH ), 1.69
(C-25), 16.27 (C-24), 15.04 (C-27).IR (film) vmax cm
= 3410.4, 2924.01, 1690.3, 1166.3, 1591.1, 1241.7,
MS-ESI (Cl, NH ): m/z. Calculated for [C H NO ] :
3
40 59
4
+
617.4444, found 618.3316 [M + H ].
2
2
(
(
(
s,3H, CH ), 0.97 (s,3H, CH ), 0.96 (s,3H, CH ), 0.93
3
3
3
s,3H, CH ) , 0.82 (s,3H, CH ), 0.75 (s,3H, CH ), 0.0
Synthesis of compounds 5
3
3
3
s,9H, CH -OTMS)
To a solution of compound 4 (30 mg, 1
3
equiv.) in 4 ml of biphasic solution of CCl :MeCN:H O
4
2
1
3
C-NMR (75.5 MHz, CDCl ): d = 172.0
(1:1:2) in 50 ml round bottom flask was added
1.8 mg RuCl .3H O (8.3 % mol) at 0°C and the
3
(
C=O), 150.12 (C=C, C-20), 124.1- 124.6 (C=C,
3
2
Olefin), 107.42 (C=C, C-29), 83.71 (C-3), 78.8
mixture was stirred vigorously for 5 minutes. NaIO4
(70 mg, 4.1 equiv.) was added portion wise and the
reaction mixture was stirred for 8 h at 0-5°C. After
completion of the reaction, the mixture was diluted
with 10 ml of distilled water and extracted with 15
ml DCM (3x). The combined organic layer was
(
(
Cquart, cyclohexene), 78.9 (Cquart, cyclohexene), 57.34
C-17), 55.48 (C-5), 51.16 (C-9), 50.6 (CH, Olefin),
50.41 (C-18), 46.1 (C-H, Olefin), 45.40 (C-19), 42.49
(
(
C-14), 41.76 (C-8), 39.81 (C-1), 39.62 (C-4), 38.2
C-13), 36.41 (C-10), 36.39 (C-22), 34.24 (C-7),
3
1.80 (C-16), 30.72 (C-15), 32.9 (CH -Olefin), 29.9
dried over MgSO and the solvent removed using
2
4
(
(
(
1
6
CH -Olefin), 29.68 (C-21), 28.01 (C-23), 24.26
C-12), 23.55 (C-11), 22.61 (C-6), 19.7 (C-30), 19.31
C-2), 16.71 (C-26), 16.45 (C-25), 16.23 (C-24),
rotary evaporator. The crude product was purified
by column chromatography on silica gel (ethyl
acetate: methanol, 9:1) to furnish 5 as brown solid
in 55% yield.
2
5.05 (C-27), 0.00 (OTMS), MS [Cl, NH ]: m/z (%)
3
+
24.1 [M + H ], MS-ESI (Cl, NH ): Calculated for
3
[
C H NO Si]: 623.6473, found 623.6460.
2-((2S,3R)-3-((1R,3aS,5aR,9S,11aR)-
3
9
65
3
1
- a c e t y l - 9 - h y d r o x y - 5 a , 5 b , 8 , 8 , 1 1 a -
(
2R)-benzyl 2-1R,3aS,5aR,5bR,9S,11aR)-
pentamethylicosahydro-1H-cyclopenta[a]chrysene-
3a-carboxamido)-5-oxotetrahydrofuran-2-yl)acetic
acid (5).
9
-hydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-
en-2-yl)icosahydro-1H- cyclopenta[a]chrysene-3a-
carboxamido) propanoate (8).
Rf = 0.60 (SiO , hexane/ethylacetate 4:1); m.p.
2
o
Rf = 0.3 (SiO2, hexane/ethylacetate 4:1); m.p. 117-
151-153 C.
o
119 C.
1
1
H-NMR (300 MHz, CDCl ): H-NMR (300
3
,
1
H-NMR (300 MHz CDCL ); d= 8.11-8.21
MHz CDCL ); d= 4.93-5.03 (m,1H,CHN), 4.60-4.68
,
3
3
(
m,1H,NH), 7.31-7.48 (m, 5H, Bn), 5.01-5.28 (d,
(m, 1H, CHCH COOH), 3.30-3.36 (m,1H,CH-
2
2
H, J = 4 Hz, CH Bn), 4.62 (brs, 1H, H-29), 4.75
OH), 3.14-3.36 (m,1H,CH, H-19), 2.48-2.76 (m,
2H, CH CN), 2.30- 2.49 (m, 2H, CH -COO), 1.65
2
(
brs,1H, H-29), 4.11-4.48 (m, 1H, CHN), 2.96-3.01
2
2
(
m, 1H, CH-OH), 3.15-3.31 (m,1H,CHCH ), 1.70
(s,3H,CH ), 1.40 (s,3H, CH ), 1.25 (s, 3H, CH ), 1.12
3
3
3
3
(
m,3H, CH ), 1.42 (s,3H, CH ), 1.21 (s,3H, CH ),
(s, 3H, CH ), 0.97 (s,3H, CH ), 0.81 (s,3H, CH )
3 3 3
3
3
3
1
.10 (CH ), 0.97 (s,3H, CH ), 0.81 (s,3H, CH ), 0.69
3
3
3
1
3
(
s,3H, CH ),
C-NMR (75.5 MHz, CDCl ): d = 171.9
3
3
(
C=O), 169.6 (C=O), 149.71 (C=C, C-20)), 107.81
1
3
C-NMR (75.5 MHz, CDCl ): d = 175.8
(C=C, C-29), 83.53 (C-3), 78.8 (C-OH, lactone),
3
(
(
(
C=O), 172.0 (C=O), 150.9 (C=C, C-20), 136.4
Cquart), 128.7- 128.5 (Ph), 109.5 (C=C, C-29), 78.9
66.8 (CH , lactone), 56.95 (C-17), 55.35 (C-5), 50.51
(C-9), 49.65 (C-18), 45.88 (C-19), 42.36 (C-14),
2
C-3), 65.7 (CH Ph), 57.03 (C-17), 56.2 (CHN-ala),
40.78 (C-8), 38.7 (C-13), 38.88 (C-1), 38.7 (CH -CO,
2
2
5
4
3
3
5.42 (C-5), 51.05 (C-9), 50.48 (C-18), 45.40 (C-19),
2.40 (C-14), 41.60 (C-8), 39.81 (C-1), 39.88 (C-4),
8.5 (C-13), 35.41 (C-10), 36.30 (C-22), 34.24 (C-7),
1.80 (C-16), 30.62 (C-15), 29.38 (C-21), 28.2 (CH3-
lactone), 36.73 (C-10), 36.53 (C-22), 34.25 (CH2-
lactone), 34.20 (C-7), 31.82 (C-16), 30.23 (C-15),
29.68 (C-21), 28.09 (C-23), 26.73 (C-12), 23.54
(C-11), 22.67 (C-6), 19.5 (C-30), 18.27 (C-2), 16.13
(C-26), 16.08 (C-25), 15.35 (C-24), 14.78 (C-27).
ala), 28.21 (C-23), 24.46 (C-12), 23.54 (C-11), 22.61
C-6), 19.6 (C-30), 19.31 (C-2), 16.77 (C-26), 16.48
(

2
1
45
ALI et al., Orient. J. Chem., Vol. 33(1), 242-248 (2017)
-1
IR (film) vmax cm = 2978.69-2933, 3420.82, 1H-cyclopenta[a]chrysene-3a-carboxamido}-5-
714.86, 1255.07, 1499, 1367.79, 1738.28. MS oxotetrahydrofuran-2-yl)acetate (6).
+
[Cl, NH ]: m/z (%) 600.1 [M + H ], MS-ESI (Cl, NH ): Rf = 0.57 (SiO , hexane/ethylacetate 4:1);
3
3
2
1
m/z, Calculated for [C H NO ] : 599.3822, found
5
H-NMR (300 MHz CDCL ); d= 8.11-8.21 (d,
35
53
7
,
3
+
99.3460.38 [M ].
J= 4.2 Hz), 1H,NH), 7.27-7.33 (m, 5H, Bn), 5.15-5.18
(
d, 2H, J= 4 Hz, CH Bn), 4.90-4.95 (m, 1H, CHN),
2
Synthesis of compounds 6
4.69 (s,1H), 3.32-3.38 (m, 1H, CH-OH), 3.07-3.11
To a stirred solution of 5 (20 mg, 1 equiv.) in (m,1H,CH, H-19), 2.57-2.66 (m, 2H, CH CN), 2.44-
2
4
ml dry DMF was added NaHCO (28 mg, 6 equiv.), 2.56 (m, 2H, CH -COO) 1.75 (s,3H, CH ), 1.17 (s,3H,
3
2
3
followed by benzyl bromide (0.1 ml, 1.6 equiv.) CH ), 1.11 (s, 3H, CH ), 0.98 (s,3H, CH ), 0.85 (s,3H,
3
3
3
sequentially at room temperature. The reaction CH ), 0.66 (m,3H, CH ).
3
3
mixture was stirred for 24 hours at room temperature.
Then, the mixture was diluted with 4 ml EtOAc :
1
3
C-NMR (75.5 MHz, CDCl ): d = 177.2
3
H O (1:1) solution and the solution was extracted. (C=O), 173.3 (C=O), 149.78 (C=C, C-20), 132.4
2
The aqueous layer extracted with EtOAc and the (Cquart), 128.3- 128.5 (Ph), 107.21 (C=C, C-29), 81.22
combine organic layer was dried over by anhydrous (C-3), 78.5 (C-OH lactone), 68.2 (CH Ph), 56.67
2
MgSO4 and concentrated reduced pressure to (C-17), 55.60 (C-5), 53.7 (CHN- lactone), 52.34
give yellow crude oil which was purified by column (C-9), 50.58 (C-18), 45.93 (C-19), 43.21 (C-14),
chromatography on silica gel (hexane:ethyl acetate, 42.86 (C-8), 38.61 (C-1), 38.9 (C-13), 38.81 (C-4),
6
:4) to afford 6 as yellowish oil.
37.09 (C-10), 36.48 (C-H lactone), 36.41 (C-22),
4.44 (C-7), 31.91 (C-16), 29.68 (C-15), 29.11
2 - ( ( 2 S , 3 R ) - 3 - (C-21), 28.95 (C-23), 25.70 (C-12), 23.64 (C-11),
(1R,3aS,5aR,5bR,9S,11aR)-1-acetyl-9- 22.66 (C-6), 19.65 (C-2),19.5 (C-30), 16.66 (C-26),
hydroxy-5a,5b,8,8,11a-penamethylicosahydro- 16.42 (C-25), 16.21 (C-24), 14.07 (C-27).
3
B e n z y l
(
-1
IR (film) nmax cm = 2978.69-2933, 3420.82,
1
714.86, 1255.07, 1499, 1367.79, 1738.28. MS-ESI
H
29
H
(Cl, NH ):m/z Calculated for [C H NO ] :689.4292,
3 42 59 7
found 689.2452.
2
0
3
0
H C
3
Synthesis of compounds 7
21
9
1
2
2
To a stirred solution of 1 (100 mg, 1 equiv.) in
2
5
1
2
18
6
ml dry DMF was added NaHCO (110 mg, 6 equiv.),
CH31
1
13 17
14 16
3
26
COOH
8
followed by benzyl bromide (60.1 mg, 1.6 equiv.)
sequentially at room temperature. The reaction
mixture was stirred for 24 hours at room temperature.
9
2
1
5
1
4
1
0
8
2
3
CH
3
5
7
2
7
6
Then, the mixture was diluted with 4 ml EtOAc–H O
2
HO
H C
(
1:1) solution and the solution was extracted. The
3
CH
3
2
4
aqueous layer extracted with EtOAc and the combine
organic layer was dried over by anhydrous MgSO4
and concentrated reduced pressure to give crude
2
3
Fig. 1: Structure of betulinic acid
TFA.HN
BocHN
a
TMSO
TMSO
2
3
Reagents and conditions (a) TFA, DCM, 0°C, 3 hrs, quant
Scheme 1: Synthesis of trimethylsiloxy cyclohexene amine salt

ALI et al., Orient. J. Chem., Vol. 33(1), 242-248 (2017)
246
white solid product which was purified by column
chromatography on silica gel (n-hex–EtOAc, 6:4) to
afford 68% of a white solid 7.
(C-12), 23.66 (C-11), 21.94 (C-6), 19.66 (C-2), 19.6
(C-30), 16.15 (C-26), 16.11 (C-25), 15.35 (C-24),
+
14.84 (C-27). MS [Cl, NH ]: m/z (%) 547.3 [M + H ].
3
MS-ESI (Cl, NH ): m/z Calculated for [C H NO ] :
3
37 54
3
+
(
5
1R,3aS,5aR,5bR,9S,11aR)-benzyl 9-hydroxy-
a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)
546.4073, found 547.2991 [M + H]
icosahydro-1H-cyclopenta[a]chrysene-3a-
carboxylate (7)
Cytotoxicity Assay
HCT116 were seeded at 2,500 cells/ well
(96 well plate) and exposed to respective compounds
for 72 h before being subjected to fixation with 10%
cold TCA and staining with 0.4% sulforhodamine.
Plates were left to air-dry at room temperature and
bound SRB was solubilised in 10 mM Tris base
solution. The plate was read at 570nm using a
spectrophotometer. A dose-response curve was
plotted from which the IC50 value was determined.
IC50 was defined as concentration of compound that
corresponded to 50% cell viability when compared
to control.
Rƒ = 0.5 ( SiO‚ , hexane/ ethyl acetate),
o
m.p. 162-164 C. ¹H NMR ( 300 MHz, CDClƒ ) :
d = 7.30-7.50 ( m, 5H, BnH), 5.12 (d, J= 2.2 Hz,
CH Ph), 4.73(brs,1H, H-29), 4.61(brs,1H, H-29),
2
3
.26-3.32 (m, 1H, CHO), 3.03-3.15 ( m, 1H, CH,
H-19), 2.26-2.55 (m, 2H, CH ), 1.69 (s,3H, CH ),
2
3
0.97 (s,3H, CH ), 0.96 (s,3H, CH ), 0.93 (s,3H, CH ),
3 3 3
0
.82 (s,3H, CH ), 0.75 (s,3H, CH )
3
3
¹
³C NMR (75.5MHz, CDClƒ ) : d= 175.91
C=O), 150.20 (C=C, C-20), 136.51 (Cquart), 128.7-
28.2 ( Ph), 107.94 (C-29), 78.96 (C-3), 70.10
CH Ph), 57.04 (C-17), 55.40 (C-5), 50.59 (C-9),
(
1
(
RESULTS AND DISCUSSION
2
4
9.99 (C-18), 46.58 (C-19), 42.50 (C-14), 40.82
(
(
2
C-8), 38.88 (C-1), 38.7 (C-13), 38.60 (C-4), 37.22
C-10), 36.32 (C-22), 34.92 (C-7), 31.53 (C-16),
9.69 (C-15), 28.36 (C-21), 27.99 (C-23), 25.71
Scheme 1 shows the key starting materials
for the synthesis of betulinic-g-butyrolactone
derivatives. NBoc trimethyl siloxycyclohexene 2
H
N
a
b
C
COOH
O
TMSO
HO
TFA.HN
HO
1
TMSO
4
3
O
O
H
N
O
H
O
N
C
O
c
C
O
O
O
COOH
HO
C
HO
O
O
5
6
Reagents and conditions: a) dry DMF, BnBr, NaHCO , 24 hrs, rt. 68% (b) DCM, HOBt (1.1 equiv.), HBTU,
3
(
1 equiv.) DiPEA (1.1 equiv, 0°C), 85%
Scheme 2: Synthesis of betulinic acid-g-butyrolactone analogs

247
ALI et al., Orient. J. Chem., Vol. 33(1), 242-248 (2017)
underwent deprotection Boc reaction by using
trifluoro acetic acid (TFA, 30%) in DCM at 0 C to
reaction of trimethyl siloxycyclohexene scaffold,
the configuration of both asymmetric centre on the
butyrolactone skeleton is cis configuration . The
possibility of cis confirmation may be due to the
presence of ruthenium tetraoxide which could create
interactions on the similar face between nitrogen lone
pair to metal and chelation of metal and carboxylate
oxygen atom.
o
9
generate trimethyl siloxycyclohexene ammonium
salt 3 in quantitative yield. The target compound,
betulinic-g-butyrolactone ester 6, was synthesized
10
in three steps reaction starting from compound 3 .
The ammonium salt was incorporated with betulinic
acid using standard peptide coupling procedure to
afford betulinic-trimethylsiloxycyclohexene peptide
4
in 69% yield. Betulinic acid was preactivated by
Benzylic protection of carboxylic acid 5
was envisioned by treating 5 with benzyl bromide in
the present of sodium hydrogen carbonate as base
to give betulinic-g-butyrolactone ester 6. In addition,
betulinic acid underwent benzylation reaction after
being treated with benzyl bromide, BnBr to afford
7. Betulinic-D-alanine peptide could be synthesized
by applying peptide synthesis procedure furnished
coupling reagent, HOBt/ HBTU and in the presence
of strong base DIPEA.
Subsequently, peptide 4 underwent
lactonization by ruthenium tetraoxide, RuO in the
presence of sodium periodate as an oxidant to
give the corresponding betulinic-g-butyrolactone
carboxylic acid 5. The RuO catalytic oxidative
4
10
peptide 8 in a good yield .
4
system was generated in situ from 8.3 % of
RuCl .3H O and 4.1 equivalent of NaIO in biphasic
Cytotoxic Activity
3
2
4
solution of CCl :MeCN:H O with ratio of (1:1:2). The
The inhibitory activity of the synthesized
4
2
conversion of cyclohexene silyl ether with co-solvent
system was completed in 8 hours at 0°C.
compounds against HCT116 was evaluated by
cytotoxic assay. In general, compound 1 had
emerged as the potent anticancer agent, with an
IC50 of 3.0 µg/mL. This was followed by compound
7, 8, and 4, with IC50 value 25.33, 36.67 and 50.00
In accordance to previous study on
lactonisation reaction involving oxidative cleavage
O
C
a
1
O
HO
7
b
O
H
N
C
O
O
HO
8
Reagents and conditions (a) DCM, HOBt (1.1 equiv.), HBTU, (1 equiv.) DiPEA (1.1 equiv, 0°C), 69%. (b)
RuCl .3H O, NaIO , CCl :MeCN:H O, 8 hrs, 0°C, 55%. (c) dry DMF, BnBr, NaHCO , 24 hrs, rt, 72%
3
2
4
4
2
3
Scheme 3: Synthesis of betulinic acid ester and betulinic acid peptide

ALI et al., Orient. J. Chem., Vol. 33(1), 242-248 (2017)
248
µg/mL respectively. The IC50 of compound 5
and 7 were beyond the range of concentration
tested. Betulinic-g-butyrolactone carboxylic acid,
activities against a human colorectal carcinoma cell
lines (HCT116).The results of preliminary biological
activity showed that two synthesized compounds (6
and 8) possessed potential cytotoxicities. Further
structure exploration especially on the aromatic
butyrolactone ester side chains are in progress in
our lab.
5
yielded an IC50 > 100 µg/mL . Its benzyl ester
derivative 6, however, yielded IC50 at least four
times lower (IC50 value = 25.3 µg/mL). Eventhough
none of the compounds exhibited comparable
activity to that of positive control (5-FU), the results
showed that incorporation of betulinic acid with
g-butyrolactone ester moiety contributed to the
inhibition of HCT116.
ACkNOꢃLEDꢀEMENT
The authors are thankful to the Universiti
Teknologi MARA Malaysia (grant no:600-RMI/DANA
5/3/RIF 690/2012 and FRGS grant no: 600-RMI/
FRGS 5/3(3/2013) for financial support.Mohd Salleh
Rofie from Integrative Pharmacogenomic Institute
(i-PROMISE), Universiti Teknologi MARA for mass
analysis.
CONCLUSION
In summary, several novel of betulinic-g-
butyrolactone and betulinic acid derivatives were
synthesized and tested for their in vitro cytotoxic
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