Boc-lysinated-betulonic acid: A potent, anti-prostate cancer agent

Article abstract of DOI:10.1016/j.bmc.2006.05.048

Betulonic acid, derived from betulinol, a pentacyclic styrene, has shown a highly specific anti-prostate cancer activity in in vitro cell cultures. However, due to the lack of solubility of betulonic acid in aqueous medium, its potent anti-cancer activity in vivo has not been determined to the fullest extent. The present study describes the chemical synthesis of hydrophilic Boc-lysinated-betulonic acid, which has improved its solubility in an aqueous biocompatible solvent. Evaluation in cytotoxicity assays, Boc-lysinated-betulonic acid dissolved in phosphate-buffered saline (PBS) containing 22% ethanol and 4% human serum albumin, has shown 95.7% inhibition of LNCaP prostate cancer cells in culture after 72 h incubation at a concentration of 100 μM, but with little effect on normally proliferating fibroblast cells. In the in vivo assay, male athymic mice transplanted with human prostate LNCaP xenografts were injected with Boc-lysinated-betulonic acid intraperitoneally at a dose of 30 mg/kg daily for 17 days. The treated mice exhibited 92% inhibition of tumor growth as compared to controls. Histological sections of the tumors showed that Boc-lysinated-betulonic acid arrested mitosis and induced apoptosis, which was confirmed by TUNEL assay, Yo-Pro-1 staining, and the release of cleaved caspase-3 from the ex vivo in tumor culture. These studies, for the first time, demonstrate that a non-toxic hydrophilic lysinated derivative of betulonic acid and its solubility in a biocompatible aqueous medium has enhanced the bioavailability of the drug and has thus unleashed its full anti-prostate cancer activity.

Full text of DOI:10.1016/j.bmc.2006.05.048

Bioorganic & Medicinal Chemistry 14 (2006) 6349–6358
Boc-lysinated-betulonic acid: A potent, anti-prostate cancer agent
Brij B. Saxena,a,* Lei Zhu,b Meirong Hao,a Eileen Kisilis,a Meena Katdare,c
Ozgur Oktem,d Arkadiy Bomshteyna and Premila Rathnama
aDivision of Reproductive Endocrinology, Department of OB-GYN, Weill Medical College of Cornell University,
New York, NY 10021, USA
bMemorial Sloan-Kettering Cancer Institute, New York, NY 10021, USA
cDepartment of Surgery, Weill Medical College of Cornell University, New York, NY 10021, USA
dCenter for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY 10021, USA
Received 6 April 2006; revised 19 May 2006; accepted 23 May 2006
Available online 13 June 2006
Abstract—Betulonic acid, derived from betulinol, a pentacyclic styrene, has shown a highly specific anti-prostate cancer activity in
in vitro cell cultures. However, due to the lack of solubility of betulonic acid in aqueous medium, its potent anti-cancer activity
in vivo has not been determined to the fullest extent. The present study describes the chemical synthesis of hydrophilic Boc-lysinat-
ed-betulonic acid, which has improved its solubility in an aqueous biocompatible solvent. Evaluation in cytotoxicity assays,
Boc-lysinated-betulonic acid dissolved in phosphate-buffered saline (PBS) containing 22% ethanol and 4% human serum albumin,
has shown 95.7% inhibition of LNCaP prostate cancer cells in culture after 72 h incubation at a concentration of 100 lM, but with
little effect on normally proliferating fibroblast cells. In the in vivo assay, male athymic mice transplanted with human prostate
LNCaP xenografts were injected with Boc-lysinated-betulonic acid intraperitoneally at a dose of 30 mg/kg daily for 17 days. The
treated mice exhibited 92% inhibition of tumor growth as compared to controls. Histological sections of the tumors showed that
Boc-lysinated-betulonic acid arrested mitosis and induced apoptosis, which was confirmed by TUNEL assay, Yo-Pro-1 staining,
and the release of cleaved caspase-3 from the ex vivo in tumor culture. These studies, for the first time, demonstrate that a non-toxic
hydrophilic lysinated derivative of betulonic acid and its solubility in a biocompatible aqueous medium has enhanced the bioavail-
ability of the drug and has thus unleashed its full anti-prostate cancer activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The anti-cancer activity of terpenoids has been
described earlier.4–6 Pentacyclic styrenes have shown
anti-tumor activity against carcinosarcoma growth
and Epstein-Barr virus in lymphoblastoid Raji cells7,8
as well as in nasopharynx carcinosarcoma.9 The in vivo
anti-cancer activity of terpenoids was also shown
against Walker-256 carcinosarcoma, as tested in mice
and rats.10 It is noteworthy that betulinol, a pentacy-
clic alcohol of the group of styrenes as well as its
other derivatives, has shown minimal cytotoxic effect
on normally proliferating fibroblast cells and on
non-target tissues.11
Prostate cancer is the most frequently diagnosed can-
cer and is a leading cause of cancer morbidity and
mortality in men in the United States with an esti-
mated incidence of more than 916 new cases and
115 deaths per day.1 The use of chemotherapeutic
agents in the treatment of cancer has become a wide-
ly used practice. However, the adverse effects of the
cytotoxic drugs to the non-targeted normal cells have
remained a major concern.2,3 Development of new
drugs with high anti-prostate cancer activity and with
low adverse effects is the most urgent demand to
treat cancer.
The structure of betulinol is based on a 30-carbon
skeleton comprising of four six-membered rings and
one five-membered ring, which has three sites available
for chemical modification, at C-3, C-20, and C-28, to
yield its derivatives for structure–function studies.
Betulonic acid, a derivative of betulinol, has shown high
cytotoxicity in vitro against LNCaP, PC-3, and
DU-145 prostate cancer cells.12–15
Keywords: Boc-lysinated-betulonic acid; Human prostate cancer cells;
Xenografts; Athymic Mice; Anti-prostate cancer agent.
*
Corresponding author. Tel.: +1 212 746 3067; fax: +1 212 746
4964; e-mail: brs2003@med.cornell.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.048

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B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
Betulonic acid is soluble in organic solvents. However,
due to the lack of solubility of betulonic acid in
biocompatible aqueous solvents, the anti-cancer
activity of betulonic acid in vivo has not been
determined to the fullest extent. In the present study,
Boc-lysinated-betulonic acid (hereafter, referred to as
lysinated-betulonic acid) has been chemically synthe-
sized as a hydrophilic derivative of betulonic acid and
solubilized in a biocompatible aqueous solvent, namely,
phosphate-buffered saline (PBS) containing 22% ethanol
and 4% human serum albumin. The anti-cancer activity
of both betulonic acid and its lysinated derivative was
investigated in in vitro MTT cytotoxicity assays as well
as in in vivo growth inhibition assay of human prostate
LNCaP tumor xenografts in athymic mice. The mecha-
nism of action of lysinated-betulonic acid was explored
by histological examination, Yo-Pro-1 staining, TUN-
EL assay, and by the release of cleaved caspase-3 from
the ex vivo tumor cultures, indicating that the lysinat-
ed-betulonic acid arrested mitosis and caused apoptosis
of prostate LNCaP tumor cells.
2.2. In vitro studies: inhibition of growth of human
prostate cancer cells in culture by betulonic acid dissolved
in DMSO
As shown in Table 1A, in the MTT assay betulonic acid,
at a concentration of 10 lM, inhibited the growth of
LNCaP, DU-145, and PC-3 prostate cancer cell lines
in culture with little cytotoxicity to normally proliferat-
ing fibroblast cells. Of the three human prostate cancer
cell lines, the effects of betulonic acid on LNCaP cells
at different concentrations as well as at different incuba-
tion time periods (Table 1B) showed that betulonic acid
inhibited the growth of LNCaP cells in a dose-depen-
dent manner at 24, 48, and 72 h of incubation. Betulonic
acid showed the highest growth inhibition at 88.4% of
LNCaP cells at a concentration of 10 lM after 72 h
incubation.
As shown in Figure 1, in the AIG assay, betulonic acid
exhibited significant inhibition in cellular growth and
tumorigenicity of all three prostate cancer cell lines at
a concentration of 10 lM in DMSO after 14 days of
incubation. The colony formation rate in the soft agar
significantly decreased to 42% (P < 0.01) for LNCaP
(Fig. 1B), 40% (P < 0.05) for DU145 (Fig. 1B), and
74% for PC-3 cells (P < 0.05) (Fig. 1B), as compared
with their corresponding controls. Interestingly, the
untreated LNCaP cells grew as three-dimensional tubu-
lar structures representing a high metastatic nature;
whereas the betulonic acid treated cells completely
lacked this activity. It is noteworthy that betulonic acid
at the same concentration displayed little effect on nor-
mal fibroblast cells (Fig. 1A).
2. Results
2.1. Synthesis and characterization of betulonic acid and
lysinated-betulonic acid
The synthesis of betulonic acid from betulinol using
hydrogen peroxide as an oxidizing agent yielded an
88% recovery and 99% purity as compared to
only 30% recovery and 80% purity using Jones
reaction.16–18. The purity of betulonic acid and lysi-
nated-betulonic acid was substantiated by a single
peak with retention times of 11.044 and 10.936 min,
respectively, as determined by gas chromatography
(GC) (Suppl. Fig. 1a and b). The confirmation of
the chemical structure of betulonic acid and lysinat-
ed-betulonic acid as determined by NMR spectrosco-
py confirmed their pentacyclic styrene nature. The
molecular mass of betulonic acid and lysinated-betu-
lonic acid was determined by a Quattro II triple
quadrupole instrument with electrospray ionization
mass spectrometric analysis (MALDI) in the positive
mode. The [M+H]+ and [M+NH4]+ ions m/z 455 and
m/z 472 were determined for betulonic acid (Suppl.
Fig. 2a and b). The MSMS spectra showed an m/z
of 544, 471, and 455 for betulonic acid. The lysinated
ester of betulonic acid appeared to be a single com-
pound, with m/z 697 singly protonated ion (Suppl.
Fig. 3a–c). At a higher resolution, more slowly
recorded ESI-MS scan, the monoisotopic molecular
mass of the neutral compound was computed to be
696.5 0.2 Da. The average molecular weight of
betulonic acid and of the lysinated derivative was
estimated to be approximately 469 and 663, respec-
tively. The NMR and mass data validated that, with-
in the acceptable limits of experimental error, one
lysine residue was conjugated to one molecule of
betulonic acid. The NMR spectroscopy and mass
spectroscopy characterization of betulonic acid and
its lysinated derivative is presented in the supplemen-
tary data.
2.3. Comparison of growth inhibition of human prostate
LNCaP cells by lysinated-betulonic acid dissolved in
DMSO and PBS: 22% ethanol/4% albumin in the
MTT assay
As shown in Table 2A, the lysinated-betulonic acid dis-
solved in DMSO inhibited the growth of LNCaP cells
to, approximately, 40% at a concentration of 10 lM
Table 1. Cytotoxicity of betulonic acid determined by MTT assay
Cell
Incubation time (h, 10 lM)
24 48
1A
LNCaP
38.0 8.0
75.0 9.0
DU-145
PC-3
Fibroblasts
5.91 4.03 30.83 14.82
10.39 4.44 47.42 10.95
0.0
0.0
Concentration (lM)
Incubation time (h)
48
24
72
1B
10
24.2 10.8
70.4 19.1
69.5 20.2
81.0 12.6
88.4 7.96
80.8 15.8
100
The data are shown as means SD of three independent experiments.
Percent growth inhibition of human prostate cancer cells and fibro-
blast cells by betulonic acid at a concentration of 10 lM.
Percent growth inhibition of LNCaP cells by betulonic acid at different
concentrations.

B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
Control Betulonic cid
6351
A
B
LNCaP
120
100
80
60
40
20
0
100
74
42
40
LNCaP
DU145
Control
LNCap
DU-145
PC-3
PC3
Fibroblast
Figure 1. Effect of betulonic acid at a concentration of 10ꢀ5 M on colony formation rate of prostate cancer cell lines and fibroblast as determined in
the anchorage-independent growth (AIG) assay: (A) growth of cells after incubation with betulonic acid for 14 days, (B) colony formation percent
compared to the control.
Table 2. Cytotoxicity of lysinated-betulonic acid determined by MTT
assay
at the concentration of 10 lM, however, it increased to
95.7% at 100 lM after 72 h of incubation (Table 2B).
Lysinated-betulonic acid at a concentration of 100 lM
Concentration
(lM)
Incubation time (h)
48
also caused significant inhibition of growth of DU-145
and PC-3 prostate cancer cells in MTT assay at 24, 48,
and 72 h of incubation with little cytotoxicity to normal-
ly proliferating fibroblast cells (Table 2C), indicating
that the improved solubility of lysinated-betulonic acid
in the PBS/ethanol/albumin solvent provided higher
availability of the drug.
24
72
2A
10
19.0 14.4
76.5 22.6
36.75 19.2
82.75 14.3
40.5 33.6
79.25 16.5
100
2B
10
16.33 5.51
50.33 13.65
16.0 9.54
88.0 6.24
24.3 13.7
95.7 3.8
100
Cells
Incubation time (h, 100 lM)
48 72
2.4. In vivo effect of lysinated-betulonic acid dissolved in
PBS/EtOH/albumin on LNCaP prostate cancer cell
xenografts in male athymic mice
24
2C
DU-145
PC-3
Fibroblasts
71.0 1.61
53.0 1.68
0
100
0
93.3
0
Visible tumors developed by day 12 post-subcutaneous
implantation of approximately 7 million LNCaP cells
in matrigel in male athymic mice. As shown in Figure
2A, the average tumor volume in control mice increased
continuously from the day 1 until day 17 of injections
with a net increase of 433 177 mm3. In contrast, the
average tumor volume in mice injected with lysinated-
betulonic acid decreased on day 3 and then remained
almost constant at, approximately, 40 2 mm3 until
day 17. The inhibition of tumor growth in the treated
mice was approximately 92% as compared to the tumor
volume in untreated control mice on day 17 (Fig. 2A).
The difference in the tumor volumes between the control
group and the lysinated-betulonic acid treated mice is
clearly visible in the photographs of mice in Figure 2B
and C, respectively. These results were reproduced in a
repeat experiment under the same conditions. There
79.0 0.42
12
77.0 0.39
18
The data are shown as means SD of three independent experiments.
Percent growth inhibition of DU-145, PC-3 and fibroblast by lysi-
nated-betulonic acid in PBS/ethanol/albumin at a concentration of
100 lM.
Percent growth inhibition of LNCaP cells by lysinated-betulonic acid
in medium/DMSO.
Percent growth inhibition of LNCaP cells by lysinated-betulonic acid
in PBS/ethanol/albumin.
after 48 as well as 72 h of incubation. However, at a con-
centration of 100 lM, the inhibition of growth increased
to 80% at all incubation times. It is noteworthy that
lysinated-betulonic acid dissolved in PBS/ethanol/albu-
min solvent showed similar cytotoxicity on LNCaP cells

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B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
700
600
500
400
300
200
100
0
A
Lysinated-Betulonic Acid
Control
1
2
3
4
5
6
7
8
9
10
11
12 13
14 15 16
17 Days
Control mice on day 17
C
Lysinated-Betulonic Acid Treated Mice on day 17
B
1
2
1
2
3
4
3
4
Figure 2. (A) Daily average tumor volume of human prostate cancer LNCaP cell xenografts in athymic mice injected with lysinated-betulonic acid
dissolved in PBS containing ethanol and human serum albumin compared to the control mice injected with the solvent alone; (B) photographs of
control mice with LNCaP xenografts on day 17; (C) photographs of mice with LNCaP xenograft treated with lysinated-betulonic acid on day 17.
was an 89% inhibition of tumor growth in the lysinated-
betulonic acid injected mice as compared to the control
group.
4A for days 0, 5, and 9. As shown in Figure 4B, the
tumor from one of the drug treated tumor also
showed growth, however, after the addition of the
drug daily from day 5 in the culture medium, the tu-
mor tissue decreased in size up to day 12, indicating a
direct inhibitory effect of the lysinated-betulonic acid
on the growth of the tumor. Whereas, as shown in
Figure 4C, the tumor tissue from the other drug treat-
ed mice did not show any growth presumably because
of the absence of any viable cells.
2.5. Immunohistochemical and histological studies of
tumor tissues
Frozen sections of the tumors identified specific antigens
PSA and PSMA in tumor cells of both lysinated-betu-
lonic acid treated and control groups of mice. The
hematoxylin–eosin stained section of the tumor tissue
from control mice revealed mitotic cancer cells indicat-
ing growth with large vesicular nuclei and prominent
nucleoli, both characteristics of cell viability (Fig. 3A).
In contrast, the tumor cells of the lysinated-betulonic
acid treated mice were devoid of mitotic division and
metabolically less active (Fig. 3B), exhibiting dark
chromatin and inapparent nucleoli. The TUNEL assay
detected apoptotic cells in the hematoxylin–eosin
stained sections of the tumor tissue. The appearance of
apoptotic cells in the sections of the tumor tissue of
the drug treated mice produced a brown stain indicative
of apoptosis (Fig. 3D). The cells in the control group did
not show any brown stain, revealing lack of apoptotic
cells (Fig. 3C).
The profile of tumor growth for 12 days is shown in
Figure 4D. The tumor tissue of the control mice grew
continuously from day 1 to day 12 with a 215.23%
increase in the tumor size. The tissue from one treated
mouse showed 68.3% growth on day 5, however, after
the addition of the drug in the culture medium, the
tumor size decreased to 13.6% on day 12. The tumor
tissue from the other treated mice did not grow at all
(Fig. 4D).
On day 12, both control and drug treated tumor cells
were double stained with Hoechst 33342 and Yo-Pro-
1, and representative images of tumor tissues were
obtained by confocal laser scanning microscopy
(Fig. 4E–G). In the tissue from the control mice, nuclei
of the cells did not stain green, indicating the absence of
apoptosis, whereas proliferating viable cells on the
periphery of the tumor tissue stained blue with Hoechst
33342 (Fig. 4E). On the other hand, the images in Figure
4F and G showed that the nuclei in the drug treated
tumor cells were stained green by the Yo-Pro-1, indicat-
ing apoptosis.
2.6. Effects of lysinated-betulonic acid on the ex vivo
growth of LNCaP tumor tissue in culture
Portions of the tumor tissue harvested from control as
well as from lysinated-betulonic acid injected mice
were grown in culture medium for 12 days. The tumor
tissue from the control mice grew as shown in Figure

B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
Lysinated-Betulonic Acid Treated
6353
B
A Control
D
Control
C
Lysinated-BetulonicAcid Treated
Figure 3. Cross-section of the tumor stained with hematoxylin–eosin: (A) from control mouse; (B) from lysinated-betulonic acid injected mouse;
TUNEL assay of cross section of the tumor tissue: (C) from control mouse; (D) from lysinated-betulonic acid-injected mouse. Insets show cells at a
higher magnification.
2.7. Detection of cleaved caspase-3 in tumor tissue
prostate carcinoma, in part due to increased resistance
of androgen-independent cells to apoptosis.23 However,
the severe side effects of chemotherapy have remained a
major problem.
Tumor tissues from both drug treated and control
samples were subjected to SDS–PAGE, and the cleaved
caspase-3 was detected in Western blots using cleaved
caspase-3 monoclonal antibody. As shown in Figure
4H, the cleaved caspase-3 was visible in drug treated tu-
mor sample, whereas no cleaved caspase-3 release was
detected in the control tissue.
Betulonic acid showed the highest cytotoxic effect on
LNCaP human prostate cancer cells in cell culture as
compared to other betulinol derivatives. Betulonic acid
was synthesized by a modified method using H2O2 as
the oxidizing agent, resulting in higher yield and purity,
compared with the previous method using Jones’
reagent.17 Betulonic acid showed high cytotoxicity in
vitro in MTT assay and significant inhibition of colony
and tumor growth in AIG assay on LNCaP, DU-145,
and PC-3 human prostate cells, with little effect on
normally proliferating fibroblast cells.
3. Discussion
Metastatic prostate carcinoma is associated with a high
morbidity and mortality rate with a medium survival of,
approximately, 12–15 months.19 Available treatment
alternatives include, radiotherapy after radical retropu-
bic prostatectomy, radical prostatectomy, external beam
radiation, prostate brachytherapy, and androgen abla-
tion of the prostate.20 Until recently, despite androgen
suppression, no cytotoxic agent has been able to change
the progression of metastatic prostate cancer. Androgen
ablation therapy remains the main course of treatment
with advanced disease. However, it has no effect on hor-
mone-independent cancer cells.21,22 Chemotherapeutic
agents result in less than a 10% response in advanced
The hydrophobic nature of betulonic acid and conse-
quent lack of solubility in aqueous solvents had
remained a limitation in achieving full expression of its
anti-cancer activity in vivo. Hence, a lysinated derivative
of betulonic acid was synthesized to yield a hydrophilic
compound. The chemical conjugation of lysine to
betulonic acid was facilitated by the use of selectively
protected amino groups of the lysine to avoid the intra-
molecular interference and permit the formation of a

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B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
Day 5 Day 9
Day 0
day 12
A
B
E
F
G
C
Ex Vivo Growth Area of the Tumor in Culture
Western Blot:
D
H
250
200
150
100
50
A: Control
Lysinated-Betulonic
Acid Treated
Control
B: Drug Treated
C: Drug Treated
Vinculin
Drug added
Cleaved
0
Caspase-3
0
1
2
3
4
5
6
7
8
9
10 11 12
-50
Day
Figure 4. Effects of lysinated-betulonic acid at a concentration of 5.5 · 10ꢀ4 M on ex vivo tumor tissues. (A) Growth of ex vivo tumor tissues from
control mouse in medium alone in in vitro culture for 12 days, selective photographs of day 0, 5, and 9 are shown. (B and C) Growth of ex vivo tumor
tissues from mouse treated with lysinated-betulonic acid from day 5 to day 12, (D) graph of daily growth of ex vivo cultures of LNCaP tumor tissue
excised from xenografts in culture from the control and the drug treated mice for 12 days. (E–G) Confocal immunofluorescence of tumor tissues
stained with Yo-Pro-1: (E) from control mouse, (F and G) from lysinated-betulonic acid treated mice. The blue stain indicates viable and dividing
cells at the periphery, whereas the green stain inside the tumor indicates apoptotic cells. (H) Detection of cleaved caspase-3 by Western blot from
tumor tissue of lysinated-betulonic acid injected mice compared to the control.
covalent bond exclusively between the C-28 of betulonic
acid and the free amine group at C-6 of the lysine. Re-
lease of the ester group at the C-terminal of the lysine
yielded the hydrophilic lysinated-betulonic acid
with enhanced solubility in aqueous medium. The solu-
bility of lysinated-betulonic acid was only 0.7 mg/mL
in 20% DMSO, while the solubility of betulonic acid
was 0.5 mg/mL. However, the solubility of the
lysinated-betulonic acid increased to 5.0 mg/mL in the
PBS/ethanol/albumin solvent compared to 1.8 mg/mL
of betulonic acid in the same solvent system.
The higher activity of lysinated-betulonic acid against
prostate cancer was further confirmed by using xeno-
graft tumors in vivo. Lysinated-betulonic acid, at a dose
of 30 mg/kg body weight, showed up to 92% growth
inhibition of the LNCaP xenograft tumors transplanted
in athymic male mice (Fig. 2A). The mice had been
treated for 17 days. However, it is tempting to speculate
that if the experimental mice were continued to be treat-
ed further, the tumors might have been further sup-
pressed. The mice injected with lysinated-betulonic
acid showed little change in body weight or vital signs
or visible toxic effect during the entire period of treat-
ment. The results of two independent in vivo experi-
ments in a total of 16 male athymic mice confirm and
validate the anti-cancer efficacy of lysinated-betulonic
acid, solubilized in the PBS/ethanol/albumin, in the inhi-
bition of LNCaP xenograft tumor growth.
Anti-prostate cancer activity of lysinated-betulonic acid
was determined using LNCaP prostate cancer cells,
which are diagnostic of human prostate cancer.24
Lysinated-betulonic acid dissolved in the PBS/ethanol/
albumin solvent showed higher inhibition of LNCaP cell
growth at a concentration of 100 lM after 72 h incuba-
tion as compared to lysinated-betulonic acid dissolved in
DMSO (Table 2A and B). It may be mentioned that
lysinated-betulonic acid at 100 lM concentration also
causes significant inhibition of growth of hormone-inde-
pendent DU-145 and PC-3 cells in the MTT assay with
little cytotoxicity to normally proliferating fibroblast
cells (Table 2C).
Anti-cancer effect of the lysinated-betulonic acid was
further demonstrated by histological studies of the
tumor, showing the absence of dividing cells in the
tumors of treated mice, whereas the tumor tissues from
control mice showed abundant cell division with a mitot-
ic index of 3–12 (Fig. 3A and B). From our preliminary
studies, the mechanism of action of lysinated-betulonic

B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
6355
30
30
30
29
29
29
20
20
19
20
21
21
21
19
19
H
H
H
H2O2
Reaction A
12
26
22
12
12
26
22
18
22
18
PCC
18
17
17
17
11
9
11
9
11
9
28
28
28
13
13
25
13
25
25
26
oxidation
H
CH2OH
oxidation
H
COOH
16
H
CHO
1
4
1
4
1
4
14
14
14
16
16
2
2
2
10
5
8
10
5
8
10
5
8
15
15
15
H
H
H
27
3
27
3
27
3
7
7
7
HO
O
O
6
6
6
H
H
H
23
23
23
24
24
24
Betulonic Acid
Betulone Aldehyde
Betulinol
BocHN
BocHN
BocHN
6
6
6
Reaction B
Reaction C
5
3
5
3
OMe
1
OH
1
OMe
4
2
4
2
2
CbzHN
CbzHN
Methylation
H2N
1
O
O
O
Pd/C, H2
(Boc-Lys(Cbz)-OMe)
(Boc-Lys(Cbz)-OH)
Boc-Lys-OMe
30
29
20
21
19
BocHN
H
12
26
22
18
H
Boc HN
OMe
17
6
11
9
28
OMe
13 H
COOH
25
2
H2N
HN
1
4
1
14
16
2
H
O
10
5
8
15
O
H
27
3
HOBt, DCC
O
7
H
O
6
H
O
23
24
H
Boc-Lysinated Ester Betulonic
Acid
Boc-Lys-OMe
Betulonic Acid
BocHN
H
LiOH·H2
O
OH
HN
H
O
O
H
O
H
Boc-Lysinated Betulonic Acid
Hereafter, referred to as Lysinated-Betulonic Acid
Figure 5. Chemical synthesis of betulonic acid and the lysinated-betulonic acid.
acid was demonstrated to be predominantly apoptosis as
shown by specific Yo-Pro-1 staining of the ex vivo tumor
tissues (Fig. 4E–G) and the detection of caspase-3 en-
zyme from the tumors treated with the drug (Fig. 4H).
The TUNEL assay, which detects the breakdown of
DNA strands of the tumor cells, further supported that
lysinated-betulonic acid caused the apoptotic death of
LNCaP cancer cells (Fig. 3C and D).
and have indicated that lysinated-betulonic acid has a
promise alone or in conjunction with other drugs, radi-
ation, and surgical treatment modalities to provide an
improved therapy of prostate cancer.
5. Materials and methods
5.1. Chemicals
4. Conclusion
Prostate cancer cells, namely LNCaP, DU145, and
PC-3, and corresponding culture media were purchased
from American Type Culture Collection (ATCC,
Manassas, VA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrasodium bromide (MTT) kits were also
obtained from ATCC. Matrigel was purchased from
BD Biosciences (Bedford, MA). Yo-Pro-1 and Hoechst
33342 stains were purchased from Molecular Probes
(Carlsbad, CA). Rabbit polyclonal antibody to
caspase-3 was obtained from Cell Signaling (Beverly,
MA). Streptavidin-HRP and diaminobenzidine (DAB)
kits were obtained from Ventana Medical Systems
(Tucson, AZ). Terminal deoxynucleotidyl Transferase
(TdT) and Biotin-16-dUTP were purchased from Roche
(Indianapolis, IN). ECL protein detection kit and
The results of the present study demonstrate that the
newly synthesized hydrophilic lysinated derivative of
betulonic acid soluble in PBS containing 22% ethanol
and 4% albumin showed significant inhibition of growth
of human prostate cancer cells in vitro as well as
dramatic inhibition of growth of LNCaP xenograft
tumors in male athymic mice in vivo. The enhanced
anti-prostate cancer activity of lysinated-betulonic acid
may be attributed to a higher solubility and consequent-
ly bioavailability of the drug in vivo. These studies have
provided a much needed breakthrough to undertake
dose–response, toxicity, and drug delivery studies of
lysinated-betulonic acid as an anti-prostate cancer drug

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B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
nitrocellulose membranes were obtained from Bio-Rad
(Hercules, CA). The BCA protein assay kit and RIPA
were purchased from Pierce (Rockford, IL). Na-Butyl-
oxycarbonyl-Ne-benzyloxycarbonyl-lysine and all other
chemicals were obtained from Sigma–Aldrich (St. Louis,
MO). Injectable grade (98%) ethanol and 4% pure
human serum albumin were obtained from American
Regent and Biocell, respectively.
3.41 mg/mL, respectively, and diluted with culture
medium containing 10% fetal bovine serum (FBS)
to yield a final concentration of 1.0 mM, in 20%
DMSO. Lysinated-betulonic acid was also solubilized
in ethanol with a concentration of 5.0 mg/mL and
diluted with phosphate-buffered saline (PBS) contain-
ing 4% human serum albumin to yield a concentra-
tion of 1.0 mM containing 14% ethanol for in vitro
cytotoxicity assays in cell culture, and to 2.2 mM
concentration in PBS containing 4% albumin and
22% ethanol for the in vivo assay in athymic male
mice.
5.2. Mice
Athymic mice, 13 weeks of age, were purchased from
The National Cancer Institute (NCI) and were housed
in a pathogen-free environment under controlled condi-
tions of light and humidity with free access to food and
water. All mice studies were performed according to the
guidelines of the Institutional Animal Care and Use
Committee and Research Animal Resource Center of
Weill Medical College of Cornell University, New York
(protocol #0402-209A).
5.4. In vitro cytotoxicity of betulonic acid and lysinated-
betulonic acid on human prostate cancer cells by MTT
assay
Human prostate cancer cell lines LNCaP, PC-3, and
DU-145 as well as human fibroblast cell lines were
obtained from ATCC and were, respectively, main-
tained in RPMI-1640, F-12K, MEM, and DMEM
supplemented with 10% fetal bovine serum at 37 ꢁC
under 5% CO2 in a humidified atmosphere in an incu-
bator. The wells of a 96-well culture plate were pre-
coated with 3% lysine to achieve optimal cell
adhesion. Approximately, 60,000–100,000 cells in
0.1 mL culture medium per well were plated. The cells
were incubated with 10 lL of the drug solutions with
a final concentration of 10 and 100 lM for 24, 48,
and 72 h. Ten microliters of 3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyl tetrazolium bromide was added into
each well at the end of each incubation period and
placed in the incubator. The cells were periodically
examined under an inverted microscope for the
appearance of an intracellular punctate purple precip-
itate, formazan, which indicated the presence of live
cells.26 When the purple precipitate was clearly visible
under the microscope, 100 lL of the detergent was
added to all wells to dissolve the formazan. The
absorbance of each well was determined at a wave-
length of 540 nm using an automatic ELISA reader
coupled with a Titerteck Multiskan Ascent (Thermo
Labsystems, Finland).
5.3. Synthesis of lysinated-betulonic acid
Betulinol was isolated from the non-saponifiable
fraction of crude sulfate soap prepared by boiling
the outer bark of the white birch tree in sodium
hydroxide (NaOH), sodium sulfate (Na2SO4), sodium
sulfite (Na2SO3), and sodium thiosulfate (Na2S2O3) at
110–120 ꢁC. Betulinol was then crystallized in ace-
tone.25 Betulonic acid was synthesized from betulinol
by a procedure described in Figure 5, Reaction A
where betulinol was first treated with pyridinium chlo-
rochromate (PCC) in dichloromethane (CH2Cl2).
Betulonic acid was obtained by further oxidation on
betulone aldehyde using 30% aqueous hydrogen per-
oxide (H2O2). In order to increase the hydrophilicity,
lysine, a biocompatible amino acid, was conjugated
to betulonic acid. Lysine was chosen due to its ability
to form two amino bonds as well as for its long
hydrocarbon chain, which would provide increased
steric flexibility. Additionally, lysine is known to be
fairly stable in most synthetic reactions.
Esterification of Boc-Lys(Cbz-OH) generated a methyl
ester. Selective deprotection of benzoyloxycarbonyl
(Cbz) was achieved by hydrogenolysis to release the pri-
mary amine (Fig. 5, Reaction B). The conjugation reac-
tion of the deprotected lysine derivative to betulonic
acid was performed in CH2Cl2 using 1-hydroxybenzo-
triazole hydrate (HOBt) to activate the C-28 of betulon-
ic acid using 1,3-dicyclohexylcarbodiimide (DCC) as the
catalyst (Fig. 5, Reaction C). The reaction occurred
selectively between the amine and carboxyl group,
whereas the ketone and alkene groups on betulonic acid
were unaffected and yielded lysinated-betulonic acid es-
ter (Boc-Lys-OMe-betulonic acid). The methyl ester on
the C-terminus of lysine was then hydrolyzed by treat-
ment with lithium hydroxide monohydrate (LiOHÆH2O)
to obtain lysinated-betulonic acid as a white powder
(Fig. 5, Reaction C).
5.5. Anchorage-independence growth (AIG) assay
AIG assay was performed to determine the ability of
betulonic acid to inhibit tumor cell growth by forming
three-dimensional colonies in soft agar in 6-well culture
plates.27 Prostate cancer cells, LNCaP, PC-3, and
DU-145, as well as normal proliferating fibroblasts
were suspended in 2 mL of RPMI, F12K and DMEM
containing 0.33% agar, respectively. A 2 mL matrix
made up of 0.6% agar was overlaid on the agar in
the absence or presence of betulonic acid at a final con-
centration of 10 lM. The cultures were incubated at
37 ꢁC in a humidified environment under 5% CO2 for
14 days without medium change. The cultures were
fixed in Cornoy’s fixative (acetic acid/100% ethanol
1:3 v/v), the size as well as the numbers of anchor-
age-independent tridimensional colonies (P40 lm
diameter) formed were determined under 10-fold
magnification.
Betulonic acid and lysinated-betulonic acid were
dissolved in DMSO at concentrations of 2.22 and

B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
6357
5.6. In vivo effect of lysinated-betulonic acid on LNCaP
prostate cancer cell xenografts in athymic mice
tration of 5.5 · 10ꢀ4 M. Solvent alone was added to
the wells with tissue from control mice. The tissues were
cultured for 12 days. Diameter measurements and pho-
tographs of the tumors were taken daily using an Olym-
pus IX 70 inverted microscope and DP11 image
analyzer.
Human prostate LNCaP cells were cultured in T-75
culture flasks in RPMI-1640 cell culture medium with
10% FBS until 80–90% confluent. Cells were scraped
from the bottom of the flask into phosphate-buffered
saline (PBS) and collected by centrifugation. In a pilot
study, five athymic male mice were transplanted on
the left flank of the abdomen with approximately 7
million LNCaP prostate cancer cells in matrigel28 to
determine the time required to grow visible tumors.
In the following study with 12 athymic male mice, 7
million LNCaP cells in matrigel were implanted in
each mouse on the left flank area. Visible tumors grew
in 12 days following implantation. The mice were ran-
domly divided into two groups with four mice each.
Each mouse in the experimental group was injected
intraperitoneally with lysinated-betulonic acid at a
dose of 30 mg/kg body weight daily for 17 days. Mice
in the control group were injected with the solvent
alone. The experiment was repeated under the same
conditions.
5.10. Detection of apoptosis by Yo-Pro-1 immunofluo-
rescence staining
On day 12, tumor tissues cultured in a 1:1 ratio of matri-
gel and RPMI culture medium supplemented with 10%
fetal bovine serum (FBS) were incubated with 80 lg/
mL of Hoechst-33342 stain for 1 h at 37 ꢁC under 5%
CO2 and 95% air. The tissues were then incubated with
4 lM Yo-Pro-1 stain for an additional hour. Confocal
laser scanning fluorescence microscopy was used to
measure viable and apoptotic tumor cells.31
5.11. Western blot
On day 12, the ex vivo tumor tissues were lysed in
100lL RIPA buffer (0.1% sodium dodesylsulfate,
0.5% sodium deoxycholate, 1% NP-40, 150 mM NaCl,
50 mM Tris–HCl, 2.5 mM EDTA, 25 mM NaF,
10 mM sodium pyrophosphate, 1 mM sodium ortho-
vanadate, and protease inhibitors such as 100 mM
PMSF, 1 lg/mL aprotinin, 1 lg/mL leupeptin, and
1 lg/mL pepstatin). The samples were centrifuged at
16,000g for 30 min at 4 ꢁC. The protein concentration
in the supernatant was determined using Pierce’s BCA
assay kit. Aliquots of 20 lg protein samples were sub-
jected to 10% SDS–polyacrylamide gel electrophoresis
(PAGE). The protein bands were transferred onto a
nitrocellulose membrane using a semi-dry blot device.
The membrane was blocked with 5% dried milk in
Tris-buffered saline and blotted with 1:100 rabbit
polyclonal caspase-3 and mouse monoclonal vinculin
antibodies overnight. The membrane was then blotted
with a second antibody conjugated with horseradish
peroxidase (HRP) and images were developed using
an ECL protein detection kit with a chemilumines-
cence device.
Mice were weighed daily and observed for vital signs
and visible adverse effect. The change in the tumor vol-
ume was determined daily, by the aid of caliper and cal-
culated according to the formula p/6(l · w · h).29 Mice
were sacrificed by CO2 asphyxiation at the end of the
experiment on day 17. The tumor tissue was dissected
out from each mouse under sterile conditions and sub-
jected to further studies.
5.7. Immunohistochemical study of the tumor tissues
A portion of the dissected tumors from mice was
frozen immediately at ꢀ80 ꢁC in optimal cutting gel.
Frozen sections of the tumors were treated with
monoclonal antibodies to localize prostate specific
membrane bound antigen (PSMBA) and prostate spe-
cific antigen (PSA) as markers to identify prostate
cancer cells.30
5.8. Histological studies
5.12. Detection of apoptotic cells by terminal deoxy-
nucleotidyl transferase biotin-dUTP nick end labeling
(TUNEL) assay
A portion of the tumor tissue was fixed in 4% formalin
for 24 h and transferred to 70% ethanol. Eight microm-
eter thick sections of the fixed tissue were stained with
hematoxylin–eosin for histological examination for cell
types and structure.
The tumor tissues from mice were examined by the
TUNEL assay to detect 30 free hydroxyl ends (3-OH)
of DNA strands created by nucleases in apoptotic
cells.32 Sections of tumor tissues were treated for
20 min with Proteinase K (20 lg/mL in PBS), equilibrat-
ed for 10 min with TdT buffer, and incubated for 2 h in
the TdT mix containing 100 U TdT and 0.5 lL
Biotin-16-dUTP. Biotin-16-dUTP labeled in 3-OH of
DNA was detected by streptavidin–HRP and visualized
using diaminobenzidine (DAB) for color reaction
according to the manufacturer’s instructions. The
in situ molecular detection was performed using 8 lm
thick paraffin sections with an automated staining
processor (Discovery XT).
5.9. Ex vivo growth of tumor tissues in culture
A portion of the tumors excised from control and lysi-
nated-betulonic acid treated mice was used for in vitro
growth in RPMI culture medium. The tumor was cut
into small pieces of approximately 4 mm2 and immersed
into wells of a 12-well culture plate containing a mixture
of matrigel and RPMI culture medium in a 1:1 ratio.
After the matrigel solution solidified, culture medium
was added to each well and changed daily. On day 5
of the culture, tumor tissues from drug treated mice were
incubated with lysinated-betulonic acid at final concen-

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B. B. Saxena et al. / Bioorg. Med. Chem. 14 (2006) 6349–6358
12. Saxena, B. B.; Garcia, C.; Bohmstein, A.; Rathnam, P.;
Acknowledgments
Haller, I. Betulonic Derivatives for Prostate Cancer
Therapy. Chemistry Biology Interface: Synergistic
New Frontiers. November 21–26, 2004. New Delhi,
India.
We are grateful to Dattatreyudu Nori, M.D., Chairman,
Department of Radiation Oncology, Neil Bander,
M.D., Professor of Urology and Thomas Caputo,
MD, Director of Gynecologic Oncology, for advice dur-
ing the course of this study, He Liu, Ph.D. for assistance
with frozen sections of the tumors, to Krista La Perle,
Ph.D., for histological sections, to Ms. Mariya Zaless-
kaya for technical assistance, to William Bracken,
Ph.D. for NMR studies, to Ivan Haller, Ph.D., for Mass
Spectroscopic studies and Professor Esther Breslow for
laboratory facilities at New York Presbyterian Hospital
and Weill Medical College. We also thank Katia Mano-
va, Ph.D. for the TUNEL assay at the Molecular Cytol-
ogy Core Facility at Memorial Sloan Kettering Cancer
Center, New York, NY.
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