Synthesis, proapoptotic screening, and structure-activity relationships of novel aza-lupane triterpenoids

Article abstract of DOI:10.1016/j.bmcl.2010.07.120

Apoptosis is a highly regulated process by which excessive cells are eliminated in order to maintain normal cell development and tissue homeostasis. Resistance to apoptosis often contributes to failure in cancer prevention and treatment. Apoptotic cell death regulators are considered important targets for discovery and development of new therapeutic agents in oncology research. A class of novel aza-lupane triterpenoids were designed, synthesized, and evaluated for antitumor activity against a panel of cancer cell lines of different histogenic origin and for ability to induce apoptosis. 3,30-Bis(aza) derivatives were identified not only to possess improved cytotoxicity compared to the natural product betulinic acid but also to affect cell death predominantly via apoptosis, whereas the mono(aza) derivatives apparently triggered cell death via different, non-apoptotic pathway(s).

Full text of DOI:10.1016/j.bmcl.2010.07.120

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5389–5393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, proapoptotic screening, and structure–activity relationships
of novel aza-lupane triterpenoids q
Aye Aye Mar, Erika L. Szotek, Ali Koohang, William P. Flavin, David A. Eiznhamer,
*
Michael T. Flavin, Ze-Qi Xu
Advanced Life Sciences, Inc., 1440 Davey Road, Woodridge, IL 60517, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Apoptosis is a highly regulated process by which excessive cells are eliminated in order to maintain nor-
mal cell development and tissue homeostasis. Resistance to apoptosis often contributes to failure in can-
cer prevention and treatment. Apoptotic cell death regulators are considered important targets for
discovery and development of new therapeutic agents in oncology research. A class of novel aza-lupane
triterpenoids were designed, synthesized, and evaluated for antitumor activity against a panel of cancer
cell lines of different histogenic origin and for ability to induce apoptosis. 3,30-Bis(aza) derivatives were
identified not only to possess improved cytotoxicity compared to the natural product betulinic acid but
also to affect cell death predominantly via apoptosis, whereas the mono(aza) derivatives apparently trig-
gered cell death via different, non-apoptotic pathway(s).
Received 29 June 2010
Revised 23 July 2010
Accepted 26 July 2010
Available online 3 August 2010
Keywords:
Betulinic acid
Aza-lupane triterpenoid
Cytotoxicity
Ó 2010 Elsevier Ltd. All rights reserved.
Apoptosis
29
Apoptosis is a highly regulated process by which excessive or
harmful cells are eliminated in order to maintain normal cell
development and tissue homeostasis.1,2 Defects in the apoptotic
cellular machinery and the apoptosis inducing pathway(s) often
result in uncontrolled cell proliferation, tumor development, and
other malignant diseases.2 Resistance to apoptosis contributes
greatly to the failure in cancer prevention and treatment.3 There-
fore, apoptotic cell death regulators are considered important tar-
gets for discovery and development of new therapeutic agents in
oncology research. Betulinic acid, 3b-hydroxy-lup-20(29)-en-28-
oic acid (1), is a phytochemical widely distributed in food, medic-
inal herbs, and other plants throughout the world and is identified
to possess a wide range of biological properties including broad
growth inhibitory activity and the ability to induce apoptosis in
the most prevalent human cancer cells.4 The betulinic acid-in-
duced apoptosis is mediated by mitochondria.5 The mitochondrial
outer membrane is initially permeabilized, rendering the release of
apoptogenic proteins, for example, cytochrome c, Smac, and apop-
tosis inducing factor (AIF), followed by the activation of caspase
cascade.6 Betulinic acid (1) has also been reported to induce cell
death by selective proteosome-dependent inhibition of the speci-
ficity protein transcription factors which regulate vascular endo-
thelial growth factor (VEGF) and survivin expression.7
30
H
17
28
CO2H
H
H
HO
3
H
1, Betulinic Acid
The analysis of cytotoxicity–structure relationships of betulinic
acid(1)anditsderivativesrevealedthatthecarboxylicacidgroupat-
tached at C17 position is essential for its cytotoxicity.4 We have re-
cently reported, however, that the C17-carboxylic acid group in
the 2-cyano-1-en-3-one system of lupane triterpenoid was not crit-
icaltoinhibitcancercellproliferationandinduceapoptosis8 andthat
elongationofthechainlengthattachedat theC17positionof1, while
maintaining the carboxylic acid group at the chain end, was detri-
mental to the cytotoxicity.9 In our continuing efforts to identify po-
tential molecules capable of modulating the process of intrinsic
mitochondrial pathway of apoptosis, we have embarked on the de-
sign and synthesis of lupane triterpenoid-based libraries that are
anticipated to possess increased ability to induce controlled cancer
cell death. Our approach involves the screening of new molecules
for in vitro cytotoxicity, apoptosis induction and caspase activation
and cytochrome c release in cell death signaling. Herein, we wish
to report our initial investigation of a novel class of aza-lupane trit-
erpenoids against cancer cell lines and their ability to induce
apoptosis.
q
Part of the results was presented at the 237th American Chemical Society
National Meeting, Salt Lake City, Utah, March 22–26, 2009. Abstract No. MEDI 273.
* Corresponding author.
E-mail address: zxu@advancedlifesciences.com (Z.-Q. Xu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.120

5390
A. A. Mar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5389–5393
The synthesis of various aza-lupane triterpenoids is shown in
atives were more cytotoxic than betulinic acid (1); (2) in contrast
to the conventional betulinic acid analogues previously reported,
the aza-compounds with C17-CH2OH (6) tended to be more consis-
tently cytotoxic than the compounds with C17-CO2H (5); (3) the
30-OH group appeared not to confer a significant effect on the inhi-
bition of cell proliferation (8 vs 6); (4) compared to the 3-aza deriv-
atives, the 30-aza substitution seemed to afford less inhibitory
compounds (9 and 10 vs 6); (5) 3,30-bis(aza) triterpenoids (11)
were among the most active compounds generated and their cyto-
toxicities were consistent across the panel cell lines tested; and (6)
the corresponding THP-protected intermediates were as cytotoxic
as, and, in a few cases, were even more inhibitory than, those with
the THP group removed (data not shown).
Compounds able to inhibit cancer cell proliferation in the MTS
assay were further investigated for apoptosis inducing activity
using the Annexin-V assay.14 The examples of mono(aza) com-
pounds are shown in Figure 1, where SK-MEL-2 cells were treated
with 7.5 lM of the test compounds for 16 h. The results indicated
that the 3-aza derivatives 5 and 6 did not significantly improve the
Scheme 1. The 3-aza derivatives (5 and 6) were readily obtained
from reductive amination of betulonic acid (3)10 and the THP-pro-
tected betulone (4),8 which were, respectively, prepared from the
corresponding betulinic acid (1) and betulin (2) according to the
published procedures. Hydroxylation of 4 via selenylation11 fol-
lowed by oxidation with H2O2 furnished the 30-hydroxy ketone
7. Without the oxidation step, the 30-phenylselenium intermedi-
ate 7a was isolated. Reductive amination of 7 led to the formation,
after removal of the THP protecting group, of 3-amino-30-hydroxy
analogues 8. Mesylation of 7 followed by nucleophilic substitution
with amines provided 30-aza-3-oxo derivatives 9, subsequent to
the removal of THP group. The 3-oxo group of 9 was reduced by
NaBH4 to generate 30-aza-3-OH compounds 10. Reductive amina-
tion of 9 afforded 3,30-bis(amino) compounds 11.
All the synthesized aza-lupane triterpenoids12 were evaluated
for their antiproliferative activity using the MTS assay13 against a
panel of cancer cell lines of different histogenic origin, including
SK-MEL-2 and A-375 of malignant melanoma, Daoy and LN-229
of cerebellar medulloblastoma, OVCAR-3 of ovarian, HT-29 of colon
and MCF-7 of breast adenocarcenoma and the results are shown in
Table 1. Based on this small set of compounds, a few observations
of structure–activity relationships can be made: (1) the aza deriv-
apoptosis induction activity when compared to betulinic acid (1),
even though 5 and 6 were generally more cytotoxic (lower IC50
)
than 1 (Table 1). The increased cytotoxicity might be due to an
uncontrolled necrotic approach. The mono(azo)derivatives 8 and
H
H
a (when R=CH2OH)
H
R
H
R
b
H
H
O
HO
H
H
H
3, R=CO2H
4, R=CH2OTHP
1, R=CO2H
2, R=CH2OH
H
R
e, f
H
R1HN
OR
H
OH
5, R=CO2H
6, R=CH2OH
H
H
H
OTHP
c, d
(when R = H)
R1HN
OH
H
H
H
O
H
H
NHR2
8
7
NHR2
H
OH
H
H
H
OH
H
HO
H
10
H
O
H
9
NHR2
SePh
H
OH
H
H
OTHP
H
H
R1HN
H
H
11
O
H
7a
Scheme 1. Reagents: (a) DHP, PPTS, MeOH; (b) CrO3, pyridine; (c) R1NH2, NaCNBH3; (d) PPTS, MeOH; (e) PhSeCl, pyridine, 30% H2O2; (f) MeSO2Cl, Et3N; (g) R2NH2; (h) NaBH4.

A. A. Mar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5389–5393
5391
Table 1
Cytotoxicity of aza-lupane triterpenoids measured as IC50 (l
M)a
Compound
R1
R2 (or R)
SK-MEL-2
A-375
Daoy
LN-229
OVCAR-3
HT-29
MCF-7
1 (betulinic acid)
5a
5b
—
H
—
CO2H
CO2H
7 (3.7)b
8
36
22
nt
14
11 (2.3)b
7
<2
29
nt
nt
40
14
28
31
nt
16
30
nt
35
HOCH2CH2
5c
CO2H
6 (0.6)b
5
6 (0.6)b
5
6
4
6
CH2CH2
CH2CH2
HO
HO
HO
O
5d
CO2H
19
nt
30
29
22
65
37
CH2
5e
CO2H
29
>75
38
>75
>75
>75
>75
O
6a
6b
H
CH2OH
CH2OH
9
2
2
19
<2
4
5
5
2
2
5
5
HOCH2CH2
<2 (0)b
6 (5.8)b
6c
CH2OH
2 (0)b
4
<2 (0)b
8
5
2
5
CH2CH2
CH2CH2
HO
HO
6d
CH2OH
17
6
18
9
4
6
17
HO
6e
8a
8b
MeOCH2CH2
H
HOCH2CH2
CH2OH
—
—
7
7
6
16
8
nt
17
37
22
14
39
19
17
65
8
15
41
34 (4.9)b
34
31 (6.3)b
29
8c
CH2CH2
CH2CH2
—
38
6
6
32
17
23
21
17
HO
HO
HO
O
8d
—
24
34
17
18
37
31
CH2
8e
—
17
>75
16
73
20
>75
75
O
8f
9a
MeOCH2CH2
—
—
38
45
18
10
34
47
38
16
40
67
47
45
40
47
CH2CH2OH
CH2CH2
9b
—
—
16
15
6
17
35
8
nt
nt
4
6
OH
OH
10a
CH2CH2OH
nt
nt
nt
nt
CH2CH2
10b
—
16
nt
16
nt
nt
nt
nt
11a
11b
H
CH2CH2OH
CH2CH2OH
6 (3.2)b
7
5
6
5 (2.9)b
13
12
16
nt
nt
<2
2
2
6
HOCH2CH2
11c
CH2CH2
CH2CH2
CH2CH2OH
5
2
<2
14
40
<2
4
HO
HO
HO
O
11d
CH2CH2OH
5
7
5
32
nt
4
4
CH2
11e
CH2CH2OH
4
5
13
32
64
5
13
O
11f
MeOCH2CH2
CH2CH2OH
8
4
5
28
<2
nt
nt
2
2
11
5
11g
H
3 (1.0)b
<2
<2 (0)b
CH2CH2
OH
OH
CH2CH2
11h
HOCH2CH2
6
5
2
6
nt
5
5
nt: not tested.
a
The IC50 values were an average of four replicate wells at each concentration tested from a single experiment, unless otherwise stated. See Ref. 13 for experimental details.
The IC50 values were an average of three separate experiments with each concentration replicated in four wells at each experiment. The standard deviations are shown in
b
parentheses.
9 appeared to enhance the apoptosis-mediated cell death; once
again, however, the necrotic population far exceeded the apoptotic
population (Fig. 1). In stark contrast, the apoptosis assay results of
the 3,30-bis(aza) triterpenoids (11) (Fig. 2) revealed not only that
all the bis(aza) compounds induce apoptosis but, more signifi-
cantly, that apoptosis appeared to be the primary route of cell
death. The apoptotic population induced by 11a, 11c, 11g, and
11h accounted for more than 60% of the cell population whereas
the necrotic population accounted for less than 15%. Similar results
were obtained when Annexin assays were performed in Daoy cells
(data not shown).
In summary, a novel class of 3,30-bis(aza) lupane triterpenoids
(11) was identified not only to possess improved cytotoxicity com-
pared to the natural product betulinic acid (1) but also to affect cell

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A. A. Mar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5389–5393
120
100
80
60
40
20
0
Necrotic Viable Apoptotic
Untreated DMSO
1
5b
5c
6a
6b
6c
8a
8c
8d
9b
Compound
Figure 1. Annexin assay: SK-MEL-2 cells were treated with 7.5
lM of test compounds for 16 h (error bars representing standard deviations of three replicates).
100
90
80
70
60
50
40
30
20
10
0
Necrotic Viable Apoptotic
Untreated DMSO
1
11a
11b
11c
11d
11e
11f
11g
11h
Compound
Figure 2. Annexin assay: SK-MEL-2 cells were treated with 7.5
lM of test compounds for 16 h (error bars representing standard deviations of three replicates).
11. (a) Sharpless, K. B.; Lauer, R. F. J. Org. Chem. 1974, 39, 429; (b) Engman, L. J. Org.
death predominantly via apoptosis. The mono(aza) derivatives (5,
6, 8, 9, and 10) apparently triggered cell death via different, non-
apoptotic pathway(s). Further studies to elucidate the mechanism
of action by these aza-lupane triterpenoids are currently underway
and will be reported in due course.
Chem. 1987, 52, 4086.
12. The newly synthesized compounds provided satisfactory MS and NMR
(500 MHz, DMSO-d6) spectra without exhibiting any discernible impurities.
For the 3-amino compound (5, 6, 8 and 11), the stereochemistry at C3 is yet to
be determined. The selected analytical data of representative compounds are
shown as follows. Compound 5c: mp 204–210 °C. 1H NMR d 0.60 (s, 3H), 0.66
(d, J = 10.4 Hz, 1H), 0.78–0.98 (m, 2H), 0.75 (s, 3H), 0.86 (s, 6H), 0.93 (s, 3H),
1.06–1.54 (m, 15H), 1.56–1.68 (m, 3H), 1.64 (s, 3H), 1.75–1.90 (m, 3H), 2.11 (d,
J = 8.4 Hz, 1H), 2.22 (t, J = 9.2 Hz, 1H), 2.55 (m, 2H), 2.80–3.00 (m, 2H), 4.56 (s,
1H), 4.68 (s, 1H), 6.64 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 9.10 (br s, 1H).
MS (APCI+) m/e 577 (M+H); Compound 6a: mp 156–163 °C. 1H NMR d 0.62 (s,
3H), 0.75 (s, 3H), 0.78–1.08 (m, 5H), 0.86 (s, 3H), 0.93 (s, 3H), 0.98 (s, 3H), 1.10–
1.38 (m, 10H), 1.45–1.68 (m, 6H), 1.63 (s, 3H), 1.82–1.92 (m, 4H), 2.12 (dd,
J = 5.3, 13.1 Hz, 1H), 2.38 (m, 1H), 3.08, 3.52 (d-AB Type, J = 4.4 Hz,
JAB = 10.6 Hz, 2H), 4.21 (t, J = 5.0 Hz, 1H), 4.54 (d, J = 1.0 Hz, 1H), 4.66 (d,
J = 2.0 Hz, 1H). MS (ESI+) m/e 442 (M+H). Compound 8c: mp 240–247 °C. 1H
NMR d 0.60 (s, 3H), 0.66 (d, J = 11.0 Hz, 1H), 0.75 (s, 3H), 0.79–1.01 (m, 4H),
0.87 (s, 3H), 0.92 (s, 3H), 0.98 (s, 3H), 1.02–1.39 (m, 11H), 1.46 (d, J = 8.5 Hz,
1H), 1.54–1.70 (m, 5H), 1.80–2.01 (m, 4H), 2.22 (m, 1H), 2.50–2.60 (m, 2H),
2.87 (m, 1H), 3.05, 3.50 (d-AB Type, J = 5.4 Hz, JAB = 10.4 Hz, 2H), 3.88 (br s, 2H),
4.20 (t, J = 5.3 Hz, 1H), 4.71 (t, J = 5.5 Hz, 1H), 4.74 (s, 1H), 4.84 (s, 1H), 6.64 (d,
J = 8.0 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 9.09 (s, 1H). MS (APCI+) m/e 578 (M+H).
Compound 9b: mp 173–182 °C. 1H NMR (500 MHz, DMSO-d6) d 0.82–1.12 (m,
3H), 0.85 (s, 3H), 0.93 (s, 3H), 0.94 (s, 3H), 0.99 (s, 3H), 1.01 (s, 3H), 1.14–1.28
(m, 3H), 1.30–1.46 (m, 9H), 1.56–1.72 (m, 4H), 1.76–2.00 (m, 4H), 2.26–2.46
(m, 3H), 2.58 (m, 2H), 2.64 (m, 2H), 3.07 (m, 3H), 3.52 (dd, J = 4.7, 10.2 Hz, 1H),
4.20 (t, J = 5.0 Hz, 1H), 4.75 (s, 2H), 6.65 (dd, J = 2.0, 6.5 Hz, 2H), 6.97 (d,
J = 8.5 Hz, 2H), 9.10 (s, 1H). MS (APCI+) m/e 576 (M+). Compound 11a: mp 190–
References and notes
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Q.; Fang, J.; He, J.; Cheng, Y. Q.; Wu, Q. L. Acta Pharmacol. Sin. 2010, 31, 66.
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M. T.; Xu, Z.-Q. Bioorg. Med. Chem. Lett. 2009, 19, 2168.
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A. A. Mar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5389–5393
5393
203 °C. 1H NMR d 0.62 (s, 3H), 0.75 (s, 3H), 0.83–1.16 (m, 5H), 0.86 (s, 3H), 0.93
(s, 3H), 0.98 (s, 3H), 1.19–1.39 (m, 11H), 1.44–1.59 (m, 6H), 1.67 (dt, J = 3.5,
12.5 Hz, 1H), 1.82–2.00 (m, 4H), 2.13 (dd, J = 4.2, 11.2 Hz, 1H), 2.29 (br s, 1H),
2.54 (q, J = 6.0 Hz, 2H), 3.07 (m, 2H), 3.08, 3.51 (AB Type, JAB = 10.5 Hz, 2H), 3.44
(t, J = 5.7 Hz, 2H), 4.20 (br s, 1H), 4.44 (br s, 1H), 4.76 (s, 1H), 4.80 (s, 1H). MS
(APCI+) m/e 501 (M+). Compound 11g: mp 195–200 °C. 1H NMR d 0.66 (s, 3H),
0.76 (s, 3H), 0.68 (d, J = 11.0 Hz, 1H), 0.78–1.03 (m, 4H), 0.89 (s, 3H), 0.92 (s,
3H), 0.98 (s, 3H), 1.02–1.27 (m, 5H), 1.31–1.50 (m, 8H), 1.57 (d, J = 10.0 Hz, 3H),
1.67 (t, J = 11.5 Hz, 1H), 1.81–1.98 (m, 3H), 2.32 (m, 3H), 2.58 (q, J = 6.5 Hz, 2H),
2.64 (q, J = 6.5 Hz, 2H), 3.08 (m, 3H), 3.51 (d, J = 10.5 Hz, 1H), 4.19 (br s, 1H),
4.74 (s, 2H), 6.65 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 9.12 (br s, 1H). MS
(ESI+) m/e 577 (M+H).
compound solutions in growth media, they were incubated for 72 h for all the
cell lines with exception for OVCAR which was treated for 120 h. Next, the MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphenyl)-
2H-tetrazolium, inner salt) reagent was added and incubation was continued
for 1.5–4 h at 37 °C. During the incubation, MTS is bioreduced to formazan by
viable cells. The inhibitory effects were obtained by measuring the absorbance
of the formazan at 490 nm on a Wallac Victor II plate reader and calculated by
subtracting the absorbance measured at the same wavelength from DMSO-
treated cells.
14. Annexin-V assay: The Guava Nexin Assay was used to measure the percentage
of cells undergoing apoptosis by treatment with the test compounds. Cells
(8.75 Â 105 per well) were plated in
a
60 mm dish the evening before
13. MTS assay: The anti-proliferative activities of the molecules were measured by
using the CellTiter 96 Aqueous Non-radioactive cell proliferation assay. Cells
treatment. Upon treatment with 7.5 lM test compound solutions in growth
media without FBS, the cells were incubated at 37 °C for 16 h. Next 10% FCS
was added to prevent cell damage and improve the efficiency of cell pelleting
during subsequent centrifugation steps. After trypsinization and cell pelleting,
the Nexin staining solution (including Annexin V and 7-ADD) was added and
incubation was continued on ice for 20 min. Apoptosis was measured on a
Guava instrument by setting the gates as described in the manufacturer’s
protocol to establish quadrants and to measure the population of viable, mid
apoptotic and late apoptotic/necrotic cells in each quadrant.
(1 Â 104 per well) were plated onto
a 96-well plate the evening before
treatment in triplicate. Test compounds were initially dissolved in DMSO at
10 mM concentration and serially diluted in growth media to various
concentrations. Each compound was tested at five different concentrations,
2, 10, 25, 50 and 75
The negative control (0
DMSO as that of the highest test concentration. Upon treatment with the test
l
M, and each concentration was replicated in four wells.
lM of test compound) contained the same amount of