J. R. Woods et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5865–5869
5867
Table 2
The incorporation of an
(thus forming 4) clearly imparted cytotoxic activity against the
cancer cell lines. It is well known that ,b-unsaturated carbonyl
a
-methylene group on eburnamonine 2
Biological evaluation in MDA-MB-231 (breast cancer), LNCaP (prostate cancer), RPMI-
8226 (myleoma), U266 (myeloma), and KMS-12BM (myeloma) cell lines
a
Cell line
LC50 (l
M)a
groups may produce cytotoxic effects following a Michael addition
from nucleophiles such as biological thiols.30 To define the struc-
(ꢀ)-Eburnamonine
41.8 (30–58)
>100
>100
>100
15-Methylene-eburnamonine (4)
ture–activity relationship of the enone of 4, the
a,b-unsaturation
MDA-MB-231
LNCaP
RPMI-8226
U266
14.1 (10–17)
9.5 (7.9–11.4)
12.2 (9.3–16.0)
4.3 (3.1–6.0
was reduced with Pd/C and H2 and analogue 9 was produced in
85% yield as a single diastereomer (Fig. 3).31 The axial assignment
of the stereochemical configuration at the C15-site (eburnamonine
numbering, see Fig. 1) was accomplished after acquisition of COSY
and NOESY. On derivative 9, the proton at the C15 position is a
quartet (J = 7.0 Hz) at 2.83 ppm and displays a NOESY crosspeak
with the axial methine proton at the C3 position. This proton
(see also Scheme 1) is a singlet at 4.16 ppm and exhibits a key
NOESY crosspeak with the axial C21 methyl group (t, J = 7.5 Hz)
at 0.98 ppm. These data support the axial assignment, because
each is on the same face of the molecule. Next, a comparison of
the cytotoxic effects of 9 and 4 in MDA-MB-231 cells demonstrates
the expected loss of biological activity from removing the unsatu-
ration at C15-position (Fig. 3B). These data validate the critical nat-
ure of the enone and this important structure–activity relationship.
To establish the reactivity of the enone towards thiol-based
nucleophiles,32 quantitative NMR studies were conducted.30,33
Quantitative NMR is a powerful analytical tool, and although the
utility of NMR for structure determination is well established, its
potential application in quantitation has not been fully appreciated
in bio-organic and medicinal chemistry. Specifically, almost all
molecules readily generate unique NMR signals, and the amplitude
(integration) of NMR signals can be acquired proportional to the
concentration. This technique eliminates the requirement of an
internal standard to calibrate concentration. Knowing the exact
concentration of a biologically active molecule and calculating
the change in concentration in relationship to time provides valu-
able kinetic data. This point is fundamental in the reactivity of
enones towards thiol-based nucleophiles, because the structure
of the enone and the nature of the solvent can cause substantial
differences in reactivity.32 Dithiothreitol (DTT) was chosen as a
representative thiol for investigation with 15-methylene-ebu-
ranmonine 4 without any deuterated solvent.34Accordingly, in an
NMR tube, DTT (10 equiv) was added in one portion to a solution
of 4 in 80% PBS buffer (pH 8.0) and 20% ethanol. The NMR sample
was immediately placed in the spectrometer and spectra were ac-
quired at intervals of 2, 8, 18, and 80 min. (Fig. 4). The 1H NMR
peaks corresponding to the methylene groups disappeared after
the addition of DTT; these data indicate that the thiol was adding
into the enone. The changes in the concentration of 4 were simul-
taneously quantified by NMR at each time interval using our previ-
ously published no-D NMR methods (Fig. 4B).33,35 At t = 0 min, the
KMS-12BM
>100
4.0 (3.3–4.8)
a
Values are given with 95% confidence interval in parentheses.
cells (Table 2).26 Our rationale for choosing these additional cell
lines is to compare prostate cancer cells to breast cancer cells
and apply our prior discovery that enone-based anti-leukemic
agents are also active against multiple myeloma cell lines.27 Agent
4 displays micromolar activity against the four cell lines and the
natural product 2 is nearly inactive even at the high dose of
100
myeloma cells compared to MDA-MB-231 (breast cancer) cells.19
Its most potent effects were LC50 = 4.3 M (U266) and LC50 = 4.0 -
M (KMS-12BM), and these low micromolar activities were an
lM. Compound 4 displays higher cytotoxicity in prostate and
l
l
excellent validation of the aforementioned synthetic studies. The
cell viability curves for four cancer cell lines are depicted in Figure 2
and present additional perspective on the side-by-side compari-
sons between 2 and 4. These viability curves, along with the previ-
ously published curves in MDA-MB-231 cells,19 display the
repeated difference between the two agents across the five cells
in that only compound 4 eliminates cancer cells in the low micro-
molar range. An additional antiproliferative study was conducted
with agent 4 in an immortalized non-cancerous cell line to exam-
ine its potential selectivity for cancer cells. Indeed, highly selective
killing of cancer cells has been demonstrated by the natural
product, piperlongumine, which bears a similar
a,b-unsaturated
amide.28 The LC50 value of 4 in FnMSC cells, an immortalized mes-
enchymal stem cell line derived from the bone marrow, is
16.71
to the LC50 value of 4.0
demonstrate decrease in potency in the non-cancerous cells com-
pared to cancer cells.
l
M.29 The direct comparison of this data to cancer cells is
l
M in KMS-12BM cells, and these data
(A)
LNCaP (prostate)
(B)
RPMI-8226 (myeloma)
150
150
100
50
0
100
50
0
concentration of 4 was 400
DTT, the concentration of 4 decreased to 330
elapsed, the concentration of 4 was 220
compound was not detected (Fig. 4C).
l
M. Two minutes after treatment with
compd 2
compd 4
compd 2
compd 4
lM. After 18 min had
l
M, and after 80 min, the
3
−
2
1
0
1
2
3
2
−
1
−
0
1
2
−
−
−
log [μM]
log [μM]
(C)
U266 (myeloma)
(D)
KMS-12BM (myeloma)
(A)
(B)
MDA-MB-231 (breast)
150
100
50
150
100
50
0
150
H
100
50
0
N
Pd/C, H2
N
4
MeOH, 85%
(dr > 20:1)
O
compd 2
compd 4
compd 2
compd 4
compd 9
compd 4
9
0
−3 −2 −1
0
1
2
−3 −2 −1
0
1
2
−3 −2 −1
0
1
2
log [μM]
log [μM]
log [μM]
Figure 2. Cell viability curves for (ꢀ)-eburnamonine (2) and 15-methylene-
eburnamonine (4). (A) LNCaP cells. (B) RPMI-8226 cells. (C) U266 cells. (D) KMS-
12BM cells.
Figure 3. Structure–activity investigation of the enone group of 4. (A) Reduction of
enone 4 to derivative 9. (B) Biological evaluation of 4 and 9 in MDA-MB-231 (breast)
cancer cells.