2
X.-F. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
R1OH
Furthermore, no catalyst or using strong base catalyst in aqueous
solvent also resulted in the unreacted starting material. To our sur-
prise, the monoepoxide 3 could be smoothly converted to 2-alky-
O
O
O
O
NaOH -H2O
O
O
H
R1O
O
R1O
O
loxyl substituted (25R)-spirostan-1,4,6-triene-3-ones
4 under
catalytic amount of NaOH in anhydrous alcohol (Scheme 1).
3
3a
4
During the synthesis and isolation of compound 4a, no ring-
opening intermediate of the monoepoxide 3a was observed.
When the reaction temperature was gradually lowered to À20 °C,
only a small amount of 4a was observed in the mixture. Elevated
temperatures could shorten the reaction time, and the highest
yield of 4a reached 87% at 50 °C under 5% NaOH in anhydrous
methanol. The higher temperatures over 50 °C or concentration
of NaOH beyond 5% resulted in reduced yields. Finally, 5% NaOH
in anhydrous alcohol was chosen as the best catalyst, and seven
target (25R)-2-alkyloxyl-spirostan-1,4,6-triene-3-ones (4a–4g)
were achieved in moderate to excellent yields (65–89%).
O
O
O
O
NaOH
OH
H
R1O
H2O
R1O
O
- H2O
O
3b
Scheme 2. Possible reaction mechanism for (25R)-2-alkyloxyl-spirostan-1,4,6-
triene-3-one (4).
Structure elucidation: All the structures of 2-alkyloxyl substituted
(25R)-spirostan-1,4,6-triene-3-ones (4a–4g) and 1,2,3-triazoles
derivatives (5a–5o) were confirmed by spectral analysis with satis-
factory spectral data. In 1H NMR spectrum of 4a–4g, the olefinic pro-
ton of H-1 in the A-ring located at 5.92–6.04 ppm as singlet, and the
single peak of H-4 located at relatively high field with chemical shift
of 6.04–6.20 ppm. Both of H-6 and H7 in the B-ring showed two sep-
arated double of doublets at 6.22–6.24 and 6.02–6.07 ppm, respec-
tively. The single peak of steroidal triazoles 5a–5o could be clearly
observed in the range of 7.55–8.15 ppm, and the proton in the
methylene groups of N–CH2 and O–CH2 were assigned to 5.03–
5.84 ppm as singletsor quartets. Thestronginfraredabsorptionpeak
at 2099 cmÀ1 was assigned to the azide group in 5d, which disap-
peared when bis-steroidal triazole 5e was formed. On the other side,
only 33 carbon resonance peaks in the 13C NMR spectra were found
for the bis-steroidal triazole 5e, which further indicated the sym-
metrical structure had been successfully synthesized. In the HR-
ESI-MS spectrum, the ion adducts of (M+Na)+ or (M+K)+ were usually
observed as the base peak ion for all the target compounds. The
structure of compound 4b was also confirmed by the X-ray crystal-
lographic analysis (CCDC number: 1050838), and its ORTEP drawing
with common atom numbering scheme was shown in Figure 1.
Biological evaluation: As our continuous interest in search of the
biological steroidal antitumor agents,28,29 all the target compound
were also evaluated for in vitro anti-proliferative activities against
three human cancer cell lines (Hep-G2, Caski and Hela) for 48 h at
37 °C by MTT assays. The growth inhibition (%) was determined in
On the basis of the results obtained, the possible mechanism for
the abnormal monoepoxide ring-opening reaction might be inter-
preted as consisting of the following steps (Scheme 2). Firstly,
the alcohol R1OH was deprotonated by NaOH to generate the
alkoxy anion, which nucleophically attacked the monoepoxide 3
to give the ring-opening intermediate 3a. After the protonating
procedure, the most stable trienone system 4 was formed under
the basic conditions through the elimination mechanism.
Click synthesis of steroidal 1,2,3-triazoles (5): Considering the
attractive features of 1,2,3-triazole moieties in medicinal and bio-
logical chemistry,21,22 a series of steroidal azoles were prepared
through Huisgen’s Cu(I)-catalyzed 1,3-dipolar cycloaddition of
(25R)-2-propynyloxy-spirostan-1,4,6-triene-3-one 4e with azides
in this work (Scheme 3). According to the literature methods, the
corresponding azides were synthesized by the substitution reac-
tion of alkyl bromides with sodium azide.23,24 We therefore
synthesized fifteen (25R)-2-[(1H-1,2,3-triazol-4-yl)methoxy]-
spirostan-1,4,6-triene-3-ones (5a–5o) via classical click chemistry
conditions using copper(II) sulfate pentahydrate, sodium ascor-
bate, tert-butanol and H2O at 80 °C in 20–93% yields.
Considering the unique characteristics and antitumor applica-
tions of the steroidal dimmers,25,26 1,4-xylyldiazide27 was also
used for the synthesis of bis-steroidal triazole derivatives. When
equimolar of 4e and 1,4-xylyldiazide were added, a steroidal tria-
zole 5d (yield 50%) and a bis-steroidal triazole 5e (yield 20%) were
spontaneously isolated in the reaction. Increasing the amount of 4e
and prolonged reaction time could result in the higher yield of the
dimer 5e (Scheme 4).
triplicate at concentrations 5, 10, 20, 40, 80 and 160 lM. All data
were presented as mean standard deviation and analyzed by
SPSS.30 The half-inhibitory concentration (IC50 value) was calcu-
lated and listed in Table 1 based on the percentage inhibition of
cell growth. Compared to the cytotoxicities against HeLa cells,
most of (25R)-2-alkyloxyl-spirostan-1,4,6-triene-3-ones (4) and
(25R)-2-[(1H-1,2,3-triazol-4-yl)methoxy]-spirostan-1,4,6-triene-
3-one (5) displayed the better anti-proliferative activities against
HepG2 and Caski cells.
O
O
O
O
a
HO
O
1
3
2
Introduction of 1,2,3-triazoles to steroidal skeleton were found
the promising cytotoxicities. Four steroidal 1,2,3-triazoles (5c, 5f,
5g, 5m) showed moderate inhibitory effects on HepG2 cells with
O
O
O
O
b
O
c
O
IC50 values of 40.2–45.6
exhibited potent anti-proliferative effects against Caski cells with
the experimental IC50 of 9.4–11.8 against Caski cells.
lM, and three triazoles of 5b, 5f and 5m
O
O
R1
lM
4
Furthermore, compound 5f were observed to possess the broadest
spectrum of cytotoxic activities for all the tested tumor cells, espe-
cially against HeLa cells (IC50 48.1 lM). These results indicated the
introduction of an unsubstituted benzyl, 2-oxopropyl or 3-hydrox-
yphenylethyl group at 1-position of the triazole resulted in increas-
ing cytostatic effect on Caski cells. The further structural exploration
and anti-tumor mechanism study for these steroidal triazoles are
under way in our research group.
4a: R1 = Me
4b: R1 = Et
4e: R1 = CHCCH2-
4f: R1 = n-Bu
4c: R1 = n-Pr 4g: R1 = i-Bu
4d: R1 = i-Pr
Scheme 1. Synthetic routes for the (25R)-2-alkyloxyl-spirostan-1,4,6-triene-3-one
(4). Reagents and conditions: (a) DDQ, 1,4-dioxane, reflux, 15 h; (b) 30% H2O2,
MeONa, MeOH, 20 °C, 12 h; (c) 5% NaOH, anhydrous R1OH, 50 °C, 3 h.