Synthesis and biological evaluation of novel triptolide analogues for anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2010.08.106

Three types of novel triptolide analogues with 9,11-olefin (3-5), five-membered unsaturated lactam ring (6-7) or A/B cis ring junction (8-14) were synthesized. Although with 9,11-olefin instead of 9,11-β-epoxide, compound 4a was much more active than the parent natural triptolide (1) with the lowest IC₅₀ value of 0.05 nM for SKOV-3 cells, clearly challenging the traditional viewpoint on the necessity of 9,11-β-epoxide group of triptolide. In addition, structure-activity relationships for three classes of compounds were studied.

Full text of DOI:10.1016/j.bmcl.2010.08.106

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6217–6221
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel triptolide analogues
for anticancer activity
⇑
Bing Zhou, Zehong Miao, Gang Deng, Jian Ding, Yaxi Yang, Huijin Feng, Yuanchao Li
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Road Zu Chong Zhi, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2010
Revised 19 August 2010
Accepted 20 August 2010
Available online 26 August 2010
Three types of novel triptolide analogues with 9,11-olefin (3–5), five-membered unsaturated lactam ring
(6–7) or A/B cis ring junction (8–14) were synthesized. Although with 9,11-olefin instead of 9,11-b-epox-
ide, compound 4a was much more active than the parent natural triptolide (1) with the lowest IC50 value
of 0.05 nM for SKOV-3 cells, clearly challenging the traditional viewpoint on the necessity of 9,11-b-epox-
ide group of triptolide. In addition, structure–activity relationships for three classes of compounds were
studied.
Keywords:
Triptolide
Ó 2010 Elsevier Ltd. All rights reserved.
Structure–activity relationships
9,11-Olefin
Five-membered unsaturated lactam ring
A/B cis ring junction
Triptolide (1) and triptonide (2) (Fig. 1), the major components
for the clinical properties of Tripterygium wilfordii Hook.
f. (TWHF)1–3, were first isolated from TWHF extracts and character-
ized as a diterpenoid triepoxide lactone containing an 18 (4?3)
abeo-abietane skeleton in 1972.4 Right after its isolation, triptolide
(1) and triptonide (2) were shown to possess potent antitumor,
antiinflammatory, immunosuppressive, and antifertile activi-
ties.4–22 Compared with some conventional chemotherapeutic
drugs, triptolide has similar or even superior anticancer activity,
especially over p53 mutated or multi-drug resistant cells.17 All
these antitumor properties mentioned above suggest that tripto-
lide should be a promising anticancer drug, however, no systematic
structure–cytotoxic activity relationship (SAR) studies have been
reported.
Previous studies on the structure–activity relationship of tripto-
lide indicated that the activation of the 9,11-b-epoxide group by
the hydrogen bonding between the 14b-hydroxyl group hydrogen
atom and the 9,11-b-epoxide oxygen atom may account for its
antitumor effect and the 9,11-b-epoxide is faithfully critical to its
anticancer activity.5 Base on the principle, for a long time no
9,11-b-epoxide modification was reported and the aim of C14
modification was just to improve the lead compound’s water-solu-
bility by carboxylation of C14b–OH to introduce water-solubility-
enhancing moieties.23,24 However, some medicinal chemists have
found recently that substituting C14-hydroxyl with other groups
such as fluoride while keeping b orientation of the substituents
16
15
O
O
O
12
8
11
9
13
14
17
20O
O
1
2
O
OH
10
5
O
3
4
O
7
O
H
6
H
18O
O
19
triptonide (2)
triptolide (1)
Figure 1. Triptolide and triptonide from Tripterygium wilfordii Hook. f.
O
O
O
O
O
O
HO
HO
O
O
R
O
O
O
O
H
H
H
HN
X
O
3, X=O, R=O
7
5
4a, X=O, R=(14R)-OH
4b, X=O, R=(14S)-OH
6, X=NH, R=O
O
O
O
O
O
H
OH
O
O
O
O
O
O
O
H
H
O
O
O
8, (7S, 8R, 12R, 13R)-epoxide
9, (7S, 8R, 12S, 13S)-epoxide
13, (7R, 8S, 12S, 13S)-epoxide
14, (7R, 8S, 12R, 13R)-epoxide
10, (12R, 13R)-epoxide
11, (12S, 13S)-epoxide
12
⇑
Corresponding author. Tel.: +86 21 50806600/3502; fax: +86 21 50807288.
E-mail address: ycli@mail.shcnc.ac.cn (Y. Li).
Figure 2. Triptolide analogues 3–14.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.106

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B. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6217–6221
or with a chiral epoxy group having a different oxygen direction at
C14 position could retain the cytotoxicity of triptolide,25,26 and the
anticancer activity of triptolide analogues may not be explained so
simply as before.5
series of analogues (compounds 8–14) with A/B cis ring junction
were designed for more comprehensive SAR studies. The SAR stud-
ies of these tripolide analogues were performed by using ovary (SK-
OV-3) and prostate (PC-3) tumor cells.
In the present study, we designed three types of analogues of tri-
ptolide 1. To begin with, to explore whether the 9,11-b-epoxide of
triptolide is faithfully critical to its anticancer activity, 9,11-b-epox-
ide modified analogues (compounds 3–5) were synthesized (Fig. 2).
Then, analogues of triptonide were sought in which five-membered
unsaturated lactone ring was replaced with the five-membered
unsaturated lactam ring (compounds 6 and 7) as amide often be-
haves as ester isostere and the water-solubility of amide is greater
than that of corresponding ester. This would have its practical sig-
nificance because good aqueous solubility of products applied is
invaluable and the clinical development of triptolide have been
challenged by water-insolubility and severe toxicity. Finally, a
The synthetic strategy followed for the preparation of the tri-
ptonide analogues 3–14 is depicted in Schemes 1–3. Considering
that direct modification of 9,11-b-epoxide on triptonide 2 was dif-
ficult owing to the presence of 12,13-a-epoxide which was very
sensitive to nucleophilic attack, we chose the triptophenolide
methyl ether 15 as the starting material which was prepared from
abietic acid according to the reported procedure27 and compound
16 was also prepared by the procedure described (Scheme 1).27
Treatment of 16 with basic H2O2 provided compound 3 in 56%
yield, along with the 12,13-b-epoxy isomer.28 Subsequent reduc-
tion of 3 with NaBH4 in MeOH furnished 4a (23%) together with
its
a-hydroxyl epimer 4b (72%) and a modification of Sharpless’
O
O
O
O
HO
HO
ref. 27
O
a
O
O
c
O
O
O
O
O
O
H
H
H
3
H
O
O
O
O
O
15
16
5
b
O
O
O
OH
OH
O
O
H
H
O
O
4b
4a
Scheme 1. Synthesis of analogues 3, 4a, 4b and 5. Reagents and conditions: (a) H2O2, NaOH, MeOH, 25 °C, 2 h, 56%; (b) NaBH4, CH3OH, rt, 0.5 h, 23% of 4a and 72% of 4b; (c)
K2OsO4Á(H2O)2, NMO, pyridine, acetone/water = 5:1, reflux, 2 days, 65%.
O
O
O
a
O
O
O
H
15
H
H
18
HN
O
O
17
b
OH
d
O
OH
OH
c
O
O
O
O
O
H
H
HN
H
e
HN
HN
21
19
20
O
O
O
g
f
O
O
O
O
O
O
O
O
O
H
H
HN
HN
H
HN
6
7
22
O
O
O
O
a
O
O
O
O
O
O
H
O
H
HN
2
7
Scheme 2. Synthesis of analogues 6 and 7. Reagents and conditions: (a) NH3ÁH2O–NH4Cl buffer solution (pH 9), H2O, reflux, 2 days, 57% of 17 and 32% of 18; (b) CrO3, HOAc
(aq), rt, 4 h, 35%; (c) BBr3, CH2Cl2, À78 °C to rt, 92%; (d) NaBH4, CH3OH, 0 °C, 0.5 h, 91%; (e) NaIO4, CH3OH/H2O = 3:1, 0 to 25 °C, 0.5 h, 85%; (f) H2O2, K2CO3, MeOH, 25 °C, 2 h,
65%; (g) mCPBA, Na2CO3, CH2Cl2/H2O = 4:3, 10 h, 45%.

B. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6217–6221
6219
O
a
O
O
c
d
OH
OH
O
O
OAc
O
O
b
OH
H
O
H
H
H
H
H
O
O
O
O
O
26
25
18
23
e
O
O
O
O
f
i
O
O
O
O
OH
O
O
O
O
O
H
H
H
24
O
O
O
8
27
10
O
O
O
O
O
g
O
j
OH
O
O
O
h
O
O
O
H
H
O
O
9
12
11
Scheme 3. Synthesis of analogues 8–12. Reagents and conditions: (a) (NH4)2Ce(NO3)6, CH3CN/H2O = 1:1, rt, 2 h, 80%; (b) BBr3, CH2Cl2, À78 °C to rt, 2 h, 90%; (c) acetic
anhydride, pyridine, 50 °C, 3 h, 98%; (d) BBr3, CH2Cl2, À78 °C to rt, 2 h, 60%; (e) NaIO4, CH3OH/H2O = 3:1, 0 to 25 °C, 0.5 h, 96%; (f) H2O2, K2CO3, MeOH, 25 °C, 2 h, 62%; (g) t-
BuOOH, (CH3)4NOH, benzene, 45 °C, 5 h, 60%; (h) NaBH4, CH3OH, 0 °C, 0.5 h, 95%; (i) mCPBA, Na2CO3, CH2Cl2/H2O = 4:3, 10 h, 62%; (j) mCPBA, Na2CO3, CH2Cl2/H2O = 4:3, 10 h,
56%.
original procedure29 using potassium osmate, pyridine and NMO
(N-methylmorpholine-N-oxide) in aqueous acetone afforded
9,11-b-diol 5 in 65% yield by dihydroxylation of 3.
affording 26 in 59% yield for the two steps. Periodate oxidation
of 26 afforded epoxy dienone 27 in 96% yield which upon treat-
ment with H2O2–K2CO3 in MeOH furnished 12,13-b-epoxide 8
as a single diastereomer. In addition, epoxidation of 27 with
tert-butyl hydroperoxide in benzene in the presence of tetrameth-
Then, we switched to the construction of the five-membered
unsaturated lactam ring triptonide’s analogues. Treatment of 15
with NH3ÁH2O–NH4Cl buffer solution provided lactam 17 and com-
pound 18 with the desired A/B cis ring junction (Scheme 2). Unfor-
tunately, attempts to direct ammonolysis of 2 with NH3ÁH2O–NH4Cl
buffer solution led to decomposition. Benzylic oxidation of 17 with
CrO3 in AcOH/H2O28 provided the ketone 19 that was demethylated
to afford phenol 20. Reduction of 20 with NaBH4 gave exclusively
the C-7b alcohol 21.28 The prototypic oxidation course described
by Adler et al.30 was applied in the conversion of 21 to epoxy die-
none 22, which was treated with basic H2O2, giving compound 6
in 65% yield, along with the 12,13-b-epoxy isomer.28 Exposure of
6 to mCPBA underwent selective b-face epoxidation to afford com-
pound 7 having triepoxides as the sole epoxidation product in 45%
yield.
ylammonium hydroxide31 gave the 12,13-
a-epoxy isomer 9 in
60% yield, along with 8 in 5% yield. Borohydride reduction of 9
afforded exclusively C14-b alcohol 12 in 95% yield. Further epox-
idation of 8 with mCPBA afforded triepoxides 10 as a single dia-
stereomer in 62% yield. Likewise, treatment of 9 with mCPBA
gave 11 in 56% yield.
In order to get the 7,8-a-epoxide analogues, 23 was first treated
with Collins reagent to afford ketone 28 which was demethylated
to give 29 that upon treatment with NaBH4 in MeOH furnished
exclusively C7-a alcohol 30 as a single diastereomer with an over-
all yield of 70% (Scheme 4). Periodate oxidation of 30 provided
compound 31 in 90% yield which upon treatment with H2O2–
K2CO3 in MeOH gave 13 and 14 in 15% and 60% yields, respectively.
The X-ray crystallographic analysis of compounds 13 and 14 con-
firmed their structures to be as shown in Figures 3 and 4.32 Direct
epoxidation of compounds 13 and 14 with some oxidizing agents,
such as dimethyldioxorane, mCPBA, or hydrogen peroxide–sodium
hydroxide, failed to give a triepoxy compound 32.
Treatment of 18 with ammonium ceric nitrate in H2O/CH3CN
gave, as shown in Scheme 3, exclusively the benzyl alcohol 23.
Direct demethylation of 23 with BBr3 generated the dehydration
product 24, so protection of hydroxyl with acetic anhydride pro-
vided compound 25 which was treated with BBr3 in CH2Cl2,
OH
OH
O
a
c
O
b
OH
O
O
OH
O
O
O
O
H
H
H
H
O
O
O
O
23
28
30
29
d
O
O
O
O
O
O
Ox.
O
e
O
O
O
O
O
O
O
O
O
H
H
O
O
H
H
O
32
O
13
14
31
Scheme 4. Synthesis of analogues 13 and 14. Reagents and conditions: (a) Collins reagent, CH2Cl2, rt, 3 h, 89%; (b) BBr3, CH2Cl2, À78 °C to rt, 2 h, 96%; (c) NaBH4, CH3OH, 0 °C,
0.5 h, 82%; (d) NaIO4, CH3OH/H2O = 3:1, 0 to 25 °C, 0.5 h, 90%; (e) H2O2, K2CO3, MeOH, 25 °C, 2 h, 15% of 13 and 60% of 14.

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B. Zhou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6217–6221
from 0.05 nM to 29 lM (Table 1). Among them, compounds 3
and 4a exhibited the highest potency, with the lowest IC50 value
(3 nM and 0.05 nM for SKOV-3 cells). However, compound 5 with
the 9,11-b-dihydroxyl was shown to be only weakly cytotoxic.
The data indicates that the 9,11-b-epoxide group of triptolide is
not unchangeable even in order to generate analogues of potent
anticancer activity. Our result apparently challenges the classical
structure-activity relationship of triptolide that considers 9,11-b-
epoxide group to be essential for its anticancer activity5 and the
present result indicates that the activity of triptolide (1) series
seems to owe more to the C-ring three-dimensional structure
which may easily and inevitably be affected by the introduction
of new groups or by changes of the groups attached to ring C or
by the changes of C-ring structure itself as compound 1–4a, having
9,11-b-epoxide or 9,11-olefin, are significantly cytotoxic while the
cytotoxicity of compound 5, having 9,11-b-dihydroxyl, reduced
greatly against the two cell lines. On the other hand, the series of
analogues (6 and 7) with five-membered unsaturated lactam ring
were found to be still effective against the two cell lines with the
Figure 3. X-ray single crystal structure of compound 13.
very low IC50 value (0.02 lM for SKOV-3 cells) and more soluble
in water than triptonide (2). Besides, the series of analogues
(8–14) with A/B cis ring junction nearly completely lost their cyto-
toxicity against PC-3, and only compound 13 retained weak
potency against SKOV-3 line. Based on the above results, we pre-
sumed that the trans A/B-ring junction is important for retaining
cytotoxic activity.
In summary, three types of novel triptolide analogues (3–14)
were prepared. The results suggested the possible effect of the C-
ring three-dimensional structure, the plane structure of five-mem-
bered unsaturated D-ring and the trans A/B-ring junction on the
cytotoxic activity of the compounds of these series. Compound
4a was much more active than the parent natural triptolide (1)
with the lowest IC50 value of 0.05 nM for SKOV-3 cells, which sug-
gested that the 9,11-b-epoxide group of triptolide is not unchange-
able even in order to generate analogues of potent anticancer
activity.
Figure 4. X-ray single crystal structure of compound 14.
As shown above, we obtained three types of novel triptolide
analogues with 9,11-olefin, five-membered unsaturated lactam
ring or A/B cis ring junction and we evaluated the in vitro antican-
cer effects of those target compounds (3–14) against two human
tumor cell lines derived from ovary (SK-OV-3) and prostate
(PC-3) using sulforhodamine B (SRB) assays.33 The result revealed
that although there is no 9,11-b-epoxide group in our new ana-
logues, the series of 9,11-olefin analogues (3, 4a, 4b and 6) were
all effective against those two cell lines, with IC50 values ranging
Acknowledgements
This work was supported by National Science & Technology
Major Project ‘‘Key New Drug Creation and Manufacturing Pro-
gram”, China (Number: 2009ZX09102-026).
Supplementary data
Table 1
Supplementary data (synthesis, specific rotation, 1H NMR, 13C
NMR, MS, HRMS, bioassay method34 and HPLC analyses of final
compounds) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2010.08.106.
In vitro anticancer activity of triptolide analogues
in SK-OV-3 and PC-3 cells
a
Compound
IC50
SK-OV-3
(lM)
PC-3
1
2
3
4a
4b
5
6
7
0.006
0.008
0.003
0.00005
0.5
>100
0.02
0.02
95
0.02
0.6
2.7
0.01
29
>100
0.9
1.4
>100
29
>100
>100
>100
>100
>100
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