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the existence of trans-conjugated triolefin. Its acyloxyl
group is assisted with dH 1.999 (3H, s), dC 21.3 (C-200),
and dC 169.8 (C-100) by the HSQC spectrum. Ascriptions
of other protons and carbons atoms are shown in Table
2. Compound 4 was identified as 60,70-dehydro-yuanhu-
adine, namely yuanhuagine. To our knowledge, there is
no report on this structure until now.
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
Yuanhuapine (5) is a known compound. Its structure
was presumed by a combination of MS and the other
spectra.16
Figure 2. Compound 1 inhibited the relaxation activity of topo I
compared with hCPT. Lane 1, DNA alone; lane 2, DNA + topo I; lane
3, DNA + 1; lanes 4–11, 1 at concentrations of 0.78, 0.39, 0.2, 0.1, 0.05,
0.025, 0.0125, and 0.006 mM + DNA + topo I, respectively; lanes 12–
15, hCPT at concentrations of 1.0, 0.76, 0.38, and 0.2 mM + DNA +
topo I, respectively.
In order to compare the inhibitory activity against DNA
topo I and investigate the structure–activity relation-
ship, three derivatives (6, 7, and 8) of 1 were prepared
4,12
by reported methods.
inhibit the relaxation activity of topo I toward DNA.
It was also found that as the concentration of 1 reduced,
the inhibition weakened gradually, once less than the
critical concentration, there is no inhibitory activity.
Compounds 2–7 illustrated the similar situation as 1.
Their agarose-gel electrophoresis pictures are also given
in Figure 3.
Commonly, esters can easily hydrolyze in basic solution,
compound 6 was prepared as hydrolysis product of 1
under basic condition. While orthoester cannot hydro-
lyze in basic solution, it can easily hydrolyze in acidic
solution, compound 8 obtained as hydrolysis product
of 1 under acidic condition. Compound 7 was prepared
by the condensation reaction between C4,C20-hydroxyl
groups with acetone.
Through the agarose-gel electrophoresis experiments,
these daphne diterpene esters indicated potent inhibitory
activity against topo I, and the inhibitory activities were
found to be structure related. The results are shown in
Table 3, the IC50 levels of 1–5 being 40.0, 38.3, 52.7,
50.1, and 53.4 lM, respectively. Compounds 6 and 7
have inhibitory activity against topo I at IC50 levels of
11.1 and 28.1 lM, respectively, which indicate a stron-
ger inhibitory activity than those of 1–5. Most interest-
ingly, 8 gave an IC50 level of 204.2 lM, nearly inactive
(IC50 > 100 lM) (Fig. 4).
2.2. Topoisomerase I inhibitory activities
DNA topo I is a kind of important enzyme in all of the
living organisms and participates in many cellular meta-
bolic processes, such as replication, transcription,
recombination, and repair.17 It has been established as
an important target of anticarcinogen by NCI, and the
inhibitors against DNA topo I offer hope for a new
way of cancer chemotherapy. As far, camptothecin
and its analogs are the only type of anticancer agents to-
ward DNA topo I under clinical application, and the
daphne diterpene esters are another kind of topo I inhib-
itors with completely different structures compared with
camptothecin and its analogs.
Compounds 1–7 have the same basic backbone with an
orthoester group on it, they exhibited the inhibitory
activity against topo I, and different substitute groups
only influenced the inhibitory activity limitedly. As ob-
served for 8, once the orthoester group was broken, the
inhibitory activity was lost. So it can be concluded that
the orthoester group is necessary in the inhibition
against DNA topo I for daphne diterpene esters.
Although 1–7 have the same basic backbone, changes
in the branch chain affected their inhibitory activity
to some extent. The structure–activity relationship will
be comparatively discussed in the following: (1) by
transforming the electron-withdrawing hydroxyl groups
at C-5 and C-20 to ketal, the inhibitory activity can be
dramatically promoted, as for 7, it manifested the
strongest activity among these compounds. (2) Acyl-
oxyl group at C-12 position substituted for less elec-
tron-withdrawing benzyloxyl group results in stronger
inhibitory activity against topo I, for example, 1
showed stronger activity than 3, 2 showed stronger
activity than 4. For compound 6, the hydroxyl group
at C-12 is a less electron-withdrawing group compared
with the ester groups in 1–4. This variation makes 6
have a much stronger activity than compounds 1–4.
(3) Changes at C-10 position of orthoether group give
a negligible effect on the inhibitory activity against
topo I, for example, although bearing different substi-
The inhibitory activities against topo I of these com-
pounds were evaluated by agarose-gel electrophoresis
experiments. In order to verify the activities of these
compounds, compound 1 was chosen for agarose-gel
electrophoresis experiment and compared with the
known topo I inhibitor hydroxycamptothecin (hCPT).
As shown in Figure 2, compound 1 exhibited inhibitory
activity against DNA topo I at IC50 level of 40.0 lM,
which value can br compared to that displayed by hCPT
(IC50 = 48.0 lM).
The photopictures of 1–8’s agarose-gel electrophoresis
experiment are presented in Figure 3. Take compound
1 as an example: lane 1 is DNA alone, existing in two
kinds of shapes—supercoiled DNA and a little of loos-
ened DNA; lane 2 is topo I together with DNA, and
supercoiled DNA was relaxed by topo I completely; in
lane 3, the system consisted of DNA and 1. Lane 3 is
similar to lane 1, which indicated that 1 could not com-
bine with DNA; In lanes 4–11, compound 1, DNA, and
topo I existed in the same system. Lanes 4–11 clearly
manifested that 1 is the inhibitor of topo I, which can