Novel bivalent securinine mimetics as topoisomerase I inhibitors

Article abstract of DOI:10.1039/c6md00563b

A series of novel bivalent securinine mimetics incorporating different linkers between C-15 and C-15′ were synthesized and their topoisomerase I (Topo I) inhibitory activities evaluated. It was thus revealed that mimetic R2 incorporating a rigid m-substituted benzene linker exhibits Topo I inhibitory activity three times that of parent securinine. Comprehensive structure-activity relationship analyses in combination with docking studies were used to rationalize the potent activity of these bivalent mimetics. Mechanistic studies served to confirm the deductions arising from docking studies that the active bivalent mimetics not only inhibited complexation between Topo I and DNA but also stabilized the Topo I-DNA complex itself.

Full text of DOI:10.1039/c6md00563b

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DOI: 10.1039/C6MD00563B
Novel bivalent securinine mimetics as topoisomerase I
†
inhibitors
1
1
1
2
1
1
Wen Hou, Hui Lin, ZhenꢀYa Wang, Martin G. Banwell, Ting Zeng, PingꢀHua Sun,
1
,
1,
Jing Lin, * and WeiꢀMin Chen *
1
2
. College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
. Research School of Chemistry, Institute of Advanced Studies, Australian National University,
Canberra, ACT 2601, Australia
Corresponding authors. Tel.: +86 20 8522 1367(J. Lin), +86 20 8522 4497 (W.-M. Chen).
*
Fax: +86 20 8522 4766.
E-mail addresses: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn (W.ꢀM. Chen).
†
The authors declare no competing interests.
Abstract
A series of novel bivalent securinine mimetics incorporating different linkers
between Cꢀ15 and Cꢀ15’ was synthesized and their topoisomerase I (Topo I)
inhibitory activities evaluated. It was thus revealed that mimetic R2 incorporating a
rigid mꢀsubstituented benzene linker exhibits Topo I inhibitory activity three times
that of parent securinine. Comprehensive structureꢀactivity relationship analyses in
combination with docking studies were used to rationalize the potent activity of these
bivalent mimetics. Mechanistic studies served to confirm the deductions arising from
docking studies that the active bivalent mimetics not only inhibited complexation
between Topo I and DNA but also stabilized the Topo IꢀDNA complex itself.
Keywords: Securinine; Bivalent mimetics; Topo I; Docking.

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Introduction
The topoisomerase I (Topo I) enzymes are involved in the cutting of one strand of
doubleꢀstranded and supercoiled DNA, relaxing that strand then reꢀannealing it in
1
specific ways. Given its pivotal role in replication, Topo I was recognized as a target
2
for the development of antitumor drugs in the 1980s. As a result of studies in the area
3
the natural product camptothecin (CPT) was identified as a potent Topo I inhibitor
4
5
and the semiꢀsynthetic derivatives topotecan and irinotecan are now used clinically
for treating, inter alia, ovarian, colon and lung cancers.
6
In previous studies we identified certain securinine derivatives that act as Topo I
inhibitors. Since various linked/dimeric forms of other inhibitors, notably
benzimidazoles, seem to exhibit better activity than the parent (monomeric)
7ꢀ11
systems,
we were prompted to construct novel, dimeric securinine derivatives for
the purpose of examining their Topo Iꢀinhibitory activities. Herein we describe the
1
2
synthesis, using heteroꢀMichael addition chemistry, of eleven such bivalent
securinine “mimetics” and the identification of one displaying three times the
inhibitory activity of the parent (monomeric) system. Further, we detail
comprehensive structureꢀactivity relationship analyses and docking studies that reveal
the origins of this enhanced activity. Mechanistic investigations, including
DNAꢀcleavage studies and electrophoretic mobility shift assays (EMSAs), as well as
DNAꢀinsertion assays, are also described that confirm the inferences drawn from the
docking studies.
Results and Discussion
1
. Chemical synthesis of bivalent securinine mimetics
11
Inspired by a report revealing that certain bisbenzimidazoles were more efficacious
than the corresponding monomers as Topo I inhibitors, eleven compounds, namely
R1~R6, HR1~HR3, F2 and F3 (Figure 1), were identified as the most suitable

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substrates with which to investigate whether or not bivalent securinine mimetics are
more active than the analogous monomeric systems. In order to establish any effects
that might be exerted by the linker joining the two monomeric units in these bivalent
systems, three types of mimetic were designed, namely those incorporating a rigid
linker (labeled as the R series), those with a semiꢀrigid linker (the HR series) and
those with a flexible one (the F series). Additionally, a monomer M2 (Figure 1)
embodying the same linker unit as the most active bivalent mimetic R2 but lacking
the second securinine unit was also prepared in order to study whether or not two such
units (rather than just the linker itself) was a necessary requirement for enhanced
activity.
[
Figure 1]
The concise routes shown in Scheme 1 allowed for the synthesis of all twelve
1
13
securinine derivatives and the structures of these were established using H and C
NMR spectroscopic as well as lowꢀ and highꢀresolution electrospray ionization (ESI)
mass spectrometric techniques.
[
Scheme 1]
2
. Biological Studies
2
.1 Evaluation of Topo I Inhibitory Properties
1
3
A Topo I inhibitory assay was employed as an initial screen to establish the efficacy
(or otherwise) of the abovementioned bivalent securinine mimetics. In this assay, CPT,
3
an established Topo I inhibitor, was used as a positive control. As shown in Figure 2,
when 100 ꢁM solutions of securinine and each of the mimetics was tested the amount
of the supercoiled form of DNA (the lower band in Figure 2A) present at a given point
in time varied significantly but was always evident and thus implying that the tested
compounds were acting as inhibitors of Topo I. All of the R series bivalent mimetics

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were stronger inhibitors than securinine itself with the most effective (R2) being
nearly as active as the positive control CPT and almost three times as active as the
parent compound. These results clearly demonstrate that bivalent securinine mimetics
can act as effective Topo I inhibitors. In the HR series only one, HR2, showed higher
activity (but only just) than securinine, while in the F series no enhancement of
activity (relative to securinine) was observed. This was also the case for compound
M2. These results indicate that the presence of a second securinine unit can impart
enhanced activity but that the manner in which the two units are joined is critical.
Specifically, a conformationally limiting (rigid) linker is pivotal as evidenced by the
reduction in activity in moving from a phenylꢀbased group to a cyclohexylꢀbased one
and then to a propyl linker (compare R2 with HR2 and with F2). The length of the
linker also has a significant impact on the activity. Thus, increasing the length of this
unit by inserting even one more carbon reduced activity significantly (compare R2
with R5). The relative disposition of the securinine units was also important as
evident from the variations in activity in “moving” from meta- to orthoꢀdisubstituted
linker units (compare R2 with R3) or to the para-disubstituted equivalent (compare
R2 with R1). On the basis of the data acquired so far, it would seem that a rigid linker
with a total length of seven atoms and incorporating an metaꢀdisubstituted aryl unit is
required for the construction of bivalent securinine mimetics that act as significantly
enhanced Topo I inhibitors.
[
Figure 2]
2
.2 Docking studies
In order to understand the origins of structureꢀactivity relationship profile defined
above and thus determine the reasons for the excellent Topo I inhibitory activity of
mimetic R2, docking studies were conducted on the manner in which this compound
binds to the enzyme. The structure of the enzyme employed for this purpose was
1
4
obtained from the Protein Data Bank (PDB ID: 1T8I) and complexes were generated
using SYBYLꢀ8.1 Surflexꢀdock. As indicated in Figure 3a, in the absence of DNA

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compound R2 binds effectively and directly to the active pocket of Topo I through the
formation of five hydrogen bonds (see yellow dashed lines) with the ARG362,
ARG364, LYS374 and ASN722 residues. Presumably it is this direct binding of
mimetic R2 to Topo I that prevents the normal interaction of the enzyme with DNA,
an effect observed during the course of our previously reported studies of the
6
activities of monomeric securinine derivatives. In addition, compound R2 binds
effectively to the covalent Topo I/DNA complex (Figure 3b) through the agency of six
hydrogen bonds (see yellow dashed lines) with two adjacent bases of DNA (viz.
DA113 and DA114 ꢀ blue structures) and the LYS425 residue of Topo I. πꢀπ Stacking
between the aryl group of the linker and TGP11 (in blue) also seems likely. Such a
suite of interactions suggests that the bivalent mimetic R2 could also stabilize the
Topo I/DNA convalent complex. Accordingly, we propose that the dual capacity of
compound R2 to bind directly to Topo I and to stabilize the complex of this enzyme
with DNA accounts for its potent inhibitory activity. In order to investigate whether or
not πꢀπ stacking between the aryl linker within compound R2 and the nucleoside
bases of DNA is important, congeners HR2 and F2 lacking an aromatic residue were
also subject to docking studies. As shown in Figures 3c and 3d both of these failed to
bind to the Topo I/DNA covalent complex and no hydrogen bonding and πꢀπ stacking
interactions were observed. This strongly suggests that πꢀπ stacking between the
aromatic residues of the linker associated with bivalent mimetic R2 and the nucleotide
bases of DNA has a major (beneficial) impact on activity.
In order to determine whether or not both securinine units within the bivalent
mimetics are necessary for effective binding, the monomeric congener M2
(possessing the same linker as the most active compound R2 but lacking a second
securinine residue) and the parent system (viz. securinine) were each subject to
docking analysis. As revealed in Figures 3e and 3f, neither of these reference
substrates bind effectively with the Topo I/DNA complex and thus highlighting the
importance of the presence of a bivalent motif.
Overall, then, these docking studies strongly suggest that the excellent Topo I
inhibitory activity of the bivalent mimetic R2 arises through a dual inhibitory

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mechanism involving, (i), binding of it, through πꢀπ stacking, with the covalent Topo
I/DNA complex (and thereby stabilizing the same) and, (ii), binding of the second
securinine unit within this inhibitor with Topo I (and so inhibiting the normal mode of
action of the enzyme).
[
Figure 3]
2
.3 Electrophoretic Mobility Shift Assays (EMSAs)
In order to substantiate the hypotheses arising from our docking studies, an
1
5
electrophoretic mobility shift assay (EMSA) was carried out so as to establish
whether or not compound R2 inhibits the binding of DNA with Topo I. As shown in
Figure 4, upon addition of successive aliquots of compound R2 to the Topo I/DNA
complex the concentration of the latter decreased linearly. This result clearly
demonstrates that the bivalent mimetic R2 blocks (inhibits) the complexation of Topo
I with DNA and so suggesting a high affinity of compound R2 for Topo I as depicted
in Figure 3a.
[
Figure 4]
2
.4 DNA-cleavage assay
A DNAꢀcleavage assay was also conducted in order to establish whether or not
compound R2 stabilizes the Topo I/DNA complex as predicted by the
aboveꢀmentioned docking studies. CPT served as a positive control in this assay and
on employing it (Figure 5) at increasing concentrations the quantities of nicked DNA
(see upper bands) accumulated in a linear manner, an outcome consistent with earlier
16,17
reports.
Nicked DNA bands were also observed with compound R2, especially at
1
00 ꢀM concentrations, although these were not as conspicuous as the ones observed
with CPT (an outcome consistent with the dual binding mode of R2 suggested by the
docking study). These cleavage assays support the proposition that mimetic R2 binds
to and thus stabilizes the Topo I/DNA covalent complex. In contrast, and as predicted
by the docking studies, compounds HR2, F2 and M2, failed to generate nicked DNA

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in the same assay.
[
Figure 5]
2
.5 DNA-intercalation assay
18
It has been reported that Topo I inhibitors can exert their effects by one (or more) of
three possible mechanisms, namely through DNA intercalation (as seen with ethidium
bromide or EB), through retarding formation of the normal Topo I/DNA complex or
through stabilizing this complex. In order to establish whether or not the mimetics
described here can act by the first pathway, a DNAꢀinsertion assay was carried out on
the Rꢀseries compounds and EB was used as the positive control. As shown in Figure
6
, on treating Topo I with 25 ꢁM EB the activity of the enzyme was shutdown. In
contrast, in the same assay Topo I remained able to convert supercoiled DNA (lower
bands in Figure 6) into a relaxed form (upper bands) in the presence of the Rꢀseries
mimetics. Accordingly, and given the outcomes of the other assays detailed above, we
propose that the more potent bivalent mimetics, such as compound R2, act by the
second and third inhibitory mechanisms but not by the first.
[
Figure 6]
3
Conclusions
In this study, a series of novel securinine bivalent mimetics and one monomer were
designed and synthesized for evaluation as Topo I inhibitors. Compound R2 was
found to display about three times the Topo I inhibitory activity of securinine itself
and so demonstrating that the development of bivalent securinine mimetics is a novel
and feasible strategy for constructing effective inhibitors of this crucial enzyme.
Furthermore, through the combined application of docking studies and
mechanistically relevant assays, the twoꢀfold binding mode and consequent dual
19
effects of this potent and structurally novel inhibitor (viz. R2) have been revealed.
The structureꢀactivity relationship data and docking studies reported here also provide

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a wealth of information for the further development of bivalent mimetics as
potentially valuable new Topo I inhibitors.
4
Experimental
4
.1 Chemistry
General procedures
Unless otherwise specified, proton ( H) and carbon ( C) NMR spectra were recorded
1
13
at 18 °C in baseꢀfiltered CDCl
3
on a Varian spectrometer operating at 300 MHz for
1
proton and 75 MHz for carbon nuclei. For H NMR spectra, signals arising from the
1
residual protioꢀforms of the solvent were used as the internal standards. H NMR data
are presented as follows: chemical shift (
relative integral] where multiplicity is defined as: s = singlet; d = doublet; t = triplet; q
quartet; m = multiplet or combinations of the above. The signal due to residual
CHCl appearing at 7.26 and the central resonance of the CDCl “triplet”
appearing at 77.1(6) were used to reference H and C NMR spectra, respectively.
δ) [multiplicity, coupling constant(s) J (Hz),
=
3
δ
H
3
1
13
δ
C
Lowꢀresolution ESI mass spectra were recorded on a Finnigan LCQ Advantage MAX
mass spectrometer and fitted with a 4000 Q TRAP or Agilent 6130 Quadruple LC/MS.
Highꢀresolution mass spectra (HRꢀMS) were obtained on an Agilent 6210 series
LC/MSD TOF mass spectrometer. Analytical thin layer chromatography (TLC) was
performed on aluminiumꢀbacked 0.2 mm thick silica gel 60 F254 plates as supplied by
Merck. Eluted plates were visualized using a 254 nm UV lamp and/or by treatment
with a suitable dip followed by heating. Flash chromatographic separations were
2
0
carried out following protocols defined by Still et al. with silica gel 60 (40–63 ꢀm)
as the stationary phase and using the ARꢀ or HPLCꢀgrade solvents indicated. Starting
materials and reagents were generally available from the Sigma–Aldrich, Merck, TCI,
Strem or Lancaster Chemical Companies and were used as supplied. Drying agents
and other inorganic salts were purchased from the AJAX, BDH or Unilab Chemical
Companies. Where necessary, reactions were performed under an inert atmosphere.
21
Compounds F2 and F3 were obtained using previously reported methods.

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Specific chemical transformations
5S,6S,11aS,11bS)-5-(Propylamino)-5,6,9,10,11,11a-hexahydro-8H-6,11b-methanofur
o[2,3-c]pyrido[1,2-a]azepin-2(4H)-one (KA). The title compound was prepared using
(
21
a modification of a previously reported procedure. Thus, a magnetically stirred
solution of securinine (1.90 g, 9.0 mmol, 1.0 equiv) in dichloromethane (DCM, 30
3 4
mL) maintained at ambient temperatures was treated with K PO (9.5 mg, 0.045
mmol, 0.05 equiv) then nꢀpropylamine (1.59 g, 27.0 mmol, 3.0 molar equiv.). After 8
h the now milkꢀwhite mixture was concentrated under reduced pressure and the
residue diluted with water (100 mL) then extracted with DCM (3 × 50 mL). The
combined organic phases were dried (MgSO
4
), filtered and concentrated under
reduced pressure. The residue thus obtained was subject to flash chromatography
(silica gel, 200:1 v/v DCM/MeOH) to give, after concentration of the appropriate
21
fractions (R
f
= 0.3, DCM:MeOH=200:1), compound KA (1.20 g, 49%) as a
1
paleꢀyellow oil. H NMR (300 MHz, CDCl
3
) δ 5.57 (d, J = 2.1 Hz, 1H), 3.22ꢀ3.16 (m,
1
1
H), 3.01 (dd, J = 6.3 and 4.2 Hz, 1H), 2.92ꢀ2.75 (m, 4H), 2.58ꢀ2.40 (m, 4H),
13
.89ꢀ1.79 (m, 2H), 1.59ꢀ1.25 (m, 8H), 0.85 (t, J = 7.5 Hz, 3H); C NMR (75 MHz,
) δ 174.5, 173.2, 110.9, 91.4, 63.0, 59.8, 59.6, 49.7, 48.8, 32.3, 30.4, 25.7, 23.8,
3.3, 21.5, 11.9. These data were essentially identical, in all respects, with those
CDCl
3
2
21
reported previously.
N-((5S,6S,11aS,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-methanofur
o[2,3-c]pyrido[1,2-a]azepin-5-yl)-N-propylbenzamide (M2). Benzoic acid (10 mmol)
was added to thionyl chloride (5 mL, 25 mmol) and the resulting solution heated, with
magnetic stirring, under reflux for 5 h while being maintained under an atmosphere of
nitrogen. The cooled reaction mixture was concentrated under reduced pressure and a
portion of the benzoyl chloride (0.3 mmol, 0.6 equiv.) thus obtained added, dropwise
over 0.33 h, to a magnetically stirred solution of KA (138 mg, 0.5 mmol, 1.0 equiv.)
in anhydrous DCM (10 mL) containing diꢀisoꢀpropylethylamine (DIPEA, 387 mg, 1.5
mmol, 3.0 equiv.) maintained at –20 °C. After 1 h the reaction mixture was warmed to
ca. 22 °C, stirred at this temperature for 16 h then treated with aqueous NaHCO
3
so as
to achieve pH ≥ 8. The ensuing mixture was concentrated under reduced pressure and

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the residue extracted with DCM (3 × 20 mL). The combined organic phases were
dried (MgSO ), filtered and concentrated under reduced pressure and the residue
4
subjected to flash chromatography (silica gel, 100:1 v/v DCM:MeOH) to give, after
concentration of the appropriate fractions, compound M2 (61 mg, 32%) as a brown
1
solid. H NMR (300 MHz, CDCl ) δ 7.40ꢀ7.34 (m, 3H), 7.32ꢀ7.26 (m, 2H), 5.67 (s,
3
1
H), 4.24 (t, J = 15.5 Hz, 1H), 3.27 (dd, J = 15.5 and 7.5 Hz, 1H), 3.15 (m, 1H), 2.90
(m, 5H), 2.59 (dd, J = 11.0 and 5.8 Hz, 1H), 1.92ꢀ1.72 (m, 2H), 1.63ꢀ1.44 (m, 3H),
1
3
1
1
2
3
.42ꢀ1.21 (m, 5H), 0.70 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, CDCl ) δ 172.8,
72.5, 165.1, 137.3, 129.4, 128.6, 126.2, 110.3, 90.9, 63.0, 60.0, 57.6, 48.3, 33.6, 29.7,
+
7.3, 26.4, 24.2, 24.0, 22.1, 11.2; MS (ESI, +ve) m/z 381 [(M+H) , 100%]; HRMS
+
(
ESI, +ve) m/z (M+H) calcd for C23
H
29
N
2
O
3
381.2173, found 381.2174.
Generalised procedure for the preparation of the bivalent securinine mimetics
R1~R6 and HR1~HR3
The relevant diacid (10 mmol) associated with the linker unit of the title compounds
was added to thionyl chloride (5 mL, 25 mmol) and the resulting solution heated, with
magnetic stirring, under reflux for 5 h while being maintained under an atmosphere of
nitrogen. The cooled reaction mixture was concentrated under reduced pressure and a
portion of the diacid chloride (0.3 mmol, 0.6 equiv.) thus obtained was added,
dropwise over 0.33 h, to a magnetically stirred solution of KA (138 mg, 0.5 mmol,
1
.0 equiv.) in anhydrous DCM (10 mL) containing DIPEA (387 mg, 1.5 mmol, 3.0
equiv.) maintained at –20 °C. After 1 h the reaction mixture was warmed to ca. 22 °C,
stirred at this temperature for 16 h then treated with an anhydrous NaHCO so as to
3
achieve pH ≥ 8. The ensuing mixture was concentrated under reduced pressure and
the residue extracted with DCM (3 × 20 mL). The combined organic phases were
4
dried (MgSO ), filtered and concentrated under reduced pressure and the residue
subjected to flash chromatography (silica gel, 100:1 v/v DCM:MeOH) to give the
relevant diamide. The spectral data sets acquired on each of these are given below.
1
N -((5S,6S,11aR,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-methanof
2
uro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N -((5S,6S,11aS,11bS)-2-oxo-2,4,5,6,9,10,11,11a-

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1
2
octahydro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N ,N -dipropylpht
1
halamide (R1) was obtained as a yellow solid. Yield 33%. H NMR (300 MHz,
CDCl
3
) δ 7.40 (broad s, 2H), 7.26 (broad s, 2H), 5.67 (broad s, 2H), 4.00ꢀ2.00 (m,
13
1
5H), 1.98ꢀ1.08 (m, 20H), 0.99ꢀ0.79 (m, 4H), 0.64 (broad s, 5H); C NMR (75 MHz,
CDCl ) δ 173.8, 172.7, 171.4, 138.2, 126.5, 110.2, 90.8, 62.6, 59.9, 57.8, 53.7, 48.4,
3
+
3
3.6, 29.6, 27.3, 26.3, 24.1, 22.1, 11.1; MS (ESI, +ve) m/z 683 [(M+H) , 100%];
+
HRMS (ESI, +ve) m/z [M+H] calcd for C40
51
H N
4
O
6
683.3803, found 683.3806.
1
N -((5S,6S,11aR,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-methanof
3
uro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N -((5S,6S,11aS,11bS)-2-oxo-2,4,5,6,9,10,11,11a-
1
3
octahydro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N ,N -dipropylisop
1
hthalamide(R2) was obtained a white solid. Yield 46%. H NMR (300 MHz, CDCl
3
)
δ 7.50ꢀ7.20 (m, 4H), 5.68 (s, 2H), 4.20 (broad s, 2H), 3.50ꢀ2.70 (m, 14H), 2.61 (m,
1
3
2
H), 1.90ꢀ1.76 (m, 4H), 1.59ꢀ1.16 (m, 16H), 0.70 (broad s, 6H); C NMR (75 MHz,
CDCl ) δ (major rotamer) 173.7, 172.7, 171.3, 137.8, 128.7, 126.9, 124.2, 110.2, 90.8,
2.8, 59.9, 57.8, 48.3, 33.6, 29.7, 27.3, 26.3, 24.1, 22.0, 14.0, 11.2; MS (ESI, +ve) m/z
3
6
6
+
+
51 4 6
83 [(M+H) , 100%]; HRMS (ESI, +ve) m/z [M+H] calcd for C40H N O 683.3803,
found 683.3810.
1
N -((5S,6S,11aR,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-methanof
4
uro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N -((5S,6S,11aS,11bS)-2-oxo-2,4,5,6,9,10,11,11a-
1
4
octahydro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N ,N -dipropyltere
1
phthalamide (R3) was obtained as a yellow solid. Yield 33%. H NMR (300 MHz,
CDCl ) δ 7.31 (broad s Hz, 4H), 5.65 (s, 2H), 4.19 (broad s, 2H), 3.50ꢀ2.65 (m, 14H),
3
13
2
.58 (m, 2H), 1.80 (m, 4H), 1.65ꢀ1.00 (m, 16H), 0.65 (broad s, 6H); C NMR (75
MHz, CDCl
3
) δ 173.8, 172.7, 171.4, 138.2, 126.4, 110.1, 90.8, 62.6, 59.9, 57.8, 53.6,
+
4
8.3, 33.6, 29.6, 27.2, 26.3, 24.1, 22.0, 11.1; MS (ESI, +ve) m/z 683 [(M+H) , 100%];
+
51 4 6
HRMS (ESI, +ve) m/z [M+H] calcd for C40H N O 683.3803, found 683.3800.
N-((5S,6S,11aS,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-methanofu
ro[2,3-c]pyrido[1,2-a]azepin-5-yl)-2-(4-(2-oxo-2-(((5S,6S,11aR,11bS)-2-oxo-2,4,5,6,9
,
10,11,11a-octahydro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)(propyl)
amino)ethyl)phenyl)-N-propylacetamide (R4) was obtained as a white solid. Yield

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1
3
6%. H NMR (300 MHz, CDCl
3
) δ 7.23 (m, 1H), 7.05 (m, 3H), 5.64 (s, 2H), 4.28
(
(
broadened s, 2H), 3.79ꢀ3.39 (m, 6H), 3.30ꢀ3.10 (m, 4H), 3.05ꢀ2.76 (m, 10H), 2.48
13
broadened s, 2H), 1.92ꢀ1.15 (m, 18H), 0.87 (broadened s, 6H); C NMR (75 MHz,
) δ (mixture of rotamers) 174.4, 172.8, 171.3, 135.5, 129.3, 129.0, 127.4, 110.0,
1.1, 61.6, 60.0, 57.4, 48.4, 41.1, 33.4, 26.8, 26.4, 25.3, 24.1, 22.1, 11.3; MS (ESI,
CDCl
3
9
+
+
+
ve) m/z 711 [(M+H) , 97.2%]; HRMS (ESI, +ve) m/z [M+H] calcd for C42
11.4116, found 711.4126.
,2'-(1,3-phenylene)bis(N-((5S,6S,11aS,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahyd
55 4 6
H N O
7
2
ro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N-propylacetamide) (R5)
1
was obtained as a yellow solid. Yield 32%. H NMR (300 MHz, CDCl
3
) δ 7.17 (s,
4
1
1
2
H), 5.65 (s, 2H), 4.34 (m, 2H), 3.75ꢀ3.41 (m, 6H), 3.33ꢀ3.09 (m, 4H), 3.02ꢀ2.77 (m,
13
0H), 2.53 (m, 2H), 1.76ꢀ1.22 (m, 18H), 0.88 (m, 6H); C NMR (75 MHz, CDCl ) δ
3
74.4, 172.8, 171.5, 133.7, 129.1, 110.1, 91.1, 61.6, 60.0, 57.3, 53.5, 48.4, 40.8, 33.4,
6.8, 26.3, 25.4, 24.2, 22.1, 11.2 (signals due to two carbons obscured or overlapping);
+
+
MS (ESI, +ve) m/z 711 [(M+H) , 100%]; HRMS (ESI, +ve) m/z [M+H] calcd for
711.4116, found 711.4126.
N-((5S,6S,11aS,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-methanofu
ro[2,3-c]pyrido[1,2-a]azepin-5-yl)-2-(2-(2-oxo-2-(((5S,6S,11aR,11bS)-2-oxo-2,4,5,6,9
10,11,11a-octahydro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)(propyl)
42 55 4 6
C H N O
,
amino)ethyl)phenyl)-N-propylacetamide (R6) was obtained as a white solid. Yield
1
4
3
3%. H NMR (300 MHz, CDCl
3
) δ 7.24ꢀ7.07 (m, 4H), 5.71 (m, 2H), 4.38 (m, 2H),
13
.75ꢀ2.70 (m, 20H), 2.56 (m, 2H), 1.89ꢀ1.26 (m, 18H), 0.90 (m, 6H); C NMR (75
) δ 174.1, 172.9, 171.4, 134.5, 129.5, 127.5, 110.2, 91.0, 61.5, 60.1, 57.2,
3.5, 48.4, 38.9, 33.4, 26.7, 26.4, 25.3, 24.1, 22.1, 11.2; MS (ESI, +ve) m/z 711
MHz, CDCl
3
5
+
+
[
(M+H) , 100%]; HRMS (ESI, +ve) m/z [M+H] calcd for C H N O 711.4116,
42 55 4 6
found
711.4126.
1
(
1R,2S)-N -((5S,6S,11aR,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-metha
2
nofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N -((5S,6S,11aS,11bS)-2-oxo-2,4,5,6,9,10,11,1
1
2
1
a-octahydro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N ,N -dipropyl
1
cyclohexane-1,2-dicarboxamide(HR1) was obtained as a white solid. Yield 21%. H

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NMR (300 MHz, CDCl
3
) δ 5.56 (m, 2H), 4.30 (m, 2H), 3.75ꢀ2.35 (m, 18H), 2.30ꢀ1.00
1
3
(
m, 28H), 0.89ꢀ0.80 (m, 6H); C NMR (75 MHz, CDCl ) δ (mixture of amide
3
rotamers) 175.3, 175.2, 174.6, 173.2, 172.8, 110.2, 109.9, 91.4, 91.1, 60.2, 59.9, 56.6,
4
8.5, 48.3, 41.9, 33.3, 30.1, 29.1, 27.0, 26.6, 26.5, 26.1, 24.3, 24.2, 23.3, 23.1, 22.2,
+
1
4.1, 11.3, 11.2, 11.1; MS (ESI, +ve) m/z 689 [(M+H) , 100%]; HRMS (ESI, +ve) m/z
+
[
M+H] calcd for C40
57
H N
4
O
6
689.4273, found 689.4274.
1
(
1R,3S)-N -((5S,6S,11aR,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,11b-
3
methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N -((5S,6S,11aS,11bS)-2-oxo-2,4,5,6,9,
1
3
1
0,11,11a-octahydro-8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N ,N -di
propylcyclohexane-1,3-dicarboxamide(HR2) was obtained as a yellow oil. Yield 26%.
1
H NMR (300 MHz, CDCl
.54ꢀ2.23 (m, 4H), 1.95ꢀ1.10 (m, 28H), 0.92ꢀ0.73 (m, 6H); C NMR (75 MHz,
CDCl ) δ (mixture of amide rotamers) 175.6, 175.5, 174.5, 174.4, 172.7, 172.7, 110.1,
3.5, 91.1, 91.0, 61.5, 61.4, 59.9(4), 59.8(9), 56.7, 56.6, 53.5, 48.3(7), 48.3(5), 48.3(3),
3
) δ 5.65 (broad s, 2H), 4.33 (m, 2H), 3.41ꢀ2.60 (m, 14H),
13
2
3
9
4
7.5(7), 47.5(6), 41.2, 40.7, 33.2, 29.6, 26.4, 26.2(4), 26.2(1), 22.1(7), 22.1(0),
+
1
1.2(4), 11.1(5); MS (ESI, +ve) m/z 689 [(M+H) , 100%]; HRMS (ESI, +ve) m/z
+
[
M+H] calcd for C40
57
H N
4
O
6
689.4273, found 689.4274.
1
4
(
1S,4S)-N ,N -bis((5S,6S,11aS,11bS)-2-Oxo-2,4,5,6,9,10,11,11a-octahydro-8H-6,
1
4
1
1b-methanofuro[2,3-c]pyrido[1,2-a]azepin-5-yl)-N ,N -dipropylcyclohexane-1,4-dic
1
arboxamide (HR3) was obtained as a white solid. Yield 8%. H NMR (300 MHz,
CDCl ) δ 5.65 (m, 2H), 4.28ꢀ4.05 (m, 2H), 3.35ꢀ2.25 (m, 20H), 1.90ꢀ1.05 (m, 26H),
.76 (m, 6H); C NMR (75 MHz, CDCl ) δ (mixture of rotamers) 176.2, 174.4, 172.7,
3
13
0
3
110.0, 91.0, 61.7, 60.0, 57.1, 53.5, 48.3, 48.0, 40.6, 33.2, 29.4, 28.1, 26.8, 26.4, 25.9,
+
2
4.1, 22.1, 11.1; MS (ESI, +ve) m/z 689 [(M+H) , 100%]; HRMS (ESI, +ve) m/z
+
[
M+H] calcd for C H N O 689.4273, found 689.4274.
4
0
57
4
6
4
4
.2 Modeling and Biological Evaluation
.2.1 Docking Studies
The Topo I structure incorporated within the SYBYL software package (ex. Tripos Inc.
St. Louis, MO, USA) was used and all the associated water and small molecule
ligands were removed. Modifications necessary to simulate a physiological

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22
environment were also made. Docking processes were refined using the SYBYLꢀ8.1
surflexꢀdocking module and a ligandꢀbased approach was used to generate the binding
site of the receptor. The protocol was generated with a threshold parameter of 0.5 Å
and a bloat parameter of 0 Å while all Kollman charges were appended and the
GeomX mode was applied to obtain better outcomes in the docking study. Potential
ligands were docked sequentially into the binding pocket. Default settings were used
for all other relevant parameters.
4
.2.2 Topo I inhibitory assay
23
In this assay, CPT was used as the positive control and the degree of inhibition of
calf thymus Topo I (ex. TaKaRa, Kyoto, Japan) reflected in the relative amount of
24
supercoiled pBR322 DNA (TaKaRa, Kyoto, Japan) remaining at the endpoint.
Relevant solutions were prepared as defined in the supplier’s protocols. Incubations
were carried out at 37 °C for 0.5 h then a staining solution, comprising 0.25% w/v
bromophenol blue, 0.25% w/v xylene cyanol FF and 40% v/v glycerol in doubly
distilled water, was added and so terminating the reaction. The final mixtures were
submitted to electrophoresis for 0.66 h in 1 × TAE buffer (comprising 40 mM
trisꢀacetate, 2 mM EDTA and 19.9 mM HOAc) in 1% agarose gel. DNA bands were
visualized through illumination with UV light and photographed with an Alpha
Innotech digital imaging system after staining with 5 ꢀg/mL EB in 1 × TAE buffer for
0
.5 h.
4
.2.3 Electrophoretic Mobility Shift Assays (EMSAs)
1
5
In a representative experiment, 1 ꢁL BSA was added into 20 ꢁL of the Topo I buffer.
Four units of calf thymus Topo I and compound R2 were then added and the resulting
mixture allowed to stand for 0.33 h. Finally, 0.1 ꢁg of pBR322 DNA was added to the
mixture that was then immersed in a water bath at 37 °C for 0.5 h. After this time the
mixture was immediately subjected to electrophoresis at 1 V/cm and 4 °C for 8 h on
1
4
2
% gel containing 1% EB. The DNA bands were visualized as described at 4.2.2.
.2.4 DNA-cleavage assay
0 units of Topo I, 0.5 ꢁg of supercoiled pBR322 DNA, and 50 or 100 ꢁM solutions
of CPT or the test compounds were combined to a total volume of 40 ꢁL in Topo I

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buffer (as provided by the supplier). Cleavage intermediates were trapped by adding
% sodium dodecyl sulfate (SDS) to terminate the reaction after incubation at 37 °C
1
for 0.5 h. Then 0.8 mg/mL proteinase K (pH 8.0) was added to the reaction mixture
that was then incubated at 50 °C for 10 min (so as to digest the Topo I). 10 ꢁl of
samples of the resulting solution were mixed with 2 ꢁL 6 × DNA loading buffer,
comprising 0.25% bromophenol blue, 0.25% xylene cyanol FF and 40% v/v glycerol
in doubly distilled water, then submitted to electrophoresis at 60 V and 25 °C for 0.5 h.
The gel was soaked in 1 × TAE containing 0.5 ꢁg/mL EB for 0.5 h then a second
electrophoretic procedure of 0.5 h duration was conducted so as to distinguish the
relaxed from the nicked forms of DNA. The cleavage effects were evaluated by
comparing the relative intensities of nicked and other DNA bands that had been
visualized by illumination with UV light then photographed as described at 4.2.2.
4
.2.5 DNA-insertion assay
This assay resembled the Topo I inhibitory one but varied in the amount of enzyme
25
added. EB, a DNA intercalating agent, was used as the positive control. Thus, ten
units of calf thymus Topo I (ex. TaKaRa, Kyoto, Japan), 0.1 ꢁg of negatively
supercoiled pBR322 DNA (TaKaRa, Kyoto, Japan) and 100 ꢁM of the R series
compound (or 25 ꢁM of EB) were added into a total of 20 ꢁL of Topo I buffer (as
provided by the supplier). The ensuing mixture was combined with 4 ꢁL of 6 × DNA
loading buffer after incubation at 37 °C for 0.5 h. Samples were subjected to
electrophoresis for 0.66 h in the same manner as defined at 4.2.2 and DNA bands
were visualized and recorded in the usual way.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (Grant
No. 81273362).
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Figure Captions
Figure 1. Structures of the target molecules.
Figure 2. (a) Topo I inhibitory activities of the title securinine derivatives. CPT used a
positive control. Supercoiled pBR322 DNA was incubated with Topo I in the presence
or absence of compound at 37 °C for 0.5 h. Lane A ꢀ pBR322 DNA alone. Lane B ꢀ
mixture of pBR322 DNA and Topo I. Lane C ꢀ mixture of pBR322 DNA, Topo I and
1
00 ꢁM CPT. Other lanes ꢀ mixtures of pBR322 DNA, Topo I and 100 ꢁM of test
compound; (b) Gray scale value analysis of A.
Figure 3. Binding model between test compounds and Topo I arising from docking
studies. (a) R2 and Topo I (no DNA); (b) R2 and Topo I; (c) HR2 and Topo I; (d) F2
and Topo I; (e) M2 and Topo I; (f) Securinine and Topo I.
Figure 4. (a) EMSA of compound R2. CPT used as a negative control. Lane A ꢀ
pBR322 DNA only. Lane B ꢀ mixture of pBR322 DNA and Topo I. Line C ꢀ mixture
of pBR322 DNA, Topo I and 50 or 100 ꢁM CPT. Other lanes ꢀ mixture of pBR322
DNA, Topo I and 50, 100, 200, or 400 ꢁM R2; (b) Gray scale value analysis of A.
Figure 5. (a) Topo Iꢀmediated DNA cleavage assay of compounds R2, M2, HR2, and
F2. CPT used as a positive control. Lane A ꢀ pBR322 DNA only. Lane B ꢀ mixture of
pBR322 DNA and Topo I. Other lanes ꢀ mixture of pBR322 DNA, Topo I and 50, 100
ꢁM of test compound; (b) Gray scale value analysis of A.
Figure 6. DNAꢀinsertion assay of the R series compounds. EB used as a positive
control. Supercoiled pBR322 DNA was incubated at 37 °C for 0.5 h with Topo I in the
presence or absence of EB, and with the test compounds. Lane A ꢀ pBR322 DNA
alone. Lane B ꢀ mixture of pBR322 DNA and Topo I. Lane EB ꢀ mixture of pBR322
DNA, Topo I and 25 ꢁM EB. Other lanes ꢀ mixture of pBR322 DNA, Topo I and 100
ꢁM of test compound.

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Figure 1. Structures of the target molecules.

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Figure 2. (a) Topo I inhibitory activities of the title securinine derivatives. CPT used a
positive control. Supercoiled pBR322 DNA was incubated with Topo I in the presence
or absence of compound at 37 °C for 0.5 h. Lane A ꢀ pBR322 DNA alone. Lane B ꢀ
mixture of pBR322 DNA and Topo I. Lane C ꢀ mixture of pBR322 DNA, Topo I and
1
00 ꢁM CPT. Other lanes ꢀ mixtures of pBR322 DNA, Topo I and 100 ꢁM of test
compound; (b) Gray scale value analysis of A.

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Figure 3. Binding model between test compounds and Topo I arising from docking
studies. (a) R2 and Topo I (no DNA); (b) R2 and Topo I; (c) HR2 and Topo I; (d) F2
and Topo I; (e) M2 and Topo I; (f) Securinine and Topo I.

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Figure 4. (a) EMSA of compound R2. CPT used as a negative control. Lane A ꢀ
pBR322 DNA only. Lane B ꢀ mixture of pBR322 DNA and Topo I. Line C ꢀ mixture
of pBR322 DNA, Topo I and 50 or 100 ꢁM CPT. Other lanes ꢀ mixture of pBR322
DNA, Topo I and 50, 100, 200, or 400 ꢁM R2; (b) Gray scale value analysis of A.

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Figure 5. (a) Topo Iꢀmediated DNA cleavage assay of compounds R2, M2, HR2, and
F2. CPT used as a positive control. Lane A ꢀ pBR322 DNA only. Lane B ꢀ mixture of
pBR322 DNA and Topo I. Other lanes ꢀ mixture of pBR322 DNA, Topo I and 50, 100
ꢁM of test compound; (b) Gray scale value analysis of A.

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Figure 6. DNAꢀinsertion assay of the R series compounds. EB used as a positive
control. Supercoiled pBR322 DNA was incubated at 37 °C for 0.5 h with Topo I in the
presence or absence of EB, and with the test compounds. Lane A ꢀ pBR322 DNA
alone. Lane B ꢀ mixture of pBR322 DNA and Topo I. Lane EB ꢀ mixture of pBR322
DNA, Topo I and 25 ꢁM EB. Other lanes ꢀ mixture of pBR322 DNA, Topo I and 100
ꢁM of test compound.

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Scheme 1 Synthetic route used to obtain the targeted securinine analogues.

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DOI: 10.1039/C6MD00563B
Graphical Abstract
Novel bivalent securinine mimetics as topoisomerase I inhibitors
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Wen Hou , Hui Lin , Zhen-Ya Wang , Martin G. Banwell , Ting Zeng , Ping-Hua Sun ,
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Jing Lin * and Wei-Min Chen *
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. College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
. Research School of Chemistry, Institute of Advanced Studies, The Australian National
University, Canberra, ACT 2601, Australia
Corresponding authors. Tel.: +86 20 8522 1367(J. Lin), +86 20 8522 4497 (W.-M. Chen).
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Fax: +86 20 8522 4766.
E-mail address: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn(W.-M. Chen).