Novel indolo-sophoridinic scaffold as Topo I inhibitors: Design, synthesis and biological evaluation as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2018.07.028

Based on the mechanism of action, novel scaffolds as Topo I inhibitors bearing indole and sophoridinine were designed. Preliminary docking study revealed that some molecules among the designed series possessed promising Topo I inhibitor properties. Subsequently, thirty new compounds were synthesized and characterized by ¹H NMR, ¹³C NMR, and Mass spectral analyses. The compounds were then screened for their antiproliferative and enzymatic inhibitory activities. The results affirmed the consistency between docking and activities and the rationality of the design strategy. Furthermore, compound 10b was chosen as a representative compound to test its anticancer effects in vitro and in vivo. The results showed that 10b caused prominent S phase cell cycle arrest and significantly suppressed tumor growth in HepG2 cell derived mouse model. These findings present a promising series of lead molecules which can serve as potential Topo I inhibitors for the treatment of cancer and a theoretical basis for structural modifications.

Full text of DOI:10.1016/j.ejmech.2018.07.028

European Journal of Medicinal Chemistry 156 (2018) 479e492
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel indolo-sophoridinic scaffold as Topo I inhibitors: Design,
synthesis and biological evaluation as anticancer agents
,
,
,
Yiming Xu a 1, Lichuan Wu b 1, Haroon Ur Rashid a c, Dewang Jing a, Xiaole Liang d,
Haodong Wang a, Xu Liu b, Jun Jiang a, Lisheng Wang b *, Peng Xie a
,
, **
a School of Chemistry and Chemical Engineering, Guangxi University, Nanning, 530004, China
b Medical College, Guangxi University, Nanning, 530004, China
c Department of Chemistry, Sarhad University of Science & Information Technology, Peshawar, Khyber Pakhtunkhwa, 25120, Pakistan
d Laboratory of Basic Medicine, Guangxi Medical University Nanning, 530022, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the mechanism of action, novel scaffolds as Topo I inhibitors bearing indole and sophoridinine
were designed. Preliminary docking study revealed that some molecules among the designed series
possessed promising Topo I inhibitor properties. Subsequently, thirty new compounds were synthesized
and characterized by 1H NMR, 13C NMR, and Mass spectral analyses. The compounds were then screened
for their antiproliferative and enzymatic inhibitory activities. The results affirmed the consistency be-
tween docking and activities and the rationality of the design strategy. Furthermore, compound 10b was
chosen as a representative compound to test its anticancer effects in vitro and in vivo. The results showed
that 10b caused prominent S phase cell cycle arrest and significantly suppressed tumor growth in HepG2
cell derived mouse model. These findings present a promising series of lead molecules which can serve
as potential Topo I inhibitors for the treatment of cancer and a theoretical basis for structural
modifications.
Received 22 May 2018
Received in revised form
27 June 2018
Accepted 10 July 2018
Keywords:
Indolo-sophoridinic Topo I inhibitors
Molecular docking
Binding mode
Anticancer activities
Enzymatic activity
© 2018 Published by Elsevier Masson SAS.
1. Introduction
transformation to camptothecin carboxylate by lactone E ring
opening [10e12]. Thus, development of Topo I inhibitors with new
DNA topoisomerase I (Topo I) is an essential and ubiquitous
enzyme that cleaves a single DNA strand to relax supercoiled DNA
for replication, transcription, recombination, and chromatin
remodeling [1e3]. Topo I is over-expressed in many cancer types
and can promote cancer cell proliferation [4e7]. Thus, Topo I is
recognized as important target in cancer therapy and anticancer
drug design. Therefore, developing new Topo I inhibitors has
become a hotspot research in medicinal chemistry.
Presently, the studies on Topo I inhibitors have focused on al-
kaloids, among which camptothecins are the primary Topo I in-
hibitors. Despite of their potent anticancer activity, many
limitations could be well-defined in all of the camptothecins [8,9].
In addition to their dose-limiting toxicity, camptothecins are inac-
tivated within minutes at physiological PH due to their
scaffold can be expected to overcome these shortcomings. Indolo-
carbazoles, indenoisoquinolines, and dibenzonaphthyridinones
represent a class of non-camptothecins compounds which have
been reported to possess the most extensive applications as Topo I
inhibitors (Fig. 1) [13e17].
An evident feature of the above poisons is their planar hetero-
cyclic ring structure containing nitrogen. A comparative analysis of
three different ternary structures obtained via X-ray crystal struc-
ture models indicates that camptothecin, indolocarbazole, and
indenoisoquinoline compounds are bound to the topoisomerase I-
DNA complex, and further reveals some prominent resemblance on
the mode of action of the Topo I poisons. The characteristic struc-
tures of these poisons allow them to intercalate into DNA and form
stacking interactions with base pairs by mimicking a DNA base pair.
It is vital to mention that most of the solvent accessible part of
every compound is protected by DNA (57%e70%) due to intercala-
tion binding mode, which further supports the idea that stacking
interactions may be the primary binding interaction for these Topo
I poisons [18] (Fig. 2). Thus, it is necessary to design new drug
* Corresponding author.
** Corresponding author.
E-mail addresses: lswang@gxu.edu.cn (L. Wang), xiep@gxu.edu.cn (P. Xie).
These authors have contributed equally.
1
https://doi.org/10.1016/j.ejmech.2018.07.028
0223-5234/© 2018 Published by Elsevier Masson SAS.

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Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
Fig. 1. Structures of camptothecin, indenoisoquinoline, indolocarbazole and sophoridine.
Fig. 2. The binding mode of Camptothecin (a), Indenoisoquinoline (b), Indolocarbazole (c) with Topo I-DNA complex obtained via X-ray crystal structure models and the binding
mode of Sophoridine (d) with Topo I gained via molecular docking.
skeleton bearing planar heterocyclic ring to perform
p
-
p
stacking
this study, we intended to develop Topo I inhibitors based on the
rational drug design strategy.
interactions with DNA. Besides, the study also reveals that the
poisons can take effect at high concentrations and thus cause dose-
limiting toxicities which in turn can damage normal tissues. So, in
addition to the planar heterocyclic ring mentioned above, another
moiety in newly designed scaffold that expected to render the
drugs with effects of improved metabolism, bioavailability and
better pharmacological profile should also be contained.
Sophoridine, another Topo I inhibitor, has captured our atten-
tion due to its several druggable advantages such as special
chemical scaffold, flexible structure, high solubility, and good safety
profiles compared to other Topo I inhibitors referred above
[19e22]. However, the moderate antitumor activity of sophoridine
limits its use as a single drug for clinical applications. Therefore, it is
reasonable to develop more effective drug candidates based on
sophoridinic structure [23e25]. The binding mode of sophoridine
revealed that sophoridine only interacted with Topo I rather than
DNA (Fig. 2). The aforementioned evidences provide mechanistic
foundations for the combination of planar heterocyclic ring with
sophoridine. When the new Topo I inhibitors contained the two
moieties, it may help to stabilize the covalent enzyme-DNA com-
plex by enhancing base stacking interaction and confer a number of
physiological and pharmaceutical activities in cancer therapy. In
2. Rational design of Topo I inhibitors
Indole, widely distributed in natural products, is a simple
molecule bearing bicyclic planar constructions and has similar-
ities with camptothecin, indolocarbazole, and indenoisoquinoline
compounds. In addition, it is a key component in the skeletal
structure of several well-known drugs [26,27] and indolo de-
rivatives have wide spectrum of biological activities, including
antituberculosis [28], anti-inflammatory [29,30], antitumor
[31,32], anticonvulsion and anticardiovascular effects [33,34]. We
speculated that indole as a necessary moiety of inhibitors which
could intercalate into DNA and interact with Topo I. Therefore,
indole was chosen for the synthesis of inhibitors. The simple and
small structure of sophoridine provides advantages in chemical
synthesis and offers opportunities of formulation for oral
administration. Besides, its structure-activity relationship reveals
that sophoridinic acid analogues containing a 3-ring core scaffold
are more favorable than sophoridine containing a 4-ring scaffold
ring [35]. So the ring D of sophoridine was opened and the
resultant product was allowed to combine with indole skillfully at

Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
481
3-position through Mannich reaction, which also provides us an
3.2. Chemistry
opportunity to perform further substitution at N-position of
indole moiety and side chain of sophoridinic acid. Consequently,
preliminarily docking studies of a series of preconceived indolo
sophoridinic derivatives were performed with the Topo I target
and then the desired products were synthesized and evaluated for
their anticancer activity (Scheme 1).
The docking results suggest that targeting newly designed in-
hibitor is a promising strategy for cancer therapy (Table 1). To verify
the possibility of the strategy, thirty novel N-substituted indolo
sophoridinic acid derivatives (mentioned above) were synthesized
(Scheme 2) using commercially available 1 as a starting material.
The desired product 2 was prepared via hydrolysis of 1 in aqueous
NaOH at the reflux temperature, which was then reacted with
sulfoxide chloride and alcohol to produce product 3. The iminium
intermediate was prepared by the addition of 3 to formaldehyde in
the presence of acetic acid which was subsequently reacted with
various indolo substituents to produce 4a-4e skillfully by Mannich
reaction. A series of halohydrocarbon acted as alkylating agents to
synthesize 6a-6e from 4a-4e using K2CO3 as a base. Similarly, the
desired compounds 6a-6e could also be produced through the re-
action of 3 with N-substituted indolo derivatives (5a-5e) directly.
Finally, considering the influence of pharmacology on hydropho-
bicity, the study of structure activity relationship was focused on
the side chain. The ester functionality of compounds 4a, 6e and 6j
was transformed into alcohol group resulting in the formation of
compounds 9, 10a and 10b respectively. Compound 2 was also tried
to transform to compound 11. However, the structure of compound
11 is unstable and its instability is suspected to be caused by amino
and carboxyl groups with different pH values. In this work, high
reaction yield is observed in each step, which provides the feasi-
bility of a useful application in terms of synthesis and drug devel-
opment cost. Melting points of the newly synthesized compounds
were determined by X-4 melting point apparatus. Their structures
were characterized by 1H NMR, 13C NMR and high resolution ESI-
MS spectra.
3. Results and discussion
3.1. Molecular docking
Molecular docking was first performed on proposed inhibitor 4a
with DNA Topo I complex. As we predicted, the docking score of 4a
is much better than sophoridine, indicating that the introduced
indole moiety could significantly improve affinity with Topo I. We
speculated that the primary affinity between indole moiety and
Topo I is the base-stacking force caused by the p-p electron inter-
action with the base, which could stabilize the DNA helix structure.
To further understand the influence of the stacking force caused by
the electron density, docking of 4d-4f bearing electrophilic groups
and 4b-4c carrying electron-donating groups with Topo I was
performed after variation of R1 at 5-position. Among the com-
pounds, 4b and 4f exhibited potent docking score. While consid-
ering the importance of halides in the drug designing research,
compounds 4g-4j containing a variety of halogens at different po-
sitions on the indole moiety were chosen for docking. Due to the
importance of alkylation strategy in drug design and modification,
a variety of alkyl groups were introduced on the nitrogen atom and
docking was executed. Better docking results of 6a-6d than 4a
revealed that alkyl group could enhance the activity. Concurrently,
docking of compounds 6e-6p with a variety of aryl groups was also
performed. Hydrophobicity, as a fundamental pharmacological
parameter, could influence the absorption of medicine, biological
activities, interactions with receptors and the toxicity from mo-
lecular metabolism etc. Herein, the ester group was transformed
into alcohol and carboxyl functionality and docking results of the
derivatives were also obtained (Table 1).
3.3. MTT assays
The target compounds were tested for their cytotoxic activities
in HepG2, CNE-2 and A549 cell lines with camptothecin as the
positive control using MTT assays. Structures of derivatives and
their cytotoxic activities are shown in Table 2.
Scheme 1. Design strategy for Topo I inhibitors.

482
Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
Table 1
Docking results of the derivatives with Topo I -DNA complex (PDB: 1T8I).
Ligand
Docking scorea
Glide energyb
Lipophilic EvdWc
Hbondd
Glide emodele
Sophoridine
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
7
10a
10b
11
ꢀ3.294
ꢀ5.778
ꢀ6.58
ꢀ19.341
ꢀ46.323
ꢀ52.238
ꢀ53.741
ꢀ50.663
ꢀ56.465
ꢀ50.332
ꢀ43.801
ꢀ48.354
ꢀ45.261
ꢀ45.222
ꢀ58.222
ꢀ48.926
ꢀ45.491
ꢀ51.874
ꢀ50.881
ꢀ57.906
ꢀ56.413
ꢀ60.511
ꢀ59.616
ꢀ57.156
ꢀ58.986
ꢀ59.798
ꢀ64.477
ꢀ62.288
ꢀ60.84
ꢀ2.656
ꢀ5.687
ꢀ5.74
0
0
ꢀ29.277
ꢀ68.591
ꢀ77.767
ꢀ71.203
ꢀ67.064
ꢀ78.233
ꢀ74.477
ꢀ69.349
ꢀ67.644
ꢀ66.654
ꢀ66.001
ꢀ80.574
ꢀ71.272
ꢀ71.332
ꢀ80.947
ꢀ79.884
ꢀ91.192
ꢀ89.064
ꢀ91.741
ꢀ89.282
ꢀ91.902
ꢀ96.019
ꢀ96.922
ꢀ96.667
ꢀ103.263
ꢀ100.655
ꢀ82.305
ꢀ88.986
ꢀ61.908
ꢀ83.008
ꢀ91.003
ꢀ80.947
ꢀ92.869
ꢀ0.786
ꢀ0.347
ꢀ0.35
0
ꢀ0.35
ꢀ0.807
0
ꢀ1.041
ꢀ0.981
ꢀ0.574
0
ꢀ5.415
ꢀ5.518
ꢀ4.716
ꢀ6.383
ꢀ5.705
ꢀ5.959
ꢀ4.154
ꢀ4.171
ꢀ6.878
ꢀ6.254
ꢀ6.421
ꢀ4.324
ꢀ6.928
ꢀ8.151
ꢀ6.725
ꢀ7.497
ꢀ6.841
ꢀ6.833
ꢀ7.774
ꢀ6.863
ꢀ6.659
ꢀ6.977
ꢀ7.423
ꢀ4.756
ꢀ4.331
ꢀ6.801
ꢀ6.887
ꢀ7.521
ꢀ4.324
ꢀ8.901
ꢀ4.769
ꢀ5.202
ꢀ4.573
ꢀ5.896
ꢀ4.55
ꢀ5.909
ꢀ3.844
ꢀ3.681
ꢀ6.1
ꢀ6.195
ꢀ5.667
ꢀ6.846
ꢀ6.06
ꢀ0.7
ꢀ1.379
ꢀ0.7
ꢀ8.037
ꢀ5.93
ꢀ0.156
ꢀ0.7
ꢀ6.773
ꢀ6.225
ꢀ6.749
ꢀ7.145
ꢀ7.068
ꢀ6.141
ꢀ6.766
ꢀ6.771
ꢀ3.826
ꢀ5.248
ꢀ5.082
ꢀ6.164
ꢀ7.212
ꢀ6.846
ꢀ7.668
ꢀ0.495
ꢀ0.367
0
ꢀ0.35
0
ꢀ1.056
ꢀ0.35
ꢀ1.043
ꢀ1.361
ꢀ0.012
ꢀ1.33
ꢀ0.722
ꢀ0.35
ꢀ1.379
ꢀ0.48
ꢀ53.887
ꢀ57.421
ꢀ46.036
ꢀ51.316
ꢀ57.519
ꢀ51.874
ꢀ62.819
Camptothecin
a
Docking score, including terms in the scoring function, various indexes, and ligand efficiency metrics, rank the affinity of ligands bound to the active site of a receptor.
Glide energy, revealing the Modified Coulomb-van der Waals interaction energy.
Lipophilic EvdW, indicating Lipophilic term derived from hydrophobic grid potential at the hydrophobic ligand atoms.
b
c
d
Hbond, Hydrogen-bonding term. This term is separated into differently weighted components that depend on whether the donor and acceptor are neutral, one is neutral
and the other is charged, or both are charged.
e
Glide emodel, indicating model energy. Emodel combines GlideScore, the nonbonded interaction energy, and, for flexible docking, the excess internal energy of the
generated ligand conformation.
Indole moiety was firstly introduced to sophoridine to produce
compound 4a. The cytotoxic activities results confirmed more
potent activities of compound 4a than sophoridine against three
cancer cell lines, indicating indole moiety significantly enhanced
the anticancer activity. Besides, the docking score of 4a
(with ꢀ5.778) is better than that of sophoridine (with ꢀ3.294)
(Table 2). These results verified the rationality of the strategy,
turning the activity from millimole into a micromole. The drugs
were designed to enhance the anticancer activity by stabilizing the
DNA-Topo I complex, in which the primary affinity is the base
scores were in accordance with the activity result. Among the
compounds, 4b, 4f, 4h, and 4k bear better score (ꢀ6.58, ꢀ6.383,-
5.959 and ꢀ6.878 respectively) than 4a (ꢀ5.778), while 4a
exhibited better activity than these compounds. We conjectured
that docking could simulate and predict the binding mode of
p-p
interaction between ligands and acceptor, but it is difficult to es-
timate the impact of changing electron cloud distribution on the
affinity, leading to the diversity between calculation and experi-
ment. Next, we introduced a variety of alkyl and aryl groups on the
nitrogen atom at the 1-position of indole. Compounds 6a-6d and
6e-6m were synthesized and investigated. Majority of these com-
pounds exhibited better anticancer activities than 4a. Meanwhile,
the docking score exerted high consistency with the activity indi-
cating that alkyl and aryl groups play an important role in
improving the activity and feasibility of guidance by docking. More
importantly, the mechanism might be explained by the use of the
calculated docking data. As shown in Table 1, the Lipophilic EvdWc
parameter is closely related to the docking score, the introduced
alkyl and aryl groups lowered the glide energy by decreasing the
Lipophilic EvdWc, and thus enhanced the activity. Besides, by
comparing the activity results of 6i, 6k, and 6l with 6n, 6o and 6p, it
is obvious that the existence of fluorine atom reduced the activity,
also indicated that variation in electron density had disadvantage
for stacking force. On the other hand, 6o and 6p perform better
activity than 4a, however, their docking score (ꢀ4.756, ꢀ4.331) is
not as good as that of 4a (ꢀ5.778), making it difficult once again to
stacking force caused by the interaction between
p electrons
derived from indole moiety and base pairs. Therefore, it was
imperative to focus the structure activity analysis on the electron
cloud of indole moiety. With a variety of indole structures bearing
electron-withdrawing and electron-donating groups introduced
into sophoridine, compounds 4a-4k were obtained and the influ-
ence on the bioactivity caused by the electronic cloud distribution
on the indole moiety was explored. The results showed that the
activities of all the derivatives of 4a (obtained after substitution at
R1) were poorer than the parent compound 4a itself. This led us to
the conclusion that the increase and decrease of the
p electron
cloud induced by electronic effect on the indole moiety could
weaken the base stacking force. We speculated that electron dis-
tribution on indole moiety without R1 substituents was the most
favorable condition for improving
p-p interaction furthest.
However, as illustrated in Table 1, not all the 4a-4k docking

Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
483
Scheme 2. Reagents and conditions: (a) NaOH aqueous, reflux, 8h; (b) SOCl2, MeOH, reflux, 3h; (c) HCHO, AcOH, R1 bearing indolo derivatives, H2O or CHCl3, rt; (d) NaH, halides,
DMSO, rt; (e) HCHO, AcOH, 5a-5p, H2O or CHCl3, rt; (f) (Boc)2O, TEA, CH2Cl2, rt, 5h; (g) LiAlH4, THF, rt, 2h; (h) NaHSO4$SiO2, CH2Cl2, reflux; (i) R1 bearing indolo derivatives, Halides
with R2 group, DMF, NaH, rt.
predict the dependency of the stacking force influence on the
altering electron cloud. Finally, alcohols (7, 10a and 10b) with
improved water solubility were obtained, aiming to execute the
SAR exploration on the effect caused by metabolism and stability.
The results revealed that a variation existed in the activities when
the functional group transformed from ester to alcohol. Compound
10a exhibited better activities than 6e against HepG2, CNE-2 and
A549, whereas 10b showed poorer activities than 6j against A549,
indicating that the alcohol group did not make positive contribu-
tion to all compounds. Thus, these results provided important clue
for designing new inhibitors.
relaxation of supercoiled DNA of plasmid pBR322 with CPT as
positive control (Fig. 3) [36]. The results showed that compounds
6e,
6i,
6m,
10a
and
10b
(with
good
docking
score: ꢀ8.151, ꢀ6.833, ꢀ6.977, ꢀ6.887, ꢀ7.521 respectively) could
inhibit the enzymatic activity, which are basically consistent with
the docking score, further indicating the rationality of the design
strategy. It is noteworthy that compound 10b exhibited potent
inhibitory activity against Topo I and antiproliferative activity
against tested cancer cell lines simultaneously. Thus, 10b was
chosen for further investigation.
Based on the feasibility of docking results and the binding mode
of camptothecin, indolocarbazole, indenoisoquinoline and sopho-
ridine the proposed inhibitor 4a was designed first. Then a series of
4a derivatives was constructed by docking and their bioactivities
were verified. Overall, the docking and activity results simulta-
neously revealed that indole is a necessary moiety for the inhibitors
and the introduction of alkyl and aryl groups could improve the
activity to some degree. These findings showed a correlation be-
tween biological activity and docking. Although limitation existed
and was confirmed, the analysis preliminarily verified the ratio-
nality of design. Further verification was carried out by enzymatic
activity.
3.5. Docking analysis of 10b with Topo I-DNA complex
Based on above study results, 10b was chosen as a representa-
tive for molecular docking studies in order to further understand
the binding modes of the analogues (Fig. 4). The indole rings in 10b
mimic a DNA base pair and thereby form stacking interactions with
both upstream and downstream base pairs DT10 and TGP11,
respectively. On the other hand, the sophoridinic moiety of 10b
formed hydrogen bonds with residues LYS436. Furthermore, the 4-
bromobenzyl substituent of 10b could not only form Lipophilic
EvdW interaction but can also produce p-p stacking force with base
pairs DA113 and DC112 similar to indole moiety, thus contributing
to the overall interaction. It explains why the introduction of
various benzyl groups could enhance the activities. In summary, the
above molecular simulations give us rational explanation of the
interactions between ligand 10b and DNA-Topo I, which in turn
provided valuable information for further development of DNA-
Topo I inhibitors.
3.4. Topo I inhibitory activity
To verify the rational design and explore the mechanism by
which compounds inhibited Topo I and thereby caused cytotoxicity,
fourteen compounds were examined at 500 mM by measuring the

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Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
Table 2
Structures and the in vitro cytotoxic activities of target derivatives.
Compound
R1
R2
R3
IC50(m
M)a
HepG2
CNE-2
A549
Sophoridine
/
H
/
H
H
H
H
H
H
H
H
H
H
H
/
4670 127
36.5 1.8
23.8 2.5
>50
>50
>50
>50
31.8 3.4
>50
>50
>50
5379 109
12.1 1.6
49.4 2.2
>50
15.3 1.5
>50
17.9 1.7
20.2 3.1
>50
5836 97
27.8 2.3
>50
>50
>50
>50
>50
39.9 3.8
>50
>50
>50
>50
42.8 3.4
37.4 3.6
42.8 2.3
30.3 3.3
4a
4b
4c
4d
4e
4f
4g
4h
4i
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
5-CH3
5-OCH3
5-CN
5-NO2
5-Cl
5-Br
5-F
4-F
6-F
2-COOCH3
H
H
>50
4j
17.1 2.1
26.4 3.1
43.4 2.6
19.4 1.2
8.6 1.8
13.4 2.4
4k
6a
6b
6c
6d
>50
CH3
CH2CH3
CH2CH2CH2CH3
15.2 1.2
12.8 1.9
6.2 0.8
14.4 1.0
H
H
6e
6f
6g
6h
6i
6j
6k
6l
H
H
H
H
H
H
H
H
CH2C6H5
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
5.5 0.6
21.7 1.3
12.1 2.6
8.6 3.1
6.5 2.7
4.8 1.6
6.1 1.3
8.5 1.4
3.3 0.9
14.8 2.3
12.1 1.2
3.5 1.2
4.6 1.4
4.3 0.7
4.8 0.8
6.7 1.4
43.4 3.2
49.3 2.4
42.3 5.4
29.6 1.3
5.2 1.6
3.5 0.8
7.4 0.9
3.7 0.5
CH2C6H4F-o
CH2C6H4F-m
CH2C6H4F-p
CH2C6H4Cl-p
CH2C6H4Br-p
CH2C6H4CF3-p
6m
H
COOCH3
8.6 2.1
7.8 1.2
24.0 1.8
6n
6o
6p
5-F
5-F
5-F
CH2C6H4Cl-p
CH2C6H4CF3-p
COOCH3
COOCH3
COOCH3
7.3 1.0
7.7 0.7
18.4 3.3
6.1 2.0
6.9 1.0
9.3 1.5
10.6 3.4
12.8 1.5
39.8 1.8
7
10a
10b
camptothecin
H
H
H
/
H
CH2OH
CH2OH
CH2OH
/
>50
>50
>50
CH2C6H5
CH2C6H4Br-p
/
2.3 0.6
3.1 0.8
13.2 2.6
2.2 0.7
3.5 1.3
0.1 0.04
33.7 2.4
22.1 3.5
5.5 0.7
a
Data are expressed as the mean SD of at least three independent experiments.
showing a pronounced effect. On the other hand, it is obvious that
the primary binding force between ligands and DNA-Topo I is the
base stacking force produced by the
p-p interaction from indole
moiety and DNA base upstream and downstream. The design
strategy could clearly be described by the mode and it can mutually
be argued with the experiment. The strategy provides useful in-
formation for designing better new Topo I inhibitors with bigger
planner heteroaromatic ring, connecting it with sophoridine scaf-
fold bearing more druggable advantages.
Fig. 3. Topo
I þ pBR322 DNA; lane CPT: camptothecin (100
others: various derivatives (500
M) þ Topo I þ pBR322 DNA.
I
inhibitory activities. lane DNA, pBR322 DNA; lane Topo I, Topo
m
M) þ Topo I þ pBR322 DNA; the
m
From an overall perspective, the special structure of parent
sophoridine offers many pharmacological advantages such as
simple chemical scaffold, flexibility, high solubility, and good safety
profiles to the new inhibitors. More significantly, as illustrated in
the binding mode, the structure could serve as a target carrier for
the planner indole moiety, conveyed to the DNA active site and
3.6. Cell cycle analysis
It is reported that cytotoxicity of Topo I inhibitor depended on
active DNA replication [37]. Based on compound 10b showing
positive activity in MTT assay and Topo I inhibition assay, we

Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
485
with vehicle control and sophoridine group, the tumor growth of
compound 10b group was significantly inhibited (10b vs vehicle:
p ¼ 0.003; 10b vs sophoridine: p ¼ 0.002, at day 25) (Fig. 6A, B).
The value of T/C in the 40 mg/kg 10b group and sophoridine
group was 55.99% and 93.38%, respectively (day 25, volume of
vehicle control ¼ 1185.78 137.44 mm3, volume of sophoridine
group ¼ 1204.27 141.01 mm3, volume of 10b group ¼ 737.04
109.29 mm3). There was also a significant tumor weight loss in
10b group compared with vehicle control (p ¼ 0.039) and soph-
oridine group (p ¼ 0.005). The tumor weight for each group was
0.84 0.09 g
(vehicle),
0.97 0.09 g
(sophoridine)
and
0.61 0.15 g (10b) (Fig. 6C). In the 40 mg/kg irinotecan group, the
value of T/C and tumor weight was 35.08% and 0.47 0.12 g (day
25). Although irinotecan displayed more potent antitumor ac-
tivity than 10b, irinotecan also exhibited more toxicity than 10b,
reflected by mouse body weight loss from day 19 (Fig. 6D).
4. Conclusion
Novel Topo I inhibitors were synthesized and tested for their
cytotoxic activities. Further studies verified the consistency be-
tween docking and activity (antiproliferative activity and Topo I
inhibitory activities) and the rationality of the design strategy. The
SAR analysis and molecular model revealed that (i) the stacking
force caused by indole moiety with base pairs is the primary af-
finity and its extent was dependent on the electron cloud; (ii)
introduction of an aryl or alkyl group on the nitrogen atom of the
indole enhanced the activity via improving the lipophicity. Among
the analogues, 10b was chosen as a representative for further
study due to its potent inhibitory potency against Topo I and
antiproliferative activity against HepG2 cells. Compound 10b
arrested HepG2 cells at the S phase in a dose-dependent manner.
The in vivo studies further demonstrated that 10b significantly
suppressed the growth of HepG2 xenograft tumors without
adverse toxicity as no distinct weight loss of mice was observed.
Taken together, the sophoridine scaffold of novel Topo I inhibitors
could serve not only as a “carrier” to transport the indole moiety
to the target, but it can also render many pharmacological ad-
vantages to the new inhibitors. Furthermore, the indole moiety
significantly enhanced the biological activities. The new strategy
provided a series novel Topo I inhibitors for further optimization
and development in cancer therapy.
Fig. 4. The binding mode of compound 10b with Topo I-DNA complex.
further investigated the effect of 10b on cell cycle distribution. The
results indicated that compound 10b led to S phase cell cycle arrest.
In fact, after 24 h, the cell population in S increases from 45.44% in
the untreated cells to 55.77%, 60.68% and 64.17% in the cells treated
5. Experimental
with 10 b at 1, 2.5, 5 mM, respectively (Fig. 5a). While the results
revealed that sophoridine could induce G1 cell cycle arrest (Fig. 5b).
Combining the results of cell cycle assay and molecular docking
analysis, we hypothesized that the difference between cell cycle
arrest mechanisms of 10b and sophoridine were caused by the
binding modes. It is well recognized that the effects of Topo I in-
hibitors depend on two routes: catalyst inhibition and poison in-
hibition. The catalytic inhibitors can react with Topo I and suppress
the enzymatic activity, causing the DNA duplication defeat. The
poison inhibitors can bind to the covalent Topo I-DNA complex,
consequently leading to double strand breaks and ultimately
apoptotic cell death [38]. Due to the binding model of 10b (Fig. 4)
similar with camptothecin (Figs. 2a), 10b can serve as poison in-
hibitor and induced S cell cycle arrest. While sophoridine serve as a
catalytic inhibitor, which induced G1 cell cycle arrest.
5.1. Molecular docking and molecular modeling
€
Glide model from Schrodinger software was used for the mo-
lecular docking studies. The structure of the compounds was
sketched using ChemDraw and optimized to lower energy con-
formers using Ligprep (Schrodinger LLC, New York, NY, USA). The
structure of human DNA-Topo I complex (PBD ID: 1T8I) was
downloaded from the Protein Data Bank and protein preparation
wizard was used to prepare complex for docking. OPLS3 force field
was used for optimizing the hydrogen bond network in the enzyme
structure after a series of preprocessing, such as mutation, adding
hydrogens, deleting water etc. 0.30 Å was a maximum cutoff of the
energy converged or the route mean square deviation (RMSD). The
receptor grid can be set up and generated from the Receptor Grid
Generation panel. Ultimately, the generated grid of protein struc-
ture was used to perform docking with the compounds using
Ligand Docking under the XP (extra precision) precision. Glide
(docking) score is the evaluation standard of ligand-protein
binding.
3.7. In vivo antitumor activity of 10b against HepG2 cells
Tumor xenografts derived from HepG2 cells were used to
evaluate the antitumor effect of compound 10b in vivo. Compared

486
Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
Fig. 5. Cell cycle analysis of compound 10b and sophoridine. HepG2 cells were incubated without (control) or with 10 b at different concentrations (1, 2.5, 5 mM) (a) or with
sophoridine at different concentrations (2, 4, 6 mM) (b) for 24 h and then analyzed for their cell cycle distribution using flow cytometry. The percentage of each population was
shown as mean SD. Data is the representative of three independent experiments. *p < 0.05, **p < 0.01.
Fig. 6. In vivo antitumor activity of 10b against liver cancer model in mice. (A) Image of the internal tumor tissues after anatomy; (B) Relationship curves of tumor volumes at
various times (1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d and 25d) after administration; (C) Average mass of internal tumor tissues of mice; (D) Relationship curves of mice
body weights at various times (1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d and 25d) after administration; Data expressed as the mean SD; statistical significance: compared
with vehicle by t-test, *, p < 0.05, **, p < 0.01; compared with sophoridine by t-test, #, p < 0.05, ##, p < 0.01.

Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
487
5.2. Chemistry
123.44, 123.00, 119.84, 114.25, 110.56, 63.74, 59.21, 54.73, 51.50,
51.47, 50.54, 45.56, 38.56, 34.37, 29.91, 26.92, 26.32, 25.49, 23.40,
22.30, 21.54, 19.27. HR-MS (ESI) m/z: calculated for C26H37N3O2
[MþH]þ: 424.2959, found: 424.2957.
All the chemicals were purchased from commercial sources and
used without further purification unless otherwise stated. All the
compounds were characterized by 1H NMR, 13C NMR and ESI-MS
spectral analyses. Bruker Avance 600 (600 MHz) spectrometer
was used for recording 1H and 13C nuclear magnetic resonance
(NMR) data using CDCl3 as solvent and TMS as an internal standard.
5.2.2.3. Methyl 12-N-3-(5-methoxy)indolosophoridinate dihydro-
chloride (4c). Brown solid; Yield: 85%; Mp: 116e118 ꢂC; 1H NMR
(600 MHz, Chloroform-d)
d 8.20 (s, 1H), 7.26e7.22 (m, 2H), 7.04 (d,
Chemical shifts are reported in
d
ppm relative to internal tetra-
J ¼ 2.2 Hz, 1H), 6.86 (m, 1H), 3.88 (s, 3H), 3.75e3.70 (m, 2H), 3.69 (s,
3H), 3.06e2.92 (m, 3H), 2.91e2.85 (m, 1H), 2.69 (d, J ¼ 8.0 Hz, 1H),
2.60e2.46 (m, 2H), 2.38e2.29 (m, 2H), 2.12e1.98 (m, 3H), 1.89 (m,
1H), 1.78e1.61 (m, 5H), 1.57e1.45 (m, 3H), 1.39 (m, 1H), 1.31e1.23
methylsilane standard (TMS, 0.00). Coupling constants (J) were in
d
hertz (Hz) and the peak patterns are indicated as follows: s, singlet;
d, doublet; t, triplet; m, multiplet; q, quartet; br, broad, etc. The
progress of reactions was monitored by thin-layer chromatography
(TLC). Detections were done by UV light (254 nm) illumination and/
or treatment with Bismuth potassium iodide solution. The products
were purified by flash column chromatography equipped with
commercial silica gel (300e400 mesh). Mass spectra were obtained
from a ThermoFisher LCQ Fleet (ESI). Melting points were deter-
mined in open capillary tubes on X-4 melting point apparatus
without correction.
(m, 1H), 1.11e1.02 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 174.08,
153.61, 131.84, 128.05, 123.70, 114.23, 112.29, 111.68, 101.89, 63.81,
59.22, 55.79, 54.28, 51.53, 51.03, 50.70, 45.39, 38.07, 34.28, 29.43,
27.20, 25.63, 25.33, 23.31, 22.16, 19.10. HR-MS (ESI) m/z: calculated
for C26H37N3O3 [MþH]þ: 440.2908, found: 440.2913.
5.2.2.4. Methyl 12-N-3-(5- cyan)indolosophoridinate dihydro-
chloride (4d). Brown oil; Yield: 93%; 1H NMR (600 MHz, Chloro-
form-d)
d
9.46 (s, 1H), 8.11 (d, J ¼ 1.5 Hz, 1H), 7.46e7.38 (m, 2H), 7.21
5.2.1. General procedure for compound 3
(s, 1H), 3.78e3.71 (m, 2H), 3.68 (s, 3H), 3.23e3.04 (m, 3H),
3.03e2.94 (m, 1H), 2.83e2.73 (m, 1H), 2.56 (m, 2H), 2.37e2.21 (m,
2H), 2.20e2.10 (m, 2H), 2.07e1.17 (m, 11H), 1.12 (m, 1H), 0.94e0.81
Sophoridine 1 (2.48g, 10 mmol) was hydrolyzed with 50 mL of
aqueous NaOH (2 mol/L) under reflux for 8 h, and then solvent was
evaporated to get 2 in crude form. SOCl2 (3 mL) was added to
methanol (40 mL) and stirred for 30 min in ice-bath. Product 2 was
then added portion-wise and the mixture was then refluxed for 3 h.
After completion of the reaction, the mixture was filtered and
solvent was concentrated to obtain 3.
(m, 1H). 13C NMR (151 MHz, CDCl3)
d 174.00, 138.56, 127.36, 125.88,
125.38, 124.79, 121.07, 114.73, 112.13, 101.97, 62.71, 58.79, 52.93,
51.56, 51.44, 49.92, 44.95, 36.83, 34.00, 29.69, 27.93, 24.17, 23.79,
23.09, 21.68, 18.73. HR-MS (ESI) m/z: calculated for C26H34N4O2
[MþH]þ: 435.2755, found: 435.2756.
5.2.2. General procedure for compound 4a-4k
5.2.2.5. Methyl 12-N-3-(5- nitro)indolosophoridinate dihydro-
A 50 mL flask was charged with 3 (1 mmol), acetic acid
(1.5 mmol), formaldehyde (1.1 mmol, 36% aq) and R1 bearing indole
and H2O or CH2Cl2 (3 mL). The reaction mixture was stirred at room
temperature and progress of the reaction was monitored by TLC.
After completion of the reaction, the mixture was poured into
10 mL of water and its pH was adjusted to 11e13 by diluted NaOH
(20% aq.). The mixture was then extracted with CH2Cl2 (3 ꢁ 10 mL),
the combined organic layers were dried over anhydrous MgSO4 for
12 h and filtered. The solvent was evaporated under reduced
pressure to give the crude product, which was purified by column
chromatography using silica gel (300e400 mesh), eluted with a
mixture of CH2Cl2 and MeOH (5:1e3:1).
chloride (4e). Green oil; Yield: 87%; 1H NMR (600 MHz, Chloro-
form-d)
d
8.59 (d, J ¼ 2.2 Hz, 1H), 8.12 (m, 1H), 7.43 (d, J ¼ 9.0 Hz,
1H), 7.32 (d, J ¼ 3.3 Hz, 1H), 6.69 (d, J ¼ 3.2 Hz, 1H), 4.86 (m, 2H),
3.71 (s, 3H), 3.46e3.29 (m, 2H), 3.25e3.10 (m, 2H), 3.06 (m, 1H),
2.79 (m, 1H), 2.55 (m, 2H), 2.45 (m, 2H), 2.39e2.21 (m, 2H), 2.13 (m,
1H), 1.94e1.46 (m, 8H), 1.38e1.14 (m, 3H). 13C NMR (151 MHz,
CDCl3) d 173.77, 141.96, 138.93, 131.05, 128.45, 118.21, 117.38, 110.21,
104.27, 66.45, 62.30, 59.61, 52.86, 51.68, 48.58, 45.28, 35.56, 33.45,
28.36, 26.88, 23.97, 23.08, 22.59, 22.53, 18.25. HR-MS (ESI) m/z:
calculated for C25H34N4O4 [MþH]þ: 455.2653, found: 455.2657.
5.2.2.6. Methyl 12-N-3-(5- chloro)indolosophoridinate dihydro-
chloride (4f). Brown oil; Yield: 92%; 1H NMR (600 MHz, Chloro-
5.2.2.1. Methyl 12-N-3-indolosophoridinate dihydrochloride (4a).
White solid; Yield: 91%; Mp: 154e156 ꢂC; 1H NMR (600 MHz,
form-d)
d
8.27 (s, 1H), 7.69 (d, J ¼ 2.1 Hz, 1H), 7.31 (d, J ¼ 8.6 Hz, 1H),
7.16 (m, 1H), 7.12 (s, 1H), 3.76e3.71 (m, 2H), 3.70 (s, 3H), 3.38e3.15
(m, 3H), 3.10 (d, J ¼ 13.5 Hz, 1H), 3.00 (d, J ¼ 11.7 Hz, 1H), 2.74e2.56
(m, 2H), 2.44e2.26 (m, 2H), 2.27e2.12 (m, 3H), 2.09e1.23 (m, 10H),
Chloroform-d)
d
8.34 (s, 1H), 7.79e7.73 (m, 1H), 7.36 (d, J ¼ 8.1 Hz,
1H), 7.20 (m, 1H), 7.12 (m, 1H), 7.07 (d, J ¼ 2.2 Hz, 1H), 3.78e3.71 (m,
2H), 3.69 (s, 3H), 3.05e2.91 (m, 3H), 2.83 (m, 1H), 2.68 (d, J ¼ 8.4 Hz,
1H), 2.56e2.45 (m, 2H), 2.33 (m, 2H), 2.12e1.98 (m, 3H), 1.88e1.60
(m, 6H), 1.50 (m, 3H), 1.39e1.31 (m, 1H), 1.29e1.22 (m, 1H),
1.17 (m, 1H), 0.93e0.83 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 173.96,
135.08, 128.55, 124.89, 124.73, 122.32, 119.42, 113.17, 112.29, 62.53,
60.20, 52.98, 51.59, 50.54, 49.95, 45.39, 36.01, 33.87, 28.62, 27.16,
23.22, 22.91, 22.87, 21.70, 18.44. HR-MS (ESI) m/z: calculated for
1.12e1.01 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 174.09, 136.71,
127.69, 122.97, 121.86, 120.16, 119.07, 114.58, 110.99, 63.68, 59.26,
54.53, 51.53, 51.37, 50.52, 45.49, 38.30, 34.31, 29.66, 27.09, 25.95,
25.32, 23.35, 22.18, 19.18. HR-MS (ESI) m/z: calculated for
C
25H34ClN3O2 [MþH]þ: 444.2412, found: 444.2415.
5.2.2.7. Methyl 12-N-3-(5- bromo)indolosophoridinate dihydro-
C
25H35N3O2 [MþH]þ: MS: 410.2802, found: 410.2806.
chloride (4g). White solid; Yield: 89%; Mp: 58e60 ꢂC; 1H NMR
(600 MHz, Chloroform-d)
d
9.06e8.87 (m, 1H), 7.92 (t, J ¼ 2.1 Hz,
5.2.2.2. Methyl 12-N-3-(5-methyl)indolosophoridinate dihydro-
1H), 7.24 (m, 1H), 7.21e7.15 (m, 1H), 7.05 (d, J ¼ 2.5 Hz, 1H), 3.68 (m,
5H), 3.04e2.89 (m, 3H), 2.79 (m, 1H), 2.64e2.58 (m, 1H), 2.53e2.39
(m, 2H), 2.29 (m, 2H), 2.14e1.94 (m, 3H), 1.84e1.68 (m, 3H),
1.68e1.38 (m, 6H), 1.33 (d, J ¼ 13.6 Hz, 1H), 1.24e1.17 (m, 1H),
chloride (4b). Green solid; Yield:83%; Mp: 62e64 ꢂC; 1H NMR
(600 MHz, Chloroform-d)
d 8.10 (s, 1H), 7.57e7.53 (m, 1H), 7.25 (d,
J ¼ 8.2 Hz, 1H), 7.03 (m, 2H), 3.75e3.71 (m, 2H), 3.69 (s, 3H),
3.04e2.89 (m, 3H), 2.80 (m, 1H), 2.68 (d, J ¼ 8.6 Hz, 1H), 2.53e2.43
(m, 5H), 2.33 (t, J ¼ 6.8 Hz, 2H), 2.15e1.98 (m, 3H), 1.78 (m, 3H), 1.66
(m, 3H), 1.57e1.46 (m, 3H), 1.37 (m, 1H), 1.25e1.19 (m, 1H), 1.11e1.02
1.10e1.01 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 174.04, 135.36,
129.47, 124.52, 124.24, 122.91, 114.20, 112.47, 112.19, 63.43, 59.14,
54.69, 51.68, 51.54, 50.36, 45.56, 38.55, 34.28, 29.90, 26.92, 26.31,
25.50, 23.33, 22.17, 19.25. HR-MS (ESI) m/z: calculated for
(m, 1H). 13C NMR (151 MHz, CDCl3)
d 174.07, 134.97, 128.21, 127.94,

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Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
C25H34BrN3O2 [MþH]þ: 488.1907, found: 488.1911.
were removed in vacuo to obtain desired 5a-5p without
purification.
5.2.2.8. Methyl 12-N-3-(5- fluro)indolosophoridinate dihydro-
chloride (4h). Brown solid; Yield; 88%; Mp: 136e138 ꢂC; 1H NMR
5.2.4. General procedure for compound 6a-6p
(600 MHz, Chloroform-d)
d
8.23 (s, 1H), 7.42 (m, 1H), 7.26 (m, 1H),
A 50 mL flask was charged with 3 (1 mmol), acetic acid
(1.5 mmol), formaldehyde (1.1 mmol, 36% aq), 5a-5p and H2O or
CH2Cl2 (3 mL). The reaction mixture was stirred at room tempera-
ture. The progress of the reaction was monitored by TLC. After
completion of the reaction, the mixture was poured into 10 mL
water and its pH was adjusted to 11e13 by diluted NaOH (20% aq.).
The mixture was extracted with CH2Cl2 (3 ꢁ 10 mL) and the com-
bined organic layers were dried over anhydrous MgSO4 for 12 h
followed by filtration. The solvent was evaporated under reduced
pressure to give the crude product, which was purified by column
chromatography using silica gel (300e400 mesh), eluted with a
mixture of CH2Cl2 and MeOH (5:1e3:1).
7.11 (d, J ¼ 2.2 Hz, 1H), 6.95 (m, 1H), 3.70 (d, J ¼ 2.4 Hz, 2H), 3.69 (s,
3H), 3.03e2.94 (m, 3H), 2.81 (m, 1H), 2.62 (d, J ¼ 8.5 Hz, 1H),
2.53e2.43 (m, 2H), 2.32 (m, 2H), 2.13e1.97 (m, 3H), 1.83e1.75 (m,
3H), 1.69e1.42 (m, 6H), 1.36e1.27 (m, 1H), 1.26e1.20 (m, 1H),
1.12e1.00 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 174.05, 157.51 (d,
J ¼ 233.8 Hz), 133.17, 128.10, 124.63, 114.87, 111.44, 110.28, 105.11,
63.46, 59.15, 54.63, 51.65, 51.52, 50.43, 45.52, 38.44, 34.30, 29.85,
27.00, 26.17, 25.47, 23.36, 22.09, 19.23. HR-MS (ESI) m/z: calculated
for C25H34FN3O2 [MþNa]þ: 450.2527, found: 450.2533.
5.2.2.9. Methyl 12-N-3-(4- fluro)indolosophoridinate dihydro-
chloride (4i). Yellow oil; Yield: 89%; 1H NMR (600 MHz, Chloro-
form-d)
d
8.99 (s, 1H), 7.12e7.03 (m, 3H), 6.77e6.69 (m, 1H),
5.2.4.1. Methyl 12-N-3-(N-methyl)indolosophoridinate dihydro-
3.87e3.71 (m, 2H), 3.67 (s, 3H), 3.01e2.90 (m, 3H), 2.80 (m, 1H),
2.70 (d, J ¼ 9.5 Hz, 1H), 2.55e2.46 (m, 2H), 2.32 (t, J ¼ 6.9 Hz, 2H),
2.14e1.95 (m, 3H), 1.76 (m, 3H), 1.70e1.45 (m, 6H), 1.36e1.29 (m,
1H), 1.24e1.18 (m, 1H), 1.11e1.02 (m, 1H). 13C NMR (151 MHz, CDCl3)
chloride (6a). Yellow oil; Yield: 86%; 1H NMR (600 MHz, Chloro-
form-d)
d
7.77 (m, 1H), 7.29 (d, J ¼ 8.0 Hz, 1H), 7.24 (m, 1H), 7.12 (m,
1H), 6.92 (s, 1H), 3.75 (s, 3H), 3.75e3.72 (m, 2H), 3.70 (s, 3H),
3.04e2.91 (m, 3H), 2.80 (m, 1H), 2.69 (m, 1H), 2.49 (m, 2H),
2.37e2.32 (m, 2H), 2.13e1.98 (m, 3H), 1.84e1.60 (m, 6H), 1.52 (m,
3H), 1.38e1.32 (m, 1H), 1.24e1.18 (m, 1H), 1.07 (m, 1H). 13C NMR
d
174.11, 157.32 (d, J ¼ 247.7 Hz), 139.44, 123.14, 122.07, 116.24,
113.72, 107.16, 104.40, 64.00, 59.17, 54.71, 51.49, 51.42, 50.70, 45.51,
38.52, 34.31, 29.92, 26.95, 26.26, 25.31, 23.42, 22.87, 19.28. HR-MS
(ESI) m/z: calculated for C25H34FN3O2 [MþH]þ: 428.2708, found:
428.2709.
(151 MHz, CDCl3)
d 174.01, 137.40, 128.15, 127.61, 121.47, 120.25,
118.61, 113.23, 109.01, 63.85, 59.23, 54.74, 51.49, 51.39, 50.47, 45.56,
38.53, 34.37, 32.61, 29.91, 26.95, 26.29, 25.54, 23.41, 22.25, 19.28.
HR-MS (ESI) m/z: calculated for C26H37N3O2 [MþNa]þ: 422.2778,
found: 422.2783.
5.2.2.10. Methyl 12-N-3-(6- fluro)indolosophoridinate dihydro-
chloride (4j). Brown oil; Yield: 87%;1H NMR (600 MHz, Chloroform-
d)
d
8.66 (s, 1H), 7.67 (m, 1H), 7.07e6.98 (m, 2H), 6.90e6.81 (m, 1H),
5.2.4.2. Methyl
chloride (6b). Yellow oil; Yield: 84%; 1H NMR (600 MHz, Chloro-
form-d)
1H), 7.11e7.07 (m,1H), 7.00 (s,1H), 4.14 (q, J ¼ 7.3 Hz, 2H), 3.78e3.71
(m, 2H), 3.69 (s, 3H), 3.37e3.27 (m, 2H), 3.19 (m, 1H), 3.13 (m, 1H),
3.04e2.97 (m, 1H), 2.77e2.71 (m, 1H), 2.65 (d, J ¼ 7.4 Hz, 1H),
2.44e2.28 (m, 3H), 2.25e2.05 (m, 4H), 1.89 (m, 1H), 1.79e1.58 (m,
5H), 1.55e1.41 (m, 6H), 1.16 (m, 1H). 13C NMR (151 MHz, CDCl3)
12-N-3-(N-ethyl)indolosophoridinate
dihydro-
3.70 (d, J ¼ 2.3 Hz, 2H), 3.68 (s, 3H), 3.00e2.92 (m, 3H), 2.78 (m, 1H),
2.61 (d, J ¼ 9.6 Hz, 1H), 2.49e2.42 (m, 2H), 2.30 (t, J ¼ 6.2 Hz, 2H),
2.10e1.92 (m, 3H), 1.79e1.73 (m, 3H), 1.63 (m, 3H), 1.56e1.41 (m,
3H), 1.29e1.26 (m, 1H), 1.24e1.17 (m, 1H), 1.10e0.97 (m, 1H). 13C
d
7.69e7.66 (m, 1H), 7.33 (d, J ¼ 8.2 Hz, 1H), 7.23e7.19 (m,
NMR (151 MHz, CDCl3)
d
174.08, 159.90 (d, J ¼ 236.8 Hz), 136.62,
124.25, 123.22, 120.88, 114.61, 107.69, 97.27, 63.55, 59.14, 54.68,
51.61, 51.51, 50.49, 45.51, 38.50, 34.28, 29.90, 26.95, 26.25, 25.53,
23.35, 22.13, 19.24. HR-MS (ESI) m/z: calculated for C25H34FN3O2
[MþH]þ: 428.2708, found: 428.2711.
d
173.92, 136.48, 128.07, 126.30, 121.52, 120.02, 118.73, 112.04,
109.32, 62.99, 60.21, 54.81, 52.98, 51.55, 50.17, 45.31, 40.79, 36.06,
33.98, 28.59, 27.18, 23.27, 22.95, 22.89, 21.85, 18.46, 15.47. HR-MS
(ESI) m/z: calculated for C27H39N3O2 [MþH]þ: 438.3115, found:
438.3117.
5.2.2.11. Methyl 12-N-3-(2-methyl formate)indolosophoridinate
dihydrochloride (4k). Yellow solid; Yield: 67%; Mp: 104e106 ꢂC; 1H
NMR (600 MHz, Chloroform-d)
d
8.86 (s, 1H), 7.92 (d, J ¼ 8.1 Hz, 1H),
7.41e7.36 (m, 1H), 7.34 (m, 1H), 7.15 (m, 1H), 4.43 (m, 2H),
4.24e3.97 (m, 2H), 3.69 (s, 3H), 2.94 (m, 4H), 2.68 (d, J ¼ 9.5 Hz, 1H),
2.54 (d, J ¼ 11.2 Hz, 1H), 2.42 (m, 1H), 2.34 (t, J ¼ 7.2 Hz, 2H), 2.13 (t,
J ¼ 11.2 Hz, 1H), 2.08e1.91 (m, 2H), 1.86 (m, 1H), 1.77e1.47 (m, 8H),
1.45 (t, J ¼ 7.1 Hz, 3H), 1.34e1.23 (m, 2H), 1.07 (m, 1H). 13C NMR
5.2.4.3. Methyl
chloride (6c). Yellow oil; Yield: 85%; 1H NMR (600 MHz, Chloro-
form-d)
12-N-3-(N-butyl)indolosophoridinate
dihydro-
d
7.66 (m, 1H), 7.28 (d, J ¼ 8.1 Hz, 1H), 7.16 (m, 1H), 7.05 (m,
1H), 6.94 (s, 1H), 4.04 (t, J ¼ 7.1 Hz, 2H), 3.74e3.66 (m, 2H), 3.64 (s,
3H), 3.20e3.00 (m, 4H), 2.85e2.77 (m, 1H), 2.67 (q, J ¼ 3.0 Hz, 1H),
2.55 (d, J ¼ 7.2 Hz, 1H), 2.31 (m, 2H), 2.19e1.91 (m, 5H), 1.83e1.58
(m, 6H), 1.58e1.33 (m, 5H), 1.33e1.23 (m, 2H), 1.15e1.03 (m, 1H),
(151 MHz, CDCl3)
d 173.97, 162.30, 135.89, 128.55, 125.51, 124.19,
122.61,122.13,119.99,111.53, 64.65, 60.84, 58.88, 54.24, 51.49, 51.25,
48.84, 45.33, 38.13, 34.38, 29.53, 27.13, 25.76, 25.25, 23.50, 22.60,
19.11, 14.47. HR-MS (ESI) m/z: calculated for C27H37N3O4 [MþH]þ:
468.2857, found: 468.2861.
0.90 (t, J ¼ 7.4 Hz, 3H). 13C NMR (151 MHz, CDCl3)
d 173.88, 136.69,
128.04, 126.96, 121.38, 120.05, 118.58, 112.12, 109.35, 62.97, 59.88,
53.48, 51.48, 50.67, 50.16, 45.86, 45.34, 36.75, 34.06, 32.27, 28.03,
27.98, 23.93, 23.91, 23.05, 21.87, 20.14, 18.66, 13.69. HR-MS (ESI) m/
z: calculated for C29H43N3O2 [MþH]þ: 466.3428, found: 466.3429.
5.2.3. General procedure for compound 5a-5p
NaH (216 mg, 5.40 mmol, 1.08 eq) was added portion wise to a
solution of R1 bearing indole (5.00 mmol, 1.0 eq.) in DMF (20 mL) at
0 ꢂC. The ice bath was removed and the mixture was stirred for
30 min. Halides (5.25 mmol, 1.05 eq.) were then added dropwise at
0 ꢂC and the reaction mixture was stirred at room temperature. The
progress of the reaction was monitored by TLC. After the comple-
tion of the reaction, addition of H2O (10 mL) was followed by
extraction with EtOAc (3 ꢁ 20 mL). The combined organic layers
were washed with brine and then dried over MgSO4. All volatiles
5.2.4.4. Methyl 12-N-3-(N-cyclopropyl)indolosophoridinate dihydro-
chloride (6d). Green oil; Yield: 87%; 1H NMR (600 MHz,
Chloroform-d)
d
7.78 (d, J ¼ 7.9 Hz, 1H), 7.31 (d, J ¼ 8.2 Hz, 1H), 7.20
(m, 1H), 7.12e7.09 (m, 1H), 7.08 (s, 1H), 3.96e3.87 (m, 2H),
3.79e3.72 (m, 2H), 3.67 (s, 3H), 3.02e2.91 (m, 3H), 2.79 (m, 1H),
2.66 (d, J ¼ 8.9 Hz, 1H), 2.48 (m, 2H), 2.32 (t, J ¼ 6.8 Hz, 2H),
2.14e1.99 (m, 3H), 1.78 (m, 3H), 1.66 (m, 3H), 1.58e1.44 (m, 3H),
1.36e1.30 (m, 1H), 1.29e1.16 (m, 2H), 1.07 (m, 1H), 0.65e0.55 (m,

Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
489
2H), 0.35 (m, 2H). 13C NMR (151 MHz, CDCl3)
d
173.93, 136.86,
2H), 3.68 (s, 3H), 3.07e2.93 (m, 3H), 2.86 (m, 1H), 2.70e2.64 (m,
1H), 2.53 (m, 2H), 2.35e2.28 (m, 2H), 2.16e1.96 (m, 3H), 1.89e1.60
128.23, 126.38, 121.35, 120.32, 118.59, 113.04, 109.20, 63.32, 59.23,
54.78, 51.77, 51.43, 50.52, 50.35, 45.56, 38.54, 34.32, 30.02, 26.94,
26.38, 25.57, 23.40, 22.11, 19.33, 11.36, 4.08, 4.04. HR-MS (ESI) m/z:
calculated for C29H41N3O2 [MþH]þ: 464.3272, found: 464.3277.
(m, 6H), 1.59e1.40 (m, 3H), 1.36e1.25 (m, 2H), 1.13e1.03 (m, 1H). 13
C
NMR (151 MHz, CDCl3)
d 173.95, 136.93, 136.26, 133.33, 128.87 (2),
128.42, 128.11 (2), 126.89, 121.88, 120.48, 119.09, 114.07, 109.40,
63.48, 59.26, 54.50, 51.55, 51.50, 50.39, 49.25, 45.50, 38.21, 34.28,
29.64, 27.16, 25.89, 25.29, 23.35, 22.11, 19.18. HR-MS (ESI) m/z:
calculated for C32H40ClN3O2 [MþH]þ: 534.2882, found: 534.2885.
5.2.4.5. Methyl 12-N-3-(N-benzyl)indolosophoridinate dihydro-
chloride (6e). Yellow oil; Yield: 82%; 1H NMR (600 MHz, Chloro-
form-d)
d 7.78 (m, 1H), 7.33e7.29 (m, 2H), 7.28e7.24 (m, 2H), 7.19
(m, 1H), 7.14e7.09 (m, 3H), 7.01 (s, 1H), 5.30 (s, 2H), 3.79e3.72 (m,
2H), 3.68 (s, 3H), 3.06e2.93 (m, 3H), 2.89 (m, 1H), 2.68 (d, J ¼ 8.9 Hz,
1H), 2.59e2.48 (m, 2H), 2.37e2.26 (m, 2H), 2.10e1.95 (m, 3H),
1.88e1.60 (m, 6H), 1.59e1.41 (m, 3H), 1.38e1.23 (m, 2H), 1.09 (m,
5.2.4.10. Methyl
dihydrochloride (6j). White solid; Yield: 76%; Mp: 76e78 ꢂC; 1H
NMR (600 MHz, Chloroform-d) 7.75 (m, 1H), 7.43e7.39 (m, 2H),
12-N-3-(N-4-bromobenzyl)indolosophoridinate
d
7.23e7.16 (m, 2H), 7.12 (m, 1H), 6.97 (d, J ¼ 8.4 Hz, 3H), 5.23 (s, 2H),
3.79e3.70 (m, 2H), 3.68 (s, 3H), 3.10 (m, 3H), 2.99 (m, 1H), 2.73 (m,
1H), 2.71e2.66 (m, 1H), 2.55 (d, J ¼ 7.2 Hz, 1H), 2.40e2.26 (m, 2H),
2.16e2.01 (m, 4H), 1.93e1.54 (m, 5H), 1.53e1.41 (m, 3H), 1.41e1.21
1H). 13C NMR (151 MHz, CDCl3)
d 174.00, 137.74, 137.07, 128.71 (2),
128.36, 127.52, 127.07, 126.77 (2), 121.71, 120.36, 118.91, 113.78,
109.51, 63.55, 59.10, 54.28, 51.49, 51.43, 50.42, 49.88, 45.37, 38.13,
34.31, 29.56, 27.19, 25.77, 25.22, 23.36, 22.10, 19.16. HR-MS (ESI) m/
z: calculated for C32H41N3O2 [MþH]þ: 500.3272, found: 500.3275.
(m, 2H), 1.18e1.05 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 173.93,
136.95, 136.70, 131.84, 128.45, 128.34, 127.01, 121.95, 121.44, 120.37,
119.15, 113.71, 109.47, 63.25, 59.24, 53.53, 51.52, 51.11, 50.26, 49.32,
45.19, 37.23, 34.16, 28.66, 27.73, 24.58, 24.47, 23.21, 21.97, 18.88. HR-
MS (ESI) m/z: calculated for C32H40BrN3O2 [MþH]þ: 578.2377,
found: 578.2383.
5.2.4.6. Methyl 12-N-3-(N-2-fluorobenzyl)indolosophoridinate dihy-
drochloride (6f). Yellow oil; Yield: 78%; 1H NMR (600 MHz, Chlo-
roform-d)
d
7.71 (d, J ¼ 7.9 Hz, 1H), 7.34 (d, J ¼ 8.3 Hz, 1H), 7.26 (m,
1H), 7.22 (m, 1H), 7.16e7.05 (m, 3H), 7.02 (m, 1H), 6.91 (m, 1H),
5.40e5.30 (m, 2H), 3.78 (s, 2H), 3.68 (s, 3H), 3.49e3.32 (m, 2H), 3.22
(m, 2H), 3.07 (d, J ¼ 11.9 Hz, 1H), 2.80e2.67 (m, 2H), 2.41 (m, 2H),
2.35e2.05 (m, 5H), 1.93 (d, J ¼ 12.6 Hz, 1H), 1.83e1.62 (m, 5H), 1.47
5.2.4.11. Methyl 12-N-3-(N-4-trifluoromethylbenzyl)indolosophor-
idinate dihydrochloride (6k). Orange oil; Yield: 72%; 1H NMR
(600 MHz, Chloroform-d)
d
7.80 (d, J ¼ 7.8 Hz, 1H), 7.56 (d,
(d, J ¼ 12.3 Hz, 3H), 1.22 (s, 1H). 13C NMR (151 MHz, CDCl3)
d 173.86,
J ¼ 8.0 Hz, 2H), 7.24e7.17 (m, 4H), 7.14 (m, 1H), 7.01 (s, 1H), 5.35 (s,
2H), 3.81e3.72 (m, 2H), 3.68 (s, 3H), 3.05e2.91 (m, 3H), 2.83 (m,
1H), 2.67 (d, J ¼ 8.8 Hz, 1H), 2.54e2.46 (m, 2H), 2.32 (m, 2H),
2.15e1.96 (m, 3H), 1.86e1.60 (m, 6H), 1.59e1.43 (m, 3H), 1.34e1.24
160.35 (d, J ¼ 246.6 Hz), 136.95, 129.53, 129.01, 128.07,
124.55,124.41, 124.39, 122.10, 119.97, 119.32, 115.58, 115.44, 109.65,
62.78, 54.91, 53.12, 51.56, 50.36, 49.98, 45.48, 43.72, 35.90, 33.83,
28.61, 26.94, 23.04, 22.82, 22.77, 21.86, 18.40. HR-MS (ESI) m/z:
calculated for C32H40FN3O2 [MþH]þ: 518.3177, found: 518.3172.
(m, 2H), 1.14e1.02 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 173.96,
141.88, 136.93, 129.85 (q, J ¼ 32.3 Hz), 128.46, 126.92, 126.88 (2),
125.71 (2), 125.70, 122.00, 120.58, 119.21, 114.41, 109.31, 63.54,
59.21, 54.61, 51.66, 51.48, 50.39, 49.43, 45.51, 38.36, 34.28, 29.79,
27.06, 26.08, 25.43, 23.38, 22.13, 19.21. HR-MS (ESI) m/z: calculated
for C33H40F3N3O2 [MþH]þ: 568.3145, found: 568.3149.
5.2.4.7. Methyl 12-N-3-(N-3-fluorobenzyl)indolosophoridinate dihy-
drochloride (6g). Yellow oil; Yield: 76%; 1H NMR (600 MHz, Chlo-
roform-d)
d
7.67 (d, J ¼ 7.9 Hz, 1H), 7.25e7.17 (m, 2H), 7.17e7.11 (m,
1H), 7.10e7.05 (m, 1H), 7.01 (s, 1H), 6.89 (m, 2H), 6.70 (m, 1H), 5.24
(s, 2H), 3.78e3.68 (m, 2H), 3.62 (s, 3H), 3.36e3.22 (m, 2H), 3.17 (m,
1H), 3.14e3.06 (m, 1H), 3.04e2.96 (m, 1H), 2.75e2.68 (m, 1H), 2.64
(d, J ¼ 8.7 Hz, 1H), 2.41e2.31 (m, 2H), 2.31e2.02 (m, 5H), 1.85 (m,
5.2.4.12. Methyl 12-N-3-(N-2, 6-dichlorobenzyl)indolosophoridinate
dihydrochloride (6l). Brown oil; Yield: 73%; 1H NMR (600 MHz,
1H), 1.78e1.06 (m, 9H). 13C NMR (151 MHz, CDCl3)
d 173.78, 163.01
Chloroform-d)
d
7.72 (d, J ¼ 7.9 Hz, 1H), 7.46 (d, J ¼ 8.1 Hz, 1H), 7.37
(d, J ¼ 8.0 Hz, 2H), 7.23 (t, J ¼ 7.9 Hz, 2H), 7.11 (t, J ¼ 7.5 Hz, 1H), 6.78
(s, 1H), 5.46 (s, 2H), 3.66 (q, J ¼ 8.4, 7.0 Hz, 5H), 3.06e2.91 (m, 3H),
2.85 (m, 1H), 2.58 (m, 2H), 2.45 (d, J ¼ 8.4 Hz, 1H), 2.25 (t, J ¼ 7.2 Hz,
2H), 2.11e1.90 (m, 3H), 1.86 (m, 1H), 1.80e1.32 (m, 8H), 1.27 (m, 2H),
(d, J ¼ 246.7 Hz), 140.27, 136.97, 130.31, 128.17, 122.28, 122.06,
120.08, 119.27, 114.56, 114.43, 113.68, 113.53, 109.61, 62.73, 60.17,
52.90, 51.50, 50.36, 49.94, 49.35, 45.29, 35.88, 33.87, 28.59, 27.03,
23.17, 22.86, 22.73, 21.81, 18.38. HR-MS (ESI) m/z: calculated for
1.05 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 173.88, 137.19, 136.85,
C
32H40FN3O2 [MþH]þ: 518.3177, found: 518.3179.
132.25 (2), 130.11, 128.75 (2), 128.10, 125.82, 121.68, 120.23, 118.95,
113.52, 109.49, 62.86, 59.37, 54.34, 51.52, 51.47, 50.20, 45.46, 44.92,
37.93, 34.24, 29.35, 27.33, 25.57, 24.99, 23.24, 21.97, 19.10. HR-MS
5.2.4.8. Methyl 12-N-3-(N-4-fluorobenzyl)indolosophoridinate dihy-
drochloride (6h). Yellow oil; Yield: 73%;1H NMR (600 MHz, Chlo-
(ESI) m/z: calculated for
found: 568.2491.
C
32H39Cl2N3O2 [MþH]þ: 568.2492,
roform-d)
d 7.70 (m, 1H), 7.26 (m, 1H), 7.20 (m, 1H), 7.14e7.06 (m,
3H), 7.02e6.95 (m, 3H), 5.26 (s, 2H), 3.76 (s, 2H), 3.68 (s, 3H),
3.46e3.35 (m, 2H), 3.29e3.17 (m, 2H), 3.07 (d, J ¼ 11.6 Hz, 1H),
2.76e2.62 (m, 2H), 2.40 (m, 1H), 2.36e2.26 (m, 2H), 2.25e2.04 (m,
4H), 1.96e1.88 (m, 1H), 1.82e1.59 (m, 5H), 1.53e1.39 (m, 3H),
5.2.4.13. Methyl 12-N-3-(N-2-naphthyl) indolosophoridinate dihy-
drochloride (6m). Yellow oil; Yield: 68%; 1H NMR (600 MHz, Chlo-
1.25e1.15 (m, 1H). 13C NMR (151 MHz, CDCl3)
d
173.92, 162.21 (d,
roform-d) d 7.88e7.70 (m, 4H), 7.58 (s, 1H), 7.50e7.42 (m, 2H), 7.32
J ¼ 246.0 Hz), 136.99, 133.24, 128.50 (2), 128.20, 127.30, 122.03,
120.11, 119.22, 115.65 (2), 112.71,109.66, 62.81, 54.87, 53.15, 51.54,
50.37, 50.02, 49.27, 45.48, 36.05, 33.88, 28.67, 26.97, 23.11, 22.90,
22.83, 21.74, 18.42. HR-MS (ESI) m/z: calculated for C32H40FN3O2
[MþH]þ: 518.3177, Found: 518.3181.
(d, J ¼ 8.2 Hz, 1H), 7.26 (m, 1H), 7.21e7.16 (m, 1H), 7.16e7.11 (m, 1H),
7.05 (s, 1H), 5.45 (s, 2H), 3.78e3.72 (m, 2H), 3.67 (s, 3H), 3.12 (m,
1H), 3.06e2.91 (m, 3H), 2.74e2.64 (m, 2H), 2.61e2.50 (m, 1H),
2.40e2.26 (m, 2H), 2.15e2.02 (m, 3H), 1.95 (m, 1H), 1.84e1.44 (m,
9H), 1.42e1.32 (m, 2H), 1.11 (m, 1H). 13C NMR (151 MHz, CDCl3)
d
174.02,137.18,135.12,133.32,132.82,128.62,128.38,127.76,127.71,
5.2.4.9. Methyl 12-N-3-(N-4-chlorobenzyl)indolosophoridinate dihy-
127.18, 126.35, 126.02, 125.65, 124.90, 121.82, 120.33, 119.01, 113.66,
109.60, 63.39, 58.89, 53.44, 51.49, 51.19, 50.34, 50.11, 45.03, 37.43,
34.21, 28.90, 27.58, 24.82, 24.66, 23.28, 22.01, 18.94. HR-MS (ESI) m/
z: calculated for C36H43N3O2 [MþH]þ: 550.3428, found: 550.3428.
drochloride (6i). Yellow oil; Yield: 74%; 1H NMR (600 MHz, Chlo-
roform-d)
d 7.78 (m, 1H), 7.28e7.23 (m, 2H), 7.23e7.16 (m, 2H), 7.12
(m, 1H), 7.06e7.01 (m, 2H), 6.98 (s, 1H), 5.25 (s, 2H), 3.79e3.71 (m,

490
Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
5.2.4.14. Methyl 12-N-3-(5-fluoro-N-4-chlorobenzyl) indolosophor-
idinate dihydrochloride (6n). Yellow oil; Yield: 76%; 1H NMR
(600 MHz, Chloroform-d) d 7.38 (m, 1H), 7.22e7.19 (m, 2H), 7.04 (m,
24.08, 22.09, 19.26. HR-MS (ESI) m/z: calculated for C24H35N3O
[MþH]þ: 382.2853, found: 382.2858.
1H), 7.00e6.94 (m, 3H), 6.85 (m, 1H), 5.16 (s, 2H), 3.66 (d, J ¼ 2.2 Hz,
2H), 3.62 (s, 3H), 3.02e2.89 (m, 3H), 2.85e2.79 (m, 1H), 2.57 (m,
1H), 2.51 (m, 1H), 2.45e2.39 (m, 1H), 2.31e2.21 (m, 2H), 2.10e1.90
5.2.6. General procedure for compound 10a-10b
LiAlH4 (2.0 equ.) was slowly added to the THF solution of ester
(1.0 equiv) at 0 ꢂC and then the solution was stirred for 2 h at room
temperature. After that, a solution of NaOH (10% in water) was
carefully added until a white solid precipitated. After filtration over
MgSO4 and evaporation of the solvent, the crude alcohol was pu-
rified by chromatography (silica gel, CH2Cl2/MeOH) to give the pure
product.
(m, 3H), 1.86e1.36 (m, 9H), 1.30e1.21 (m, 2H), 1.10e1.00 (m, 1H). 13
C
NMR (151 MHz, CDCl3)
d
173.84, 157.51 (d, J ¼ 234.2 Hz), 135.96,
133.45, 128.89 (2), 128.57, 128.06 (2), 113.81, 113.78, 110.15, 110.09,
105.43, 105.27, 63.09, 59.33, 54.52, 51.73, 51.43, 50.23, 49.50, 45.52,
38.12, 34.18, 29.55, 27.16, 25.83, 25.22, 23.28, 21.95, 19.13. HR-MS
(ESI) m/z: calculated for
found: 552.2791.
C
32H39ClFN3O2 [MþH]þ: 552.2788,
5.2.6.1. 12-N-3-(N-benzyl)indolosophoridinol dihydrochloride (10a).
White solid; Yield: 94%; Mp: 56e58 ꢂC; 1H NMR (600 MHz, Chlo-
5.2.4.15. Methyl 12-N-3-(5-fluoro-N-4-trifluromethylbenzyl) indolo-
sophoridinate dihydrochloride (6o). Yellow oil; Yield: 78%; 1H NMR
roform-d)
d
7.80 (d, J ¼ 7.9 Hz, 1H), 7.33e7.24 (m, 4H), 7.19 (m, 1H),
7.15e7.10 (m, 3H), 7.00 (d, J ¼ 4.5 Hz, 1H), 5.29 (d, J ¼ 2.5 Hz, 2H),
3.78e3.74 (m, 2H), 3.59 (t, J ¼ 6.7 Hz, 2H), 3.00e2.89 (m, 3H), 2.79
(m, 1H), 2.71e2.65 (m, 1H), 2.48 (m, 2H), 2.11e1.99 (m, 3H),
1.86e1.71 (m, 3H), 1.69e1.47 (m, 6H), 1.45e1.27 (m, 3H), 1.27e1.13
(600 MHz, Chloroform-d)
d
7.53 (d, J ¼ 8.1 Hz, 2H), 7.34 (m, 1H), 7.16
(d, J ¼ 8.0 Hz, 2H), 7.10e7.04 (m, 2H), 6.90 (m, 1H), 5.32 (s, 2H),
3.75e3.67 (m, 2H), 3.65 (s, 3H), 3.35e3.26 (m, 2H), 3.21 (m, 1H),
3.11 (m, 1H), 3.00 (m, 1H), 2.69e2.60 (m, 2H), 2.40e2.24 (m, 3H),
2.22e1.62 (m, 9H), 1.47e1.39 (m, 2H), 1.25e1.13 (m, 1H). 13C NMR
(m, 2H), 1.08 (m, 1H). 13C NMR (151 MHz, CDCl3)
d 137.80, 137.06,
128.71 (2), 128.43, 127.52, 127.00, 126.76 (2), 121.69, 120.41, 118.88,
114.06, 109.50, 63.92, 62.45, 59.11, 54.58, 51.58, 50.45, 49.85, 45.47,
38.14, 33.15, 29.89, 27.06, 26.18, 25.48, 24.11, 22.00, 19.25. HR-MS
(ESI) m/z: calculated for C31H41N3O [MþH]þ: 472.3322, found:
472.3324.
(151 MHz, CDCl3)
d
173.82, 157.62 (d, J ¼ 234.9 Hz), 141.38, 133.53,
130.01 (q, J ¼ 32.4 Hz), 128.94, 128.57, 126.88 (2), 125.77 (2), 125.76,
113.05, 110.52, 110.25, 105.18, 62.45, 59.98, 52.88, 51.48, 50.77,
49.91, 49.75, 45.26, 35.96, 33.83, 28.58, 27.24, 23.31, 22.90 (2), 21.60,
18.44. HR-MS (ESI) m/z: calculated for C33H39F4N3O2 [MþH]þ:
586.3051, found: 586.3057.
5.2.6.2. 12-N-3-(N-4-bromobenzyl)indolosophoridinol
chloride (10b). White solid; Yield: 93%; Mp: 72e74 ꢂC; 1H NMR
(600 MHz, Chloroform-d)
dihydro-
5.2.4.16. Methyl 12-N-3-(5-fluoro-N-2, 6-dichlorobenzyl) indoloso-
phoridinate dihydrochloride (6p). Brown oil; Yield: 71%; 1H NMR
(600 MHz, Chloroform-d) d 7.43e7.37 (m, 3H), 7.31 (m, 2H), 7.00 (m,
d
7.80 (d, J ¼ 7.8 Hz, 1H), 7.45e7.40 (m,
2H), 7.23e7.17 (m, 2H), 7.13 (m, 1H), 7.01e6.95 (m, 3H), 5.23 (s, 2H),
3.76 (q, J ¼ 13.4 Hz, 2H), 3.60 (m, 2H), 3.02e2.87 (m, 3H), 2.79 (m,
1H), 2.68 (m, 1H), 2.48 (m, 2H), 2.14e1.98 (m, 3H), 1.87e1.72 (m,
3H), 1.70e1.48 (m, 6H), 1.48e1.27 (m, 3H), 1.27e1.15 (m, 2H), 1.09
1H), 6.85 (s, 1H), 5.49 (d, J ¼ 3.0 Hz, 2H), 3.69 (s, 5H), 3.40e3.31 (m,
2H), 3.25 (m, 1H), 3.17e3.04 (m, 2H), 2.64 (m, 2H), 2.50e2.43 (m,
1H), 2.40e2.12 (m, 6H), 2.12e1.99 (m, 2H), 1.94e0.79 (m, 8H). 13
C
(m, 1H). 13C NMR (151 MHz, CDCl3)
d 136.92, 136.84, 131.84 (2),
NMR (151 MHz, CDCl3)
d
173.72, 157.53 (d, J ¼ 235.0 Hz), 136.80 (2),
128.48, 128.42 (2), 126.78, 121.87, 121.40, 120.52, 119.08, 114.42,
109.38, 63.92, 62.45, 59.06, 54.57, 51.62, 50.42, 49.30, 45.46, 38.09,
33.16, 29.90, 27.05, 26.18, 25.49, 24.12, 21.99, 19.25. HR-MS (ESI) m/
z: calculated for C31H40BrN3O [MþH]þ: 550.2428, found: 550.2424.
133.86, 131.88, 130.31, 128.86 (2), 128.18, 127.84, 112.47, 110.28,
110.23, 104.89, 62.17, 60.50, 53.12, 51.56, 50.51, 49.84, 45.52, 45.23,
35.86, 33.88, 29.70, 27.01, 23.06, 22.88, 22.70, 21.60, 18.37. HR-MS
(ESI) m/z: calculated for
found: 586.2391.
C
32H38Cl2FN3O2 [MþH]þ: 586.2398,
5.3. In vitro cytotoxicity assay
5.2.5. General procedure for compound 9
The HepG2, CNE-2 and A549 cell lines were obtained from
4a (2 mmol) in CH2Cl2 (10 mL) was added to (Boc)2O (2.2 mmol)
and TEA (1 mL) and the mixture was stirred at room temperature.
After 5 h, the organic solvent was concentrated in vacuo to produce
7. Then LiAlH4 (4 mmol) was slowly added to the THF solution of
7 at 0 ꢂC and the solution was stirred for 2 h at room temperature. A
solution of NaOH (10% in water) was then carefully added to the
reaction mixture until a white solid precipitated. After filtration
over MgSO4 and evaporation of the solvent, compound 8 in crude
form was obtained. To a solution of 8 in CH2Cl2 (15 mL), NaH-
SO4$SiO2 (600 mg) was added and the mixture was refluxed. After
completion of the reaction, the mixture was cooled and filtered. The
concentrated filtrate was chromatographed over silica gel using a
mixture of CH2Cl2 and MeOH (5:1e3:1) to get 9.
American Type Culture Collection (ATCC). The suspension (100 mL/
well) with evaluated cells (3e4 ꢁ 104 cell/mL) and DMEM culture
medium of 10% fetal bovine serum (FBS) was seeded into 96-well
plates. After a 24 h incubation period in 5% CO2, compounds with
different concentrations (1, 5,10, 20, 50 mM), made by serial dilution
in culture medium (DMEM of 10% fetal bovine serum) of stock
solutions of test compounds prepared in DMSO, were added and in
incubation for 48 h. Final concentration of DMSO was less than 0.1%
in each well. Then 20 mL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, St. Louis, MO, USA)
was added to each well, followed by incubation for 4 h at 37 ꢂC.
Supernatant from each well was carefully removed and 150 mL
DMSO was added to each well for the colorimetric reaction. Finally,
the optical density was measured at the 490 nm wavelength on an
enzyme-linked immunosorbent assay microplate reader and each
concentration was tested in threefold.
5.2.5.1. 12-N-3-indolosophoridinol dihydrochloride (7). White solid;
Yield: 81%; Mp: 77e79 ꢂC; 1H NMR (600 MHz, Chloroform-d)
d 8.24
(s, 1H), 7.78 (m, 1H), 7.37 (m, 1H), 7.21 (m, 1H), 7.13 (m, 1H), 7.07 (d,
J ¼ 2.3 Hz, 1H), 3.77e3.72 (m, 2H), 3.61 (m, 2H), 3.02e2.89 (m, 3H),
2.79 (m, 1H), 2.68 (m, 1H), 2.52e2.43 (m, 2H), 2.11e2.01 (m, 3H),
1.85e1.72 (m, 3H), 1.68e1.48 (m, 6H), 1.44e1.26 (m, 3H), 1.21 (m,
d 136.66, 127.74,
122.82, 121.87, 120.19, 119.08, 114.90, 110.94, 63.99, 62.69, 59.14,
54.65, 51.52, 50.53, 45.51, 38.27, 33.10, 29.91, 26.99, 26.27, 25.53,
5.4. Topo I inhibitory activity
Topo I enzymatic activity was determined by measuring the
decreased mobility of the relaxed isomers of supercoiled pBR322
DNA. Topo I and pBR322 were purchased from Takara Bio Inc. One
unit of enzyme was defined as the amount that completely relaxes
2H), 1.07 (m, 1H). 13C NMR (151 MHz, CDCl3)

Y. Xu et al. / European Journal of Medicinal Chemistry 156 (2018) 479e492
491
0.5
performed in a final volume of 20 mL reaction volume containing
0.5 g pBR322 DNA and 1 unit of human Topo I with or without our
m
g of supercoiled pBR322 DNA in 30 min at 37 ꢂC. The assay was
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