Scaffold Diversity Inspired by the Natural Product Evodiamine: Discovery of Highly Potent and Multitargeting Antitumor Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b00910

A critical question in natural product-based drug discovery is how to translate the product into drug-like molecules with optimal pharmacological properties. The generation of natural product-inspired scaffold diversity is an effective but challenging strategy to investigate the broader chemical space and identify promising drug leads. Extending our efforts to the natural product evodiamine, a diverse library containing 11 evodiamine-inspired novel scaffolds and their derivatives were designed and synthesized. Most of them showed good to excellent antitumor activity against various human cancer cell lines. In particular, 3-chloro-10-hydroxyl thio-evodiamine (66c) showed excellent in vitro and in vivo antitumor efficacy with good tolerability and low toxicity. Antitumor mechanism and target profiling studies indicate that compound 66c is the first-in-class triple topoisomerase I/topoisomerase II/tubulin inhibitor. Overall, this study provided an effective strategy for natural product-based drug discovery. (Figure Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00910

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Scaffold Diversity Inspired by Natural Product Evodiamine:
Discovery of Highly Potent and Multi-targeting Antitumor Agents
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a,b,
†
a,
†
a,
†
a
a
Shengzheng Wang , Kun Fang , Guoqiang Dong , Shuqiang Chen , Na Liu ,
a
a
c
a
a,
*
Zhenyuan Miao , Jianzhong Yao , Jian Li , Wannian Zhang and Chunquan Sheng
a
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
School of Pharmacy, Fourth Military Medical University, 169 Changle West Road,
Xi'an, 710032, P.R. China
b
c
School of Pharmacy, East China University of Science & Technology, 130 Meilong
Road, Shanghai 200237, P.R. China
ꢀ AUTHOR INFORMATION
Corresponding Author
*
Phone: 86ꢀ21ꢀ81871239. Eꢀmail: shengcq@hotmail.com
Author Contributions
†
S.W., K.F. and G.D. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ABSTRACT
A critical question in natural productsꢀbased drug discovery is how to translate them
into drugꢀlike molecules with optimal pharmacological properties. The generation of
natural productsꢀinspired scaffold diversity is an effective but challenging strategy to
investigate broader chemical space and identify promising drug leads. Extending our
efforts on natural product evodiamine, a diverse library containing eleven
evodiamineꢀinspired novel scaffolds and their derivatives were designed and
synthesized. Most of them showed good to excellent antitumor activity against
various human cancer cell lines. In particular, 3ꢀchloroꢀ10ꢀhydroxyl thioꢀevodiamine
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(
66c) showed excellent in vitro and in vivo antitumor efficacy with good tolerability
and low toxicity. Antitumor mechanism and target profiling study indicate compound
6c is the firstꢀinꢀclass triple topoisomerase I/topoisomerase II/tubulin inhibitor.
6
Overall, this study provided an effective strategy for natural productꢀbased drug
discovery.
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INTRODUCTION
Natural products (NPs) have been an important source of bioactive molecules for the
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development of therapeutic drugs. NPs have evolved to target multiple proteins
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and often possess diverse biological activities, which led to a combination of
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therapeutic effects and toxicity. The scaffolds of NPs can be regarded as ‘‘bioactive’’
or “privileged” scaffolds in chemical space because they have been naturally selected
in evolution to specifically interact with a diversity of biological targets. However, the
probability of developing an isolated NP directly into a therapeutic drug is relatively
low. In most cases, NPs are used as biologically preꢀvalidated starting points for drug
discovery and development. Thus, a critical question to be answered is how to
translate NPs into drugꢀlike molecules with optimized pharmacological properties.
The most commonlyꢀused method is the generation of large numbers of analogues by
semiꢀsynthetic or total synthetic approaches. However, modifying NPs by simple
derivatization or functional group transformations only achieved limited chemical
diversity. Chemical space and structureꢀactivity relationships (SARs) around NPs
cannot be fully investigated. Scaffold diversity inspired by NPs allows for increased
probability and quality to identify promising drug leads. To achieve this goal, several
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new strategies have been introduced, such as biologyꢀoriented synthesis (BIOS)
,
.
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,
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diversityꢀoriented synthesis (DOS) , and diverted total synthesis (DTS)
Applications of these innovative methods indicated that the creation of NPꢀinspired
scaffold diversity was proven to be a powerful tool to investigate new chemical space
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and identify novel lead compounds for drug discovery.
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Evodiamine (1, Figure 1) is a quinazolinocarboline alkaloid isolated from the fruit
of Evodia rutaecarpa Bentham. Evodiamine has diverse biological activities
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including antiꢀinflammatory,
antiꢀobesity,
and antitumor effects. Among
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them, the antitumor activity of evodiamine has been extensively investigated.
Evodiamine showed cytotoxicity against a variety of human cancer cellꢀlines by
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inducing apoptosis.
Moreover, it was a naturally multiꢀtargeting antitumor
molecule, which exerted the antitumor activity by various molecular mechanism such
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as caspaseꢀdependent
and ꢀindependent pathways, sphingomyelin pathway,
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calcium/JNK signaling, PI3K/Akt/caspase and FasꢀL/NFꢀκB signaling pathways .
Due to its broadꢀspectrum and multiꢀtargeting antitumor profile, evodiamine
represents a good starting point for the development of novel antitumor agents.
Figure 1. Chemical structures of evodiamine and its highly potent derivatives
Previously, we identified topoisomerase I (Top1) as one of the molecular targets of
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evodiamine by structureꢀbased virtual screening. Subsequently, a number of
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substituted evodiamine derivatives were designed and synthesized.
Interestingly,
several analogues, such as 3ꢀfluoroevodiamine (2), 10ꢀhydroxylevodiamine (3),
3ꢀfluoroꢀ10ꢀhydroxylevodiamine (4), and 3ꢀaminoꢀ10ꢀhydroxylevodiamine (5),
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showed excellent antitumor activity at low nanomlar range. Moreover, these
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evodiamine derivatives could significantly inhibit the tumor growth in the A549 and
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HCT116 tumor xenograft models. Interestingly, antitumor mechanism studies
revealed that the evodiamine derivatives acted by dual inhibition of Top1 (poisons)
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37ꢀ39
and Top2 (catalytic inhibitors). The above work in combination with other reports
preliminary clarified the SAR of substitutions on evodiamine scaffold. In order to
achieve a deep understanding of chemical space around evodiamine, it is highly
desirable to generate evodiamineꢀinspired scaffold diversity and investigate their
biological activities.
CHEMISTRY
Chemical synthesis of evodiamineꢀinspired diverse scaffolds and their derivatives
are depicted in Schemes 1-9. First, the Eꢀring phenyl group of evodiamine was
replaced by various heterocycles including thiophene (12a-c, 15, 26a-b), pyrrole
(
29a-c) and furan (32). Among them, the synthesis of compounds 12a-c and 15 was
3
3, 34
based on the coupling method similar to our previous report (Scheme 1).
Substituted 3,4ꢀdihydroꢀβꢀcarboline (intermediates 8a-b) was prepared by reacting
substituted tryptamine 6a-b with ethyl formate and followed by intramolecular ring
3
3
closure in the presence of POCl
intermediate 9 (or 13) with triphosgene and followed by methylation in the presence
of CH I. Coupling of carbolines 8a-b with intermediate 11 (or 14) afforded
3
. Intermediate 11 (or 14) was prepared by reacting
3
compounds 12a-b (or 15). Hydroxyl substituted derivative 12c was obtained by
demethylation of compound 12a in the presence of BBr and DCM.
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Scheme 1. Reagents and Conditions: (a) Ethyl formate, reflux, 6 h, yield 99%; (b) POCl , DCM, 0
3
°C, 6 h, yield 73ꢀ75%; (c) Triphosgene, THF, reflux, 4 h; (d) CH I, KOH, DMF, 2 h, two steps
yield 52%; (e) CH Cl , rt, 3 h, yield 78ꢀ79%; (f) BBr , DCM, ꢀ78 °C, 2.5 h, yield 49%.
2
2
3
Starting from methyl 2ꢀmercaptoacetate (16) and methyl acrylate (17), intermediate
4
0, 41
2
4 was prepared via seven steps according to the reported methods.
compound 26a was prepared by reacting intermediate 24 and 8c in the presence of
POCl under the microwave condition and followed by reduction in the presence of
NaBH
Scheme 3).
Then,
3
33
4
(Scheme 2). Using a similar protocol, scaffolds 29 and 32 were prepared
(
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Scheme 2
HS
COOMe
COOMe
a
b
c
16
S
COOMe
COOMe
S
O
COOMe
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19
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7
Boc
NH
Boc
NH2
N
COOMe
e
f
d
S
S
S
S
O
O
O
O
N
OH
O
O
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23
H
N
O
h
g
O
N
i
S
O
NH
O
N
N
S
O
O
H
2
4
N
25
H
8c
O
O
j
O
N
HO
N
N
N
S
N
N
S
H
H
2
6a
26b
Scheme 2. Reagents and Conditions: (a) Piperidine, 50 °C, 2 h, yield 99%; (b) NaH, THF, reflux,
5
h, yield 35%; (c) NH OH, HCl, BaCO , MeOH, reflux, Overnight, yield 98%; (d) HCl,
2
3
etherꢀMeOH, rt, 24 h, yield 91%; (e) (Boc)
2
O, DMAP, DCM, Overnight, yield 40%; (f) MeI, NaH,
DMF, 6 h, yield 84%; (g) CF
3
COOH, CH
2
Cl
2
, rt, 2.5 h, yield 98%; (h) POCl
3
, toluene,
microwave, 130 °C,45 min; (i) NaBH
4
, EtOH, rt, 1 h, two steps yield 27%; (j) BBr , DCM, ꢀ78
3
°C, 2.5 h, yield 43%.
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Scheme 3. Reagents and Conditions: (a) (Boc)
MeI, NaH, DMF, 6 h, yield 84ꢀ89%; (c) CF COOH, CH
toluene, microwave, 130 ºC, 45 min; (e) NaBH , EtOH, rt, 1h, yield 36ꢀ56% over two steps; (f)
BBr , CH Cl , ꢀ78 ºC, 2.5 h, yield 56%.
2
O, DMAP, CH
2 2
Cl , overnight, yield 40ꢀ45%; (b)
3
2
2
Cl , rt, 2.5 h, yield 94ꢀ97%; (d) POCl ,
3
4
3
2
2
The synthetic method of compound 36b was according to the procedure reported by
4
2
You’s group (Scheme 4). First, 5ꢀmethoxylꢀtryptamine (33) and phthalic anhydride
(34) were refluxed in toluene to afford intermediate 35. In the presence of TfOH,
intramolecular cyclization of intermediate 35 afforded compound 36a. Treatment of
compound 35 with Hantzsch ester 37 and chiral phosphoric acid 38 in dioxane
afforded compound 36b. The absolute configuration of compound 36b was in
42
accordance with the literature data .
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Scheme 4. Reagents and Conditions: (a) Toluene, reflux, 5 h; (b) TfOH, DCM, 0 ºC, 30 min, yield
49% over two steps; (c) Hantzsch ester 37, chiral phosphoric acid 38, dioxane, rt, 3 h, yield 87%.
Structural modification for Cꢀring piperdine analogue of evodiamine was focused on
ring expansion to sevenꢀmembered ring and in combination with replacing ABꢀring
indole with benzofuran (compounds 46, 47, 54a-b). Starting from azepanꢀ2ꢀone (39),
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intermediate 43 was prepared via three steps according to the reported method. In
the presence of POCl , intermediates 43 and 44 were reacted under the microwave
condition to afford quaternary ammonium salt 45. After reduction by NaBH
3
4
,
compound 46 was obtained (Scheme 5). The synthesis of compound 54a (Scheme 6)
was similar to the synthetic method of compound 46. Hydroxyl substituted derivatives
(47, 54b) were obtained by demethylation of the corresponding methoxyl derivatives
in the presence of BBr and DCM.
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Scheme 5
HCl
NHNH2
MeO
H
H
H
N
O
N
O
N
O
MeO
a
b
42
NH
N
c
Br
Br
N
H
43
O
3
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NH
O
OMe
O
O
MeO
MeO
N
N
e
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N
N
N
H
N
H
d
4
5
46
O
HO
O
N
MeO
f
N
N
N
N
H
H
N
4
7
46
Scheme 5. Reagents and Conditions: (a) PCl , ZnI , Br , N , CHCl , 0 ºC, 6 h, yield 70%; (b)
5
2
2
2
3
Piperidine, N
2
, reflux, 4.5 h, yield 91%; (c) EtOH, H
SO
2 4
, reflux, 5 h, yield 53%; (d) POCl
3
,
,
toluene, microwave, 130 ºC, 45 min; (e) NaBH
4 3
, EtOH, rt, 1 h, yield 38% over two steps; (f) BBr
CH Cl , ꢀ78 ºC, 2.5 h, yield 46%.
2
2
Scheme 6. Reagents and Conditions: (a) Acetone, 6 h, 20 °C, yield 47%; (b) AcOH, reflux, 4 h,
yield 47%; (c) K CO , MeI, DMF, 4 h, yield 99%; (d) POCl , Toluene, microwave, 130 °C, 45
2
3
3
min; (e) NaBH
4
3 2 2
, EtOH, rt, 1 h, yield 65% over two steps; (f) BBr , CH Cl , ꢀ78 °C, 2.5 h, yield
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6%.
The synthetic method for oxoꢀevodiamine derivatives is outlined in Scheme 7 and
Scheme 8. Firstly, substituted 2ꢀhydroxyꢀbenzoyl chloride (56) was prepared by
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reacting intermediate 55 with SOCl in DCM. Then, intermediate 56 and substituted
2
3
,4ꢀdihydroꢀβꢀcarboline (8) were reacted at room temperature to afford derivatives 57.
Reduction of the nitro group of compounds 57l and 57ac using 10% Pd/C and H
2
afforded the amino derivatives 57an and 57ao. Compounds 57ap-57au were
synthesized by acylation or reductive amination of compounds 57an and 57ao. In the
presence of BBr3 and DCM, hydroxyl substituted derivatives (58a-58p) were
prepared by demethylation of the corresponding methoxyl derivatives. Treatment of
compounds 57 with Lawesson’s reagent afforded thiocarboyl derivatives 59a-59c.
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Scheme 7
N
O
R1
O
1
O
9
N
N
H
8
10
R1
4
OH
OH
a
Cl
OH
R2
R2
3
R2
b
N
H
O
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R1
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R1
R2
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7a
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
57q 10-Cl
57r 10-OMe
H
H
R1
R2
R1
R2
1-OMe
2-Cl
2-Me
2-CF3
2-OCH3
2-F
3-OCH3
3-F
3-Cl
3-Br
3-NO2
1,3-2Cl
1,3-2I
4-F
1
1
7c
7d
7e
57s 10-OMe 1-OCH3
57ah
H
57t
10-OMe
2-Cl
2-Me
2-CF3
57ak 10-OMe
57al 10-OMe
57am10-OMe
57u 10-OMe
57v 10-OMe
57w 10-OMe
57x 10-OMe
57y 10-OMe
57z 10-OMe
57aa 10-OMe
57ab 10-OMe
57ac 10-OMe
2
2
2
2
57f
5
5
5
7g
7h
7i
2-OCH3 57ai
H
H
2-F
3
3
3-OCH3
3-F
3-Cl
3-Br
3-NO2
3
3
57j
57aj
5
5
5
5
7k
7l
7m
7n
4
4
57ad 10-OMe 1,3-2Cl
57o
7p
57ae 10-OMe
57af 10-OMe
7ag 10-OMe
1,3-2I
4-F
4-CH3
5
4-CH3
5
O
O
R
N
R
N
O
c
d
NO2
NH2
N
O
N
H
H
R = H, OMe
57an: R = H
7ao: R = OMe
5
57l, 57ac
R1
R2
5
5
5
7ap
7aq
7ar
H
H
H
H
H
H
3-NHCOCH(CH2)2
3-NHCOCH3
3-NHCH CH3
O
R1
N
2
R2
57as
3-NHCH CH CH
2
2
3
N
H
O
5
5
7at
7au
3-NHCH CH(CH )
2
2 2
3-N(CH CH CH )
2
2
3 2
Scheme 7. Reagents and Conditions: (a) SOCl , DCM, reflux, 6 h, yield 98%; (b) Intermediate 8,
2
DCM, rt, 6 h, yield 33%~56%; (c) H
2
, Pd/C, EtOAc, yield 97%; (d) 57apꢀ57aq, acyl chloride,
CN, MeOH, yield 59%~82%.
3
Et N, DCM, yield 65%~73%; 57ar-57au, aldehyde, NaBH
3
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9
Scheme 8
O
O
1
R1
N
R3
N
4
a
4
3
R2
3
R2
O
N
O
N
H
H
2
2
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
R and R contain one or two methoxyl groups)
1 2
58a-p
57
R3
R2
R3
H
H
H
R2
R3
8i OH
8j OH
58k OH
58l OH
R2
58a
55b
1-OH
2-OH
3-OH
H
1-OH
2-Cl
2
5
5
3-OH
3-NH2
3-Cl
3-Br
1,3-2Cl
58o
58p
OH
OH
5
5
5
5
5
5
8c
3
3
8d OH
8e OH
OH
8g OH
8h OH
5
5
8m OH
8n OH
8f
4-CH3
2-CH3
2-CF3
4
O
N
S
b
N
4
2
59a R = H
59b R = 2-CH3
59c R = 4-CH3
3
R
N
O
N
O
R
H
H
1
57
59a-c
Scheme 8. Reagents and Conditions: (a) BBr , DCM, ꢀ78 °C～0 °C, 6 h, yield 32%~68%; (b)
3
Lawesson’s reagent, PhMe, reflux, 6 h. yield 73%~80%.
The chemical synthesis of thioꢀevodiamine derivatives (63a-66f) was outlined in
Scheme 9. Starting from substituted 2ꢀaminoꢀbenzoic acids (60), various
4
4
2
ꢀmercaptoꢀbenzoic acids (61) were prepared according to reported protocols. Then,
intermediate 61 and SOCl were refluxed in DCM for 1 h to obtain various
2
2
ꢀmercaptoꢀbenzoyl chloride (62). Derivatives 63a-63h were obtained by reacting
intermediate 62 and substituted 3,4ꢀdihydroꢀβꢀcarboline (8) at room temperature. In
the presence of H and Na WO , the sulfur atom of compounds 63a and 63d was
2
O
2
2
4
oxidized to the sulfone (63i and 63j). Under the microwave condition, intermediates
64 (commercial available) and 8b were irradiated at 120 °C for 30 min to obtain
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound 65. Hydroxyl substituted derivatives (66a-66f) were obtained by
demethylation of the corresponding methoxyl derivatives in the presence of BBr
3
and
DCM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 9
R1
N
O
O
O
N
a
OH
NH2
OH
SH
b
Cl
SH
62
H
8
R2
R2
R2
c
61
60
R1
H
H
R2
H
3-F
3-Cl
H
3-F
3-Cl
3-OMe
3-Br
6
6
3a
3b
O
R1
N
63c
3d
63e
3f
63g
3h
H
6
OMe
OMe
OMe
OMe
OMe
N
S
R2
H
6
63a-h
6
O
O
R
N
d
R
N
N
H
S
N
S
O
H
O
63a, 63d
63i: R = H
63j: R = OMe
O
O
N
N
e
OH
+
N
N
S
N
SH
H
H
N
8b
64
65
X
S
S
R
H
3-F
O
O
66a
66b
MeO
N
N
HO
f
6
6c
66d
6e
66f
S
S
S
SO2
3-Cl
N
H
X
X
N
H
3-OMe
3-OH
H
R2
R
6
66a-66f
X = S, SO2
2 2 2
Scheme 9. (a) 1.1. NaNO , HCl, H O; 1.2. potassium ethylxanthate, H O, 80 °C/HCl (aq); 1.3.
NaOH (aq), Na S O /HCl; (b) SOCl , DCM, reflux, 1 h; (c) DCM, rt, 6 h, yield 36%~55% over
2
2
4
2
three steps; (d) H
O
2 2
, Na
2
WO
4
, AcOH, 55°C, 3 h, yield 63%~78%; (e) microwave, 120°C, 30 min,
yield 30%; (f) BBr
3
, DCM, ꢀ78°C～0°C, 6 h, yield 40%~70%.
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9
RESULTS AND DISCUSSION
Evodiamine-inspired New Scaffolds Design. A series of evodiamineꢀinspired new
scaffolds were designed accordingly to chemical diversity and synthetic possibility
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
Figure 2). First, the Eꢀring phenyl of evodiamine was replaced by various
heterocycles including thiophene, pyrrole and furan. Second, the Dꢀring
2
1
,3ꢀdihydropyrimidinꢀ4(1H)ꢀone scaffold was converted into fiveꢀmembered
Hꢀpyrrolꢀ2(5H)ꢀone ring. Moreover, replacing Dꢀring Nꢀmethyl with oxygen or
sulfur afforded oxoꢀevodiamine and thioꢀevodiamine. Third, the Cꢀring piperdine was
enlarged to sevenꢀmembered ring and in combination with replacing ABꢀring indole
with benzofuran. As a result, eleven totally new scaffolds were designed and
synthesized. Considering that 10ꢀhydroxyl group was important for improving the
3
4
antitumor activity of evodiamine, 10ꢀhydroxyl or 10ꢀmethoxyl group was introduced
on the new scaffolds to investigate the difference of SARs between various scaffolds.
Figure 2. Evodiamine inspired scaffold diversity.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In vitro Antitumor Activity of Evodiamine-inspired New Scaffolds. The
evodiamineꢀinspired new scaffolds were assayed for growth inhibitory activities
toward human cancer cellꢀlines A549 (lung cancer), MDAꢀMBꢀ435 (breast cancer)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
33
and HCT116 (colon cancer) using the MTT assay. Evodiamine and camptithecin
were used as reference drugs. As shown in Table 1, several new scaffolds (e.g. 26b,
5
4a, 57a, 64a, 66a) showed good to excellent antitumor activity with a broad
spectrum. The Eꢀring thiophene scaffold 12b only showed weak antitumor activity.
Introduction of a methoxyl group on Aꢀring (compound 12a) led to the loss of
antitumor activity. After demethylation of 12a, the hydroxyl derivative 12c showed
improved antitumor activity towards the A549 (IC50 = 15.8 ꢀM) and HCT116 (IC50
=
34
23.6 ꢀM) cell lines. These SARs were similar to that of evodiamine. Furthermore,
movement of the sulfur atom in the thiophene ring (15, 26a and 26b) resulted in the
increase of the antitumor activity. Compound 26b showed good activity against A549
(IC50 = 3.9 ꢀM), HCT116 (IC50 = 4.8 ꢀM) and MDAꢀMBꢀ435 (IC50 = 32.3 ꢀM) cell
lines. The pyrrole scaffold (29a-c) also showed broadꢀspectrum antitumor activity
with a similar SAR as to evodiamine, but they were less active than the thiophene
scaffold 26b. In contrast, the furan scaffold 32 was only weakly active. Similarly, the
Dꢀring modified scaffolds 36a and 36b also showed poor antitumor activity. When
the Cꢀring of evodiamine was enlarged to sevenꢀmembered ring, the methoxyl
derivative 46 was totally inactive and the correspondingly hydroxyl derivative 47
showed moderate antitumor activity (IC50 range: 32.4 ꢀM ~ 69.2 ꢀM). Interestingly,
further changing the indole substructure of 46 to benzofuran (compound 54a) led to
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significant increase of the antitumor activity (IC50 range: 5.3 ꢀM to 28.4 ꢀM).
However, demethylation of compound 54a yielded an inactive hydroxyl derivative
5
4b, whose SAR was different from that of other scaffolds. The results indicated that
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
antitumor mechanism might be changed after scaffold morphing. Among the
evodiamineꢀinspired scaffolds, oxoꢀ and thioꢀevodiamine showed the best antitumor
activity. The unsubstituted oxoꢀevodiamine (IC50 range: 1.4 ꢀM to 4.0 ꢀM) and
thioꢀevodiamine (IC50 range: 0.23 ꢀM ~ 0.52 ꢀM) were far more potent than the
original evodiamine scaffold. Introduction of 10ꢀmethoxyl group on them all led to
decrease of the antitumor activity. For the corresponding hydroxyl derivatives,
1
0ꢀhydroxylꢀoxoꢀevodiamine (58d) showed similar activity to its original template. In
contrast, 10ꢀhydroxylꢀthioꢀevodiamine (66a) was highly active with IC50 values
against A549, HCT116, and MDAꢀMBꢀ435 cell lines of 0.05 ꢀM, 0.04 ꢀM and <
0.003 ꢀM, respectively, which was comparable or superior to camptothecin (CPT).
Due to excellent antitumor activity of oxoꢀ and thioꢀevodiamine scaffolds, they were
subjected for further SAR investigations.
Table 1. In vitro antitumor activity of evodiamineꢀinspired scaffolds (IC50, µM)
Compounds
2a
12b
2c
A549
>200
HCT116
>200
MDA-MB-435
>200
1
110.7 ± 13.2
15.8 ± 1.1
26.9 ± 3.1
32.3 ± 2.7
3.9 ± 0.20
>200
28.5 ± 3.0
5.0 ± 0.11
>200
192.5 ± 15.7
23.6 ± 2.0
14.1 ± 1.6
94.9 ± 8.9
4.8 ± 0.62
76.5 ± 6.2
54.1 ± 5.1
15.4 ± 2.0
177.8 ± 1.8
>200
>200
>200
1
15
36.2 ± 2.5
49.0 ± 4.3
32.3 ± 2.9
206.7 ± 19.8
42.0 ± 5.3
11.7 ± 0.91
181.6 ± 19.4
>200
26a
26b
2
2
9a
9b
2
9c
32
6a
3
>200
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
4
4
54a
5
5
5
58d
6b
6
7
193.1 ± 18.2
>200
32.4 ± 3.3
6.2 ± 0.41
>200
30.9 ± 3.5
>200
59.8 ± 6.4
5.3 ± 0.55
>200
>200
>200
69.2 ± 5.9
28.4 ± 3.1
>200
3.0 ± 0.23
>200
1.4 ± 0.20
0.23 ± 0.03
17.8 ± 1.5
<0.003
4b
7a
7r
2.4 ± 0.22
>200
3.1 ± 0.28
>200
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4.0 ± 0.31
0.37 ± 0.01
50.1 ± 5.2
0.05 ± 0.004
>200
2.3 ± 0.19
0.52 ± 0.04
0.5 ± 0.03
0.04 ± 0.05
>200
6
6
6
4a
4d
6a
Evodiamine
CPT
22.1 ± 2.3
1.00 ± 0.06
0.10 ± 0.001
0.09 ± 0.005
SAR of the Oxo-evodiamine Derivatives. Based on the scaffold of
oxoꢀevodiamine, a number of derivatives were designed and synthesized. First,
various substitutions were introduced on the Eꢀring (57b-p). The 1ꢀmethoxyl
derivative (57b) showed better activity against the MDAꢀMBꢀ435 cell line (IC50
=
2
.21 ꢀM) than the original scaffold 57a (IC = 3.0 ꢀM), but it was less active for the
5
0
other two cancer cell lines. Moving the methoxyl group to position 2 (57f) and 3 (57h)
all led to decreased activity. All the 2ꢀsubstituted (57c-g) and 4ꢀ substituted (57o-p)
derivatives only showed moderate antitumor activity. In contrast, the 3ꢀchloro analogy
5
7j showed good activity particularly for the A549 cell line (IC50 = 0.05 ꢀM). The
3
ꢀfluoro (57i) and 3ꢀbromo (57k) and 1,3ꢀdisubstituted (57m-n) derivatives were less
potent, whereas the 3ꢀnitro derivative 57l was totally inactive. When the nitro group
of 57l was reduced to the amine, compound 57an showed good antitumor activity
(
IC50 range: 2.7 ꢀM ~ 9.1 ꢀM). However, further substitution or acylation of 57an led
to the decrease of the activity (57ap-au). On the other hand, annulation the Eꢀring
with phenyl at different positions (57ah-am) was also unfavorable for the activity.
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Second, the carbonyl group of oxoꢀevodiamine (57a) was converted into
thiocarbonyl group (59a-c). However, these thiocarbonyl derivatives generally
showed decreased antitumor activity. Third, a series of 10ꢀhydroxyl (58a-p) as well as
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
0ꢀmethoxyl (57s-57ag) oxoꢀevodiamine derivatives were synthesized. In general, the
methoxyl derivatives only showed moderate antitumor activity with the exception of
compound 57aa. The 3ꢀchloroꢀ10ꢀmethoxyl analogy 57aa was highly active against
A549 cell line (IC50 = 0.05 ꢀM), which was much better than all the reference drugs
including CPT, topotecan (TPT) and irinotecan (IRT). Notably, the 3ꢀchloro derivative
5
7j also showed excellent antitumor activity, indicating that the 3ꢀchloro substitution
was important for the activity.
Interestingly, significant improvement of the antitumor activity was observed for
the hydroxyl derivatives. Moreover, the hydroxyl group was more favorable at Aꢀring
than Eꢀring. For example, 1ꢀhydroxyl (58a), 2ꢀhydroxyl (58b) and 3ꢀhydroxyl (58c)
derivatives were moderately active. In contrast, most of the 10ꢀhydroxyl derivatives
showed significantly better activity. Particularly, compounds 58g, 58k, 58l and 58n
were the most active oxoꢀevodiamine derivatives. The IC50 value of compounds 58g,
58k, 58l and 58n against MDAꢀMBꢀ435 cell line was lower than 3 nM, which was
comparable to CPT and superior to TPT and IRT. For the HCT116 cell line,
compounds 58g, 58l and 58n were far more potent than the reference drugs with an
IC50 value of lower than 3 nM. SAR analysis revealed that the 1,10ꢀdihydroxyl
substitution (58e) was unfavorable and 3,10ꢀdihydroxyl substitution (58i) resulted in
slight increase of the activity. On the basis of 10ꢀhydroxyl oxoꢀevodiamine (58d),
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
further introduction of 2ꢀmethyl (58g), 3ꢀamino (58j), 3ꢀchloro (58k), 3ꢀbromo (58l)
and 4ꢀmethyl (58n) led to substantial improvement of the antitumor activity, whereas
further annulations of Eꢀring phenyl group (58o-p) had little effect.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. In vitro antitumor activity of the oxoꢀevodiamine derivatives (IC , µM)
50
Compounds
7b
7c
7d
A549
26.8 ± 2.5
58.6 ± 5.3
62.5 ± 6.3
25.8 ± 2.5
174.2 ± 16.1
95.0 ± 8.8
14.8 ± 2.3
6.6 ± 0.46
0.05 ± 0.002
1.6 ± 0.07
>200
MDA-MB-435
2.21 ± 0.21
40.6 ± 4.1
25.5 ± 1.7
23.3 ± 2.2
71.2 ± 7.5
6.7 ± 0.57
25.6 ± 2.3
5.8 ± 0.61
1.4 ± 0.09
13.8 ± 1.2
>200
HCT116
16.90 ± 1.7
34.17 ± 3.3
97.23 ± 9.2
21.87 ± 1.9
52.34 ± 5.6
170.81 ± 23.1
>200
5
5
5
57e
57f
57g
57h
5
7i
46.10 ± 4.2
0.6 ± 0.05
3.7 ± 0.39
>200
57j
5
7k
7l
7m
5
5
1.8 ± 0.11
>200
6.7 ± 0.66
24.4 ± 2.1
31.0 ± 3.6
38.1 ± 0.32
2.1 ± 0.19
52.7 ± 5.6
8.7 ± 0.79
>200
14.2 ± 1.3
>200
57n
57o
35.1 ± 2.8
>200
71.0 ± 7.8
>200
5
5
7p
7q
3.49 ± 0.29
>200
5.59 ± 0.44
80.1 ± 8.3
4.6 ± 0.48
198.0 ± 18.7
26.2 ± 2.3
>200
5
5
7s
7t
3.8 ± 0.33
>200
5
7u
7v
7w
5
50.5 ± 4.7
>200
31.7 ± 32.6
>200
5
57x
57y
>200
37.1 ± 3.6
23.7 ± 1.9
156.7 ± 13.1
10.1 ± 1.0
9.5 ± 0.85
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7z
13.1 ± 1.2
0.05 ± 0.003
2.6 ± 0.1
>200
25.1 ± 2.3
1.0 ± 0.11
5.4 ± 0.48
>200
36.4 ± 3.2
2.4 ± 0.19
7.1 ± 0.77
>200
5
7aa
7ab
5
57ac
58ad
58ae
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7.7 ± 0.68
>200
13.9 ± 1.4
182.3 ± 17.5
145.00 ± 13.9
3.5 ± 0.26
176.1 ± 10.2
>200
21.0 ± 1.3
>200
58af
>200
>200
5
8ag
8ah
10.9 ± 1.3
>200
18.4 ± 1.6
169.0 ± 17.3
>200
5
5
8ai
8aj
7ak
187.9 ± 17.6
19.1 ± 2.3
>200
5
21.6 ± 3.2
>200
21.6 ± 2.3
>200
5
57al
>200
58.0 ± 5.7
>200
34.8 ± 3.3
31.00 ± 4.0
9.1 ± 0.88
132.4 ± 12.8
11.7 ± 1.7
19.6 ± 2.3
>200
57am
>200
57an
57ao
57ap
57aq
2.7 ± 0.22
113.7 ± 10.9
28.4 ± 2.5
20.1 ± 1.8
>200
2.3 ± 0.21
42.8 ± 3.8
9.4 ± 6.9
5.8 ± 0.62
39.4 ± 3.7
16.7 ± 1.9
5.3 ± 5.1
2.3 ± 0.21
>200
5
7ar
7as
5
173.2 ± 14.9
>200
>200
57at
>200
57au
58a
>200
>200
103.3 ± 10.7
17.5 ± 1.6
16.2 ± 1.2
>200
42.3 ± 4.8
29.3 ± 2.7
11.5 ± 1.2
66.2 ± 5.9
7.1 ± 0.58
<0.003
58b
54.1 ± 4.5
1.9 ± 0.23
53.4 ± 5.0
18.7 ± 1.6
<0.003
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5
8c
8e
5
8f
8g
8h
10.6 ± 0.98
6.5 ± 0.54
14.7 ± 1.2
5
5
10.9 ± 1.8
10.5 ± 1.1
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8i
8j
8k
3.8 ± 0.32
29.1 ± 2.1
5.2 ± 0.44
0.06 ± 0.005
8.6 ± 0.72
1.8 ± 0.09
5.6 ± 0.43
3.1 ± 0.26
7.7 ± 0.69
65.2 ± 8.0
11.0 ± 1.6
0.7 ± 0.06
7.4 ± 0.82
20.1 ± 2.8
1.2 ± 0.14
0.5 ± 0.06
<0.003
0.8 ± 0.08
0.6 ± 0.03
0.06 ± 0.004
<0.003
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58l
58m
58n
<0.003
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7.0 ± 0.84
<0.003
0.3 ± 0.02
<0.003
5
8o
8p
9a
9b
9c
5.1 ± 0.47
6.3 ± 0.61
5.6 ± 0.48
44.4 ± 4.8
3.8 ± 0.35
<0.003
5.1 ± 0.47
2.7 ± 0.30
4.8 ± 0.39
60.3 ± 5.8
4.5 ± 0.41
0.02 ± 0.001
0.4 ± 0.03
6.3 ± 0.77
5
5
5
5
CPT
TPT
IRT
0.7 ± 0.09
18.6 ± 1.2
SAR of the Thio-evodiamine Derivatives. Due to promising antitumor profile of
thioꢀevodiamine (63a) and 10ꢀhydroxylꢀthioꢀevodiamine (66a), a series of substituted
derivatives were investigated. As shown in Table 3, the introduction of 3ꢀfluoro (63b)
and 3ꢀchloro (63c) substituents on compound 63a was unfavorable for the antitumor
activity. Moreover, all the 3ꢀsubstitutedꢀ10ꢀmethoxyl analogues (63e-h) showed
moderate antitumor activity. Notably, after demethylation, the corresponding
1
0ꢀhydroxyl derivatives (66b-e) were highly active at low nanomolar range. The IC50
values of compounds 66b-e against HCT116 and MDAꢀMBꢀ435 cell lines were lower
than 3 nM. In particular, the 3ꢀchloroꢀ10ꢀhydroxyl derivative 66c revealed excellent
inhibitory activity against all the three cancer cell lines (IC50 range: 0.02 ꢀM to <
0
.003 ꢀM), highlighting its promising feature as a novel antitumor candidate.
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Further modification was focused on oxidation of the sulfur atom to sulfone (63i, 63j,
and 66f). Unfortunately, these sulfone derivatives were almost inactive. Moreover,
replacement of phenyl group with pyridinyl (65) also led to loss of the activity.
Table 3. In vitro antitumor activity of the thioꢀevodiamine derivatives (IC , µM)
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Compounds
63b
A549
>200
MDA-MB-435
>200
HCT116
>200
6
6
3c
3e
2.1 ± 0.12
1.2 ± 0.10
2.3 ± 1.9
11.3 ± 1.0
8.2 ± 0.79
>200
29.1 ± 2.6
6.5 ± 0.55
2.7 ± 0.31
20.0 ± 2.1
2.7 ± 0.32
>200
38.8 ± 3.9
3.9 ± 0.29
44.1 ± 5.0
122.0 ± 13.7
84.7 ± 8.8
>200
6
3f
3g
3h
3i
6
6
6
63j
65
>200
>200
>200
>200
>200
>200
66b
1.2 ± 0.11
0.02 ± 0.001
0.3 ± 0.02
0.7 ± 0.08
>200
<0.003
<0.003
6
6c
6d
6e
6f
<0.003
<0.003
6
<0.003
<0.003
6
<0.003
<0.003
6
46.0 ± 4.3
>200
>200
Evodiamine
CPT
>200
22.12 ± 1.9
0.02 ± 0.003
0.4 ± 0.02
6.3 ± 5.6
0.7 ± 0.06
7.4 ± 0.69
20.1 ± 2.7
<0.003
TPT
0.7 ± 0.05
18.6 ± 1.7
IRT
Antitumor Spectrum of the Selected Oxo- and Thio-evodiamine Derivatives. In
vitro antitumor assay revealed that a number of oxoꢀ and thioꢀevodiamine derivatives
showed highly potent activity against A549, HCT116 and MDAꢀMBꢀ435 cell lines.
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To further evaluate their antitumor spectrum, several compounds (58i, 58k, 66c, 66d,
and 66e) were tested against a variety of human cancer cell lines including SW579
(thyroid cancer), SKꢀHepꢀ1 (liver cancer), PCꢀ3 (pancreatic cancer), NCIꢀH446 (small
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cell lung cancer), MV3 (melanoma cells), K562 (leukemia cells) and BEL7404 (liver
cancer). As depicted in Table 4, all the compounds showed good to excellent
antitumor activity against the tested cancer cell lines with the exception of the
NCIꢀH446 cell line. They were particularly effective against the K562 and BEL7404
cell lines with IC50 values lower than 3 nM, which were more potent than the positive
control CPT. Among them, compound 66e showed the best antitumor activity. It was
highly active against PCꢀ3, K562 and BEL7404 cell lines with IC50 values lower than
3 nM. Moreover, it was also active against SKꢀHepꢀ1 and MV3 cell lines with IC50
values of 0.49 ꢁM and 0.14 ꢁM, respectively. These results confirmed that oxoꢀ and
thioꢀevodiamine derivatives had a broad antitumor spectrum, particularly for PCꢀ3,
K562 and BEL7404 cell lines, which were superior or comparable to CPT.
Table 4. In vitro antitumor spectrum of the selected evodiamine derivatives (IC50
,
µM)
Compounds
SW579
5.15 ± 0.48
5.56 ± 0.51
6.9 ± 0.59
7.3 ± 0.63
3.5 ± 0.23
0.71 ± 0.69
SK-Hep-1
1.1 ± 0.09
1.8 ± 0.11
2.1 ± 0.19
2.2 ± 0.13
0.49 ± 0.03
0.96 ± 0.78
PC-3
NCI-H446
>200
MV3
1.2 ± 0.09
1.1 ± 0.13
1.3 ± 0.11
1.2 ± 0.08
0.14 ± 0.01
<0.003
K562
<0.003
<0.003
<0.003
<0.003
<0.003
<0.003
BEL7404
0.06
5
8i
0.84 ± 0.07
0.40 ± 0.03
0.37 ± 0.02
58k
66c
>200
<0.003
>200
<0.003
6
6d
6e
CPT
0.43
±
0.03
>200
<0.003
6
<0.003
16.6 ± 1.4
>200
<0.003
2.5 ± 0.12
0.14 ± 0.01
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The Effect of Selected Evodiamine Derivatives on Apoptosis and Cell Cycle
Distribution in A549 Cells. To test the effect of novel oxoꢀ and thioꢀevodiamine
derivatives on the induction of apoptosis, compounds 58k and 66a were evaluated by
cell morphology and fluoresceneꢀactivated cell sorting (Figure 3). After treating with
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ꢁM of compounds 58k or 66a for 36 h, the majority of A549 cells became small,
round, and floating. In contrast, a normal shape and wellꢀordered cytoskeleton was
observed in the untreated control group and DMSO group. After treating with
compounds 58k or 66a, the percentage of apoptotic cells was 6.7% and 6.0%,
respectively. In contrast, the percentage of apoptotic cells in control and DMSO group
was 0.7% and 0.5%, respectively. These results indicated that oxoꢀ and
thioꢀevodiamine derivatives could effectively induce the cell morphology change of
A549 cells and the apoptosis of cancer cells.
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2
Evodiamine was reported to arrest PCꢀ3 cells at the G /M phase. Herein, the
effect of oxoꢀ and thioꢀevodiamines 58k and 66a on cell cycle progression of A549
cells was evaluated by flow cytometric method (Figure 4 and Figure S1). The results
showed that a G
2
/M arrest was initiated after a 12 h exposure to compounds 58k and
/M
66a. After treating with compounds 58k and 66a at 5 ꢁM for 36 h, the radios in G
2
phase of the cell cycle were 63.7% and 68.2%, respectively. In contrast, the radios of
cells untreated or treated with DMSO in G /M phase of the cell cycle were 11.9% and
2
8
.0%, respectively. Thus, the two compounds could significantly arrest the A549 cell
cycle at the G /M phase.
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Figure 3. (A) The effect of evodiamine derivatives on human cancer cell morphology.
A549 cells were treated with DMSO or 5 ꢀM of compounds 58k or 66a for 36 h, and
representative photographs were captured under a light microscope. (B) Evodiamine
derivativesꢀinduced cell apoptosis. A549 cells were treated with DMSO or 5 ꢀM of
compounds 58k or 66a for 48 h. Apoptosis was examined by flow cytometry (n = 3).
Representative photographs from three independent experiments were displayed.
Figure 4. A549 cells treated with 58k (5 ꢀM) or 66a (5 ꢀM) for 0ꢀ36 h were assayed
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by flow cytometry after staining with PI. Cells untreated or treated with DMSO were
used for comparison.
Top1 Inhibitory Activity and the Binding Modes. Previously, Top1 was found to
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be one of the antitumor targets of evodiamine derivatives.
They act by stabilizing
a covalent Top1ꢀDNA complex called the cleavable complex. To further evaluate the
inhibitory effect of oxoꢀ and thioꢀevodiamine derivatives on Top1ꢀmediated DNA
cleavage, representative compounds (i.e. compounds 58d, 58i, 58j, 58k, 63a, 63j, 66a,
66c, 66d, and 66e) were assayed. As shown in Figure 5, all the tested compounds
showed potent inhibitory activity against Top1ꢀmediated relaxation of supercoiled
DNA at the concentration of 200 ꢁM. At the concentration of 100 ꢁM, most
thioꢀevodiamine derivatives (compounds 63a-66e) retained potent inhibitory activity,
but the oxoꢀevodiamine derivatives showed decreased activity (see Table S1 for
inhibition rate). Moreover, good Top1 inhibitory activity was still observed for the
thioꢀevodiamine derivatives at the concentration of 50 ꢁM. The above results
indicated that thioꢀevodiamine derivatives showed higher inhibitory activity than the
oxoꢀevodiamine derivatives against Top1ꢀmediated DNA relaxation.
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Figure 5. Top 1 inhibitory activity of the evodiamine derivatives. (A) Inhibition of
Top1 relaxation activity at 200 µM. Lane 1, supercoiled plasmid DNA; Lane 2, DNA
+
Top1; Lane 3, DNA + Top1 + CPT; Lanes 4ꢀ13, DNA + Top1 + evodiamine
derivatives (58d, 58i, 58j, 58k, 63a, 63j, 66a, 66c, 66d, and 66e, respectively). (B)
Inhibition of Top1 relaxation activity at 100 µM. The lanes are the same as the assay
at 200 µM. (C) Inhibition of Top1 relaxation activity at 50 µM. Lanes 4ꢀ10, DNA +
Top1 + evodiamine derivatives (58k, 63a, 63j, 66a, 66c, 66d, and 66e, respectively).
Next, the binding modes of compounds 58k and 66c were investigated by
molecular docking and molecular dynamics (MD) simulations. Gold 5.0.1 and
3
4
Desmond with the same parameter as our previously reports were used for
molecular modeling. As depicted in Figure 6, compounds 58k and 66c formed
baseꢀstacking interactions with the surrounding DNA base pairs. Their Aꢀring C10
hydroxyl groups formed a hydrogen bond with Glu356. Moreover, an additional
hydrogen bond was observed between the Eꢀring C3 hydroxyl group of compound
5
8k and Thr718. Evodiamine and its substituted analogues shared a unique “L type”
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conformation in the active site of Top1. In contrast, oxoꢀ and thioꢀevodiamine
derivatives displayed “plannar” configuration in the binding site, which was
beneficial to form stronger baseꢀstacking interactions with surrounding DNA base
pairs and thus enhance the Top1 inhibitory activity.
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58
59
60
Figure 6. Schematic representation of the proposed binding mode for compounds 58k
(A) and 66c (B) with Top1ꢀDNA (PDB code: 1T8I). The figures were generated using
Pymol (http://www.pymol.org/).
Top2 Inhibitory Activity and the Binding Modes. Previously, Top2 was
identified as another molecular target of evodiamine derivatives using the target
3
4
prediction in combination of enzymatic inhibitory assay. To further evaluate the
inhibitory effect of the oxoꢀ and thioꢀevodiamine derivatives on Top2, representative
compounds (i.e. 58d, 58i, 58j, 58k, 63a, 63j, 66a, 66c, 66d, 66e, and 66f) were
assayed. As shown in Figure 7, only compounds 58i, 63j, 66c and 66e showed
moderate to good Top2 inhibitory activity at the concentration of 100 µM. However,
the other compounds were almost inactive at the same condition. In comparison with
34
substituted evodiamine derivatives, the new scaffolds showed decreased Top2
inhibitory activity, indicating that new mechanism (targets) might be involved in their
excellent antitumor activity .
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Figure 7. Top2 inhibitory activity of the evodiamine derivatives. (A) Inhibition of
Top2 relaxation activity at 100 µM. Lane 1, supercoiled plasmid DNA; Lane 2, DNA
+ Top2; Lane 3, DNA + Top2 + etoposide; Lanes 4ꢀ14, DNA + Top2 + evodiamine
derivatives (58d, 58i, 58j, 58k, 63a, 63j, 66a, 66c, 66d, 66e, and 66f, respectively).
To investigate the binding modes between evodiamine derivatives and Top2α,
compounds 58i, 66c, and 66e were subjected to molecular docking and molecular
dynamic simulations. As depicted in Figure 8, all compounds were observed to be
wellꢀfitted into the ATPꢀbinding domain of Top2α. The Aꢀring C10 hydroxyl group
and Dꢀring C5 carbonyl group formed a hydrogen bond with Arg98 and Asn150,
respectively. For compounds 58i and 66e, the Eꢀring C3 hydroxyl group formed
2
+
coordination interaction with Mg ion and increased the binding energy with Top2α,
which was consistent with the fact that Eꢀring C3 hydroxyl was beneficial for
increasing the Top2α inhibitory activity.
Figure 8. Schematic representation of the proposed binding mode for compounds 58i
(A), 66c (B) and 66e (C) in the ATPꢀbinding domain of Top2α (PDB code: 1ZXM).
The figures were generated using Pymol (http://www.pymol.org/).
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