Scaffold Hopping of Natural Product Evodiamine: Discovery of a Novel Antitumor Scaffold with Excellent Potency against Colon Cancer

Article abstract of DOI:10.1021/acs.jmedchem.9b01626

Inspired by the natural product evodiamine, a novel antitumor indolopyrazinoquinazolinone scaffold was designed by scaffold hopping. Structure-activity relationship studies led to the discovery of compound 15j, which shows low nanomolar inhibitory activity against the HCT116 cell line. Further antitumor mechanism studies indicated that compound 15j acted by the dual inhibition of topoisomerase 1 and tubulin and induced apoptosis with G2 cell-cycle arrest. The quaternary ammonium salt of compound 15j (compound 15js) exhibited excellent in vivo antitumor activity (TGI = 66.6%) in the HCT116 xenograft model with low toxicity. Indolopyrazinoquinazolinone derivatives represent promising multitargeting antitumor leads for the development of novel antitumor agents.

Full text of DOI:10.1021/acs.jmedchem.9b01626

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Article
Scaffold Hopping of Natural Product Evodiamine: Discovery of a Novel
Antitumor Scaffold with Excellent Potency against Colon Cancer
Lei Wang, Kun Fang, Junfei Cheng, Yu Li, Yahui Huang, Shuqiang
Chen, Guoqiang Dong, Shanchao Wu, and Chunquan Sheng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01626 • Publication Date (Web): 27 Dec 2019
Downloaded from pubs.acs.org on December 27, 2019
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Scaffold Hopping of Natural Product Evodiamine: Discovery of a
Novel Antitumor Scaffold with Excellent Potency against Colon
Cancer
Lei Wanga, b, †, Kun Fang a, †, Junfei Cheng a, †, Yu Li , Yahui Huang , Shuqiang Chen ,
a
a
a
a, *
a, *
a, b,c*
Guoqiang Dong , Shanchao Wu , Chunquan Sheng
a
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, China
b
School of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Qiuyang
Road, Fuzhou, Fujian 350122, China
c
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,
China
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ABSTRACT
Inspired by the natural product evodiamine,
a
novel antitumor
indolopyrazinoquinazolinone scaffold was designed by scaffold hopping.
Structure-activity relationship studies led to the discovery of compound 15j, which
shows low nanomolar inhibitory activity against the HCT116 cell line. Further
antitumor mechanism studies indicated that compound 15j acted by the dual
inhibition of topoisomerase 1 (Top1) and tubulin and induced apoptosis with G2
cell-cycle arrest. The quaternary ammonium salt of compound 15j (compound 15js)
exhibited excellent in vivo antitumor activity (TGI = 66.6%) in the HCT116 xenograft
model with low toxicity. Indolopyrazinoquinazolinone derivatives represent
promising multitargeting antitumor leads for the development of novel antitumor
agents.
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INTRODUCTION
1
Natural products (NPs) are critical in antitumor drug discovery and development. To
date, approximately 83% (113 out of 136) of anticancer small molecules approved by
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the FDA are NPs or NP derivatives. For instance, camptothecin (CPT), a typical
topoisomerase 1 (Top1) inhibitor isolated in 1966 from the Chinese tree Camptotheca
acuminnata, shows potent antitumor activity. The modification of natural CPT
3
resulted in several clinical antitumor drugs, such as topotecan (TPT) and irinotecan
IRT)4.
(
Historical analysis of NP-based drug discovery indicates that successful examples
2
of the direct development of unmodified NPs are still rare, mainly due to insufficient
levels of activity, high structural complexity, unfavorable physicochemical and
5
pharmacokinetic properties, significant toxicity and so on. Therefore, the structural
optimization of NPs to improve their pharmacological activities and druggabilities is
always required before they can be developed into clinical drugs.5, 6 Strategies for the
structural modification of NPs mainly include designing NP derivatives through
7
step-by-step structure-activity relationships (SAR) analyses, structural simplification
7
of structurally complex NPs, and the generation of NP-based compound libraries
8
with scaffold diversity . In particular, the design of NP-based novel molecular
scaffolds by scaffold hopping is an effective strategy to better navigate the drug-like
chemical space around NPs and identify drug candidates.8
Evodiamine (1, Figure 1) is the main alkaloid component of the traditional Chinese
9
herb Wu-Chu-Yu, and it has diverse biological activities. In recent years, evodiamine
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has attracted broad research interest as an antitumor lead compound. Evodiamine
has a multitargeting antitumor effect and acts by the induction of apoptosis and the
inhibition of invasion and metastasis.11-13 Previously, we performed systematic
structural optimizations of evodiamine and identified highly active evodiamine
derivatives.14-16 Moreover, diverse evodiamine-inspired scaffolds were designed,
leading to the discovery of several novel evodiamine derivatives (2-4) with potent in
vitro and in vivo antitumor activities (Figure 1).14-16 Thus, designing novel scaffolds
based on evodiamine offers an effective and promising strategy for developing novel
antitumor agents. Thus, the chemical space around the evodiamine skeleton must be
extensively explored. Furthermore, the in vivo antitumor activities of our previously
identified oxo- and thio-evodiamine derivatives (2-4) remain to be improved.
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O
O
N
N
R2
N
C
B
B
R1
N C
N
N
N
N
X
H
H
O
Evodiamine (1)
2 R = OH, R = OH, X = O
R = OH, R = OH, X = S
R = Cl, R = OH, X = S
Indolopyrazinoquinazolinone
(15a)
1
2
3
4
1 2
1
2
Figure 1. Chemical structures of evodiamine, oxo- and thio-evodiamine derivatives
and indolopyrazinoquinazolinone 15a.
To this end, an evodiamine-based scaffold hopping strategy was applied to design a
novel indolopyrazinoquinazolinone scaffold (15a) by reversing the connectivity of the
B/C rings of evodiamine (Figure 1). Systematic SAR studies led to compound 15j,
which was shown to possess excellent antitumor potency. Interestingly, the quaternary
ammonium salt of 15j (compound 15js) showed potent in vivo antitumor efficacy in
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HCT116 colon cancer xenograft models with low toxicity.
CHEMISTRY
The chemical synthesis of various substituted indolopyrazinoquinazolinone
derivatives is depicted in Schemes 1-5. First, substituted 1H-indole-2-carboxylic
acids 5 and ethanol were refluxed in the presence of concentrated sulfuric acid to
afford various ethyl 1H-indole-2-carboxylates 6. Then, intermediates 6 were reacted
with 2-bromoacetonitrile in the presence of sodium hydride (NaH) and
N,N-dimethylformamide (DMF) at room temperature, and subsequent intramolecular
ring closure provided intermediates 8. Starting from substituted 2-aminobenzoic acids
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, methyl 2-(methylamino)benzoate intermediates 13 were synthesized through
esterification, N-Boc protection, N-methylation and hydrolysis. The coupling of
intermediates 8 and 13 in the presence of POCl under microwave conditions
3
16
produced corresponding dehydro-intermediates 14. Finally, target compounds 15a-z
and 15aa-ab were obtained by reduction with NaBH (Scheme 1).
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Scheme 1a
O
O
O
O
a
c
O
b
R1
R1
R1
R1
N
H
OH
N
O
N
N
NH
H
CN
6
a-f
7a-f
8a-f
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Boc
Boc
NH2
O
NH2
O
HN
O
NH
O
N
O
OH
d
O
e
f
g
O
O
O
R2
R2
R2
R2
R2
9a-j
10a-j
11a-j
12a-j
13a-j
23
22
6
9
R2
5
4
16
R
2
2
N
1
N
1
R1
R1
N
2
N
15 20
N
N
3
6
O
O
10
15a-x
O
h
i
11
1
4a-x
19
5
4
16
1
N
N
X
X
R1
R1
2
N
15
N
N
N
3
O
1
5y-z, 15aa-ab
14y-z, 14aa-ab
15m R = 1-OCH , R = 21-NH
2
15a R = H, R = H
1 2
1
3
2
1
5b R = 1-OCH , R = H
15n R = 1-OCH , R = 22-OCH
1 3 2 3
15o R = 1-OCH , R = 21-CH
1 3 2 3
15p R = 1-Br, R = 21-F
1 2
15q R = 1-Br, R = 22-OCH
1 2 3
15r R = 1-Cl, R = 21-F
15s R = 1-CH , R = 21-F
1 3 2
15t R = 1-OCF , R = 21-F
15u R = 1-cyclobutoxyl, R = 21-F
1
3
2
15c R = 1-Cl, R = H
1
2
1
6
15d R = 2-Cl, R = H
1
2
1
1
5y R = 1-OCH , X =
5z R = 1-OH,
1
3
1
1
1
1
1
5e R = 1-F, R = H
1
2
X =
N
S
1
5f R = 1-CH , R = H
15
1
3
2
1
2
5g R = 1-Br, R = H
1
2
1
6
5h R = H, R = 22-OCH
15aa R = 1-OCH , X =
1 3
15ab R = 1-OH,
1
2
3
1
3
2
5i R = H, R = 21-OCH
X =
1
2
3
1
2
1
15
15j R = 1-OCH , R = 21-F
15v R = 1-cyclopentyloxyl, R = 21-F
1 2
15w R = 1-cyclohexyloxyl,R = 21-F
1 2
15x R = 1-OCD , R = 21-F
1 3 2
1
3
2
1
5k R = 1-OCH , R = 21-Cl
1
3
2
1
5l R = 1-OCH , R = 21-Br
1
3
2
a
Reagents and conditions: (a) EtOH, H SO , reflux, 10 h; (b) BrCH CN, NaH, DMF, rt, overnight,
2
4
2
yield 45-62% over two steps; (c) NaBH , CoCl , MeOH, THF, reflux, 6 h, yield 39-55%; (d)
4
2
MeOH, SOCl , reflux, overnight; (e) (Boc) O, DMAP, CH Cl , rt, overnight, yield 31-56%; (f)
2
2
2
2
MeI, NaH, DMF, rt, 6 h, yield 61-85%; (g) CF COOH, CH Cl , rt, 2 h, yield 82-96%; (h) POCl ,
3
2
2
3
toluene, microwave, 130 °C, 45 min; (i) NaBH , EtOH, rt, 1 h, yield 23-62% over two steps.
4
Intermediates 18 were prepared by reacting various benzaldehydes 16 with ethyl
-azidoacetate 17, followed by intramolecular cyclization in the presence of
2
1,2-dichlorobenzene at high temperature to afford substituted ethyl
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1H-indole-2-carboxylates 19. Then, target compounds 23a-c were prepared following
synthetic methods similar to those described for derivatives 15a-z (Scheme 2).
Scheme 2a
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1
1
1
1
1
1
1
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2
2
2
2
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0
O
O
N3
O
O
O
O
NH
O
O
R1
1
7
O
b
R1
c
R1
R1
N
N
H
a
N3
1
6a-b
18a-b
19a-b
20a-b
O
R2
R2
R2
N
H
N
N
21
R1
e
R1
N
N
N
N
d
O
O
22a-b
23a R = 1-OCH , 2-OCH , 3-OCH , R = H
1 3 3 3 2
23b R = 2-SCH , R = 21-F
1
3
2
2
3c R = 1-OCH , R = 21,22-diOCH
1 3 2 3
aReagents and conditions: (a) Na, MeOH, 0 °C, 6 h, yield 53-72%; (b) 1,2-dichlorobenzene, 180
C, 2 h, yield 65-78%. (c) BrCH CN, NaH, DMF, rt, overnight; NaBH , CoCl , MeOH, THF,
°
2
4
2
reflux, 6 h, yield 45-62% over two steps; (d) POCl , toluene, microwave, 130 °C, 45 min; (e)
3
NaBH , EtOH, rt, 1 h, yield 36-37% over two steps.
4
Target compounds 24, 25a-b, 26-27, 28a-b, 29a-b and 30 were synthesized as
shown in Scheme 3. 1-Hydroxy-substituted derivative 24 was obtained by
demethylation of 15b in the presence of BBr and DCM at -78 °C. The reactions of
3
compounds 15b and 15f with benzoyl peroxide and N-bromosuccinimide (NBS)
produced 9-bromine derivatives 25a and b. Treatment of compound 15e with
Lawesson’s reagent gave thiocarbonyl derivative 27. The Vilsmeier reactions17 of
compounds 15j and 15o afforded corresponding formylated derivatives 28a and 28b.
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Compound 26 was obtained from the reductive amination of compound 28a.
Additionally, reduction reactions were used to prepare compounds 29a and 29b.
Scheme 3a
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HO
N
a
15b
N
N
O
24
R3
b
f
1
1
5b
5f
25a R = CH , R = H, R =Br
1 3 2 3
R1
N
R2 25b R = OCH , R = 21-F, R = Br
1
3
2
3
N
N
N
N
28a
26 R1 = OCH3, R2 = 21-F, R3 =
O
S
F
N
c
1
5e
N
N
27
HO
O
O
O
N
N
15j
d
R2
e
R2
15o
N
N
N
N
O
O
2
8a R = 21-F
29a R = 21-F
2
2
28b R = 21-CH3
29b R = 21-CH3
2
2
HN
O
O
O
O
O
O
F
N
N
F
F
13c
e
N
N
N
N
N
NH
d
O
O
6j
14w
30
a
Reagents and conditions: (a) BBr , CH Cl , -78 °C, 2.5 h, yield 43%; (b) benzoyl peroxide, NBS,
3
2
2
CCl , reflux, 3.5 h, yield 24-26%; (c) Lawesson’s reagent, toluene, reflux, 4 h, yield 37%; (d)
4
POCl , DMF, rt, 2 h, yield 82-86%; (e) NaBH , MeOH, rt, 1 h, yield 92-93%; (f) methyl
3
4
piperazine, NaBH CN, CH COOH, DCM, rt, overnight, yield 63%.
3
3
The structural modification of the C-ring was focused on alkylation of the side
chain and ring expansion to a seven-membered ring (Schemes 4 and 5). The
7
methylation of intermediate 31 in the presence of (Me Si) NHNa and MeI gave
3
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intermediate 32.18 The Michael addition of intermediate 37 and acrylonitrile 38
smoothly provided intermediate 39.19 The following methods for the synthesis of
derivatives 36a-b and 43 were similar to those used to obtain compounds 15a-z.
Scheme 4a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
O
O
R2
NH
O
O
O
O
O
O
O
O
a
b
34a-b
c
N
N
N
N
NH
CN
CN
31
32
33
O
O
N
N
R2
R2 36a R = 21-H
2
d
3
6b R = 21-F
N
2
N
N
O
O
35a-b
a
Reagents and conditions: (a) (Me Si) NHNa, MeI, THF, rt, overnight, yield 44%; (b) NaBH ,
3
2
4
CoCl , MeOH, THF, reflux, 6 h, yield 36%; (c) POCl , toluene, microwave, 130 °C, 45 min; (d)
2
3
NaBH , EtOH, rt, 1 h, yield 32-35% over two steps.
4
Scheme 5a
NH
O
O
O
O
O
O
O
O
O
O
a
b
N
O
N
N
NH
c
CN
H
3
8
CN
3
7
39
40
N
O
N
HO
N
d
e
N
N
N
N
N
N
O
O
O
41
42
43
aReagents and conditions: (a) Benzyltrimethylammonium hydroxide, 1,4-dioxane, reflux, 1 h,
yield 72%; (b) NaBH , CoCl , MeOH, THF, reflux, 6 h, yield 36%; (c) POCl , toluene,
4
2
3
microwave, 130 °C, 45 min; (d) NaBH , EtOH, rt, 1 h, yield 32% over two steps; (e) BBr ,
4
3
CH Cl ,
-78
°C,
2.5
h,
yield
48%.
2
2
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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
RESULTS AND DISCUSSION
Design, in Vitro Antitumor Activity and SARs of Indolopyrazinoquinazolinone
Derivatives. The indolopyrazinoquinazolinone scaffold (15a) was designed by
changing the connectivity between the B-ring and the C-ring of evodiamine. Based on
the scaffold of 15a, a number of derivatives were designed and synthesized according
to their synthetic accessibility and chemical diversity (Figure 2). In total, 45 new
compounds were designed and synthesized by introducing various substituents on the
A, B, and E rings and variations of the C/D ring structures. The growth inhibitory
activities of indolopyrazinoquinazolinone derivatives were assayed against human
cancer cell lines HCT116 (colon cancer), MDA-MB-435 (breast cancer), and A549
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
2
0
(lung cancer) using the CCK8 assay. Evodiamine and CPT were used as reference
drugs. Compared with natural evodiamine, indolopyrazinoquinazolinone 15a showed
improved activity against the HCT116 and A549 cell lines (Table 1). Further
modification of compound 15a led to the discovery of several derivatives with
excellent activities against the HCT116 cell line.
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R2
N
R1
N
N
O
1
5a-i, 15p-x, 23a-b, 24
R
MeO
N
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
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
N
N
O
15j-o, 23c
O
Scaffold
Hopping
R1
N
N
D
N
SAR
R2
C
E
MeO
N
A
B
A
B
D
N
E
N C
N
N
N
H
Reversing
Piperidine
Ring
O
O
Evodiamine (1)
HCT116 IC50 > 200 µM
Indolopyrazinoquinazolinone (15a)
HCT116 IC50 = 47.5 µM
25b, 26, 28a-b, 29a-b, 30
MeO
N
N
N
MeO
O
N
F
F
N
N
42
N
O
N
N
15j
S
HCT116 IC50 = 2 nM
27
Figure 2. Design of indolopyrazinoquinazolinone derivatives.
First, various substituents were introduced on the A-ring (15a-g, 23a and 24) or
E-ring (15h-i). Among them, 1-methyl derivative 15f showed the best antitumor
activity, and it was particularly active against the HCT116 cell line (IC = 0.03 M,
5
0
Table 1). When the 1-methyl group of compound 15f was replaced with 1-chloro (15c)
and 1-bromo (15g), the antitumor activities against the HCT116 cell line were
significantly decreased (Table 1). In contrast, the 1-fluoro analogue (15e) was
completely inactive. Unfortunately, the movement of the chloro substituent from C1
(
15c) to C2 (15d) or 1,2,3-trimethyl substitution (23a) resulted in total loss of the
antitumor activity (Table 1). Additionally, a 22-methoxy substituent (15h) was
unfavorable for activity.
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3
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6
7
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
Table 1. In vitro antitumor activities of A- or E-ring-modified derivatives (IC , µM)
5
0
R2
N
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
R1
N
N
O
Compd
5a
5b
5c
15d
5e
5f
5g
5h
15i
3a
R1
H
R2
H
H
H
H
H
H
H
HCT116 MDA-MB-435 A549
1
48.5
12.0
4.4
>200
20.2
13.5
>200
>200
13.3
15.9
>200
168.2
>200
16.8
22.1
2.7
21.5
2.7
1
1-OCH3
1-Cl
2-Cl
1-F
1
27.7
>200
>200
>200
15.2
24.3
15.6
>200
14.5
>200
2.8
>200
>200
0.03
7.3
1
1
1-CH3
1-Br
H
1
1
22-OCH3
124.2
49.2
>200
44.3
>200
0.02
H
21-OCH3
2
1,2,3-trimethoxy
H
H
-
2
4
1-OH
1
-
-
CPT2
-
Second, A,E-ring di-substituted derivatives were designed and synthesized. In
general, 21-fluoro-substituted derivatives (15j, 15r, 15s, 15t, and 23b) had better
antitumor activities than other 21-substituted derivatives (15k, 15l, 15m and 15o). In
1
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particular, 1-methoxy-21-fluoro analogue 15j was highly active against the HCT116
cell line (IC = 0.002 μM, Table 2), and its activity was superior to that of CPT. The
5
0
results suggested that the 21-fluoro group was important for antitumor activity. Based
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
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
on compound 15j, the replacement of the 1-methoxy group with bromo (15p), chloro
(15r), cycloalkoxy (15u-w) and methylthio (23b) groups led to substantial decreases
in the antitumor activity, while the 1-methyl (15s) and 1-trifluoromethoxy (15t)
derivatives retained excellent activity against the HCT116 cell line (Table 2).
Therefore, 1-methoxy and 21-fluoro groups were the best combination for antitumor
activity.
Table 2. In vitro antitumor activities of A- and E-ring-modified derivatives (IC ,
5
0
µM)
R2
N
R1
N
N
O
R1
R2
MDA-MB-4
Compd
HCT116
A549
3
5
1
5j
5k
5l
15m
5n
5o
5p
1-OCH3
1-OCH3
1-OCH3
1-OCH3
1-OCH3
1-OCH3
1-Br
21-F
21-Cl
0.002
61.3
1.0
158.8
>200
>200
>200
30.8
1
>200
>200
>200
>200
>200
97.6
1
21-Br
>200
>200
180.5
19.4
21-NH2
22-OCH3
21-CH3
21-F
1
1
>200
>200
1
28.5
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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
15q
5r
5s
15t
5u
15v
5w
5x
23b
3c
1-Br
22-OCH3
>200
>200
142.9
1.0
>200
53.0
1
1-Cl
1-CH3
21-F
6.7
0.06
1
21-F
22.8
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
1-OCH3
1-cyclobutoxy
1-cyclopentyloxy
1-cyclohexyloxy
1-OCD3
2-SCH3
1-OCH3
H
21-F
0.83
>200
>200
>200
50.14
11.43
85.8
95.52
98.54
>200
173.50
31.33
8.3
1
21-F
34.15
57.85
66.82
0.01
21-F
1
21-F
1
21-F
21-F
9.4
2
21,22-diOCH3
>120.60
48.5
23.82
>200
22.1
>200
21.5
15a
1
H
-
-
>200
0.02
>200
2.8
CPT
-
-
2.7
Third, various substituents were introduced on the B- or C-rings (25a-b, 26, 28a-b,
9a-b, 30 and 36a-b). Based on highly potent compounds 15j and 15f, the addition of
2
a bromo group at the C9 position (25a and 25b) resulted in improved antitumor
activity against the A549 cell line, whereas the potencies against the other two cell
lines were lower (Table 3). Introducing methyl (compound 30), formyl methylene
(28a), ethanol (29a) or N-methyl piperidyl methylene (26) moieties at the C9 position
of compound 15j also led to obviously lower activities against the HCT116 cell line
(
Table 3). Moreover, the addition of a dimethyl group (36a-b) to the C10 position of
compounds 15b and 15j led to the loss of the antiproliferative activity (Table 3).
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Table 3. In vitro antitumor activities of B-ring-modified derivatives (IC , µM)
5
0
R3
R1
N
R2
N
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
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
N
R4
O
R5
MDA-MB
435
Compd
R1
R2
R3
R4
R5 HCT116
A549
-
2
5a
5b
26
8a
8b
9a
9b
30
6a
6b
CH3
H
F
F
F
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3.3
9.2
12.3
89.1
5.82
>200
70.5
22.8
41.5
132.20
86.4
10.7
>200
22.1
2.7
14.1
12.3
32.87
59.2
>200
6.6
2
OCH3
OCH3
OCH3
Br
N
N
5.5
2
CHO
CHO
>200
18.1
5.6
2
OCH3 CH3
OCH3
OCH3 CH3 CH OH
2
F
CH OH
2
2
5.8
72.1
13.24
180.9
>200
21.5
>200
2.8
2
OCH3
F
H
F
H
-
CH3
H
H
H
-
21.93
6.2
3
OCH3
CH3 CH3
CH3 CH3
3
OCH3
1.5
1
5a
1
H
-
H
-
H
-
48.5
>200
0.02
CPT
-
-
-
-
-
Finally, the modification of the scaffold was also investigated. However, when the
scaffold carbonyl group was converted to a thiocarbonyl, the resulting compound (27)
was completely inactive (Table 4). In addition, the replacement of the E-ring phenyl
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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
group with a pyrrole (15y-z) or thiophene (15aa and 15ab) was unfavorable for the
antitumor activity (Table 4). Pyrazole derivatives 15y-z were almost inactive, and
thiophene derivatives 15aa and 15ab showed moderate antitumor activity with IC50
values in the range of 9.5 M to 105.9 M (Table 4). These results indicated that the
introduction of a heterocycle at the E-ring might lead to decreased antitumor activity,
and detailed SARs remained to be further investigated. Similarly, when the C-ring
was enlarged to a seven-membered ring (compounds 42 and 43), no improvement in
the antitumor activity was observed (Table 4).
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 4. In vitro antitumor activities of scaffold-modified derivatives (IC , µM).
5
0
R
O
N
N
F
X
N
N
1
N
N
O
n
Y
5js
MDA-MB-43
5
Compd
R
Y
X
n
HCT116
A549
15y
15z
OCH3
OH
O
O
1
1
189.7
>200
>200
>200
>200
>200
N
N
1
5aa
OCH3
OH
O
O
S
1
1
1
2
2
9.5
44.6
66.8
47.0
30.3
S
S
1
5ab
105.9
>200
56.0
2
7
F
>200
196.4
>200
>200
32.0
42
OCH3
OH
O
O
4
3
>200
111.4
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9
1
5a
H
-
O
-
1
-
48.5
87.15
>200
0.02
>200
>200
22.1
2.7
21.5
>200
>200
2.8
1
5js
1
-
-
-
-
-
-
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
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
CPT
-
-
-
The
above
analyses
suggested
preliminary
SARs
for
the
indolopyrazinoquinazolinone antitumor scaffold that can be summarized as follows
Figure 3). (1) The installation of methoxy or methyl substituents at the C1 position is
(
preferred, whereas the introduction of halogen atoms (F, Cl, and Br) at this position is
unfavorable. (2) C21 fluoro substitution is of great importance for antitumor activity.
The combination of C21-fluoro and C1-methoxy substitution resulted in the most
potent compound (15j). (3) Ring expansion of the C-ring to a seven-membered ring is
not beneficial. (4) The conversion of the D-ring carbonyl group into the thiocarbonyl
group has a negative effect on the antitumor activity. (5) Substitution at the C9
position is unfavorable.
5
C9: substitution is disadvantageous.
2
3
22
C21-substitution is preferred, F is of
great importance for the antitumor
activity.
C21-F and C1-OCH3 are the best
combination.
9
1
2
N
C1: OCH , CH3 are preferred,
3
21
1
halogens atoms (F, Cl, Br)
are unfavorable.
2
N
N
O
1
10
Thiophene and pyrrole are unfavored.
9
3
Enlarging the C-ring is unfavorable.
4
C=O > C=S, C=S is inactive.
Figure 3. Summary of the SARs of indolopyrazinoquinazolinone derivatives.
Antitumor Spectrum of Compound 15j. The above results showed that a number
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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
of derivatives offered highly potent activity against the HCT116 cell line. In an
attempt to investigate the antitumor spectrum of this scaffold, compound 15j was
selected for assays against a variety of human cancer cell lines, including K562
(leukemia cells), PC-3 (pancreatic cancer), MV3 (melanoma cells), BEL7404 (liver
cancer), SK-Hep-1 (liver cancer), SW579 (thyroid cancer), and NCI-H446 (small-cell
lung cancer). As shown in Table 5, compound 15j exhibited good to excellent
antitumor activities against the tested cancer cell lines, with the exception of the
NCI-H446 cell line. Notably, compound 15j was particularly effective against the
K562 cell line, with an IC value lower than 3 nM. Moreover, it also showed good
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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antiproliferative activity against the PC-3 (IC = 0.3 µM) and BEL7404 cell lines
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(IC = 0.1 µM), and its activity was superior to that of CPT (IC = 2.4 µM and 0.4
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µM, respectively). These results revealed that compound 15j has a broad antitumor
spectrum.
Table 5. In vitro antitumor spectrum of compound 15j (IC , µM)
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SK-Hep-
NCI-H4
46
Compd
K562
PC-3
MV3
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SW579
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2.4
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6.1
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>200
>200
CPT
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Inhibitory Activities of Compound 15j against Top1, Top2 and Tubulin.
Previously, evodiamine and its derivatives were identified as dual Top1/Top2
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cleavage through the stabilization of the transient Top1- or Top2-DNA cleavable
complexes. To further evaluate the inhibitory effects of indolopyrazinoquinazolinone
derivatives on Top1 and Top2, representative compounds (i.e., 15a, 15j, 15c and 15f)
were assayed. CPT and etoposide (Eto) were employed as positive controls. As shown
in Figure 4A, all the tested compounds were active against Top1-mediated relaxation
of supercoiled DNA at a concentration of 100 M. However, they were inactive
against Top2-mediated DNA relaxation at the same concentration. The above results
confirmed that these derivatives were Top1 inhibitors. Further Top1-mediated DNA
cleavage assays of compound 15j were conducted to identify whether the derivatives
were poisons or catalytic inhibitors. Similar to CPT (a Top1 poison), compound 15j
could also increase the intensity of the nicked band (Figure 4B), suggesting that it
was a Top1 poison.
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Our previous work also revealed that 3-chloro-10-hydroxy thio-evodiamine was the
first triple Top1/Top2/tubulin inhibitor.16 Notably, indolopyrazinoquinazolinone
derivatives only exhibited moderate Top1 inhibitory activities, but their
antiproliferative activities were comparable or superior to those of CPT, suggesting
they might act on additional targets. Given the structural similarity between
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-chloro-10-hydroxy thio-evodiamine and the indolopyrazinoquinazolinone
derivatives, the tubulin inhibitory activity of compound 15j was determined using a
concentration-activity assay16 with colchicine as the positive control. Similar to
colchicine, compound 15j was found to inhibit tubulin polymerization in a
concentration-dependent manner with an IC50 value of 26.3 M (Figure 4C),
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implying that compound 15j was a microtubule depolymerization agent. Moreover,
visual evidence for the disruption of microtubules can be concurrently obtained using
fluorescein mouse anti-α-tubulin analysis. Herein, the tubulin polymerization
inhibitory effects of compound 15j were further explored by immunofluorescent
staining of HCT116 cells using paclitaxel as the control. As shown in Figure 4D, the
microtubule networks in untreated HCT116 cells had a normal arrangement with
microtubules extending from the central regions of the cell to the cell periphery. In
contrast, after treatment with different concentrations of compound 15j for 12 h, the
microtubules in the cytosol were clustered around the cell nucleus. These findings
indicated that compound 15j disrupted the microtubule organization of HCT116 cells.
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Figure 4. (A) Top1 and Top2 inhibitory activities of the selected
indolopyrazinoquinazolinone derivatives. Inhibition of Top1 relaxation activity at 100
µM: Lane 1, supercoiled plasmid DNA; Lane 2, DNA + Top1; Lane 3, DNA + Top1
+ CPT; Lanes 4-7, DNA + Top1 + test compound (15a, 15j, 15e, and 15f,
respectively). Inhibition of Top2 relaxation activity at 100 µM: Lane 1, supercoiled
plasmid DNA; Lane 2, DNA + Top2; Lane 3, DNA + Top2 + etoposide; Lanes 4-7,
DNA + Top2 + test compound (15a, 15j, 15e, and 15f, respectively). (B)
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Top1-mediated DNA cleavage assay gel of compound 15j using supercoiled pUC19.
Lane 1, supercoiled plasmid DNA (pUC19); lane 2, DNA + Top1; lane 3, DNA +
Top1 + CPT (100 μM); lanes 4−7, DNA + Top1 + 15j (100, 50, 25, and 12.5 μM). (C)
Tubulin inhibitory activities of compound 15j and colchicine. Following treatment
with different concentrations of 15j and colchicine, the in vitro polymerization of
tubulin at 37 °C was continuously monitored by recording the absorbance at 340 nm
over 20 min. The data are the average of two independent experiments. Compound
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5j showed concentration-dependent inhibition of tubulin polymerization. (D)
Tubulin polymerization inhibitory effects of 15j by an immunostaining assay.
HCT116 cells were treated with paclitaxel and 15j at different concentrations for 12 h.
The nuclei were stained with DAPI (blue), and the microtubules were stained with
mouse anti-α-tubulin (green). The images were taken using a TSC SP8 laser confocal
microscope (Leica, Germany).
Compound 15j was also analyzed for its off-target effects in 30 antitumor targets
(Figure 5A), which did not show any affinity for the examined proteins (< 20%
inhibition at 5 M). The above results suggest that compound 15j might selectively
act on both Top1 and tubulin. The combination use of Top1 and tubulin inhibitors has
a synergistic antitumor effect in cancer patients.21 However, only a few dual
Top1/tubulin inhibitors have been reported.16, 22 This work provides a new type of
dual Top1/tubulin inhibitor and further validated its efficacy in antitumor
chemotherapy. Additionally, it should be noted that additional targets might account
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for the antitumor activity of the indolopyrazinoquinazolinone derivatives, and their
multitargeting antitumor mechanisms require further exploration.
The Binding Modes of Compound 15j with Top1/Tubulin. To better understand
the interactions of compound 15j with Top1 and tubulin, molecular docking studies
were performed using our well-established docking protocols.15, 16 As depicted in
Figure 5B, compound 15j displayed a “planar” configuration and formed
base-stacking interactions with the TGP11 base pair in the active site of Top1. As
shown in Figure 5C, compound 15j fit well within the colchicine-binding domain of
tubulin. The oxygen atom of the D-ring carbonyl formed a hydrogen bond with Ala
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50, and the E-ring formed a salt bridge with the surrounding residue Lys 254.
Figure 5. (A) The inhibition rates of compound 15j against a panel of 30 antitumor
targets. G9a, lysine methyltransferase G9a; Sirtuin1, NAD-dependent deacetylase
sirtuin-1; TNKS1, tankyrase 1; TNKS2, tankyrase 2; PARP, poly-ADP-ribose
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polymerase; JMJD3, jumonji domain-containing 3; SHP2, phosphatase shatterproof 2;
DNMT3a/3L, DNA methyltransferase 3a/3L; PRMT1, protein arginine
N-methyltransferase 1; EZH2, enhancer of zeste homolog 2; SET8, lysine
methyltransferase enzyme SET8; CDK, cell cycle-dependent kinase; MMP, matrix
metalloproteinase; PKAC-α, protein kinase A catalytic subunit α; HDAC3, histone
deacetylase 3; Abl, Abelson tyrosine kinase; PLK1, polo-like kinase 1; EGFR,
epidermal growth factor receptor; JMJD2A, jumonji domain-containing protein 2A;
PI3K (p110α/p85α), phosphoinositide-3-kinase (p110α/p85α); FAK, focal adhesion
kinase; PDE4A1A, phosphodiesterase 4A1A; PDE4D7, phosphodiesterase 4D7;
PDE4A4, phosphodiesterase 4A4. (B) Proposed binding modes for compound 15j
with the Top1-DNA complex (PDB code: 1T8I). (C) Proposed binding modes for
compound 15j with the colchicine-binding domain of tubulin (PDB code: 1SA0). The
figures were generated using Pymol (http://www.pymol.org/).
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Compound 15j-Induced Apoptosis and G2 Cell-cycle Arrest in HCT116 Cells.
The effect of compound 15j on the induction of apoptosis in HCT116 cells was
evaluated by flow cytometry. As shown in Figure 6A, compound 15j induced
apoptosis of HCT116 cells in a concentration-dependent manner. After exposure to
various concentrations (0.04, 0.2 and 1 M) of compound 15j for 24 h, the
percentages of apoptotic cells were 32.52%, 45.97% and 52.00%, respectively. In
contrast, the percentage of apoptotic cells in the untreated control was 0.86%. These
results demonstrated that compound 15j could effectively inhibit cancer cell growth
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through apoptosis induction. The cell-cycle arrest caused by compound 15j in
HCT116 cells was determined by flow cytometry, using an equivalent volume of
DMSO as the control (Figure 6B). After treatment with 4, 12 and 36 nM compound
15j for 24 h, the percentage of cells in the G2 fraction increased from 17.73%,
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5.49% to 84.13%, with a concomitant decrease in the proportion of cells in other
phases of the cell cycle, suggesting G2 cell-cycle arrest in HCT116 cells.
Figure 6. The induction of apoptosis and cell-cycle arrest by compound 15j in
HCT116 cells. (A) The effects of compound 15j on the induction of apoptosis. The
cells were exposed to increasing concentrations of the test compound for 24 h and
analyzed by annexin V/propidium iodide double staining. The data are representative
of two independent experiments. (B) The effect of compound 15j on the cell cycle as
determined by flow cytometry. HCT116 cells were treated with compound 15j at 4,
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2 or 36 nM for 24 h. The data are representative of three independent experiments.
In Vivo Antitumor Efficacy. Inspired by the excellent in vitro antitumor activity
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of compound 15j, its in vivo antitumor efficacy was further evaluated in an HCT116
xenograft mouse model. However, after intraperitoneal (IP) administration of the test
compound for 21 consecutive days at a dose of 4 mg/kg, compound 15j only
exhibited moderate antitumor activity with a tumor growth inhibition (TGI) rate of
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7.0% (Figure 7A, P < 0.01). The test compound was well tolerated by the treated
mice, and no significant losses of body weight were observed (Figure 7B). In contrast,
TPT caused a significant loss of body weight at a dose of 0.5 mg/kg.
Although compound 15j had excellent growth inhibitory activity against the
HCT116 cell line (IC = 2 nM), its marginal in vivo antitumor efficacy may be due to
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its poor aqueous solubility (1.5 g/mL at pH 7.4) under physiological conditions. To
address this limitation, we synthesized the quaternary ammonium salt of compound
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5j (compound 15js), which showed a 500-fold higher aqueous solubility (850.4
g/mL at pH 7.4). Then, the in vivo antitumor efficacy of compound 15js was

evaluated in the HCT116 xenograft mouse model. After IP administration on 21
consecutive days at a dose of 16 mg/kg, compound 15js exhibited remarkable
antitumor potency with a TGI rate of 66.6% (Figure 7C), which was comparable to
the clinical antitumor agent TPT (TGI = 68.5%). In addition, the body weights of the
mice in the three groups were recorded to evaluate the toxicity of the test compounds.
As shown in Figure 7D, compound 15js had little effect on the body weight of the
treated mice, suggesting that its toxicity was significantly lower than that of TPT.
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Figure 7. Antitumor efficacies of compounds 15j and 15js in the xenograft model. (A)
The efficacy of compound 15j in the HCT116 xenograft model at a dose of 4 mg/kg.
(B) The change in the body weight of nude mice treated with compound 15j. (C) The
efficacy of compound 15js in the HCT116 xenograft model at a dose of 16 mg/kg. (D)
The change in the body weight of nude mice treated with compound 15js. Data are
presented as the mean  SEM; n = 6 nude mice per group: (*) P < 0.05, (**) P < 0.01,
(***) P < 0.001 versus the control group, determined with Student’s t test.
CONCLUSION
In summary, a novel indolopyrazinoquinazolinone antitumor scaffold was
identified by scaffold hopping from evodiamine. Systematic structural optimization
and SAR studies led to the discovery of a highly active indolopyrazinoquinazolinone
derivative (15j) with low nanomolar inhibitory activity against the HCT116 cell line.
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Further mechanistic studies revealed that it acted by the dual inhibition of Top1 and
tubulin. Compound 15js (the quaternary ammonium salt of 15j) exhibited excellent in
vivo antitumor activity, representing a promising lead compound for the development
of new antitumor agents. Additionally, scaffold hopping was shown to be an effective
approach to improve the druggability of evodiamine. Further structural optimization
and antitumor mechanism studies are currently underway in our laboratories.
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EXPERIMENTAL SECTION
Chemistry. General Methods. H NMR and 13C NMR spectra were recorded on
Bruker AVANCE300 or AVANCE600 spectrometers (Bruker Company, Germany)
using TMS as an internal standard and DMSO-d or CDCl as the solvent. Chemical
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shifts are given in ppm (δ). The mass spectra were recorded on an Esquire 3000
LC-MS mass spectrometer. Silica gel thin-layer chromatography separations were
performed on precoated plates GF-254 (Qingdao Haiyang Chemical, China). All
solvents and reagents were analytically pure, and no further purification was needed.
All starting materials were commercially available. The purity of each compound was
analyzed by HPLC (Agilent Technologies 1260 Infinity) using MeOH/H O as the
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mobile phase at a flow rate of 0.6 mL/min on a C18 column (Agilent 20RBA ×
SB-C18, 5 μm, 4.6 mm × 150 mm). All final compounds exhibited purities greater
than 95%.
Ethyl 1-(cyanomethyl)-5-methoxy-1H-indole-2-carboxylate (7a). To a solution of
intermediate 5a (5.00 g, 25.90 mmol) in EtOH (80 mL) was slowly added
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concentrated sulfuric acid (4 mL), and the mixture was refluxed for 12 h. After the
reaction, the solution was cooled to room temperature, and the resulting brown solid
was separated by filtration and washed with water to afford crude intermediate 6a,
which can be directly used in the next step without further purification. Then, 6a was
dissolved in DMF (40 mL), and NaH (60% dispersion in mineral oil, 1.10 g, 27.40
mmol) was slowly added at 0 °C. After the reaction was stirred at 0 °C for 30 min,
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-bromoacetonitrile (4.38 g, 36.50 mmol) was added in one portion. The mixture was
stirred at room temperature for 12 h and then poured into ice-water (250 mL). The
aqueous layer was extracted with ethyl acetate (3 × 150 mL), and the combined
organic layers were washed with brine, dried over sodium sulfate and concentrated
under reduced pressure. The residue was purified by column chromatography on silica
gel (PE: EtOAc = 10: 1) to afford intermediate 7a (4.2 g, yield 62% over two steps) as
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a white solid. H NMR (600 MHz, DMSO-d ) δ: 8.09 (s, 1H), 7.44 (d, J = 9.0 Hz,
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1H), 7.13 (s, 1H), 6.94 (m, 2H), 6.15 (t, J = 5.8 Hz, 2H), 3.76 (s, 3H), 3.62 (m, 1H);
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C NMR (150 MHz, CDCl ) δ: 161.82, 155.57, 134.10, 126.97, 126.86, 117.62,
3
114.96, 112.12, 110.51, 103.43, 61.13, 55.75, 32.44, 14.29; HRMS (ESI, positive)
+
m/z calcd for C H N O [M + H] : 259.1077; found 259.1076.
1
4
15
2
3
8
-Methoxy-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one (8a). Intermediate 7a (2.00
g, 7.70 mmol) and CoCl (2.00 g, 15.40 mmol) were dissolved in dry MeOH (30 mL)
2
and THF (15 mL), and NaBH (1.85 g, 77.00 mmol) was slowly added in an ice bath.
4
After the mixture was heated to 60 °C for 4 h, the mixture was filtered through a plug
of Celite and washed with DCM. Then, the filtrate was concentrated and purified by
2
9
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