Synthesis and biological evaluation of novel benzylidene-succinimide derivatives as noncytotoxic antiangiogenic inhibitors with anticolorectal cancer activity in vivo

Article abstract of DOI:10.1016/j.ejmech.2019.06.094

A novel series of benzylidene-succinimide derivatives were synthesized, characterized and evaluated for their cytotoxicities against HCT116, and SW480 cancer cells and NCM460 normal human cells. Their antiangiogenic capabilities were evaluated using a chick chorioallantoic membrane (CAM) assay. The compound, XCF-37b, was selected as the most potent antiangiogenic inhibitor with noncytotoxicity to evaluate the pharmacological effects on human umbilical vein endothelial cells (HUVECs) and cancer cells in vivo and in vitro. The results showed that XCF-37b inhibited HT29-cell colon tumor growth in vivo, without showing cytotoxicity against the five other cancer cell lines in vitro. Experiments confirmed that XCF-37b had obvious antiangiogenic activity by HUVEC migration and invasion and rat aortic ring angiogenesis ex vivo. Mechanism studies showed that XCF-37b inhibited the AKT/mTOR and VEGFR2 signaling pathways, as evidenced by decreased expressions of phosphor-AKT (p-AKT), p-mTOR, p-VEGFR2 (Tyr175), p-Src (Tyr416), p-FAK (Tyr925), and p-Erk1/2 (Thr202/Tyr204). Moreover, XCF-37b significantly decreased the protein expressions of matrix metalloproteinase-2 (MMP-2), MMP-9 and hypoxia-inducible factor-1α (HIF-1α). XCF-37b generally regulated angiogenic inhibition through several regulatory pathways, without significantly interfering with colorectal cancer cell growth.

Full text of DOI:10.1016/j.ejmech.2019.06.094

Accepted Manuscript
Synthesis and biological evaluation of novel benzylidene-succinimide derivatives as
noncytotoxic antiangiogenic inhibitors with anticolorectal cancer activity in vivo
Kaixiu Luo, Yafeng Bao, Feifei Liu, Chuanfan Xiao, Ke Li, Conghai Zhang, Rong
Huang, Jun Lin, Jihong Zhang, Yi Jin
PII:
S0223-5234(19)30621-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.094
EJMECH 11497
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 April 2019
Revised Date: 5 June 2019
Accepted Date: 30 June 2019
Please cite this article as: K. Luo, Y. Bao, F. Liu, C. Xiao, K. Li, C. Zhang, R. Huang, J. Lin, J. Zhang, Y.
Jin, Synthesis and biological evaluation of novel benzylidene-succinimide derivatives as noncytotoxic
antiangiogenic inhibitors with anticolorectal cancer activity in vivo, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.094.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Abstract
Synthesis and biological evaluation of novel benzylidene-succinimide derivatives
as non-cytotoxic anti-angiogenic inhibitors with anti-colorectal cancer activity in
vivo
Kaixiu Luo,‡a Yafeng Bao,‡b Feifei Liu, ‡b Chuanfan Xiao,‡a , Ke Li,c Conghai Zhang,a Rong
Huang,a Jun Lin,*a Jihong Zhang*b and Yi Jin*a

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel benzylidene-succinimide derivatives
1
2
3
as noncytotoxic antiangiogenic inhibitors with anticolorectal cancer activity in
vivo
4
5
6
Kaixiu Luo,‡a Yafeng Bao,‡b Feifei Liu, ‡b Chuanfan Xiao,‡a , Ke Li,c Conghai Zhang,a Rong
Huang,a Jun Lin,*a Jihong Zhang*b and Yi Jin*a
7
8
a. Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry Education and Yunnan
Province, School of Chemical Science and Technology, Yunnan University, Kunming, 650091, P.
R. China.
9
10
11
12
13
14
15
16
b. Laboratory of Molecular Genetics of Aging and Tumor, Medical School, Kunming University of
Science and Technology, Kunming, 650500, P. R. China.
c. Biomedical Department, Yunnan Cancer Hospital, the Third Affiliated Hospital of Kunming
Medical University, Kunming, 650118, P. R. China.
‡ the authors contributed equally to this work.
17
18
19
20
* Corresponding author. Tel./fax: +86-871-6503-3119. E-mail: linjun@ynu.edu.cn (J. Lin);
zhjihong2000@126.com (JH. Zhang); jinyi@ynu.edu.cn (Y. Jin).
21
22
23
Keywords: arylmethylene succinimide; angiogenesis; colon cancer; noncytotoxic; mTOR; matrix
metalloproteinase; hypoxia-inducible factor-1α.
24
25
26
27
28
1

ACCEPTED MANUSCRIPT
29
Abstract
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
A novel series of benzylidene-succinimide derivatives were synthesized, characterized and
evaluated for their cytotoxicities against HCT116, and SW480 cancer cells and NCM460 normal
human cells. Their antiangiogenic capabilities were evaluated using a chick chorioallantoic
membrane (CAM) assay. The compound, XCF-37b, was selected as the most potent antiangiogenic
inhibitor with noncytotoxicity to evaluate the pharmacological effects on human umbilical vein
endothelial cells (HUVECs) and cancer cells in vivo and in vitro. The results showed that XCF-37b
inhibited HT29-cell colon tumor growth in vivo, without showing cytotoxicity against the five other
cancer cell lines in vitro. Experiments confirmed that XCF-37b had obvious antiangiogenic activity
by HUVEC migration and invasion and rat aortic ring angiogenesis ex vivo. Mechanism studies
showed that XCF-37b inhibited the AKT/mTOR and VEGFR2 signaling pathways, as evidenced
by decreased expressions of phosphor-AKT (p-AKT), p-mTOR, p-VEGFR2 (Tyr175), p-Src
(Tyr416), p-FAK (Tyr925), and p-Erk1/2 (Thr202/Tyr204). Moreover, XCF-37b significantly
decreased the protein expressions of matrix metalloproteinase-2 (MMP-2), MMP-9 and
hypoxia-inducible factor-1α (HIF-1α). XCF-37b generally regulated angiogenic inhibition through
several regulatory pathways, without significantly interfering with colorectal cancer cell growth.
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
2

ACCEPTED MANUSCRIPT
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
1. Introduction
Colorectal cancer (CRC) is ranked as one of the three most common cancers and one of the
three leading causes of cancer-related deaths worldwide.1-3 Despite the developments in clinical
oncology in recent decades, CRC recurrence and chemotherapy resistance remain critical
problems in effectively treating patients with CRC.4, 5 Because approximately 50% of patients
do not respond to CRC treatment, new therapeutic medications are urgently needed to improve
the curative efficacy and decrease the toxicity of ineffective treatments in patients with CRC.6
Several mechanisms have been proposed as chemotherapeutic strategies for treating
patients with CRC, including cytotoxic chemotherapy and targeted therapies across multiple
lines of treatment,7 clearance of cancer stem cells,8 epigenetic modification,9 and CRC
antiangiogenesis.10, 11 Tumor angiogenesis plays a crucial role in regulating invasion and
metastasis.12, 13 Dozens of proangiogenic factors have been identified as potential targets for
developing angiogenic inhibitors. Reducing the expressions of angiogenesis-related proteins,
such as the vascular endothelial growth factor (VEGF) family proteins, has been demonstrated
to significantly suppress endothelial survival, mitogenesis, differentiation, migration, and
vascular permeability14-16 and to decrease the expressions of the metastasis-associated proteins,
matrix metalloproteinase-2 (MMP-2) and MMP-9.17 In addition, VEGF receptor and several
signaling cascades, such as Ras-MAPK, Scr-FAK, and AKT-mTOR, dominate in tumor
initiation and progression and regulate essential cellular functions, including survival,
proliferation and angiogenesis.18, 19 These pathways are also abnormal in many cancers,
including CRC.20, 21 For example, blocking AKT-mTOR overactivity has antitumor effects on
colon cancer cells.22 Moreover, hypoxia-inducible factor-1α (HIF-1α) is a transcriptional factor
controlling genes involved in glycolysis, angiogenesis, invasion, and metastasis of tumor
progression.23 Under hypoxic conditions, the stabilized HIF-1α/HIF-1β dimer translocates into
the nucleus to activate the transcription of target genes including VEGF;24 thus, regulating the
nuclear translocation of HIF-1α effectively inhibits tumor angiogenesis. However, current
antiangiogenic clinical medications, such as ramucirumab, sorafenib, and regorafenib, still have
limitations in antiangiogenic therapies including cytotoxicity, drug resistance, reduced drug
delivery, increased hypoxia, and tumor metastasis. Treating malignant tumors effectively with
low toxicity remains a major challenge and is urgently needed. The original concept of
3

ACCEPTED MANUSCRIPT
95
96
antiangiogenesis therapy involved “tumor starvation” via reduced vascularization as the primary
mechanism.12 Therefore, to inhibit tumor angiogenesis without producing cytotoxicity, novel
angiogenic inhibitors must be developed.25-28 Herein, we designed and synthesized a novel
series of benzylidene-succinimide derivatives, which showed obvious antiangiogenic activity
upon biological evaluation. These compounds had no obvious cytotoxicity on several cancer
cell lines and normal human colon epithelial cells.
97
98
99
100
101
102
103
104
105
106
107
108
109
2. Results and discussion
2.1. Compound design strategy
Michael conjugate addition is an effective method of elaborating interactions between drugs
and receptor proteins.29-31 Maleimides are excellent Michael acceptors, which usually form
succinimide derivatives. Substances bearing a succinimide fragment have been found in many
natural products and in some candidate drugs (Fig. 1), such as moiramide B and andrimide, which
are highly specific antibiotics,32 longimide A/B, which has antitumor activity,33 and GNX-686,
which has antiangiogenic properties.34
110
111
Fig. 1. Representative bioactive compounds bearing a succinimide fragment
112
113
114
115
116
117
118
119
Michael addition of thiols or amines to endocyclic C=C bonds of maleimides is the
site-specific drug-protein bioconjugation under physiological conditions for several marketed drugs
(Fig. 2a, route i).35 However, this methodology has a major drawback in that the resulting
thio-succinimidyl adducts 3 are prone to involving an exchange reaction with more active
nucleophilic substrates (e.g., cysteine residues of the proteins in vivo; Fig. 2a, route ii).36-39 To
overcome the instabilities of the adducts 3, a method was developed to form relatively stable
ring-opened thiol adducts via hydrolysis under a positive environment (Fig. 2a, route iii).36, 37
However, this approach is not an ideal application because it introduces an additional negative
4

ACCEPTED MANUSCRIPT
120
121
122
123
124
125
126
127
128
129
130
131
132
charge in the ring-opened products (6 and 7), which may make it difficult for cellular internalization
of cell-penetrating medications. Consequently, developing stable Michael-adduct-forming
maleimide derivatives is highly attractive to pharmaceutical chemists. Recently, Kalia et al.
reported that exocyclic olefinic maleimides can form highly stable thio-Michael adducts that resist
thiol exchange under physiological conditions (Fig. 2b).40 Although some active molecules bearing
a benzylidene-succinimide unit show potent GPR119 agonistic activity,41 inhibit DDX and HIV-1
replication,42 and have oral activity for treating induced acute liver injury,43 to our knowledge,
benzylidene-succinimide derivatives remain unreported in tumor antiangiogenesis applications.
Thus, after previously exploring potential angiogenic inhibitors,44-46 we aimed to design
benzylidene-succinimide derivatives with potent antiangiogenic activity and antitumor properties in
vivo. As many of the marketed antiangiogenic medications have a terminal amide modification (e.g.,
sorafenib,47 regorafenib,48 and sunitinib49), we reasoned that a benzylidene-succinimide fragment
bearing a terminal amide unit (Fig. 2c) would possess this pharmacological attribute.
133
134
Fig 2. a) Conventional methodology for bioconjugation between a maleimide and a protein using endocyclic
135
136
olefinic maleimide. b) Kalia’s approach using exocyclic olefinic maleimides. c) Molecular design for the current
work.
137
2.2. Chemistry
5

ACCEPTED MANUSCRIPT
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
Scheme 1 illustrates the general synthetic approach for benzylidene-succinimide derivatives
(h). First, 4-(arylamino)but-2-enoic acid (c) was prepared using maleic anhydride and amine in
acetone or ethyl acetate solvent with a yield of over 97%. To prevent self-polymerization of the
intermediate (c), the reaction rate and temperature were controlled by slowly adding substituted
aniline in an ice bath. The intermediate (c) then underwent the condensation ring-closure reaction
catalyzed by triethylamine in an acetic anhydride solvent to form N-substituted maleimide. To
obtain the optimum yield in this step, we investigated the reaction conditions at different dosage
ratios of 4-(phenylamino)but-2-enoic acid (c1), acetic anhydride and triethylamine at different
temperatures. The results showed that the optimum yield (over 90%) could be obtained at a mixing
(molar) ratio of 1:6:0.5 at 50°C for the three substrates. Referring to Kalia’s method,40 the catalytic
condition of 4-nitrophenole and 1,4-addition of PPh3 to N-substituted maleimide (d) followed by the
Wittig
reaction
with
3-carboxybenzaldehyde
(e)
can
obtain
the
3-carboxybenzylidene-N-substituted-succinimide (f) at room temperature with good yields of 75–
95%. The double bond in the products was assigned to E configuration by NOE analysis (Fig. 1S,
SI).50, 51 In this step, we screened the reaction effect in different solvents and found that the reaction
yields in non-proton solvents (e.g., DCM, THF or toluene) were not ideal, but in proton solvents
(e.g., EtOH or MeOH), the yields could reach over 90%. Finally, the target products (g) were
prepared by a condensation reaction of intermediate (f) and alkyl/arylamine. Because the
intermediate is poorly soluble (f), the effects of different solvent combinations (e.g., DMF+DCM or
DMF+MeOH) and condensation agents (e.g., HOBT+EDCI or HOBT+TBTU) were investigated.
The experimental results showed that the benzylidene-succinimide derivatives (g) can be obtained
in medium-to-high yield (50–90%) at room temperature using a mixed solvent (DMF:DCM=1:5)
and HOBT+TBTU (molar ratio = 1:1) as the condensation reagent.
161
Scheme 1. General preparation methods for 2-aryl benzoxazoles derivatives.
162
6

ACCEPTED MANUSCRIPT
163
164
2.3 In vitro cytotoxicity assay
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
We first synthesized twenty 3-amidobenzylidene-N-phenyl-succinimide compounds (Table 1, g1–
g20) with different amide substituents, which were evaluated for cell growth cytotoxicities against
two colon cancer cell lines (HCT116 and SW480) using the standard MTT assay, with cisplatin as
the reference control. Neither the alkyl amides (g5–g8) nor most aryl amides exhibited obvious
cytotoxicities against the growth of both cancer cell types. However, when the substituents of
arylamino were 4-ethoxy aniline, 3-(methylthio)aniline, or 3-ethylaniline, respectively, compounds
g13 (IC50= 84.83µM and 91.45 µM), g15 (IC50= 65.23 µM and 74.29 µM) and g20 (IC50= 81.21µM
and 88.64 µM) showed inhibition in both HCT116 and SW480 cells.
To investigate the effect of different N-substituted succinimides on inhibitory activities, three
N-substituted succinimide compounds (N-p-tolyl g21–g24, N-butyl g25–g29, and N-benzyl g30–
g33) were prepared (Table 2, g21–g33). The in vitro assay results showed that most of the
compounds with these three N-substituents had no significant cytotoxicities. Notably, the
compounds bearing 4-ethoxy aniline or a 3-ethylaniline group in the arylamino moiety (g23, g25,
g31, and g32) exhibited cytotoxicities. We also prepared the hydrogenated product (g34) of
compound g24, which showed no cytotoxicity against these cell lines.
Finally, we evaluated the cytotoxicities of the synthesized compounds on the NCM460 cell line
(normal human colon epithelial cells). No compounds showed cytotoxicities against this cell line,
confirming the feasibility of designing the benzylidene-succinimide derivatives as noncytotoxic
antiangiogenic inhibitors.
7

ACCEPTED MANUSCRIPT
184
Table 1. Cytotoxic effects in vitro of synthesized compounds g1-g20.
a
in vitro anti-proliferative effects (IC50, µM)
Cpd
Structure
HCT116
5.59±0.23
SW480
NCM460
Control
Cisplatin (DDP)
10.02±0.37
17.10±0.30
g1
g2
g3
g4
g5
g6
g7
g8
g9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8

ACCEPTED MANUSCRIPT
g10
g11
g12
g13
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
84.83±3.65
91.45±2.18
g14
>100
>100
>100
g15
g16
g17
g18
g19
g20
65.23±1.85
74.29±0.87
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
81.21±2.75
88.64±1.82
9

ACCEPTED MANUSCRIPT
185
186
187
a IC50 values are presented as the mean values of at least three independent experiments.
Table 2. Cytotoxic effects in vitro of synthesized compounds g21-g33.
a
in vitro anti-proliferative effects (IC50, µM)
Cpd
Structure
HCT116
5.31±0.28
SW480
NCM460
14.37±0.42
Contral
Cisplatin (DDP)
9.54±0.17
g21
g22
g23
>100
>100
96.85±3.57
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
41.53±2.73
65.34±3.68
92.98±4.62
83.25±2.73
>100
g24
>100
(XCF-37b)
g25
g26
g27
g28
g29
75.32±3.94
>100
>100
>100
>100
>100
>100
>100
10

ACCEPTED MANUSCRIPT
g30
g31
g32
g33
g34b
95.05±4.27
>100
91.48±4.18
73.21±3.27
>100
>100
>100
>100
>100
>100
80.60±2.69
41.70±1.74
>100
>100
>100
188
189
a IC50 values are presented as the mean values of at least three independent experiments.
b The compound was prepared by double-bond hydrogenation of XCF-37b (see experimental section).
190
191
192
193
194
195
196
197
198
199
200
201
202
203
2.4 Antiangiogenesis screening via chick chorioallantoic membrane (CAM) assay
The CAM assay, an inexpensive, reliable, simple, and convenient biological method, was selected
to initially evaluate the synthesized compounds for their angiogenic inhibitory effects. First,
compounds g1–g20 were screened to study the extent of new blood vessel formation, which
represents the antiangiogenic strength. Fig. 3a shows the inhibitory effects compared with normal
saline (the blank control) and sunitinib (the positive control). The antiangiogenic activities of the
compounds were semiquantitatively analyzed using GraphPad Prism 5.0 (Fig. 3b). The results
showed that the five compounds g4–g9 exhibited some antiangiogenic activities, but none were as
active as the positive control. Subsequently, the compounds g21–g34 were screened using the CAM
assay (Fig. 4a). The results showed that g24 (XCF-37b) and g29 exhibited the same antiangiogenic
activities as sunitinib. Exposure to XCF-37b drastically impaired neovascularization with the
absence of vascular networks (Fig. 4b). Quantitative analysis revealed that 20 nmol XCF-37b
reduced the number of blood vessels by 96.4% compared with the saline control. These results
confirmed the antiangiogenic potential of XCF-37b through an ex vivo assay. An in vivo mouse
11

ACCEPTED MANUSCRIPT
204
205
206
207
208
Matrigel plug model of angiogenesis was compared with the control. VEGF-supplemented
Matrigels appeared red, indicating the formation of a functional vasculature inside them; however,
with XCF-37b treatment, the red color in the Matrigels diminished (Fig. 4c). Intriguingly, the
hydrogenated product g34 of XCF-37b showed moderate antiangiogenic activity. This result
implied that the double bond of benzylidene-succinimide contributed to antiangiogenic effect.
209
210
211
Fig. 3. a) Effect of compounds g1–g16 on CAM blood vessel growth (10 nM/eggs). b) Quantification of the effect
of compounds g1–g20 on tube formation shown in the bar graph. The bars show the mean ± SD (n=3) values.
212
12

ACCEPTED MANUSCRIPT
213
214
215
216
217
218
219
220
Fig. 4. a) Effect of compounds g21ꢀg34 on CAM blood vessel growth (10 nM/eggs). b) Quantification of the
effect of compounds g21–g34 on tube formation shown in the bar graph. The bars show the mean ± SD (n=3). c)
In vivo angiogenesis assay, Matrigel alone or with VEGF indicated concentrations of compounds were injected
into the flanks of Balb/c nude mice, and 15 days later, the mice were sacrificed, and the Matrigel plugs were
removed, weighed and photographed.
221
222
2.5 XCF-37b inhibited HUVEC and other cancer cell proliferation
13

ACCEPTED MANUSCRIPT
223
224
225
226
227
228
229
230
231
232
Suppression of HUVEC growth and proliferation is another critical aspect of angiogenesis. To
assess the antiangiogenic activity of XCF-37b in vitro, the inhibitory effects on VEGF-induced
endothelial cell proliferation were evaluated using an MTT assay. VEGF plays an important role in
neoangiogenesis in endothelial cells.52 XCF-37b inhibited HUVEC cell proliferation
concentration-dependently. Moreover, XCF-37b significantly inhibited VEGF-induced
proliferation of HUVECs with an IC50 value of 8.56 µM compared with those without VEGF
stimulation (IC50 = 26.58 µM; Table 3).
To determine whether XCF-37b possesses any cytotoxicity against other human cancer cell lines, a
growth inhibition assay was performed, and the results showed IC50 values >100 µM in HCT116,
SW480, HT29, PC-3 and HepG2 cells (Table 3).
233
234
Table 3. In vitro antiproliferative effects of compound XCF-37b. a
Compound
Sunitinib
DDP
HUVEC
6.46±0.23
NT
HUVEC+VEGF
3.63±0.24
NT
HCT116
NT
SW480
NT
HT29
NT
PC-3
NT
HepG2
NT
7.062±1.786 9.338±1.544 7.23±0.155 6.04±0.586 2.79±0.226
>100 92.985±0.325 >100 >100 >100
XCF-37b 26.58±2.17
8.56±2.0
235
236
a IC50 values are presented as the mean values from at least three independent experiments.
237
238
239
240
241
242
243
244
245
246
247
248
2.6 XCF-37b suppressed HUVEC migration and invasion
Endothelial cell migration and invasion are key events in angiogenesis. To investigate the effect
of XCF-37b in endothelial cell migration, a wound healing migration assay was performed.
HUVEC cells were wounded with tips, followed by a 12-h starvation, then treated with XCF-37b (1
µM and 2.5 µM) for 6, 12, and 24 h. The control cells nearly completely migrated to fill the initial
clear area after 24 h, while XCF-37b treatment significantly reduced VEGF-induced HUVEC cell
migration both dose- and time-dependently (Figure 5b). XCF-37b (2.5 µM) suppressed HUVEC
migration by 30%, 50% and 60% at 6, 12, and 24 h, respectively, compared with the control.
Similarly, XCF-37b also induced dose-dependent suppression of the VEGF-induced invasion of
HUVEC cells (Figure 5a). XCF-37b treatment at 1 µM, 5 µM, 10 µM and 20 µM suppressed
HUVEC cell invasion by 20%, 40%, 50% and 70%, respectively. This study demonstrated that
XCF-37b suppressed angiogenesis by inhibiting endothelial cell migration and invasion.
14

ACCEPTED MANUSCRIPT
249
250
Fig. 5 XCF-37b inhibited HUVEC proliferation, migration, and invasion.
251
252
253
254
255
256
257
258
259
260
261
262
At the later stage of angiogenesis, endothelial cells rearrange themselves into tubes to form
capillaries. To further determine the antiangiogenic effects of XCF-37b on VEGF-induced
endothelial cell capillary-like structure formation, a two-dimensional Matrigel tube formation assay
was applied. HUVECs seeded on the Matrigel surface became elongated and formed capillary-like
structures, and VEGF significantly enhanced the capillary-like network. However, XCF-37b
concentration-dependently abrogated the capillary-like network formation. We observed that 1 µM,
2.5 µM and 5 µM of XCF-37b treatment inhibited HUVEC capillary tube formation by 30%, 73%,
and 80%, respectively, after 6 h compared with the VEGF control. We investigated the effects of
XCF-37b on microvessel sprouting from vascular tissues using an ex vivo rat aortic ring assay.
Microvessels emerging from cultured rat aorta embedded in the Matrigel mimicked several stages
of angiogenesis, including endothelial proliferation, migration and tube formation. As Fig. 6a
shows, VEGF significantly initiated microvessel sprouting and formed a complex microvessel
15

ACCEPTED MANUSCRIPT
263
264
265
266
267
268
269
270
network around the aortic rings. However, treatment with XCF-37b at 1 µM, 5 µM, 10 µM, or 20
µM dramatically dose-dependently suppressed capillary sprouting, with 95% inhibition observed at
20 µM, suggesting that XCF-37b suppresses VEGF-induced angiogenesis ex vivo. In addition,
XCF-37b at 1–5 µM alone had no obvious cytotoxicity on HUVECs after incubation with cells for
48 h. The results implied that the inhibition of VEGF-induced formation in the cells was not due to
cytotoxicity (Fig. 6b).
Formation of the VEGF-induced elongated and robust tube-like structures in the HUVEC cells was
concentration-dependently abrogated by XCF-37b.
271
272
Fig. 6 XCF-37b inhibited ex vivo and in vivo angiogenesis. a) In the rat aortic ring assay, rat aortic ring sections
were embedded in Matrigel and cultured in the opti-MEM medium. After 1 day of starvation, treatments were
started and continued with the indicated compound concentrations every 3 days for 7 days. On day 7, images were
taken using inverse microscopy. b) Using a tube-formation assay, HUVECs suspended in M200 medium with 10%
FBS were seeded in solidified Matrigel and incubated with test compounds for 6 h. The cells were then imaged.
273
274
275
276
277
16

ACCEPTED MANUSCRIPT
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
2.7 Apoptotic effect of XCF-37b on HUVEC and cancer cells
To further determine whether the cytotoxic effect was attributable to the decreasing HUVEC
viability, annexin-V/PI analysis was performed to detect apoptosis in VEGF-stimulated HUVECs in
the presence of XCF-37b. Cells were treated with increasing concentrations of XCF-37b (1 µM, 5
µM, 10 µM, and 20 µM). As Fig. 7 shows, XCF-37b did not significantly alter the VEGF effects on
the percentage of apoptotic cells. We also detected the apoptotic protein marker PARP and
caspase-3 after XCF-37b treatment using Western blot. No PARP or caspase-3 cleavage was
observed with increasing concentrations of XCF-37b, suggesting that XCF-37b exerts
antiproliferative activity without causing cytotoxic effects in HUVECs.
No apoptosis was observed after different concentrations of XCF-37b treatment (1 µM, 5 µM, 10
µM, and 20 µM) for 24 h. HT29 cells showed no apoptosis after XCF-37b (5 µM) exposure at 24,
48, and 72 h. Moreover, we observed no PARP or caspase-3 cleavage in the HT29 cells (Fig. 8).
No prominent apoptosis was observed after XCF-37b treatment in HUVEC or HT29 cells,
indicating that the XCF-37b-induced inhibitions of cell proliferation, migration, and invasion were
not cytotoxicity-related.
293
294
Fig. 7 Apoptosis of XCF-37b in HUVEC cells
17

ACCEPTED MANUSCRIPT
295
296
Fig. 8 Apoptosis of XCF-37b in HT29 cells
297
298
299
300
301
302
303
304
305
306
307
308
2.8 XCF-37b attenuated the VEGFR2 signaling pathway in HUVEC cells
VEGFR2 signaling is essential to vascular endothelial cell function. VEGFR2 binds with VEGF to
phosphorylate VEGFR2, which then activates various downstream signaling cascades, such as
MAPK/ERK, which is responsible for endothelial cell migration, proliferation, and survival. To
understand the molecular mechanisms of XCF-37b-mediated antiangiogenic properties, we
examined the pathways and signaling cascades using Western blot. As Fig. 9a shows, XCF-37b
concentration-dependently suppressed VEGFR2 phosphorylation in VEGF-stimulated HUVECs.
Dramatic downregulations of phospho-FAK (Tyr925), phospho-Src (Tyr416) phospho-AKT
(Ser473) and phospho-ERK (Thr202/Tyr204), phospho-STAT3 (Tyr705), which are downstream
targets of VEGFR2, were observed with 1, 5 and 10 µM of XCF-37b treatment. However, total
FAK, Src, AKT, and ERK STAT3 remained unchanged (Fig. 9a). The results revealed that
XCF-37b inhibited angiogenesis by directly targeting VEGF-stimulated VEGFR2 activation in
18

ACCEPTED MANUSCRIPT
309
310
311
312
313
314
315
316
317
318
319
endothelial cells and further suppressed VEGFR-2 downstream signaling pathways. AKT is a
serine/threonine kinase that plays an important role in cell growth, proliferation, migration, protein
synthesis, and angiogenesis. Activation of the AKT/mTOR/p70S6K pathway is required for
endothelial cell proliferation and migration. Next, we examined expression of the AKT-mTOR
signaling pathway after XCF-37b exposure and found that p-mTOR (Ser 2448) levels were reduced
together with p-AKT. XCF-37b dose-dependently decreased the levels P70S6K (Thr389) in
endothelial cells (Fig. 9b). These results showed that XCF-37b may inhibit endothelial cell
proliferation by downregulating the AKT/mTOR/p70S6K pathway.
These studies demonstrated that XCF-37b is a potent inhibitor of tumor angiogenesis in vitro and ex
vivo. XCF-37b inhibited VEGFR2 activation, thereby suppressing the AKT and ERK-mediated
angiogenic signaling pathways.
320
19

ACCEPTED MANUSCRIPT
321
322
323
324
325
Fig. 9 XCF-37b inhibited VEGFR2 signaling pathways in HUVEC cells. The cells were starved overnight, then
pretreated with the indicated concentrations of XCF-37b (1, 5, 10, and 20 µM) for 1.5 h, followed by the addition
of VEGF (50 ng/mL) for another 30 min. Cell pellets were collected for immunoblotting. a) Effect of XCF-37b on
VEGFR-2, Src, FAK, Erk1/1, and STAT3. b) Effect of XCF-37b on mTOR, AKT, and p70S6K. c, d, and e)
Quantitative evaluation of enzymatic activities after XCF-37b treatment on gelatin zymography.
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
2.9 XCF-37b blocked the AKT/mTOR/P70S6K pathway in HT 29 cells
AKT, mTOR, and STAT3 phosphorylation was significantly dose-dependently reduced in HT29
cancer cells (Fig. 10a).
HIF-1α, a transcription factor, is consistently overexpressed in various cancer cells and is closely
correlated with angiogenesis, tumor growth and metastasis53. VEGF is an HIF-1-responsive gene.
HIF-1α binds to the hypoxia-responsive element of the VEGF promoter and activates VEGF gene
expression at the transcriptional level54. VEGF and its family members are essential for inducing
tumor angiogenesis, and aberrant expression of VEGF is a key regulator in hypoxia-induced
angiogenesis55. Our study showed that inhibiting HIF activity reduced tumor growth. HIF-1α and
VEGF were dramatically decreased when concentration-dependently treated with XCF-37b in
HT29 cancer cells (Fig. 10b). HIF-1 is a key regulator of VEGF expression, suggesting that
XCF-37b decreased VEGF expression via blocking the HIF-1 pathway. Additional RT-PCR
analyses showed that XCF-37b did not reduce the VEGF mRNA levels; however, the HIF-1α
mRNA level was reduced concentration-dependently.
Cancer cells secrete various angiogenic factors that change the adjacent endothelial cells and
promote tumor angiogenesis, leading to tumor growth and progression56, 57. MMPs (especially
MMP-2/9) degrade the extracellular matrix and are critical for cancer cell movement. XCF-37b
treatment dose-dependently decreased the MMP-9 and MMP-2 expressions (Fig. 10c). Gelatin
zymography examination revealed that XCF-37b treatment significantly reduced the secreted MMP
2/9 activity.
The data indicated that XCF-37b showed distinct antiangiogenic activity against tumor migration
by downregulating MMP-9, MMP-2 activity, and the VEGF/HIF-1α signaling pathway.
20

ACCEPTED MANUSCRIPT
348
349
Fig. 10 XCF-37b inhibited VEGFR2 signaling pathways in HT29 cells. a) Effect of XCF-37b on AKT-mTOR
signaling, HT29 cells were treated with the indicated concentrations of XCF-37b for 24 h, and cell lysates were
subjected to immunoblotting. b) Effect of XCF-37b on HIF-α, VEGF and MMP-9. c) Effect of MMP-9 and
MMP-2. d, e) Quantitative evaluation of MMP-9, VEGF and HIF-1α enzymatic activities after XCF-37b
treatment on gelatin zymography.
350
351
352
353
354
355
356
357
358
359
360
361
2.10 In vitro kinase screening for XCF-37b
To investigate the potential protein target for the most active XCF-37b compound, XCF-37b’s
kinase inhibitory profile was tested at 10 µM over a panel of 28 oncogenic kinases at Kebai
Biotechnology Co., Ltd. (Nanjing, Jiangsu, China).58 Fig. 11 shows that XCF-37b displayed good
inhibitory activity (above 90%) against two kinases: VEGFR-2 (95.4%) and mTOR (93.8%).
Moreover, it exerted moderate inhibitory activity (50–70%) towards AKT1, ERK1, ERK2 and
P70S6K and modest activity against some other kinases with an inhibition percentage of <40%.
21

ACCEPTED MANUSCRIPT
362
363
Fig. 11 Inhibition percentages of compound XCF-37b over 28 oncogenic kinases at 10 µM. Data are mean (± SD)
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
for three independent experiments.
2.11 XCF-37b inhibited tumor growth and angiogenesis in xenograft murine models
To elucidate the antitumor effects of XCF-37b in vivo, HT29 colon xenograft murine models were
used. XCF-37b was administered o.p. every other day for 3 weeks. The results showed that
XCF-37b suppressed HT29 subcutaneous tumor growth (Fig. 11). The relative tumor volume in the
XCF-37b group (60 mg/kg) was reduced by 53.51%. XCF-37b treatment did not significantly
decrease the mouse body weight compared with that of the control. We further investigated the
angiogenic effect in the tumor via staining with the CD31 endothelial marker. XCF-37b treatment
markedly reduced the development of newly developed vessels in the tumors (Fig. 12). The mean
vessel density in tumors treated with XCF-37b was reduced to 5%, which is similar to the effect of
sunitinib. H&E staining showed that tumors exhibited large areas of late apoptotic and necrotic cells
after XCF-37b treatment. Consistent with the in vitro results, the p-mTOR levels were reduced in
the tumors after XCF-37b treatment. In addition, XCF-37b treatment reduced cell proliferation in
tumors as shown by Ki-67 staining. XCF-37b treatment caused an apoptotic effect in tumors on the
TUNEL assay. The results implied that XCF-37b suppressed tumorigenesis by targeting
angiogenesis.
22

ACCEPTED MANUSCRIPT
381
382
Fig. 12 XCF-37b inhibited tumor growth and neoangiogenesis in HT29 colon xenografts. a) In vivo tumor growth
inhibition and body weight. b) Immunohistological assessments of H&E, CD-31, Ki-67, p-mTOR, and
quantitative analysis; and c) Apoptotic effect of XCF-37b treatment in tumors via TUNEL assay
383
384
385
386
387
388
389
390
391
392
393
394
2.12 Molecular docking study
To rationalize the observed VEGFR-2 and mTOR kinase inhibitor activity from a three-dimensional
(3D) structural perspective, XCF-37b was docked in the kinase domain using the GoldDock 5.0
module of Discovery Studio 3.5. Fig. 13 illustrates the 3D-interaction model of XCF-37b with
VEGFR-2 and mTOR, while Supplementary Figs. 2S and 3S show the corresponding 2D
interaction model. The docking model demonstrates that XCF-37b was located deep inside the
extended hydrophobic pocket formed by the DFG out conformation of VEGFR-2 and the
ATP-binding site of mTOR. The binding energies using the protocol (MM-PBSA-BINDING
ENERGY) of XCF-37b inside VEGFR-2 and mTOR were -25.37 kJ/mol and -30.76 kJ/mol,
23

ACCEPTED MANUSCRIPT
395
396
397
398
399
400
401
402
403
404
405
406
407
respectively. In VEGFR-2, two hydrogen-bonding interactions were observed: in the hinge region
of the kinase, the amide oxygen of the benzamide moiety H-bonded to residue Lys868, and in the
adenine region, the amide oxygen of the succinimide scaffold formed H-bonds with residue Cys919.
Additionally, the N-phenyl ring of the succinimide was H-π stacked with residue Leu840, which
enhanced the binding interaction to VEGFR-2 kinase. The para-methoxy of the benzamide moiety
extended to a narrow hydrophobic subpocket of the allosteric site of VEGFR-2, which was
embraced by residues Leu1019, Asp1046, and Leu889. In mTOR, two hydrogen-bonding
interactions were observed: the amide oxygen of the benzamide moiety H-bonded to residue
Val2240, and the amide oxygen of the succinimide scaffold formed H-bonds with residue Asp2357.
Intriguingly, three H-π and one π-π interaction occurred between XCF-37b and mTOR kinase, in
which N-phenyl rings of the succinimide stacked with Asp2357, a double bond stacked with
Tyr2225, and the para-methoxy of the benzamide moiety stacked with Trp2239. These results
indicated that compound XCF-37b is a dual inhibitor of VEGFR-2 and mTOR kinases.
408
409
Fig. 13 Putative binding modes of compound XCF-37b in kinases were predicted by molecular modeling. The 3D
images were prepared with Accelrys Discovery Studio, and the dashed lines illustrate atom pairs within hydrogen
bonds; a) Binding mode of XCF-37b in VEGFR-2 kinase; b) Binding mode of XCF-37b in mTOR kinase.
410
411
412
413
414
3. Conclusions
In summary, a new class of benzylidene-succinimide derivatives was prepared and tested by
24

ACCEPTED MANUSCRIPT
415
416
417
418
419
420
421
422
423
assessing its potential to inhibit angiogenesis. The biological evaluation identified several
structurally distinct antiangiogenic inhibitors that caused no cytotoxic effects in HUVECs or
HCT116, SW480 and NCM460 cancer cells. Among the series compounds, XCF-37b suppressed
HUVEC migration and invasion and rat aortic ring angiogenesis ex vivo, possibly by suppressing
angiogenesis-associated factors. XCF-37b inhibited HT29 colon tumor growth in vivo. This
compound inhibited the AKT/mTOR and VEGFR2 signaling pathway, as evidenced by decreased
expressions of phosphor-AKT (p-AKT), p-mTOR, p-VEGFR2 (Tyr175), p-Src (Tyr416), p-FAK
(Tyr925), and p-Erk1/2 (Thr202/Tyr204). Moreover, XCF-37b significantly decreased the protein
expressions of MMP-2, MMP-9 and HIF-1α. These results will help further optimize and develop
new medication candidates for clinical studies as novel angiogenic inhibitors.
424
425
426
4. Experimental
427
428
4.1 Chemistry
All chemicals and reagents were of commercial grade and used without further purification.
429
430
431
432
433
434
435
436
437
438
439
The reactions were monitored by thin-layer chromatography (TLC) using silica gel GF254. Column
chromatography was performed with 200–300 mesh silica gel. All yields refer to isolated products
after purification. The intermediates and the products synthesized were fully characterized by
spectroscopic data. The nuclear magnetic resonance (NMR) spectra were recorded with a Bruker
DRX-500 NMR spectrometer (1H: 500 MHz, 13C: 125 MHz) using CDCl3 or DMSO-d6 as solvents.
Chemical shifts (δ) are expressed in parts per million (ppm), and J values are given in hertz (Hz). IR
spectra were recorded with a Fourier transform-infrared (FT-IR) Thermo Nicolet Avatar 360
spectroscope using a KBr pellet. High-resolution mass spectrometry (HRMS) was performed by
liquid chromatography/mass selective detector time-of-flight (LC/MSD TOF) using an Agilent
instrument. The melting points were measured using an XT-4A melting point apparatus without
correction.
440
441
442
443
444
445
446
4.1.1 Synthesis of N-substituted phenyl maleimide
Maleic anhydride (0.05 mol) was dissolved in 0.2 mol ethyl acetate and stirred in an ice bath.
After the maleic anhydride completely dissolved, 0.05 mol substituted aniline (compounds a) was
added to 0.2 mol ethyl acetate solution. The yellow solid was slowly precipitated in the reaction.
After stirring for 1 h in an ice bath, the resultant solution was filtered and washed. N-substituted
phenyl maleamic acid was obtained with a yield over 97%. Next, 0.05 mol maleamic acid was
25

ACCEPTED MANUSCRIPT
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
dissolved in 0.2 mol acetic anhydride while adding 0.025 mol of triethylamine. The solution was
heated and stirred in an oil bath at 55–65°C for 1 hour. When the reaction cooled, the resultant
solution was added to isovolumetric deionized water and left to stand overnight. The precipitated
yellow solid was filtered and washed. N-substituted phenyl maleimide was obtained with a yield of
over 88%.
4.1.2 Synthesis of N-butyl maleimide and N-benzyl maleimide
Maleic anhydride (60 g; 0.61 mol) and 90 mL acetone were mixed in a 500-mL four-port bottle.
A mixture of butylamine 57 mL (0.58 mol) or benzylamine 64 mL (0.58 mol) in acetone 90 mL was
then added slowly into the solution under nitrogenous conditions and rapidly agitated. After the
titration was complete, the reaction was stirred for 1 h. Next, 85 mL of acetic anhydride, 36 mL of
triethylamine, and 1.8 mL of 1:6 nickel sulfate aqueous solution were added to the reaction system.
The reaction temperature was raised to 70°C, and the reflux reaction lasted 4 hours. After cooling to
room temperature, the reactants were washed to near neutral using water, saturated Na2CO3 solution
and water again. After drying and distillation, the product yield was over 88%.
4.1.3 General procedure for synthesizing N-substituted benzylidene pyrrolidine dione derivatives
(compounds f)
N-substituted maleimide (0.014 mol; compound d) was dissolved in 40 mL alcohol and stirred.
After N-substituted maleimide was completely dissolved, 0.012 mol triphenylphosphine was added
to the reaction for 4–5 minutes. Next, 3-carboxybenzaldehyde (0.01 mol) was added rapidly and
stirred at room temperature for 6 hours. White solids were precipitated, filtered and washed. The
product was obtained, and the yield was over 90%.
4.1.4 General procedure for synthesizing benzylidene-succinimide derivatives (compounds
g1-g33)
N-substituted benzylidene pyrrolidine dione (0.5 mol), 0.6 mmol TBTU, and 0.6 mmol EDCI were
dissolved in 2 mL DCM and 0.75 mmol DIEA mixture solution and stirred for 30 minutes. Next, 0.5
mol of amine or methylamino acid in DCM (8 mL) solution was slowly added to the solution and
stirred at room temperature for 4–10 hours with the TLC monitoring reaction. After the reaction was
26

ACCEPTED MANUSCRIPT
477
478
479
480
481
482
complete, the resultant solution was washed with dilute hydrochloric acid and saturated sodium
bicarbonate solution, then dried with anhydrous sodium sulfate, followed by column
chromatography separation. The target product was obtained by crystallization. The yield ranged
from 72–90%.
4.1.5 Spectral data of compounds g1-g33
483
484
485
486
487
488
489
490
491
3-((2,5-Dioxo-1-phenylpyrrolidin-3-ylidene)methyl)-N-phenylbenzamide (g1)
Yellow solid; M.p. 231-232 °C; 1H NMR (500 MHz, DMSO-d6) δ = 10.38 (s, 1H, HN), 8.23 (s, 1H,
HAr2), 8.03 (d, J = 8.1 Hz, 1H, HAr2), 7.94 (d, J = 7.8 Hz, 1H, HAr2), 7.82 (d, J = 7.2 Hz, 2H, HAr),
7.73 – 7.63 (m, 2H, HAr and C=CH), 7.54 (t, J = 7.6 Hz, 2H, HAr), 7.46 (t, J = 7.4 Hz, 1H, HAr),
7.43 – 7.34 (m, 4H, HAr), 7.14 (t, J = 7.4 Hz, 1H, HAr), 3.98 (d, J = 2.4 Hz, 2H, CH2). 13C NMR
(126 MHz, DMSO-d6) δ = 173.77, 170.22, 165.41, 139.42, 136.16, 134.61, 133.28, 132.95, 132.24,
129.75, 129.62, 129.40, 129.21, 129.05, 128.66, 127.50, 126.76, 124.23, 120.84, 34.45; HRMS
(TOF ESI+): m/z calcd for C24H18N2O3 [M+Na]+, 405.1210, found, 405.1211.
492
493
3-((2,5-Dioxo-1-phenylpyrrolidin-3-ylidene)methyl)-N-(4-fluorophenyl)benzamide (g2)
494
495
496
497
498
499
500
501
Yellow solid; M.p. 235-236 °C; 1H NMR (500 MHz, DMSO-d6) δ = 10.43 (s, 1H, HN), 8.22 (d, J =
2.1 Hz, 1H, HAr2), 8.03 (d, J = 7.7 Hz, 1H, HAr2), 7.93 (d, J = 7.8 Hz, 1H, HAr2), 7.87 – 7.79 (m,
2H, HAr), 7.72 – 7.64 (m, 2H, HAr and C=CH), 7.54 (t, J = 7.6 Hz, 2H, HAr), 7.49 – 7.43 (m, 1H,
HAr), 7.40 (dd, J = 7.8, 1.6 Hz, 2H, HAr), 7.28 – 7.18 (m, 2H, HAr), 3.98 (d, J = 2.4 Hz, 2H, CH2);
13C NMR (125 MHz, DMSO-d6): δ = 173.72, 170.17, 165.32, 159.76, 157.85, 135.99, 135.76,
134.63, 133.29, 132.23, 129.70, 129.62, 129.35, 129.19, 128.64, 127.47, 126.77, 122.74, 122.68,
115.69, 115.52, 34.44; HRMS (TOF ESI+): m/z calcd for C24H17FN2O3 [M+Na]+, 423.1115, found,
423.1115.
502
503
3-((2,5-Dioxo-1-phenylpyrrolidin-3-ylidene)methyl)-N-p-tolylbenzamide (g3)
504
505
506
Yellow solid; M.p. 231-232 °C; 1H NMR (500 MHz, DMSO-d6) δ = 10.29 (s, 1H, HN), 8.21 (s, 1H,
HAr2), 8.02 (d, J = 8.1 Hz, 1H, HAr2), 7.92 (d, J = 8.2 Hz, 1H, HAr2), 7.70 – 7.65 (m, , 4H, HAr3,2
and C=CH), 7.54 (t, J = 7.7 Hz, 2H, HAr3), 7.46 (t, J = 7.4 Hz, 1H, HAr3), 7.40 (d, J = 8.1 Hz, 2H,
27

ACCEPTED MANUSCRIPT
507
508
509
510
HAr1), 7.19 (d, J = 8.2 Hz, 2H, HAr1), 3.97 (d, J = 2.4 Hz, 2H, CH2), 2.30 (s, 3H, CH3); 13C NMR
(126 MHz, DMSO-d6) δ = 173.77, 170.21, 165.17, 136.87, 136.20, 134.58, 133.21, 132.94, 132.24,
129.68, 129.60, 129.43, 129.35, 129.21, 128.66, 127.49, 126.72, 120.86, 34.44, 20.89; HRMS
(TOF ESI+): m/z calcd for C25H20N2O3 [M+Na]+, 419.1366, found, 419.1366.
511
512
3-((2,5-Dioxo-1-phenylpyrrolidin-3-ylidene)methyl)-N-(4-methoxyphenyl)benzamide (g4)
513
514
515
516
517
518
519
520
Yellow solid; M.p. 231-232 °C; 1H NMR (500 MHz, DMSO-d6) δ = 10.16 (s, 1H, HN), 8.13 (s, 1H,
HAr2), 7.93 (d, J = 7.7 Hz, 1H, HAr2), 7.83 (d, J = 7.8 Hz, 1H, HAr2), 7.63 (d, J = 8.9 Hz, 2H,
HAr1), 7.59 – 7.56 (m, 2H, HAr2 and C=CH), 7.45 (t, J = 7.5 Hz, 2H, HAr3), 7.38 (d, J = 7.8 Hz,
1H, HAr3), 7.31 (d, J = 7.6 Hz, 2H, HAr3), 6.88 (d, J = 9.0 Hz, 2H, HAr1), 3.89 (d, J = 2.5 Hz, 2H,
13
CH2), 3.68 (s, 3H, OCH3); C NMR (126 MHz, DMSO-d6) δ = 174.24, 170.69, 165.43, 156.58,
136.70, 135.07, 133.63, 133.45, 132.94, 132.77, 130.09, 129.80, 129.70, 129.14, 127.98, 127.19,
122.97, 114.67, 56.07, 34.95; HRMS (TOF ESI+): m/z calcd for C25H20N2O4 [M+Na]+, 435.1315,
found, 435.1317.
521
522
1-phenyl-3-(3-(pyrrolidine-1-carbonyl)benzylidene)pyrrolidine-2,5-dione (g5)
1
523
524
525
526
527
528
529
Yellow solid; M.p. 210-212 °C; H NMR (500 MHz, Chloroform-d) δ = 7.66 (t, J = 2.5 Hz, 1H,
HAr2), 7.61 (s, 1H, HAr2), 7.51 (t, J = 7.1 Hz, 2H, HAr), 7.48 – 7.39 (m, 3H, HAr, C=CH), 7.35 (d,
J = 7.6 Hz, 1H, HAr), 7.31 (dd, J = 8.4, 1.3 Hz, 2H, HAr), 3.71 (d, J = 2.4 Hz, 2H, COCH2), 3.60 (t,
J = 7.0 Hz, 2H, NCHα), 3.37 (t, J = 6.6 Hz, 2H, NCHβ), 1.99 – 1.89 (m, 2H, CH2αCH2α), 1.85 (q, J =
13
6.5 Hz, 2H, CH2βCH2β); C NMR (126 MHz, Chloroform-d) δ = 173.16, 170.19, 168.99, 138.71,
134.89, 134.59, 132.33, 131.81, 129.57, 129.23, 129.06, 126.83, 124.51, 50.07, 46.74, 34.66, 26.85,
24.83; HRMS (TOF ESI+): m/z calcd for C22H20N2O3 [M+Na]+, 383.1366, found, 383.1367.
530
531
3-(3-(morpholine-4-carbonyl)benzylidene)-1-phenylpyrrolidine-2,5-dione (g6)
532
533
534
535
536
537
538
Yellow solid; M.p. 218-220 °C; 1H NMR (500 MHz, Chloroform-d) δ = 7.65 (s, 1H, HAr2), 7.56 –
7.48 (m, 2H, HAr), 7.47 – 7.38 (m, 4H, HAr and C=CH), 7.34 (t, J = 7.4 Hz, 1H, HAr), 7.30 (d, J =
7.9 Hz, 2H, HAr), 3.69 (d, J = 2.6 Hz, 2H, COCH2), 1.21 (ddd, J = 14.6, 9.3, 5.5 Hz, 4H,
CH2OCH2), 0.81 (qd, J = 11.8, 9.6, 4.0 Hz, 4H, CH2NCH2); 13C NMR (126 MHz, Chloroform-d) δ
= 172.53, 169.60, 169.25, 136.41, 134.54, 134.02, 131.18, 129.38, 129.09, 128.72, 128.57, 126.33,
124.47, 66.78, 34.15; HRMS (TOF ESI+): m/z calcd for C22H20N2O4 [M+Na]+, 399.1315, found,
399.1317.
539
28