Structure–activity relationship study of DEL-22379: ERK dimerization inhibitors with increased safety

Article abstract of DOI:10.1007/s11030-020-10088-0

Abstract: Aberrant activation of ERK signaling pathway usually leads to oncogenesis, and small molecular agents targeting this pathway are impeded by the emergence of drug resistance due to reactivation of ERK signaling. Compound DEL-22379 has been reported to inhibit ERK dimerization which was unaffected by drug-resistant mechanism reactivating the ERK signaling. Here, we discussed a structure–activity relationship study of DEL-22379. Forty-seven analogues were designed and synthesized. Each synthesized compound was biologically evaluated for their inhibitory rates on several tumor cell lines and compounds with high inhibitory rates were further evaluated for IC₅₀ values. The structure–activity relationship of idolin-2-one scaffold and the impact of Z/E configuration on potency were discussed. Potential safety of two synthesized analogues was investigated and in silico docking study of five compounds was performed to understand the structural basis of ERK dimerization inhibition. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-020-10088-0

Molecular Diversity
https://doi.org/10.1007/s11030-020-10088-0
ORIGINAL ARTICLE
Structure–activity relationship study of DEL‑22379: ERK dimerization
inhibitors with increased safety
Yang Yang1 · Yuanzheng Zhou1 · Lei Tao1 · Tao Yang1,2 · Yinglan Zhao1 · Youfu Luo1
Received: 14 February 2020 / Accepted: 9 April 2020
© Springer Nature Switzerland AG 2020
Abstract
Aberrant activation of ERK signaling pathway usually leads to oncogenesis, and small molecular agents targeting this
pathway are impeded by the emergence of drug resistance due to reactivation of ERK signaling. Compound DEL-22379
has been reported to inhibit ERK dimerization which was unafected by drug-resistant mechanism reactivating the ERK
signaling. Here, we discussed a structure–activity relationship study of DEL-22379. Forty-seven analogues were designed
and synthesized. Each synthesized compound was biologically evaluated for their inhibitory rates on several tumor cell
lines and compounds with high inhibitory rates were further evaluated for IC50 values. The structure–activity relationship
of idolin-2-one scafold and the impact of Z/E confguration on potency were discussed. Potential safety of two synthesized
analogues was investigated and in silico docking study of fve compounds was performed to understand the structural basis
of ERK dimerization inhibition.
Graphic abstract
Keywords Indolin-2-one · Synthesis · ERK dimerization inhibitor · Anti-cancer activity · Structure–activity relationship
Yang Yang, Yuanzheng Zhou and Lei Tao have contributed
equally to this work.
Introduction
Electronic supplementary material The online version of this
The mitogen-activated protein kinase cascade (ERK sign-
aling pathway) plays an essential role in eukaryotic cel-
lular functions ranging from proliferation to diferentia-
tion [1–3]. After pathway activation by the extracellular
stimulation, the RAS family proteins frstly form a GTP-
binding complex, following the sequential phosphorylation
of downstream kinase module: RAF, MEK, and ERK [4,
5]. Once phosphorylated, ERK can activate a broad spec-
trum of nuclear and cytoplasmic substrates [4, 6]. Aber-
rant activation of this pathway usually leads to various
diseases especially oncogenesis [2, 7]. Through the past
decades, eforts have been made to exploit small molecular
article (https://doi.org/10.1007/s11030-020-10088-0) contains
supplementary material, which is available to authorized users.
* Yinglan Zhao
zhaoyinglan@scu.edu.cn
* Youfu Luo
luo_youfu@scu.edu.cn
1
Cancer Center, West China Hospital, Sichuan University
and Collaborative Innovation Center for Biotherapy,
Chengdu, Sichuan, China
2
Laboratory of Human Diseases and Immunotherapies, West
China Hospital, Sichuan University, Chengdu, Sichuan,
China
Vol.:(0123456789)
1 3

Molecular Diversity
inhibitors targeting this pathway for antineoplastic therapy,
and some of these agents have already progressed to clinic,
such as vemurafenib and dabrafenib [4, 8]. However, the
emergence of drug resistance hampered the efcacy of
such agents due to diverse mechanism, most of which
involve the reactivation of ERK signaling [2, 9]. Thus,
novel agents with diferent mode of action that can circum-
vent such reactivation are urgently needed.
Results and discussion
Analogues synthesis
Three series of DEL-22379 analogues 5a~5k (series 1),
9a~9q (series 2) and 16a~16s (series 3) were prepared in
this study. Synthesis of analogues 5a~5k was carried out
by synthetic route depicted in Scheme 1. Compounds 5a
and 5b were obtained directly by aldol reaction of starting
materials indolin-2-one (3a) or 5-nitroindolin-2-one (3b)
with 5-methoxy-1H-indole-3-carbaldehyde (4), respec-
tively, while 5c was obtained from reduction of compound
5b. Analogues 5d~5k were synthesized by aldol reaction
of 5-methoxy-1H-indole-3-carbaldehyde (4) with interme-
diates 3d~3k, which were products of amide condensation
of acids 2d~2k and 5-aminoindolin-2-one (1) reduced from
5-nitroindolin-2-one (3b).
Upon phosphorylation, ERK monomers form homodi-
mers [10]. The dimerized ERK proteins are able to trans-
port into nucleus or stay in cytoplasm to activate cyto-
plasmic substrates [11, 12]. It has been demonstrated that
interfering ERK dimerization is sufcient to impede tumor
progression [2, 8, 12]. A small molecule, DEL-22379
(Fig. 1a), has been reported to inhibit ERK dimerization
without alter ERK phosphorylation level [2]. Besides, this
molecule is unafected by drug-resistant mechanism reac-
tivating the ERK signaling [2], suggesting aiming at the
inhibition of ERK dimerization a promising solution to
cancer therapy.
Synthesis of analogues 9a~9q follows the similar syn-
thetic route (Scheme 2) of series 1. Amide condensation was
frstly performed to couple 5-aminoindolin-2-one (1) and
3-(piperidin-1-yl)propanoic acid (6) to aford intermediate
7, followed by aldol reaction of intermediate 7 with aromatic
aldehyde 8a~8q to give analogues 9a~9q.
The inspiring work of DEL-22379 encouraged us to
investigate the ERK dimerization inhibitory potency of
indolin-2-one scafold. Our group designed and synthe-
sized three series of indolin-2-one compounds (Fig. 1b)
with modifcation on C-3 position and C-5 position of
DEL-22379. MTT experiment was conducted to determine
the inhibitory rates of synthesized compounds on several
tumor cell lines, and then IC50 of selected compounds was
evaluated. The structure–activity relationship (SAR) of
indolin-2-one scafold was discussed.
The similar strategy (Scheme 3) was also utilized to syn-
thesize analogues 16a~16s. Compound 16a was prepared
through aldol reaction of starting materials indolin-2-one (3a)
and 1-naphthaldehyde (8h), while compounds 16d~16j was
obtained from aldol reaction of 1-naphthaldehyde (8h) and
intermediates 3d~3j used in Scheme 1. Same method in the
synthesis of intermediates 3d~3k was used to condense acid
Fig. 1 a Structure of DEL-
22379. b Structure of syn-
thesized compounds 5a~5k,
9a~9q, 16a~16s
1 3

Molecular Diversity
Scheme 1 Synthesis of analogues 5a~5k. Reagents and conditions: (I). 80% aqueous solution of hydrazine hydrate, FeCl3, activated carbon,
EtOH, 78 °C; (II). HATU, Et3N, DCM, 25 °C; (III). piperidine, MeOH, 64 °C
Scheme 2 Synthesis of analogues 9a~9q. Reagents and conditions: (I). HATU, Et3N, DCM, 25 °C; (II). piperidine, MeOH, 64 °C
10b~10c, 10k~10p with 5-aminoindolin-2-one (1) to give
intermediates 11b~11c, 11k~11p, followed by aldol con-
densation with 1-naphthaldehyde (8h) yielding compounds
16b~16c, 16k~16p. Intermediate 11q, 11r and 11s were pre-
pared through diferent ways. Intermediate 11q was prepared
through amide condensation of 2-oxoindoline-5-carboxylic
1 3

Molecular Diversity
Scheme 3 Synthesis of analogues 16a~16s. Reagents and conditions: (I). HATU, Et3N, DCM, 25 °C; (II). K2CO3, NaI, N,N’-dimethylforma-
mide (DMF), 90 °C; (III). DMF, 25 °C; (IV). piperidine, MeOH, 64 °C
acid (12) and 2-(piperidin-1-yl)ethan-1-amine (13); interme-
diate 11r was obtained through a nucleophilic substitution
reaction by coupling 5-aminoindolin-2-one (1) and 1-(3-chlo-
ropropyl)piperidine (14), intermediate 11s was prepared by
reacting 5-aminoindolin-2-one (1) with di(1H-imidazol-1-yl)
methanone (CDI, 15) then with 2-(piperidin-1-yl)ethan-1-
amine (13). Then aldol condensation with 1-naphthaldehyde
(8h) was conducted to give fnal product 16q, 16r and 16s.
The Z/E confguration of all synthesized compounds was
determined by 2D ROESY NMR spectrum (Supporting
Information 1, only 2D ROESY spectrum of 5g, 5c, 5j and
9a~9q was showed for the rest of the compounds in series 1
and all compounds in series 3 forming the same confgura-
tion, respectively) judging by whether the olefnic hydrogen
has a co-signal with hydrogen at C-4 position of indolin-
2-one or the C-4 position hydrogen has a co-signal with
hydrogen on aromatic ring of aldehyde moiety (Fig. 2). Most
analogues of series 1 (5a~5b, 5d~5i, 5k) with a 5-methoxy-
1H-indole-3-methylene at the C-3 position formed Z iso-
mers except for DEL-22379, 5c and 5j (they formed a Z/E
mixture), while all analogues of series 3 (16a~16s) with a
napthalene-1-methylene at the C-3 position formed E iso-
mers. Analogues of series 2 formed diferent confguration
due to diferent substitution of C-3 position, 9b, 9c, 9d, 9e,
9g, 9l~9n, 9q were Z/E mixtures; 9c was a Z isomer; 9h~9k,
9o, 9p formed E isomers.
analogues 5a~5k were synthesized. It was documented that
tumor cell lines harboring RAS mutant like A549, DLD1,
HCT116 or harboring BRAF mutant like A375 were more
sensitive to ERK dimerization inhibitors than RAS/BRAF
wild-type tumor cell lines [2]. Thus, tumor cell lines A549,
A375, DLD1 and HCT116 were chosen and subsequently
subjected to 10 μM 5a~5k and DEL-22379, and the inhibi-
tory rates were determined (Table 1). When 3-(piperidin-
1-yl)propanamide side chain of DEL-22379 was replaced
by small group like a hydrogen atom (5a), a nitro group (5b)
or an amino group (5c), the mean inhibitory rates reduced.
A shorter side-chain replacement like acetamide (5d), pro-
pionamide (5e), cyclopropanecarboxamide (5f), or oxygen
atom subsititution like 3-morpholinopropanamide (5j) leads
to sharp decreases on inhibitory rates. When using phenylic
group on C-5 position side chain (5g, 5i and 5k), the inhibi-
tory rates also declined a lot. Notably, substituting with a
cinnamide (5h) making this compound a planar molecule
maintained high inhibitory rates on all four tumor cells,
this may due to the non-specifc binding (of-target) of the
Michael acceptor contained by cinnamide moiety.
Modifcation on C‑3 position aromatic ring of DEL‑22379
(series 2: analogues 9a~9q), inhibitory rates determination
and IC50 evaluation of selected compounds
After the exploration on C-5 position side chain of DEL-
22379, we wondered how the chemical structure of C-3
position aromatic ring would afect the efcacy of this scaf-
fold. Seventeen analogues 9a~9q with various aromatic
rings on C-3 position were prepared and inhibitory rates
(at 10 μM) were determined (Table 2) on four tumor cell
lines mentioned above. Single aromatic ring substitutions
like thiophene-3-methylene (9a), thiophene-2-methylene
Inhibitory rates, IC50 and SAR study
Modifcation on C‑5 position side chain of DEL‑22379 (series
1: analogues 5a~5k) and inhibitory rates determination
We started our investigation with modifying the C-5 position
side chain on indolin-2-one scafold of DEL-22379, and 11
1 3

Molecular Diversity
Fig. 2 The 2D ROESY NMR spectrum (part) of compound 5g (a) and compound 9h (b), Z/E confguration was judging from the existing of co-
signal of olefnic hydrogen with hydrogen at C-4 position and C-4 position hydrogen with hydrogens on aromatic ring of aldehyde moiety
(9b) and pyridine-2-methylene (9d) weakened the efcacy
of this scafold, while 3,5-dimethyl-1-H-pyrrole-2-meth-
ylene (9c), which was designed following the structure
of approved anti-cancer drug (sunitinib) that also contain
an indolin-2-one scafold, maintained~60% mean inhibi-
tory rate. Deletion of methoxy group (9e) leaded to a
slight decrease on inhibitory rates, but substitution with
a 1-H-indole-5-methylene (9f) and a 1-H-indole-7-meth-
ylene (9g) resulted in signifcant reduction. When using
a napthalene-1-methylene (9h) and a 4-fuoronapthalene-
1-methylene (9i), high inhibitory rates were observed.
Quinoline-4-methylene (9j), napthalene-2-methylene (9l)
and quinoxaline-2-methylene (9n) substitutions also lead
to efcacy decline, but adding methoxy groups to 9j (9k)
and 9l (9m) enhanced their efcacy (same in DEL-22379
and 9e). Three benzene rings fusing leaded to lower (9p)
or higher (9o) inhibitory rates, while two benzene rings
linking (9q) resulted in efcacy attenuation.
1 3

Molecular Diversity
Table 1 Inhibitory rates of
analogues 5a~5k (series 1) on
four tumor cell lines A549,
A375, DLD1 and HCT116
Compounds
R5
R6
Configuration
Inhibitory rates (at 10μM)
A549 A375 DLD1 HCT Mean
(%),
(%),
(%),
116 (%), rate (%),
DEL-22379
Z/E = 3/1
88
91
71
94
72
48
76
46
40
16
59
81
40
24
37
86
57
38
51
30
18
– 6
36
72
26
18
33
Z
42
13
40
14
2
56
38
37
30
11
58
54
52
28
17
5
5a
5b
5c
5d
5e
5f
Z
Z/E = 4/1
Z
Z
Z
– 7
8
– 37
Z
42
74
31
15
19
36
68
17
18
20
5g
5h
5i
Z
67
18
14
56
Z
Z/E = 4/1
Z
5j
5k
To further investigate the potency of analogues with
rather high inhibitory rates, ten analogues (5h, 9c, 9e,
9h, 9i, 9k, 9m, 9o, 9p, 9q) and DEL-22379 were selected
and IC50 values on tumor cell lines A549, A375, DLD1,
HCT116 along with human hepatocyte LO2 were evaluated
(Fig. 3,see Table S1 in Supporting Information 1 for IC50
values). Compounds 5h, 9c, 9e, 9h exhibited comparable
potency on all tumor cell lines comparing to DEL-22379;
compounds 9m, 9p and 9q showed a slight decrease on
some tumor cell lines; IC50 of compounds 9i and 9k on
A549 increased to more than 20 μM, while compounds
9o showed a slightly enhanced tumor inhibitory efcacy.
Although the anti-tumor efcacies of analogues of series 1
and 2 did not increase up to any orders of magnitude, but
interestingly, compound 9h with a napthalene-1-methylene
on C-3 position exhibited an about fvefold–tenfold selec-
tivity between tumor cell lines and human liver cell LO2,
which encouraged us to make further exploration on this
analogue.
Further exploration of C‑5 position side chain based
on 9h (series 3, analogues 16a~16s), inhibitory rates
determination and IC50 evaluation of selected compounds
In order to seek more appropriate C-5 position side chain for
better analogues with higher selectivity and potency, nine-
teen analogues 16a~16s were designed based on 9h and syn-
thesized, inhibitory rates were determined against the same
tumor cells (Table 3). Varying degrees of decreases were
observed when substituted with side chain used in series 1
(16a, 16d, 16e, 16f, 16g, 16h, 16i, 16j), and even efcacy of
Michael acceptor cinnamide (16h) declined to 45% inhibi-
tory rate. Using of a 3-cyclohexylpropanamide (16l) also
reduced the potency of this scafold, but using of a 3-thio-
morpholinopropanamide (16k) kept a rather high inhibitory
rate. Replacing the piperidine ring with a pyrrolidine ring
(16n) or a diethylamine (16p) leaded to slight decrease.
Attenuation of the side chain length (16b, 16m, 16o) leaded
to markedly decrease in inhibitory rate, while increase in
1 3

Molecular Diversity
Table 2 Inhibitory rates of
analogues 9a~9q (series 2) on
three tumor cell lines A549,
A375, DLD1 and HCT116
Compounds
R7
R8
Configuration
Inhibitory rates (at 10μM)
A549 A375 DLD1 HCT Mean
(%),
(%),
(%),
116 (%), rate (%),
DEL – 22379
Z/E = 3/1 88
91
71
94
22
71
70
23
86
24
50
59
23
Z/E = 1/1
Z/E = 3/2
Z
30
32
29
30
16
57
64
20
28
41
74
19
9a
9b
9c
9d
Z/E = 1/2
Z/E = 2/1
68
34
23
26
37
40
69
31
24
40
86
13
15
84
44
45
88
96
52
97
62
56
55
94
64
53
68
14
29
66
74
16
56
42
81
16
95
69
80
85
26
32
94
93
35
77
55
97
35
98
90
94
76
30
32
68
75
36
75
48
64
36
93
59
60
9e
9f
E
Z/E = 1/6
9g
9h
9i
E
E
E
9j
E
9k
9l
Z/E = 1/5
Z/E = 1/4
Z/E = 1/3
E
9m
9n
9o
9p
9q
E
Z/E = 1/4
the side chain length (16c) maintained comparable potency.
Finally, we changed the linker type of the connecting part
of side chain to indolin-2-one scafold (16q, 16r, 16s), 16r
and 16s exhibited slightly lower inhibitory rates, while 16q
exhibited a higher inhibitory rate.
A375, DLD1, HCT116, and LO2 (Fig. 4). Compounds 16k,
16n, 16p, 16q and 16r exhibited decreased potency and all
lost selectivity. Only 16s showed similar IC50 values and
selectivity comparing to 9h. Throughout structure–activity
relationship of analogues of all three series, we concluded
that some fused aromatic ring substitution at C-3 position,
a 4~5 atoms length side chain linker, an alkyl group with
IC50 of six compounds (16k, 16n, 16p, 16q, 16r, 16s)
along with DEL-22379 and 9h were evaluated on A549,
1 3

Molecular Diversity
administrations of an extreme dose (500 mg/kg) in Balb/c
mice. During 15 days observation, the animals showed no
abnormality. Meanwhile, we did not observe a loss of body
weight in 9h- or 16s-treated mice (Fig. 8). The result of
hematological evaluation indicated that 9h and 16s treat-
ment did not alter the counts of white blood cells (WBC),
red blood cells (RBC), hemoglobin (HGB) or hematocrit
(HCT) levels, etc. (Fig. 9). In addition, neither biochemical
parameters altered after 9h or 16s treatment (Fig. 10). Above
results suggested that 9h and 16s were relative safe agents
for oral administration.
In silico docking
In an attempt to understand the structural basis how our
newly synthesized compounds inhibited ERK dimeriza-
tion, in silico docking of selected compounds with ERK
was performed. It has been reported that phosphorylated
ERK dimers had a diferent conformation comparing to
unphosphorylated ERK dimers [13, 14], since ERK phos-
phorylation promote ERK homodimers formation and the
fact that DEL-22379 could not disrupt already performed
ERK dimers [2], we decided to perform our docking study
on phosphorylated ERK monomer. Compounds 9h, 9o, 16s,
E-DEL-22379 and Z-DEL-22379 was selected to dock to
available phosphorylated ERK monomer structure (PDB ID:
5V60) [15], the result of 16s was depicted in Fig. 8 (see Fig-
ure S1–S4 in Supporting Information 1 for docking results
of 9o, 16s, E-DEL-22379 and Z-DEL-22379). Compound
16s bounded to the dimer interface of phosphorylated ERK
monomer (Fig. 11a) with its indolin-2-one scafold and the
side chain part insert into the cleft of the protein surface of
this monomer (Fig. 11b). The benzene ring of indolin-2-one
interacted with the benzoyl groups of Phe 181 and Phe 329
through π−π stacking, the nitrogen atoms of indolin-2-one
and urea linkage resembling hydrogen bond interactions
with Asp 177 and Asp 335, while nitrogen atom of piperi-
dyl group form an ionic bond interaction with TPO 183, the
phosphorylated residue Thr 183 (Fig. 11c, d). The other four
compounds adopted similar mode of action with phosphoryl-
ated ERK monomer suggesting compounds of this scafold
could interfere ERK dimerization by binding to the interface
of phosphorylated ERK.
Fig. 3 IC50 values of selected compounds. Each tumor cell line was
treated with various concentrations (0~20 μM) of selected com-
pounds for 48 h, respectively. Cell viability was detected by CCK-8
assay and IC50 values were calculated by Graph-Pad Prism 7 soft-
ware using XY modeling. Data are expressed as the mean S.D. from
three experiments
nitrogen at the end of C-5 position side chain of indolin-
2-one scafold are essential for anti-tumor efcacy (Fig. 5).
Relationship between analogue confgurations and efcacy
Throughout all three series, we noticed that analogues with
high anti-tumor efcacy most come up with an E confgu-
ration like 9h, 9i, 9k, 9o, 9p and analogues of series 3 or
a mixture with more E isomer like 9m and 9q. Only a few
compounds with Z confguration or mixture with more Z
isomer exhibited comparable efcacy comparing to DEL-
22379, like 5h (Z), 9c (Z), 9e (Z/E=2/1). We presumed that
aromatic rings on C-3 position with an E confguration make
more contribution to increase the anti-tumor potential of this
scafold than Z. Compound DEL-22379 obtained from pur-
chasing or synthesized was a Z/E mixture (Z/E = 3/1), we
wondered whether a pure E isomers of DEL-22379 would
possess a higher potency. The Z/E mixed DEL-22379 was
heated in dioxane at 110 °C for 8 h to form a>95% E isomer
(Fig. 6), then IC50 was evaluated (Fig. 7). Only selectivity
increase between tumor cells and normal cell were observed
but not anti-tumor potency. Consider that compounds with
high selectivity were all E isomers (9h, 16s, E-DEL-22379),
we concluded that analogues of this scafold with E confgu-
ration were more likely to display selectivity than Z isomers.
Conclusion
In summary, three series analogues of DEL-22379 were
designed, synthesized and biologically evaluated. The impact
of C-3 position aromatic rings and C-5 position side chain
of indolin-2-one scafold on anti-tumor efect was explored.
Compounds 9c, 9e, 9h, 9o in series 2 and compound 16s in
series 3 were found to possess a comparable or increased
Acute toxicity study
To further investigate the safety of 9h and 16s, we con-
ducted an acute toxicity test and investigated whether 9h
and 16s could induce hematological changes after two oral
1 3

Molecular Diversity
Table 3 Inhibitory rates of
analogues 16a~16s (series 3)
on four tumor cell lines A549,
A375, DLD1 and HCT116
Compounds
R9
R10
Configuration
Inhibitory rates (at 10μM)
A549 A375 DLD1 HCT Mean
( ),
( ),
( ),
116 ( ), rate ( ),
DEL-22379
9h
Z/E = 3/1
88
91
71
94
94
23
43
95
44
49
32
37
54
36
35
97
53
57
81
48
85
97
82
93
86
68
16
23
62
29
30
18
20
45
15
24
58
39
33
56
32
52
80
54
64
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
26
18
1
88
26
18
53
29
10
19
15
35
3
66
– 1
30
76
5
16a
16b
16c
16d
16e
16f
25
38
41
19
18
52
5
22
1
9
16g
16h
16i
40
17
21
85
47
35
65
51
61
83
60
54
– 1
39
44
44
23
50
22
41
92
55
89
16j
8
11
17
26
5
16k
16l
16m
16n
16o
16p
16q
16r
21
48
19
22
16s
1 3

Molecular Diversity
Experimental section
Chemistry
All reagents were directly used as purchased without
further purification. All solvents were dried according
to standard methods before use, solvents used in optical
spectroscopic studies were HPLC grade. All NMR spec-
trum were recorded on a Bruker Avance (Varian Unity
Inova) 500 MHz spectrometer in DMSO-d6 with tetra-
1
methylsilane (TMS) as internal standard. H NMR and
13C NMR spectra were recorded respectively at 400 MHz
1
and 100 MHz. H NMR and 13C NMR were analyzed by
MestReNova Software while 2D ROESY were analyzed
by Topspin Software. High resolution mass spectrum
(HRMS) was performed on an Agilent LC/MSD TOF
system G3250AA. Analytical thin layer chromatography
(TLC) was performed on silica gel 60 F254 precoated
plates (0.25 mm) from Qingdao Haiyang Inc., and compo-
nents were visualized by ultraviolet light (254 nm). Sili-
cycle silica gel 300–400 (particle size 40–63 μm) mesh
was used for all flash column chromatography experi-
ments. Melting points of all compounds were determined
using SGW X-4 micromelting point apparatus (Shanghai
jingke industrial co.,ltd).
Fig. 4 IC50 values of selected compounds. Data are expressed as the
mean S.D. from three experiments
Preperation of 5‑aminoindolin‑2‑one (1)
To a suspension of 5-nitroindolin-2-one (5g, 1 eq.) in EtOH
(50 mL), activated carbon (1 g) and FeCl3 (1 g) were added.
The mixture was heated to 78 °C, and stirred for 10 min.
Then 80% aqueous solution of hydrazine hydrate (8 eq.)
was added dropwise into the reaction mixture in 5 min, the
resulting mixture was allowed to stirred at 78 °C for 8–10 h
and then cooled to room temperature. The mixture was fl-
tered to remove residue of activated carbon, the fltrate was
concentrated under vacuum to aford crude product, which
was purifed by recrystallization in EtOH (about 15 mL)
to give 5-aminoindolin-2-one as a pale yellow solid (yield
91.9%).
Fig. 5 Structure-activity relationship of indolin-2-one scafold
potency comparing to DEL-22379. We concluded the SAR
that some fused aromatic ring substitution at C-3 position,
a 4~5 atoms length side chain linker, an alkyl group with
nitrogen at the end of C-5 position side chain are essential for
anti-tumor efcacy. Compounds 9h and 16s exhibited highly
selectivity and further acute toxicity study suggested these two
compounds were potential safe agents. In Silico Docking study
suggested these analogues of indolin-2-one scafold inhibited
ERK by bound to protein’s cleft of dimerization interface.
This study presented a preliminary insight into the anti-tumor
potency of this chemical structure, and we consider that our
future work should consist in the further confrmation of ERK
dimerization inhibition mechanism of synthesized compounds
and the evaluation of pharmacodynamics and pharmacokinet-
ics of hit compound.
General procedure A for the synthesis of intermediates
3d~3k, 7, 11b~11c, 11k~11p, 11q
To a solution of corresponding acids 2d~2k, 6, 10b~10c,
10k~10p, 12 (600 mg, 1 eq.) in dichloromethane (DCM)
(7 mL), triethylamine (1.4 eq.) and 1-[Bis(dimethylamino)
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hex-
afuorophosphate (HATU) (1.3 eq.) were added. The reac-
tion mixture was stirred at room temperature for 20–30 min,
then corresponding amines 1, 13 (1.1 eq.) was added into the
1 3

Molecular Diversity
Fig. 6 Comparison of 1H NMR spectrum (part) of purchased or synthesized DEL-22379 (top) and transformed DEL-22379 (below)
reaction mixture. The resulting mixture was stirred at room
temperature for 6 h. After the reaction was fnished, the sol-
vent was evaporated under vacuum, then purifed by column
chromatography to give intermediate 3d~3k, 7, 11b~11c,
11k~11p, 11q (yield 13.2%~51.4%).
General procedure B for the synthesis of compounds
5a~5b, 5d~5k, 9a~9q, 16a~16s
To a solution of the corresponding starting materials 3a~3b
or corresponding intermediates 3d~3k, 7, 11b~11c, 11k~11s
(200 mg, 1 eq.) in MeOH (5 mL), piperidine (1.5 eq.) and
corresponding aromatic aldehydes 4, 8a~8q (1.2 eq.) were
added. The reaction mixture was heated to refux and stirred
for 1–8 h, then cooled to room temperature. The mixture was
added with water (10 mL) and extracted with DCM (10 mL)
for three times, the combined organic layer was washed with
brine and dried over anhydrous sodium sulfate. Then puri-
fed by column chromatography to give products 5a~5b,
5d~5k, 9a~9q, 16a~16s (yield 17.5%~63.8%).
N‑(2‑oxoindolin‑5‑yl)acetamide (3d) Following general
procedure A, starting from acetic acid (2d) and 5-aminoin-
dolin-2-one (1) to aford intermediate 3d as a white solid
1
(yield 51.4%). H NMR (400 MHz, DMSO-d6) δ 10.25 (s,
Fig. 7 IC50 values of DEL-22379 (Z/E mixture) and DEL-22379 (E
isomer). Data are expressed as the mean S.D. from three experi-
ments
1H), 9.77 (s, 1H), 7.50 (d, J=0.8 Hz, 1H), 7.30 (dd, J=8.4,
1.6 Hz, 1H), 6.72 (d, J=8.0 Hz, 1H), 3.45 (s, 2H), 2.00
1 3

Molecular Diversity
Fig. 8 Body weight of tumor-bearing mice treated with 9h and 16s. The data were expressed as the mean SD (n ≧ 6)
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 176.75, 168.22,
139.58, 133.95, 126.45, 118.84, 116.88, 109.28, 36.52,
24.31.
J=8.4 Hz, 1H), 3.44 (s, 2H), 1.74 (m, 1H), 0.75 (m, 4H).
13C NMR (100 MHz, DMSO-d6) δ 176.73, 171.53, 139.51,
134.04, 126.47, 118.80, 116.88, 109.29, 36.52, 14.89, 7.36
(2C).
N‑(2‑oxoindolin‑5‑yl)propionamide (3e) Following general
procedure A, starting from propionic acid (2e) and 5-ami-
noindolin-2-one (1) to aford intermediate 3e as a white
solid (yield 45.7%). 1H NMR (400 MHz, DMSO-d6) δ 10.26
(s, 1H), 9.69 (s, 1H), 7.51 (d, J=0.8 Hz, 1H), 7.33 (dd,
J=8.0, 1.6 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H), 3.45 (s, 2H),
2.28 (q, J=7.6 Hz, 2H), 1.07 (t, J=7.6 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 176.77, 171.98, 139.51, 133.97,
126.43, 118.89, 116.94, 109.28, 36.51, 29.90, 10.28.
N‑(2‑oxoindolin‑5‑yl)‑3‑phenylpropanamide
(3g) Fol-
lowing general procedure A, starting from 3-phenylpro-
panoic acid (2g) and 5-aminoindolin-2-one (1) to aford
1
intermediate 3g as a white solid (yield 17.4%). H NMR
(400 MHz, DMSO-d6) δ 10.27 (s, 1H), 9.76 (s, 1H), 7.50 (d,
J=0.8 Hz, 1H), 7.30 (m, 2H), 7.25 (m, 3H), 7.18 (m, 1H),
6.73 (d, J=8.4 Hz, 1H), 3.44 (s, 2H), 2.90 (t, J=7.2 Hz,
2H), 2.59 (t, J=8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 176.83, 170.42, 141.67, 139.63, 133.79, 128.76 (2C),
128.69 (2C), 126.47, 126.38, 119.00, 117.02, 109.31, 38.32,
36.49, 31.41.
N‑(2‑oxoindolin‑5‑yl)cyclopropanecarboxamide (3f) Fol-
lowing general procedure A, starting from cyclopropane-
carboxylic acid (2f) and 5-aminoindolin-2-one (1) to aford
1
intermediate 3f as a white solid (yield 40.4%). H NMR
N‑(2‑oxoindolin‑5‑yl)cinnamamide (3h) Following general
procedure A, starting from cinnamic acid (2h) and 5-ami-
noindolin-2-one (1) to aford intermediate 3h as a pale yel-
(400 MHz, DMSO-d6) δ 10.26 (s, 1H), 10.03 (s, 1H), 7.50
(d, J=0.8 Hz, 1H), 7.33 (dd, J=8.4, 2.0 Hz, 1H), 6.72 (d,
1 3

Molecular Diversity
Fig. 9 Efects of oral gavage of 9h and 16s on blood routine in Balb/c
mice. WBC (white blood cell count), #NEUT (neutrophile granu-
locyte count), #LYMPH (lymphocyte count), #MONO (monocyte
count), #EOS (eosnophils granulocyte count), #BASO (basophilic
granulocyte count), #LUC (large unstained cell count), %NEUT (neu-
trophilic granulocyte percentage), %LYMPH (lymphocyte percent-
age), %MONO (monocyte percentage), %EOS (eosnophils granu-
locyte percentage), %BASO (basophilic granulocyte percentage),
%LUC (large unstained cell percentage), RBC (red blood cell count),
HGB (hemoglobin), HCT (hematocrit), MCV (mean corpuscular vol-
ume), MCH (mean corpuscular hemoglobin), MCHC (mean corpus-
cular hemoglobin concentration), RDW (red cell distribution width),
PLT (platelet count), MPV (mean platelet volume)
Fig. 10 Efects of oral gavage of
9h and 16s on blood biochem-
istry in Balb/c mice. ALP2L
(alkaline phosphatase), ALTL
(alanine aminotransferase),
ASTL (aspartate aminotrans-
ferase), CK (creatine kinase),
LDHI2 (lactic dehydrogenase),
UREAL (urea), CREA (creati-
nine), TP2 (total protein), ALB2
(albumin), GLB (globulin),
HDLC3 (high-density lipo-
protein), A/G (albumin/
globulin), GLUC3 (blood glu-
cose), CHO2I (cholesterol),
TRIGL (triglyceride), K (K+),
NA (Na+), CL (Cl−), UA2
(uric acid), BILT3 (bilirubin),
LDLC3 (low density lipopro-
tein), BUN (blood urea nitro-
gen)
1 3

Molecular Diversity
Fig. 11 In silico docking of 16s with phosphorylated ERK monomer.
Compound 16s bounded to the dimer interface of phosphorylated
ERK monomer (a and b); The amino acid residues involved were His
176, Asp 177, His 178, Phe 181, Leu 182, TPO 183, Phe 329, Leu
332 and Asp 335 (c and d)
low solid (yield 26.0%). 1H NMR (400 MHz, DMSO-d6) δ
10.32 (s, 1H), 10.11 (s, 1H), 7.60 (m, 4H), 7.43 (m, 4H),
6.81 (m, 2H), 3.49 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
δ 176.75, 163.57, 140.02, 139.95, 135.31, 133.90, 130.11,
129.47 (2C), 128.11 (2C), 126.64, 122.99, 119.05, 116.94,
109.45, 36.55.
3‑morpholino‑N‑(2‑oxoindolin‑5‑yl)propanamide (3j) Fol-
lowing general procedure A, starting from 3-morpholino-
propanoic acid (2j) and 5-aminoindolin-2-one (1) to aford
1
intermediate 3i as a white solid (yield 26.5%). H NMR
(400 MHz, DMSO-d6) δ 10.27 (s, 1H), 9.89 (s, 1H), 7.51
(d, J=0.8 Hz, 1H), 7.32 (dd, J=8.4, 2.0 Hz, 1H), 6.73
(d, J=8.4 Hz, 1H), 3.57 (m, 4H), 3.45 (s, 2H), 2.60 (t,
J=7.2 Hz, 2H), 2.43 (m, 6H). 13C NMR (100 MHz, DMSO-
d6) δ 176.75, 170.06, 139.63, 133.85, 126.49, 118.89,
116.92, 109.31, 66.67 (2C), 54.76, 53.50 (2C), 36.52, 34.31.
N‑(2‑oxoindolin‑5‑yl)‑3‑(3,4,5‑trimethoxyphenyl)propana‑
mide (3i) Following general procedure A, starting from
3-(3,4,5-trimethoxyphenyl)propanoic acid (2i) and 5-ami-
noindolin-2-one (1) to aford intermediate 3i as a colorless
oil (yield, 43.8%). 1H NMR (400 MHz, DMSO-d6) δ 10.28
(s, 1H), 9.77 (s, 1H), 7.53 (s, 1H), 7.35 (dd, J=8.0, 0.8 Hz,
1H), 6.76 (d, J=8.4 Hz, 1H), 6.56 (s, 2H), 3.74 (s, 6H),
3.63 (s, 3H), 3.46 (s, 2H), 2.88 (t, J=7.6 Hz, 2H), 2.61 (t,
J=8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 176.85,
170.57, 153.20 (2C), 139.69, 137.39, 136.20, 133.80,
126.49, 119.07, 117.08, 109.33, 105.90 (2C), 60.35, 56.13
(2C), 38.43, 36.47, 31.85.
2‑(4‑methoxyphenyl)‑N‑(2‑oxoindolin‑5‑yl)acetamide
(3k) Following general procedure A, starting from
2-(4-methoxyphenyl)acetic acid (2k) and 5-aminoindolin-
2-one (1) to aford intermediate 3k as a white solid (yield
1
33.9%). H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H),
9.97 (s, 1H), 7.50 (s, 1H), 7.33 (dd, J=8.4, 1.6 Hz, 1H), 7.24
(m, 2H), 6.88 (m, 2H), 6.72 (d, J=8.4 Hz, 1H), 3.72 (s, 3H),
3.52 (s, 2H), 3.44 (s, 2H). 13C NMR (100 MHz, DMSO-
1 3

Molecular Diversity
d6) δ 176.73, 169.43, 158.46, 139.73, 133.84, 130.46 (2C),
128.60, 126.49, 119.00, 116.99, 114.19 (2C), 109.29, 55.50,
42.85, 36.51.
2-one (1) to aford intermediate 11l as a white solid (yield
1
28.4%). H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H),
9.69 (s, 1H), 7.51 (d, J = 0.8 Hz, 1H), 7.31 (dd, J = 8.4,
2.0 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 3.44 (s, 2H), 2.26
(t, J = 7.6 Hz, 2H), 1.65 (m, 5H), 1.48 (q, J = 7.2 Hz, 2H),
1.16 (m, 4H), 0.88 (m, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 176.75, 171.46, 139.53, 133.99, 126.43, 118.88,
116.94, 109.26, 37.29, 36.52, 34.40, 33.19, 33.09 (2C),
26.59, 26.25 (2C).
N‑(2‑oxoindolin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(7) Following general procedure A, starting from 3-(piperi-
din-1-yl)propanoic acid (6) and 5-aminoindolin-2-one (1) to
1
aford intermediate 7 as a colorless oil (yield 33.4%). H
NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 10.03 (s, 1H),
7.50 (d, J=0.8 Hz, 1H), 7.31 (dd, J=8.4, 1.6 Hz, 1H), 6.74
(d, J=8.4 Hz, 1H), 3.45 (s, 2H), 2.77 (m, 2H), 2.56 (m, 6H),
1.56 (m, 4H), 1.43 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
δ 176.75, 169.63, 139.70, 133.75, 126.53, 118.90, 116.91,
109.35, 54.34, 53.76 (2C), 36.52, 33.55, 25.28 (2C), 23.81.
N‑(2‑oxoindolin‑5‑yl)‑2‑(pyrrolidin‑1‑yl)acetamide
(11m) Following general procedure A, starting from 2-(pyr-
rolidin-1-yl)acetic acid (10m) and 5-aminoindolin-2-one (1)
to aford intermediate 11m as a colorless oil (yield 30.3%).
1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.54 (s,
1H), 7.54 (d, J=0.8 Hz, 1H), 7.37 (dd, J=8.0, 2.0 Hz, 1H),
6.74 (d, J=8.4 Hz, 1H), 3.45 (s, 2H), 3.20 (s, 2H), 2.57
(m, 4H), 1.74 (m, 4H). 13C NMR (100 MHz, DMSO-d6)
δ 176.84, 168.83, 139.87, 133.22, 126.44, 119.45, 117.40,
109.27, 59.91, 54.15 (2C), 36.49, 23.92 (2C).
N‑(2‑oxoindolin‑5‑yl)‑2‑(piperidin‑1‑yl)acetamide
(11b) Following general procedure A, starting from
2-(piperidin-1-yl)acetic acid (10b) and 5-aminoindolin-
2-one (10b) to aford intermediate 11b as a colorless oil
1
(yield 24.9%). H NMR (400 MHz, DMSO-d6) δ 10.29 (s,
1H), 9.50 (s, 1H), 7.54 (d, J=0.8 Hz, 1H), 7.37 (dd, J=8.0,
2.0 Hz, 1H), 6.74 (d, J=8.4 Hz, 1H), 3.46 (s, 2H), 3.02 (s,
2H), 2.44 (m, 4H), 1.55 (m, 4H), 1.40 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 176.75, 168.51, 139.94, 133.10,
126.50, 119.32, 117.28, 109.26, 63.08, 54.55 (2C), 36.49,
25.94 (2C), 24.02.
N‑(2‑oxoindolin‑5‑yl)‑3‑(pyrrolidin‑1‑yl)propanamide
(11n) Following general procedure A, starting from
3-(pyrrolidin-1-yl)propanoic acid (10n) and 5-aminoindo-
lin-2-one (1) to aford intermediate 11n as a colorless oil
1
(yield 21.1%). H NMR (400 MHz, DMSO-d6) δ 10.26 (s,
1H), 9.92 (s, 1H), 7.50 (d, J=0.8 Hz, 1H), 7.32 (dd, J=8.4,
1.6 Hz, 1H), 6.72 (d, J=8.4 Hz, 1H), 3.45 (s, 2H), 2.70
(t, J=7.2 Hz, 2H), 2.45 (m, 6H), 1.68 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) δ 176.75, 170.09, 139.58, 133.91,
126.48, 118.83, 116.87, 109.30, 53.87 (2C), 52.09, 36.52,
36.45, 23.62 (2C).
N‑(2‑oxoindolin‑5‑yl)‑4‑(piperidin‑1‑yl)butanamide
(11c) Following general procedure A, starting from
4-(piperidin-1-yl)butanoic acid (10c) and 5-aminoindolin-
2-one (1) to aford intermediate 11c as a pale yellow oil
1
(yield 18.7%). H NMR (400 MHz, DMSO-d6) δ 10.26 (s,
1H), 9.73 (s, 1H), 7.51 (d, J=0.8 Hz, 1H), 7.32 (dd, J=8.4,
1.2 Hz, 1H), 6.72 (d, J=8.4 Hz, 1H), 3.44 (s, 2H), 2.31 (m,
8H), 1.73 (m, 2H), 1.50 (m, 4H), 1.37 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 176.73, 171.04, 139.52, 133.98,
126.40, 118.87, 116.93, 109.25, 58.30, 54.32 (2C), 36.52,
34.70, 25.78 (2C), 24.39, 22.66.
2‑(diethylamino)‑N‑(2‑oxoindolin‑5‑yl)acetamide
(11o) Following general procedure A, starting from dieth-
ylglycine (10o) and 5-aminoindolin-2-one (1) to aford
intermediate 11o as a colorless oil (yield 24.9%). 1H NMR
(400 MHz, DMSO-d6) δ 10.28 (s, 1H), 9.47 (s, 1H), 7.54
(d, J=0.8 Hz, 1H), 7.37 (dd, J=8.4, 2.0 Hz, 1H), 6.73 (d,
J=8.0 Hz, 1H), 3.45 (s, 2H), 3.10 (s, 2H), 2.58 (q, J=7.2 Hz,
4H), 1.01 (t, J=6.8 Hz, 6H). 13C NMR (100 MHz, DMSO-
d6) δ 176.75, 169.79, 139.95, 132.98, 126.54, 119.22,
117.19, 109.28, 57.91, 48.37 (2C), 36.49, 12.48 (2C).
N‑(2‑oxoindolin‑5‑yl)‑3‑thiomorpholinopropanamide
(11k) Following general procedure A, starting from 3-thio-
morpholinopropanoic acid (10k) and 5-aminoindolin-2-one
(1) to aford intermediate 11k as a white solid (yield 22.3%).
1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 9.83 (s,
1H), 7.50 (d, J=0.8 Hz, 1H), 7.31 (dd, J=8.4, 2.0 Hz, 1H),
6.73 (d, J=8.4 Hz, 1H), 3.45 (s, 2H), 2.67 (m, 6H), 2.59 (m,
4H), 2.42 (t, J=7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 176.75, 170.15, 139.64, 133.83, 126.50, 118.91,
116.94, 109.31, 55.37, 54.68 (2C), 36.53, 34.18, 27.70 (2C).
3‑(diethylamino)‑N‑(2‑oxoindolin‑5‑yl)propanamide
(11p) Following general procedure A, starting from
3-(diethylamino)propanoic acid (10p) and 5-aminoindolin-
2-one (1) to aford intermediate 11p as a colorless oil (yield
1
18.8%). H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H),
9.98 (s, 1H), 7.50 (d, J=0.8 Hz, 1H), 7.31 (dd, J=8.4,
2.0 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H), 3.45 (s, 2H), 2.79 (t,
J=6.8 Hz, 2H), 2.57 (q, J=6.8 Hz, 4H), 2.42 (t, J=6.8 Hz,
2H), 1.00 (t, J=6.8 Hz, 6H). 13C NMR (100 MHz, DMSO-
3‑ c yclohex yl‑N‑(2‑ oxoindolin‑5‑yl)propanamide
(11l) Following general procedure A, starting from
3-cyclohexylpropanoic acid (10l) and 5-aminoindolin-
1 3

Molecular Diversity
d6) δ 176.74, 170.12, 139.62, 133.84, 126.51, 118.84,
(Z)‑3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)indolin‑2‑one
(5a) Following general procedure B, starting from starting
materials (3a) and 5-methoxy-1H-indole-3-carbaldehyde
(4) to aford compound 5a as a Z isomer, orange solid (yield
58.2%), melting point 257 °C. 1H NMR (400 MHz, DMSO-
d6) δ 11.88 (s, 1H), 10.48 (s, 1H), 9.43 (d, J=2.8 Hz, 1H),
8.15 (s, 1H), 7.93 (d, J=7.2 Hz, 1H), 7.74 (d, J=2.4 Hz,
1H), 7.41 (d, J=8.8 Hz, 1H), 7.14 (td, J=7.6, 0.8 Hz, 1H),
6.99 (td, J=7.2, 0.8 Hz, 1H), 6.86 (m, 2H), 3.89 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 168.60, 155.50, 139.47,
134.47, 131.22, 129.56, 128.07, 127.00, 126.34, 120.82,
119.18, 118.82, 113.41, 112.85, 111.85, 109.37, 101.21,
56.09. HRMS (Q-TOF): calculated for C18H14N2O2 [M]:
290.1055. Found [M+H]+: 291.1130.
116.88, 109.33, 48.74, 46.62 (2C), 36.52, 34.06, 11.89 (2C).
2‑oxo‑N‑(2‑(piperidin‑1‑yl)ethyl)indoline‑5‑carboxam‑
ide (11q) Following general procedure A, starting from
2-oxoindoline-5-carboxylic acid (12) and 2-(piperidin-1-yl)
ethan-1-amine (13) to aford intermediate 11q as a white
solid (yield 22.6%). 1H NMR (400 MHz, DMSO-d6) δ 10.62
(s, 1H), 8.21 (m, 1H), 7.71 (m, 2H), 6.85 (d, J=8.0 Hz, 1H),
3.53 (s, 2H), 3.30 (m, 2H), 2.40 (m, 6H), 1.49 (m, 4H), 1.37
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 177.12, 166.34,
146.77, 128.05, 127.80, 126.10, 123.82, 108.90, 58.27,
54.56 (2C), 37.37, 36.07, 26.03 (2C), 24.49.
5‑((3‑(piperidin‑1‑yl)propyl)amino)indolin‑2‑one (11r) To
a solution of 5-aminoindolin-2-one (1) (300 mg, 1 eq.) in
dry DMF (5 mL) was added K2CO3 (1.1 eq.), NaI (1.1 eq.)
and 1-(3-chloropropyl)piperidine (14) (1.1 eq.). The reac-
tion mixture was stirred at 90 °C for 8 h, then cooled to
room temperature. 20 mL water was added and the mix-
ture was extracted with DCM (15 mL) for three times, the
combined organic layer was washed with brine, dried over
sodium sulfate, and concentrated under vacuum to aford
crude product, which was purifed through silica gel column
chromatography to give intermediate 11r as a colorless oil
(Z)‑3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑5‑nitroindo‑
lin‑2‑one (5b) Following general procedure B, starting
from starting materials (3b) and 5-methoxy-1H-indole-
3-carbaldehyde (4) to aford compound 5b as a Z isomer,
orange solid (yield 54.9%), decomposed at 370 °C. 1H NMR
(400 MHz, DMSO-d6) δ 12.12 (s, 1H), 11.21 (s, 1H), 9.51
(d, J=3.2 Hz, 1H), 8.90 (d, J=2.0 Hz, 1H), 8.51 (s, 1H),
8.10 (dd, J=8.8, 2.4 Hz, 1H), 7.91 (d, J=2.0 Hz, 1H), 7.44
(d, J=8.8 Hz, 1H), 7.02 (d, J=8.8 Hz, 1H), 6.90 (dd, J=8.4,
2.0 Hz, 1H), 3.92 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 168.79, 155.91, 144.62, 142.27, 136.04, 132.20, 131.34,
129.83, 127.11, 123.25, 116.10, 114.81, 113.57, 113.02,
112.32, 109.16, 102.02, 56.25. HRMS (Q-TOF): calculated
for C18H13N3O4 [M]: 335.0906. Found [M+H]+: 336.0976.
1
(yield 25.8%). H NMR (400 MHz, DMSO-d6) δ 9.93 (s,
1H), 6.56 (d, J=8.0 Hz, 1H), 6.52 (d, J=0.8 Hz, 1H), 6.35
(dd, J=8.4, 2.4 Hz, 1H), 3.33 (s, 2H), 2.96 (t, J=6.8 Hz,
2H), 2.37 (m, 6H), 1.67 (m, 2H), 1.51 (m, 4H), 1.40 (m,
2H). 13C NMR (100 MHz, DMSO-d6) δ 176.27, 144.94,
133.87, 127.14, 110.78, 110.28, 109.87, 56.96, 54.45 (2C),
42.64, 36.68, 26.23, 25.85 (2C), 24.41.
(Z/E)‑5‑amino‑3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)
indolin‑2‑one (5c) To a suspension of 5b (200 mg, 1 eq.)
in EtOH (10 mL), activated carbon (40 mg) and FeCl3
(40 mg) were added. The mixture was heated to 78 °C, and
stirred for 10 min. Then 80% aqueous solution of hydra-
zine hydrate (8 eq.) was added dropwise into the reaction
mixture in 5 min, the resulting mixture was allowed to
stirred at 78 °C for 8–10 h, then cooled to room tempera-
ture. The mixture was fltered to remove residue of acti-
vated carbon, the fltrate was concentrated under vacuum
to aford crude product, which was purifed through silica
gel column chromatography to give compound 5c as a
Z/E mixture (Z/E = 4:1), red solid (yield 17.5%), melting
point 266 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.85 (s,
1/5H), 11.79 (s, 4/5H), 10.02 (s, 4/5H), 9.97 (s, 1/5H),
9.38 (d, J = 2.8 Hz, 4/5H), 8.11 (d, J = 1.2 Hz, 1/5H), 7.88
(s, 4/5H), 7.76 (s, 1/5H), 7.58 (d, J = 2.0 Hz, 4/5H), 7.41
(m, 1H), 7.22 (d, J = 1.6 Hz, 1/5H), 7.15 (d, J = 2.0 Hz,
1/5H), 7.12 (d, J = 1.6 Hz, 4/5H), 6.86 (m, 1H), 6.56 (m,
1H), 6.44 (m, 1H), 4.60 (m, 2H), 3.87 (s, 12/5H), 3.81 (s,
3/5H). 13C NMR (100 MHz, DMSO-d6) δ 168.62, 155.35,
143.49, 143.16, 134.13, 131.17, 130.86, 129.91, 129.40,
127.09, 126.83, 126.52, 120.16, 114.43, 113.64, 113.40,
1‑(2‑oxoindolin‑5‑yl)‑3‑(2‑(piperidin‑1‑yl)ethyl)urea
(11s) To a solution of di(1H-imidazol-1-yl)methanone
(CDI, 15) (300 mg, 1.1 eq.) in dry DMF (5 mL), 5-ami-
noindolin-2-one (1) (1 eq.) was added. The mixture was
stirred at room temperature for 3 h. Then 2-(piperidin-1-yl)
ethan-1-amine (13) (1 eq.) was added dropwise into the
reaction mixture and stirred for another 3 h. After the reac-
tion was fnished, 20 mL water was added, and the mix-
ture was extracted with DCM (15 mL) for three times, the
combined organic layer was washed with brine, dried over
sodium sulfate, and concentrated under vacuum to aford
crude product, which was purifed through silica gel column
chromatography to give intermediate 11 s as a white solid
1
(yield 18.9%). H NMR (400 MHz, DMSO-d6) δ 10.16 (s,
1H), 8.50 (s, 1H), 7.32 (d, J=0.8 Hz, 1H), 7.10 (dd, J=8.0,
1.6 Hz, 1H), 6.66 (d, J=8.0 Hz, 1H), 6.01 (m, 1H), 3.41 (s,
2H), 3.18 (q, J=6.0 Hz, 2H), 2.38 (m, 6H), 1.52 (m, 4H),
1.41 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 176.65,
155.92, 138.09, 135.22, 126.51, 117.48, 115.91, 109.36,
58.58, 54.41 (2C), 36.80, 36.58, 25.82 (2C), 24.37.
1 3

Molecular Diversity
112.76, 111.57, 110.87, 109.70, 105.77, 100.79, 100.72,
56.00, 55.83. HRMS (Q-TOF): calculated for C18H15N3O2
[M]: 305.1164. Found [M + H]+: 306.1239.
(Z)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑oxoin‑
dolin‑5‑yl)‑3‑phenylpropanamide (5g) Following general
procedure B, starting from intermediate (3g) and 5-meth-
oxy-1H-indole-3-carbaldehyde (4) to aford compound 5g
as a Z isomer, yellow solid (yield 28.1%), melting point
(Z)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑oxoin‑
dolin‑5‑yl)acetamide (5d) Following general procedure B,
starting from intermediate (3d) and 5-methoxy-1H-indole-
3-carbaldehyde (4) to aford compound 5d as a Z isomer,
yellow solid (yield 35.5%), melting point 325 °C. 1H NMR
(400 MHz, DMSO-d6) δ 11.90 (s, 1H), 10.42 (s, 1H), 9.76
(s, 1H), 9.43 (s, 1H), 7.99 (s, 1H), 7.87 (d, J=1.2 Hz, 1H),
7.60 (d, J=2.4 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 7.28 (dd,
J=8.0, 1.2 Hz, 1H), 6.87 (dd, J=8.4, 2.0 Hz, 1H), 6.77
(d, J=8.0 Hz, 1H), 3.88 (s, 3H), 2.04 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 168.72, 168.32, 155.50, 135.79,
134.67, 132.98, 131.22, 129.40, 127.87, 126.18, 120.02,
119.01, 113.52, 112.94, 112.15, 111.65, 109.14, 100.76,
56.00, 24.14. HRMS (Q-TOF): calculated for C20H17N3O3
[M]: 347.1270. Found [M+H]+: 348.1344.
1
255 °C. H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H),
10.43 (s, 1H), 9.77 (s, 1H), 9.44 (d, J = 2.0 Hz, 1H), 8.00
(s, 1H), 7.89 (d, J = 1.2 Hz, 1H), 7.61 (d, J = 2.0 Hz, 1H),
7.42 (d, J = 8.8 Hz, 1H), 7.30 (m, 5H), 7.20 (m, 1H), 6.87
(dd, J = 8.8, 2.4 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 3.89 (s,
3H), 2.95 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 8.0 Hz, 2H). 13
C
NMR (100 MHz, DMSO-d6) δ 170.44, 168.73, 155.51,
141.79, 135.78, 134.72, 132.96, 131.24, 129.41, 128.81
(2C), 128.75 (2C), 127.89, 126.41, 126.20, 119.93,
119.02, 113.52, 112.92, 111.94, 111.66, 109.17, 100.81,
56.02, 38.30, 31.50. HRMS (Q-TOF): calculated for
C27H23N3O3 [M]: 437.1739. Found [M + H]+: 438.1818.
(Z)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑oxoin‑
dolin‑5‑yl)cinnamamide (5h) Following general proce-
dure B, starting from intermediate (3h) and 5-methoxy-
1H-indole-3-carbaldehyde (4) to aford compound 5h
as a Z isomer, yellow solid (yield 24.7%), melting point
(Z)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑oxoindo‑
lin‑5‑yl)propionamide (5e) Following general procedure B,
starting from intermediate (3e) and 5-methoxy-1H-indole-
3-carbaldehyde (4) to aford compound 5e as a Z isomer,
yellow solid (yield 30.3%), melting point 296 °C. 1H NMR
(400 MHz, DMSO-d6) δ 11.90 (s, 1H), 10.42 (s, 1H), 9.69 (s,
1H), 9.44 (d, J=2.4 Hz, 1H), 8.00 (s, 1H), 7.91 (d, J=1.2 Hz,
1H), 7.61 (d, J=2.0 Hz, 1H), 7.42 (d, J=8.8 Hz, 1H), 7.30
(dd, J=8.4, 1.6 Hz, 1H), 6.87 (dd, J=8.8, 2.4 Hz, 1H), 6.78
(d, J=8.4 Hz, 1H), 3.89 (s, 3H), 2.33 (q, J=7.6 Hz, 2H),
1.12 (t, J=7.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 172.02, 168.73, 155.51, 135.71, 134.69, 133.07, 131.23,
129.41, 127.85, 126.17, 119.96, 119.07, 113.50, 112.92,
112.05, 111.66, 109.14, 100.81, 56.03, 29.79, 10.33. HRMS
(Q-TOF): calculated for C21H19N3O3 [M]: 361.1426. Found
[M+H]+: 362.1502.
1
283 °C. H NMR (400 MHz, DMSO-d6) δ 11.93 (s, 1H),
10.48 (s, 1H), 10.09 (s, 1H), 9.45 (d, J = 2.0 Hz, 1H), 8.05
(s, 2H), 7.62 (m, 4H), 7.45 (m, 5H), 6.87 (m, 3H), 3.90 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 168.73, 163.73,
155.53, 140.03, 136.01, 135.37, 134.77, 132.98, 131.24,
130.11, 129.50 (2C), 129.42, 128.12 (2C), 128.06, 126.32,
123.02, 119.81, 118.93, 113.54, 112.97, 111.87, 111.69,
109.31, 100.79, 56.01. HRMS (Q-TOF): calculated for
C27H21N3O3 [M]: 435.1583. Found [M + H]+: 436.1658.
(Z)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑ox‑
oindolin‑5‑yl)‑3‑(3,4,5‑trimethoxyphenyl)propanamide
(5i) Following general procedure B, starting from inter-
mediate (3i) and 5-methoxy-1H-indole-3-carbaldehyde
(4) to aford compound 5i as a Z isomer, yellow solid
(yield 28.4%), melting point 292 °C. 1H NMR (400 MHz,
DMSO-d6) δ 11.91 (s, 1H), 10.42 (s, 1H), 9.76 (s, 1H),
9.43 (s, 1H), 7.99 (s, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.59
(d, J = 2.4 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.30 (dd,
J = 8.4, 2.0 Hz, 1H), 6.87 (dd, J = 8.8, 2.4 Hz, 1H), 6.78
(d, J = 8.0 Hz, 1H), 6.59 (s, 2H), 3.88 (s, 3H), 3.76 (s, 6H),
3.63 (s, 3H), 2.89 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 8.4 Hz,
2H). 13C NMR (100 MHz, DMSO-d6) δ 170.54, 168.71,
155.51, 153.22 (2C), 137.46, 136.21, 135.80, 134.72,
132.93, 131.23, 129.40, 127.88, 126.19, 119.93, 119.01,
113.53, 112.92, 111.96, 111.64, 109.19, 106.00 (2C),
100.78, 60.43, 56.25 (2C), 56.01, 38.44, 31.95. HRMS
(Q-TOF): calculated for C30H29N3O6 [M]: 527.2056.
Found [M + H]+: 528.2129.
(Z)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑oxoindo‑
lin‑5‑yl)cyclopropanecarboxamide (5f) Following general
procedure B, starting from intermediate (3f) and 5-methoxy-
1H-indole-3-carbaldehyde (4) to aford compound 5f as a Z
isomer, yellow solid (yield 25.6%), melting point 290 °C. 1H
NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 10.42 (s, 1H),
10.01 (s, 1H), 9.44 (d, J=2.4 Hz, 1H), 8.00 (s, 1H), 7.92 (d,
J=0.8 Hz, 1H), 7.61 (d, J=1.6 Hz, 1H), 7.42 (d, J=8.8 Hz,
1H), 7.28 (dd, J=8.0, 1.2 Hz, 1H), 6.87 (dd, J=8.8, 2.4 Hz,
1H), 6.78 (d, J=8.0 Hz, 1H), 3.89 (s, 3H), 1.80 (m, 1H),
0.80 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 171.69,
168.73, 155.51, 135.72, 134.71, 133.11, 131.23, 129.42,
127.90, 126.20, 119.98, 119.07, 113.49, 112.89, 112.06,
111.67, 109.17, 100.88, 56.05, 14.78, 7.25 (2C). HRMS
(Q-TOF): calculated for C22H19N3O3 [M]: 373.1426. Found
[M+H]+: 374.1507.
1 3

Molecular Diversity
(Z/E)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑ox‑
oindolin‑5‑yl)‑3‑morpholinopropanamide (5j) Following
general procedure B, starting from intermediate (3j) and
5-methoxy-1H-indole-3-carbaldehyde (4) to aford com-
pound 5j as a Z/E mixture (Z/E=4:1), yellow solid (yield
18.8%), melting point 305 °C. 1H NMR (400 MHz, DMSO-
d6) δ 12.10 (s, 1/5H), 11.90 (s, 4/5H), 10.42 (s, 4/5H), 10.36
(s, 1/5H), 9.95 (s, 1/5H), 9.87 (s, 4/5H), 9.43 (s, 4/5H), 8.38
(d, J=0.8 Hz, 1/5H), 8.18 (s, 1/5H), 8.00 (s, 4/5H), 7.89 (d,
J=1.2 Hz, 4/5H), 7.86 (s, 1/5H), 7.59 (d, J=2.0 Hz, 4/5H),
7.41 (m, 1H), 7.31 (m, 1H), 7.21 (d, J=2.0 Hz, 1/5H), 6.88
(m, 1H), 6.80 (m, 1H), 3.88 (s, 12/5H), 3.82 (s, 3/5H), 3.58
(m, 4H), 2.65 (m, 2H), 2.45 (m, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 170.33, 170.18, 170.12, 168.72, 155.50,
155.37, 137.67, 135.75, 134.71, 133.50, 133.01, 131.53,
131.23, 130.01, 129.40, 128.81, 127.90, 126.20, 121.14,
119.76, 119.01, 114.25, 113.52, 112.92, 111.79, 111.64,
110.86, 109.68, 109.19, 100.79, 100.73, 66.70, 56.00, 55.85,
54.86, 54.80, 53.56, 53.50, 34.41, 34.19. HRMS (Q-TOF):
calculated for C25H26N4O4 [M]: 446.1954. Found [M+H]+:
447.2033.
136.80, 136.19, 134.04, 133.71, 131.84, 130.70, 129.90,
129.38, 129.04, 127.90, 126.53, 126.45, 125.18, 124.88,
121.56, 121.43, 120.80, 115.10, 111.88, 110.26, 109.82,
55.08, 54.98, 54.19, 54.10, 34.46, 26.13, 26.11, 24.51,
24.49. HRMS (Q-TOF): calculated for C21H23N3O2S [M]:
381.1511. Found [M+H]+: 382.1556.
(Z/E)‑N‑(2‑ oxo ‑3‑(thiophen‑2‑ylmethylene)indo‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9b) Following
general procedure B, starting from intermediate (7) and
thiophene-2-carbaldehyde (8b) to aford compound 9b as
a Z/E mixture (Z/E=3:2), red solid (yield 27.2%), melting
1
point 130 °C. H NMR (400 MHz, DMSO-d6): δ 10.52 (s,
1H), 10.18 (s, 2/5H), 10.02 (s, 3/5H), 8.60 (d, J=1.6 Hz,
2/5H), 8.01 (m, 11/5H), 7.89 (d, J=5.2 Hz, 3/5H), 7.82 (d,
J=3.6 Hz, 2/5H), 7.78 (s, 2/5H), 7.43 (dd, J=8.4, 2.0 Hz,
2/5H), 7.32 (dd, J=5.2, 4.0 Hz, 2/5H), 7.21 (m, 6/5H), 6.85
(d, J=8.4 Hz, 2/5H), 6.80 (d, J=8.0 Hz, 3/5H), 2.60 (m,
2H), 2.45 (m, 2H), 2.38 (m, 4H), 1.51 (m, 4H), 1.39 (m, 2H).
13C NMR (100 MHz, DMSO-d6): δ 170.45, 170.40, 169.77,
167.91, 138.74, 138.50, 137.71, 137.63, 136.88, 136.38,
134.95, 133.80, 133.63, 132.51, 129.20, 128.59, 127.88,
127.71 124.72, 124.15, 122.30, 121.41, 121.20, 120.76,
115.53, 111.96, 110.27, 109.90, 55.08, 55.01, 54.19, 54.12,
34.47, 26.15, 26.11, 24.51. HRMS (Q-TOF): calculated for
C21H23N3O2S [M]: 381.1511. Found [M+H]+: 382.1555.
(Z)‑N‑(3‑((5‑methoxy‑1H‑indol‑3‑yl)methylene)‑2‑oxoindo‑
lin‑5‑yl)‑2‑(4‑methoxyphenyl)acetamide (5k) Following
general procedure B, starting from intermediate (3k) and
5-methoxy-1H-indole-3-carbaldehyde (4) to aford com-
pound 5k as a Z isomer, yellow solid (yield 21.3%), melting
1
point 293 °C. H NMR (400 MHz, DMSO-d6) δ 11.90 (s,
(Z)‑N‑(3‑((3,5‑dimethyl‑1H‑pyrrol‑2‑yl)methylene)‑2‑oxoin‑
dolin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide (9c) Following
general procedure B, starting from intermediate (7) and
3,5-dimethyl-1H-pyrrole-2-carbaldehyde (8c) to aford
compound 9c as a Z isomer, yellow solid (yield 26.4%),
1H), 10.43 (s, 1H), 9.95 (s, 1H), 9.44 (d, J=2.4 Hz, 1H),
8.00 (s, 1H), 7.91 (d, J=1.2 Hz, 1H), 7.61 (d, J=2.0 Hz,
1H), 7.41 (d, J=8.8 Hz, 1H), 7.32 (m, 3H), 6.88 (m, 3H),
6.78 (d, J=8.4 Hz, 1H), 3.88 (s, 3H), 3.74 (s, 3H), 3.57
(s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 169.50, 168.72,
158.49, 155.51, 135.82, 134.74, 133.01, 131.22, 130.59
(2C), 129.41, 128.64, 128.01, 126.21, 119.80, 119.00,
114.20 (2C), 113.50, 112.93, 111.84, 111.68, 109.18,
100.85, 56.04, 55.52, 42.80. HRMS (Q-TOF): calculated
for C27H23N3O4 [M]: 453.1689. Found [M+H]+: 454.1783.
1
melting point 299 °C. H NMR (400 MHz, DMSO-d6) δ
13.37 (s, 1H), 10.71 (s, 1H), 9.97 (s, 1H), 7.82 (d, J=2.0 Hz,
1H), 7.36 (s, 1H), 7.22 (dd, J=8.4, 2.0 Hz, 1H), 6.80 (d,
J=8.4 Hz, 1H), 6.01 (d, J=2.0 Hz, 1H), 2.60 (t, J=7.2 Hz,
2H), 2.43 (m, 6H), 2.32 (s, 3H), 2.29 (s, 3H), 1.51 (m, 4H),
1.39 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 170.30,
169.97, 136.22, 134.64, 133.64, 132.01, 126.94, 126.25,
123.31, 118.24, 113.29, 113.08, 110.34, 109.65, 55.09,
54.17 (2C), 34.45, 26.08 (2C), 24.49, 13.99, 11.74. HRMS
(Q-TOF): calculated for C23H28N4O2 [M]: 392.2212. Found
[M+H]+: 393.2285.
(Z/E)‑N‑(2‑ oxo ‑3‑(thiophen‑3‑ylmethylene)indo‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9a) Following
general procedure B, starting from intermediate (7) and
thiophene-3-carbaldehyde (8a) to aford compound 9a as a
Z/E mixture (Z/E=1:1), orange solid (yield 27.8%), melting
1
point 123 °C. H NMR (400 MHz, DMSO-d6): δ 10.54 (s,
( Z/E) ‑ N‑ ( 2 ‑ oxo ‑ 3 ‑ ( p y r i d i n ‑ 3 ‑ y l m e t hy l e n e ) i n d o ‑
1/2H), 10.48 (s, 1/2H), 10.09 (s, 1/2H), 10.03 (s, 1/2H), 8.88
(d, J=2.4 Hz, 1/2H), 8.17 (d, J=5.2 Hz, 1/2H), 8.12 (m,
1H), 7.99 (s, 1/2H), 7.75 (dd, J=4.8, 3.2 Hz, 1/2H), 7.71 (s,
1/2H), 7.62 (dd, J=4.8, 2.8 Hz, 1/2H), 7.56 (m, 1H), 7.44
(dd, J=8.4, 1.2 Hz, 1/2H), 7.21 (dd, J=8.4, 1.2 Hz, 1/2H),
6.82 (d, J=8.4 Hz, 1/2H), 6.78 (d, J=8.4 Hz, 1/2H), 2.58
(m, 2H), 2.43 (m, 6H), 1.44 (m, 6H). 13C NMR (100 MHz,
DMSO-d6): δ 170.42, 169.65, 167.88, 138.80, 136.85,
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9d) Following
general procedure B, starting from intermediate (7) and
nicotinaldehyde (8d) to aford compound 9d as a Z/E mix-
ture (Z/E=1:2), orange solid (yield 23.9%), melting point
149 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.61 (s, 1/3H),
10.57 (s, 2/3H), 10.05 (s, 1/3H), 10.04 (s, 2/3H), 9.22 (d,
J=1.6 Hz, 1/3H), 8.89 (m, 1H), 8.66 (dd, J=4.8, 1.2 Hz,
2/3H), 8.58 (dd, J=4.8, 1.2 Hz, 1/3H), 8.12 (m, 2/3H), 8.01
1 3

Molecular Diversity
(d, J=1.6 Hz, 1/3H), 7.77 (d, J=1.6 Hz, 2/3H), 7.68 (s,
1/3H), 7.61 (s, 2/3H), 7.54 (dd, J=8.0, 4.8 Hz, 2/3H), 7.48
(m, 1H), 7.28 (dd, J=8.4, 2.0 Hz, 1/3H), 6.83 (d, J=8.4 Hz,
2/3H), 6.79 (d, J=8.4 Hz, 1/3H), 2.61 (t, J=7.2 Hz, 2/3H),
2.54 (t, J=6.8 Hz, 4/3H), 2.46 (t, J=7.2 Hz, 2/3H), 2.37
(m, 16/3H), 1.45 (m, 6H). 13C NMR (100 MHz, DMSO-d6):
δ 170.45, 170.36, 168.78, 167.60, 152.96, 150.73, 150.60,
150.27, 139.31, 138.43, 137.50, 136.87, 133.88, 133.76,
133.18, 132.62, 130.90, 130.26, 129.95, 129.49, 124.71,
124.16, 123.56, 122.33, 121.79, 121.05, 114.53, 112.71,
110.61, 110.04, 55.04, 54.89, 54.17, 54.06, 34.43, 34.33,
26.08, 26.06, 24.49, 24.45. HRMS (Q-TOF): calculated for
C22H24N4O2 [M]: 376.1899. Found [M+H]+: 377.1865.
general procedure B, starting from intermediate (7) and
1H-indole-7-carbaldehyde (8g) to aford compound 9g
as a Z/E mixture (Z/E = 1:6), orange solid (yield 25.0%),
1
melting point 151 °C. H NMR (400 MHz, DMSO-d6):
δ 11.80 (s, 1/7H), 11.52 (s, 6/7H), 10.51 (s, 1H), 10.00
(s, 1/7H), 9.95 (s, 6/7H), 8.67 (d, J = 7.6 Hz, 1/7H), 8.14
(s, 1/7H), 8.07 (d, J = 1.2 Hz, 1/7H), 7.98 (s, 6/7H), 7.79
(d, J = 1.2 Hz, 6/7H), 7.69 (m, 1H), 7.57 (d, J = 7.2 Hz,
6/7H), 7.47 (m, 1H), 7.39 (t, J = 2.8 Hz, 6/7H), 7.23 (dd,
J = 8.4, 1.6 Hz, 1/7H), 7.13 (t, J = 7.6 Hz, 6/7H), 7.07
(t, J = 8.0 Hz, 1/7H), 6.81 (d, J = 8.4 Hz, 6/7H), 6.77
(d, J = 8.0 Hz, 1/7H), 6.55 (m, 1H), 2.62 (t, J = 6.8 Hz,
2/7H), 2.51 (m, 12/7H), 2.35 (m, 6H), 1.43 (m, 6H). 13C
NMR (100 MHz, DMSO-d6): δ 170.27, 169.45, 138.99,
134.92, 133.47, 132.67, 128.94, 127.85, 126.66, 123.12,
122.12, 121.90, 119.37, 118.51, 114.84, 110.15, 102.36,
54.89, 54.02, 34.21, 26.05, 24.44. HRMS (Q-TOF): cal-
culated for C25H26N4O2 [M]: 414.2056. Found [M + H]+:
415.2119.
(Z/E)‑N‑(3‑((1H‑indol‑3‑yl)methylene)‑2‑oxoindo‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9e) Following
general procedure B, starting from intermediate (7) and
1H-indole-3-carbaldehyde (8e) to aford compound 9e as a
Z/E mixture (Z/E=2:1), yellow solid (yield 20.9%), melting
point 188 °C. 1H NMR (400 MHz, DMSO-d6): δ 12.09 (brs,
1H), 10.45 (s, 2/3H), 10.38 (s, 1/3H), 10.11 (s, 1/3H), 9.99
(s, 2/3H), 9.46 (s, 2/3H), 8.42 (d, J=2.0 Hz, 1/3H), 8.24 (s,
1/3H), 8.05 (m, 2/3H), 7.98 (m, 4/3H), 7.88 (s, 1/3H), 7.76
(d, J=7.6 Hz, 1/3H), 7.54 (m, 1H), 7.25 (m, 3H), 6.83 (d,
J=8.4 Hz, 1/3H), 6.79 (d, J=8.0 Hz, 2/3H), 2.60 (m, 2H),
2.44 (m, 6H), 1.47 (m, 6H). 13C NMR (100 MHz, DMSO-
d6): δ 170.46, 170.38, 170.26, 168.68, 137.73, 136.68,
136.37, 135.77, 134.19, 133.61, 133.23, 129.61, 128.54,
128.14, 127.82, 127.25, 126.02, 123.31, 122.99, 122.77,
121.81, 121.42, 121.38, 119.74, 119.59, 118.95, 118.46,
114.16, 112.83, 111.49, 111.46, 110.84, 109.77, 109.34,
55.14, 55.07, 54.20, 54.12, 34.57, 34.46, 26.12, 26.10,
24.53, 24.48. HRMS (Q-TOF): calculated for C25H26N4O2
[M]: 414.2056. Found [M+H]+: 415.2116.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9h) Following
general procedure B, starting from intermediate (7) and
1-naphthaldehyde (8h) to aford compound 9h as an E iso-
1
mer, orange solid (yield 39.4%), melting point 157 °C. H
NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.90 (s, 1H),
8.07 (m, 3H), 7.96 (m, 1H), 7.87 (m, 1H), 7.63 (m, 3H),
7.47 (dd, J=8.4, 1.6 Hz, 1H), 7.42 (d, J=1.6 Hz, 1H), 6.83
(d, J=8.4 Hz, 1H), 2.46 (t, J=6.8 Hz, 2H), 2.29 (m, 6H),
1.36 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 170.22,
168.89, 139.20, 133.86, 133.73, 133.54, 131.74, 131.23,
130.41, 130.06, 129.20, 127.55, 127.36, 127.06, 126.05,
124.65, 122.14, 121.48, 114.85, 110.38, 54.83, 53.97 (2C),
34.17, 26.03 (2C), 24.43. HRMS (Q-TOF): calculated for
C27H27N3O2 [M]: 425.2103. Found [M+H]+: 426.2181.
E ‑ N‑ ( 3 ‑ ( ( 1 H ‑ i n d o l ‑ 4 ‑ y l ) m e t h y l e n e ) ‑ 2 ‑ oxo i n d o ‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9f) Following
(E)‑N‑(3‑((4‑fluoronaphthalen‑1‑yl)methylene)‑2‑oxoin‑
general procedure B, starting from intermediate (7) and
1H-indole-4-carbaldehyde (8f) to aford compound 9f as an
E isomer, red solid (yield 17.7%), melting point 150 °C. 1H
NMR (400 MHz, DMSO-d6) δ 11.43 (s, 1H), 10.52 (s, 1H),
9.98 (s, 1H), 7.94 (s, 1H), 7.80 (s, 1H), 7.52 (m, 4H), 7.23 (t,
J=7.6 Hz, 1H), 6.82 (d, J=8.4 Hz, 1H), 6.47 (m, 1H), 2.61
(s, 2H), 2.40 (m, 6H), 1.47 (m, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 169.95, 169.57, 138.97, 136.56, 134.80,
133.37, 127.77, 127.39, 127.31, 125.84, 121.89, 121.71,
121.29, 120.41, 115.26, 114.11, 110.09, 100.35, 54.55,
53.85 (2C), 33.77, 25.65 (2C), 24.09. HRMS (Q-TOF): cal-
culated for C25H26N4O2 [M]: 414.2056. Found [M+H]+:
415.2137.
dolin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9i) Follow-
ing general procedure B, starting from intermediate (7)
and 4-fuoro-1-naphthaldehyde (8i) to aford compound 9i
as an E isomer, orange solid (yield 26.3%), melting point
1
136 °C. H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H),
9.90 (s, 1H), 8.18 (m, 1H), 8.00 (m, 2H), 7.86 (dd, J=8.0,
6.0 Hz, 1H), 7.73 (m, 2H), 7.47 (m, 2H), 7.33 (s, 1H), 6.82
(d, J=8.4 Hz, 1H), 2.47 (t, J=6.8 Hz, 2H), 2.30 (m, 6H),
1.36 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 170.21,
168.78, 160.32, 157.80, 139.19, 133.59, 132.94, 132.67 (d,
J=4.9 Hz), 130.39 128.74, 128.38 (d, J=4.2 Hz), 127.80 (d,
J=8.7 Hz), 125.11 (d, J=2.1 Hz), 123.52 (d, J=16.6 Hz),
122.03, 121.39, 121.11 (d, J=5.0 Hz), 114.67, 110.41,
110.19 (d, J=20.0 Hz), 54.79, 53.95 (2C), 34.17, 25.99
(2C), 24.38. HRMS (Q-TOF): calculated for C27H26N3O2
[M]: 443.2009. Found [M+H]+: 444.2076.
(Z/E)‑N‑(3‑((1H‑indol‑7‑yl)methylene)‑2‑oxoindo‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide (9g) Following
1 3

Molecular Diversity
( E ) ‑ N‑ ( 2 ‑ oxo ‑ 3 ‑ ( q u i n o l i n ‑ 4 ‑ y l m e t h y l e n e ) i n d o ‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide (9j) Following
(Z/E)‑N‑(3‑((6‑methoxynaphthalen‑2‑yl)methylene)‑2‑ox‑
oindolin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9m) Fol-
general procedure B, starting from intermediate (7) and
quinoline-4-carbaldehyde (8j) to aford compound 9j as an
E isomer, orange solid (yield 26.6%), melting point 264 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 9.87 (s,
1H), 9.01 (d, J=4.4 Hz, 1H), 8.15 (m, 1H), 7.97 (m, 2H),
7.85 (m, 1H), 7.75 (m, 1H), 7.66 (m, 1H), 7.51 (dd, J=8.4,
1.2 Hz, 1H), 7.21 (d, J=1.2 Hz, 1H), 6.83 (d, J=8.4 Hz,
1H), 2.44 (t, J=7.2 Hz, 2H), 2.27 (m, 6H), 1.35 (m, 6H).
13C NMR (100 MHz, DMSO-d6) δ 168.32, 168.22, 150.80,
148.38, 140.84, 139.74, 133.38, 132.24, 130.92, 130.59,
130.21, 127.90, 125.79, 125.16, 122.60, 121.05, 120.92,
115.20, 110.68, 52.98 (2C), 52.71, 31.40, 23.55 (2C), 22.17.
HRMS (Q-TOF): calculated for C26H26N4O2 [M]: 426.2056.
Found [M+H]+: 427.2113.
lowing general procedure B, starting from intermediate (7)
and 6-methoxy-2-naphthaldehyde (8m) to aford compound
9m as a Z/E mixture (Z/E=1:4), orange solid (yield 39.6%),
1
melting point 130 °C. H NMR (400 MHz, DMSO-d6): δ
10.56 (s, 1/5H), 10.52 (s, 4/5H), 10.04 (s, 1/5H), 10.03 (s,
4/5H), 8.81 (s, 1/5H), 8.66 (dd, J=8.8, 1.2 Hz, 1/5H), 8.26
(s, 4/5H), 8.09 (d, J=0.8 Hz, 4/5H), 8.02 (d, J=0.8 Hz,
1/5H), 7.93 (m, 8/5H), 7.87 (m, 2/5H), 7.80 (m, 4/5H), 7.75
(s, 1/5H), 7.73 (s, 4/5H), 7.42 (m, 8/5H), 7.37 (d, J=2.0 Hz,
1/5H), 7.24 (m, 6/5H), 6.84 (d, J=8.4 Hz, 4/5H), 6.79 (d,
J=8.0 Hz, 1/5H), 3.92 (s, 3H), 2.62 (t, J=6.8 Hz, 2/5H),
2.54 (t, J=6.8 Hz, 8/5H), 2.47 (t, J=6.8 Hz, 2/5H), 2.36
(m, 28/5H), 1.42 (m, 6H). 13C NMR (100 MHz, DMSO-d6):
δ 170.34, 169.46, 159.07, 139.08, 136.72, 135.42, 133.54,
130.81, 130.10, 129.85, 128.58, 127.66, 127.59, 127.31,
121.81, 121.55, 119.77, 115.03, 110.33, 106.61, 55.86,
54.88, 54.17, 54.01, 34.29, 26.07, 26.00, 24.38. HRMS
(Q-TOF): calculated for C28H29N3O3 [M]: 455.2209. Found
[M+H]+: 456.2287.
(E)‑N‑(3‑((6‑methoxyquinolin‑4‑yl)methylene)‑2‑oxoin‑
dolin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9k) Follow-
ing general procedure B, starting from intermediate (7)
and 6-methoxyquinoline-4-carbaldehyde (8k) to aford
compound 9k as an E isomer, orange solid (yield 27.5%),
1
melting point 213 °C. H NMR (400 MHz, DMSO-d6) δ
(Z/E)‑N‑(2‑oxo‑3‑(quinoxalin‑6‑ylmethylene)indo‑
10.65 (s, 1H), 9.89 (s, 1H), 8.84 (d, J=4.4 Hz, 1H), 8.05 (d,
J=9.2 Hz, 1H), 7.97 (s, 1H), 7.71 (d, J=4.4 Hz, 1H), 7.50
(m, 2H), 7.30 (d, J=1.6 Hz, 1H), 7.20 (d, J=2.8 Hz, 1H),
6.83 (d, J=8.4 Hz, 1H), 3.87 (s, 3H), 2.46 (t, J=6.8 Hz,
2H), 2.29 (m, 6H), 1.36 (m, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 170.25, 168.48, 158.25, 148.00, 144.61,
139.45, 139.24, 133.73, 132.01, 131.81, 131.11, 127.01,
122.95, 122.44, 121.49, 121.00, 115.04, 110.57, 103.00,
56.03, 54.80, 53.99 (2C), 34.24, 26.03 (2C), 24.39. HRMS
(Q-TOF): calculated for C27H28N4O3 [M]: 456.2161. Found
[M+H]+: 457.2220.
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9n) Following
general procedure B, starting from intermediate (7) and
quinoxaline-6-carbaldehyde (8n) to aford compound 9n as
a Z/E mixture (Z/E=1:3), yellow solid (yield 22.8%), melt-
ing point 195 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.66
(s, 1/4H), 10.61 (s, 3/4H), 10.07 (s, 1/4H), 10.00 (s, 3/4H),
9.09 (d, J=1.2 Hz, 1/4H), 9.02 (m, 6/4H), 8.99 (m, 1/4H),
8.97 (m, 1/4H), 8.77 (dd, J=8.8, 2.0 Hz, 1/4H), 8.38 (s,
3/4H), 8.17 (m, 6/4H), 8.11 (d, J=8.8 Hz, 1/4H), 8.05 (d,
J=1.6 Hz, 1/4H), 7.93 (s, 1/4H), 7.83 (s, 3/4H), 7.76 (d,
J=1.6 Hz, 3/4H), 7.53 (dd, J=8.4, 2.0 Hz, 3/4H), 7.30 (dd,
J=8.4, 2.0 Hz, 1/4H), 6.85 (d, J=8.4 Hz, 3/4H), 6.81 (d,
J=8.4 Hz, 1/4H), 2.62 (t, J=7.2 Hz, 2/4H), 2.48 (m, 2H),
2.40 (m, 4/4H), 2.31 (m, 18/4H), 1.35 (m, 6H). 13C NMR
(100 MHz, DMSO-d6): δ 170.48, 170.34, 168.95, 167.55,
146.99, 146.89, 146.86, 146.79, 143.27, 142.93, 142.65,
142.38, 139.47, 137.68, 136.83, 135.97, 135.29, 134.61,
133.87, 133.71, 133.50, 133.06, 131.01, 130.45, 130.09,
129.99, 129.71, 128.89, 124.89, 122.50, 122.03, 121.06,
114.72, 112.93, 110.63, 110.06, 55.06, 54.81, 54.18, 53.95,
34.44, 34.23, 26.09, 25.97, 24.50, 24.35. HRMS (Q-TOF):
calculated for C25H25N5O2 [M]: 427.2008. Found [M+H]+:
428.2036.
(Z/E)‑N‑(3‑(naphthalen‑2‑ylmethylene)‑2‑oxoindo‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9l) Following
general procedure B, starting from intermediate (7) and
2-naphthaldehyde (8l) to aford compound 9l as a Z/E mix-
ture (Z/E=1:5), orange solid (yield 32.2%), melting point
122 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.58 (s, 1/6H),
10.56 (s, 5/6H), 10.06 (s, 1/6H), 10.03 (s, 5/6H), 8.85 (s,
1/6H), 8.62 (dd, J=8.8, 1.2 Hz, 1/6H), 8.32 (s, 5/6H), 8.00
(m, 4H), 7.83 (m, 1H), 7.76 (s, 5/6H), 7.60 (m, 2H), 7.46
(dd, J=8.4, 1.6 Hz, 5/6H), 7.27 (dd, J=8.4, 1.6 Hz, 1/6H),
6.85 (d, J=8.4 Hz, 5/6H), 6.80 (d, J=8.0 Hz, 1/6H), 2.60
(m, 2H), 2.36 (m, 6H), 1.42 (m, 6H). 13C NMR (100 MHz,
DMSO-d6): δ 170.30, 169.31, 139.25, 136.31, 133.77,
133.58, 133.19, 132.29, 129.93, 129.15, 128.75, 128.24,
128.15, 127.86, 127.20, 127.05, 122.12, 121.40, 115.10,
110.42, 54.80, 54.14, 53.96, 34.17, 26.02, 25.92, 24.31.
HRMS (Q-TOF): calculated for C27H27N3O2 [M]: 425.2103.
Found [M+H]+: 426.2182.
(E)‑N‑(2‑oxo‑3‑(phenanthren‑9‑ylmethylene)indo‑
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9o) Following
general procedure B, starting from intermediate (7) and
phenanthrene-9-carbaldehyde (8o) to aford compound 9o
as an E isomer, orange solid (yield 25.3%), melting point
1
160 °C. H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H),
1 3

Molecular Diversity
9.87 (s, 1H), 8.96 (d, J=8.0 Hz, 1H), 8.90 (d, J=8.4 Hz,
1H), 8.20 (s, 1H), 8.05 (m, 3H), 7.74 (m, 4H), 7.54 (dd,
J=8.4, 2.0 Hz, 1H), 7.39 (d, J=1.2 Hz, 1H), 6.85 (d,
J=8.4 Hz, 1H), 2.37 (t, J=6.4 Hz, 2H), 2.19 (m, 6H),
1.19 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 170.22,
168.91, 139.34, 133.98, 133.54, 131.14, 130.88, 130.57
(2C), 130.12, 130.00, 129.93, 128.42, 128.27, 127.94 (2C),
127.70, 125.56, 124.09, 123.41, 122.36, 121.59, 115.33,
110.40, 54.71, 53.83 (2C), 34.02, 25.87 (2C), 24.33. HRMS
(Q-TOF): calculated for C31H29N3O2 [M]: 475.2260. Found
[M+H]+: 476.2337.
1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.11 (s,
1H), 8.07 (t, J=8.8 Hz, 2H), 7.94 (d, J=8.0 Hz, 1H), 7.84
(d, J=7.2 Hz, 1H), 7.62 (m, 3H), 7.19 (t, J=8.2 Hz, 1H),
6.90 (t, J=6.8 Hz, 2H), 6.69 (t, J=7.6 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 168.72, 143.50, 133.75, 133.68,
132.20, 131.02, 130.65, 130.24, 130.23, 129.21, 127.54,
127.19, 127.08, 125.95, 124.83, 123.05, 121.51, 121.48,
110.58. HRMS (Q-TOF): calculated for C19H13NO [M]:
271.0997. Found [M+H]+: 272.1077.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑2‑(piperidin‑1‑yl)acetamide
(16b) Following
(E)‑N‑(3‑(anthracen‑9‑ylmethylene)‑2‑ oxoindo ‑
general procedure B, starting from intermediate (11b) and
lin‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9p) Following
1-naphthaldehyde (8h) to aford compound 16b as an E iso-
1
general procedure B, starting from intermediate (7) and
anthracene-9-carbaldehyde (8p) to aford compound 9p
as an E isomer, orange solid (yield 25.2%), melting point
mer, orange solid (yield 36.3%), melting point 120 °C. H
NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.32 (s, 1H),
8.08 (m, 3H), 7.95 (m, 1H), 7.88 (d, J=7.2 Hz, 1H), 7.62
(m, 3H), 7.41 (m, 2H), 6.83 (d, J=8.0 Hz, 1H), 2.89 (s,
2H), 2.34 (m, 4H), 1.48 (m, 4H), 1.37 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 168.90, 168.37, 139.50, 133.90,
133.72, 132.70, 131.72, 131.21, 130.38, 130.10, 129.21,
127.55, 127.48, 127.04, 126.01, 124.70, 122.59, 121.45,
115.56, 110.29, 62.83, 54.42 (2C), 25.94 (2C), 23.97.
HRMS (Q-TOF): calculated for C26H25N3O2 [M]: 411.1947.
Found [M+H]+: 412.2024.
1
250 °C. H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H),
9.58 (s, 1H), 8.77 (s, 1H), 8.28 (s, 1H), 8.20 (m, 2H), 7.96
(m, 2H), 7.55 (m, 5H), 6.80 (d, J=8.4 Hz, 1H), 5.93 (d,
J=2.0 Hz, 1H), 2.28 (t, J=6.8 Hz, 2H), 2.10 (m, 6H), 1.28
(m, 2H), 1.18 (m, 4H). 13C NMR (100 MHz, DMSO-d6)
δ 169.98, 168.36, 139.28, 133.50, 132.86, 131.97, 131.32
(2C), 129.57 (2C), 128.83, 128.59 (2C), 128.53, 127.29
(2C), 126.14 (2C), 125.54 (2C), 122.32, 121.64, 115.64,
110.15, 55.38, 53.77 (2C), 33.78, 25.92 (2C), 24.42. HRMS
(Q-TOF): calculated for C31H29N3O2 [M]: 475.2260. Found
[M+H]+: 476.2332.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑4‑(piperidin‑1‑yl)butanamide
(16c) Following
general procedure B, starting from intermediate (11c) and
1-naphthaldehyde (8h) to aford compound 16c as an E
isomer, orange solid (yield 27.2%), melting point 139 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 9.54 (s,
1H), 8.07 (m, 3H), 7.96 (m, 1H), 7.87 (m, 1H), 7.63 (m,
3H), 7.52 (d, J=1.6 Hz, 1H), 7.45 (dd, J=8.4, 2.0 Hz, 1H),
6.81 (d, J=8.4 Hz, 1H), 2.25 (m, 4H), 2.16 (m, 4H), 1.61
(m, 2H), 1.43 (m, 4H), 1.34 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 171.06, 168.93, 139.11, 133.73, 133.72,
133.67, 131.68, 131.31, 130.40, 130.05, 129.20, 127.55,
127.44, 127.06, 126.11, 124.63, 122.20, 121.39, 115.07,
110.28, 58.41, 54.43 (2C), 34.69, 25.98 (2C), 24.58, 22.77.
HRMS (Q-TOF): calculated for C28H29N3O2 [M]: 439.2260.
Found [M+H]+: 440.2338.
(Z/E)‑N‑(3‑([1,1′‑biphenyl]‑4‑ylmethylene)‑2‑oxoindoli
n‑5‑yl)‑3‑(piperidin‑1‑yl)propanamide
(9q) Following
general procedure B, starting from intermediate (7) and
[1,1′-biphenyl]-4-carbaldehyde (8q) to aford compound
9q as a Z/E mixture (Z/E=1:4), orange solid (yield 31.1%),
1
melting point 212 °C. H NMR (400 MHz, DMSO-d6): δ
10.58 (s, 1/5H), 10.53 (s, 4/5H), 10.03 (s, 1H), 8.52 (m,
2/5H), 8.00 (m, 1H), 7.80 (m, 28/5H), 7.68 (s, 1/5H), 7.65
(s, 4/5H), 7.49 (m, 14/5H), 7.41 (m, 1H), 7.28 (dd, J=8.4,
2.0 Hz, 1/5H), 6.83 (d, J=8.4 Hz, 4/5H), 6.78 (d, J=8.0 Hz,
1/5H), 2.61 (t, J=7.2 Hz, 2/5H), 2.54 (m, 8/5H), 2.46 (t,
J=6.8 Hz, 2/5H), 2.36 (m, 28/5H), 1.42 (m, 6H). 13C NMR
(100 MHz, DMSO-d6): δ 170.37, 169.29, 141.75, 139.74,
139.16, 135.97, 133.83, 133.61, 133.25, 130.68, 129.55,
129.51, 128.50, 128.11, 127.38, 127.23, 127.18, 126.77,
121.99, 121.38, 114.88, 110.41, 55.06, 54.87, 54.18, 54.03,
34.44, 34.33, 26.08, 26.03, 24.49, 24.38. HRMS (Q-TOF):
calculated for C29H29N3O2 [M]: 451.2260. Found [M+H]+:
452.2340.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑oxoindolin‑5‑yl)
acetamide (16d) Following general procedure B, start-
ing from intermediate (3d) and 1-naphthaldehyde (8h)
to aford compound 16d as an E isomer, yellow solid
(yield 42.7%), melting point 270 °C. 1H NMR (400 MHz,
DMSO-d6) δ 10.62 (s, 1H), 9.68 (s, 1H), 8.07 (m, 3H),
7.96 (m, 1H), 7.87 (d, J = 7.2 Hz, 1H), 7.63 (m, 3H), 7.56
(d, J = 1.6 Hz, 1H), 7.42 (dd, J = 8.4, 2.0 Hz, 1H), 6.82 (d,
J = 8.4 Hz, 1H), 1.89 (s, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 168.92, 168.14, 139.16 133.76, 133.73, 133.69,
(E)‑3‑(naphthalen‑1‑ylmethylene)indolin‑2‑one (16a) Fol-
lowing general procedure B, starting from indolin-2-one (3a)
and 1-naphthaldehyde (8h) to aford compound 16a as an E
isomer, yellow solid (yield 63.8%), melting point 199 °C.
1 3

Molecular Diversity
131.68, 131.29, 130.38, 130.10, 129.21, 127.54, 127.43,
127.06, 126.15, 124.62, 122.05, 121.40, 114.96, 110.33,
24.17. HRMS (Q-TOF): calculated for C21H16N2O2 [M]:
328.1212. Found [M + H]+: 329.1299.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑oxoindolin‑5‑yl)
cinnamamide (16h) Following general procedure B, start-
ing from intermediate (3h) and 1-naphthaldehyde (8h) to
aford compound 16h as an E isomer, yellow solid (yield
26.7%), melting point 273 °C. 1H NMR (400 MHz, DMSO-
d6) δ 10.64 (s, 1H), 9.92 (s, 1H), 8.09 (m, 3H), 7.98 (m,
1H), 7.92 (d, J=6.8 Hz, 1H), 7.63 (m, 7H), 7.43 (m, 4H),
6.88 (d, J=8.4 Hz, 1H), 6.69 (d, J=16.0 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 168.93, 163.48, 140.08, 139.50,
135.23, 134.00, 133.75, 133.60, 131.71, 131.31, 130.50,
130.10, 129.99, 129.43 (2C), 129.23, 128.08 (2C), 127.58,
127.48, 127.10, 126.18, 124.66, 122.76, 122.24, 121.58,
114.96, 110.51. HRMS (Q-TOF): calculated for C28H20N2O2
[M]: 416.1525. Found [M+H]+: 417.1598.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑oxoindolin‑5‑yl)
propionamide (16e) Following general procedure B,
starting from intermediate (3e) and 1-naphthaldehyde
(8h) to aford compound 16e as an E isomer, yellow solid
(yield 44.0%), melting point 272 °C. 1H NMR (400 MHz,
DMSO-d6) δ 10.58 (s, 1H), 9.54 (s, 1H), 8.08 (m, 3H),
7.97 (m, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.64 (m, 3H),
7.55 (d, J = 1.6 Hz, 1H), 7.47 (dd, J = 8.4, 2.0 Hz, 1H),
6.82 (d, J = 8.4 Hz, 1H), 2.16 (q, J = 7.6 Hz, 2H), 0.98
(t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ
171.86, 168.93, 139.10, 133.75, 133.73, 133.72, 131.68,
131.32, 130.42, 130.05, 129.20, 127.56, 127.45, 127.07,
126.15, 124.63, 122.13, 121.41, 114.93, 110.32, 29.77,
10.18. HRMS (Q-TOF): calculated for C22H18N2O2 [M]:
342.1368. Found [M + H]+: 343.1443.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑3‑(3,4,5‑trimethoxyphenyl)propanamide
(16i) Following general procedure B, starting from inter-
mediate (3i) and 1-naphthaldehyde (8h) to aford com-
pound 16i as an E isomer, orange solid (yield 30.2%), melt-
1
ing point 130 °C. H NMR (400 MHz, DMSO-d6) δ 10.59
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑oxoindolin‑5‑yl)
cyclopropanecarboxamide (16f) Following general pro-
cedure B, starting from intermediate (3f) and 1-naphthal-
dehyde (8h) to aford compound 16f as an E isomer, yel-
(s, 1H), 9.63 (s, 1H), 8.07 (m, 3H), 7.93 (m, 1H), 7.88 (d,
J=7.2 Hz, 1H), 7.63 (m, 3H), 7.57 (d, J=1.2 Hz, 1H), 7.44
(dd, J=8.4, 1.6 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 6.48 (s,
2H), 3.67 (s, 6H), 3.59 (s, 3H), 2.75 (t, J=7.2 Hz, 2H),
2.47 (t, J=8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
δ 170.37, 168.92, 153.13 (2C), 139.24, 137.30, 136.13,
133.82, 133.74, 133.53, 131.68, 131.32, 130.43, 130.02,
129.20, 127.57, 127.43, 127.08, 126.11, 124.63, 122.19,
121.44, 115.00, 110.36, 105.87 (2C), 60.39, 56.15 (2C),
38.28, 31.71. HRMS (Q-TOF): calculated for C31H28N2O5
[M]: 508.1998. Found [M+H]+: 509.2079.
1
low solid (yield 31.8%), melting point 291 °C. H NMR
(400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.86 (s, 1H), 8.07
(m, 3H), 7.97 (m, 1H), 7.88 (d, J = 6.8 Hz, 1H), 7.64 (m,
3H), 7.48 (m, 2H), 6.83 (d, J = 8.2 Hz, 1H), 1.61 (m, 1H),
0.69 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 171.49,
168.95, 139.12, 133.83, 133.73, 133.68, 131.67, 131.30,
130.45, 130.00, 129.20, 127.58, 127.45, 127.09, 126.14,
124.63, 122.22, 121.44, 114.94, 110.36, 14.80, 7.34
(2C). HRMS (Q-TOF): calculated for C23H18N2O2 [M]:
354.1368. Found [M + H]+: 355.1540.
(E)‑3‑morpholino‑N‑(3‑(naphthalen‑1‑ylmethylene)‑
2‑oxoindolin‑5‑yl)propanamide (16j) Following general
procedure B, starting from intermediate (11j) and 1-naph-
thaldehyde (8h) to aford compound 16j as an E isomer,
orange solid (yield 32.7%), melting point 137 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.73 (s, 1H), 8.07 (m,
3H), 7.96 (m, 1H), 7.87 (d, J=6.8 Hz, 1H), 7.63 (m, 3H),
7.45 (m, 2H), 6.82 (d, J=9.2 Hz, 1H), 3.49 (m, 4H), 2.50
(m, 2H), 2.31 (m, 6H). 13C NMR (100 MHz, DMSO-d6)
δ 169.99, 168.90, 139.24, 133.86, 133.73, 133.49, 131.72,
131.26, 130.43, 130.06, 129.22, 127.57, 127.40, 127.08,
126.07, 124.65, 122.23, 121.45, 115.00, 110.36, 66.63 (2C),
54.58, 53.39 (2C), 34.09. HRMS (Q-TOF): calculated for
C26H25N3O3 [M]: 427.1896. Found [M+H]+: 428.2076.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑3‑phenylpropanamide (16g) Following general
procedure B, starting from intermediate (3g) and 1-naph-
thaldehyde (8h) to aford compound 16g as an E isomer,
1
orange solid (yield 30.2%), melting point 189 °C. H
NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.60 (s, 1H),
8.08 (m, 3H), 7.97 (m, 1H), 7.88 (d, J = 6.8 Hz, 1H), 7.64
(m, 3H), 7.53 (d, J = 2.0 Hz, 1H), 7.42 (dd, J = 8.4, 2.0 Hz,
1H), 7.23 (m, 2H), 7.15 (m, 3H), 6.82 (d, J = 8.4 Hz, 1H),
2.80 (t, J = 7.6 Hz, 2H), 2.46 (t, J = 8.4 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 170.27, 168.93, 141.62, 139.23,
133.83, 133.74, 133.48, 131.68, 131.31, 130.45, 130.02,
129.21, 128.71 (2C), 128.67 (2C), 127.57, 127.43, 127.09,
126.35, 126.13, 124.64, 122.27, 121.42, 115.14, 110.34,
38.24, 31.33. HRMS (Q-TOF): calculated for C28H22N2O2
[M]: 418.1681. Found [M + H]+: 419.1757.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑3‑thiomorpholino propanamide (16k) Following
general procedure B, starting from intermediate (11k) and
1-naphthaldehyde (8h) to aford compound 16k as an E iso-
1
mer, orange solid (yield 34.4%), melting point 140 °C. H
1 3

Molecular Diversity
NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.66 (s, 1H),
8.07 (m, 3H), 7.95 (m, 1H), 7.87 (d, J=7.2 Hz, 1H), 7.63
(m, 3H), 7.46 (d, J=1.6 Hz, 1H), 7.43 (dd, J=8.4, 2.0 Hz,
1H), 6.82 (d, J=8.4 Hz, 1H), 2.60 (m, 4H), 2.54 (m, 6H),
2.29 (t, J=6.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
δ 170.07, 168.90, 139.25, 133.85, 133.72, 133.46, 131.71,
131.26, 130.44, 130.05, 129.21, 127.57, 127.41, 127.08,
126.06, 124.65, 122.29, 121.44, 115.07, 110.35, 54.93,
54.60 (2C), 34.00, 27.68 (2C). HRMS (Q-TOF): calcu-
lated for C26H25N3O2S [M]: 443.1667. Found [M+H]+:
444.1719.
J=6.8 Hz, 3H), 2.47 (m, 4H), 2.35 (t, J=6.8 Hz, 2H), 1.64
(m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 169.80, 168.90,
139.22, 133.83, 133.73, 133.52, 131.71, 131.27, 130.43,
130.05, 129.21, 127.57, 127.41, 127.08, 126.07, 124.65,
122.14, 121.45, 114.93, 110.36, 53.77 (2C), 51.77, 35.86,
23.55 (2C). HRMS (Q-TOF): calculated for C26H25N3O2
[M]: 411.1947. Found [M+H]+: 412.2020.
(E)‑2‑(diethylamino)‑N‑(3‑(naphthalen‑1‑ylmethylene)
‑2‑oxoindolin‑5‑yl)acetamide (16o) Following general
procedure B, starting from intermediate (11o) and 1-naph-
thaldehyde (8h) to aford compound 16o as an E isomer,
orange solid (yield 28.4%), melting point 112 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.29 (s, 1H), 8.08
(m, 3H), 7.95 (m, 1H), 7.88 (d, J=6.8 Hz, 1H), 7.62 (m,
3H), 7.46 (d, J=2.0 Hz, 1H), 7.38 (dd, J=8.4, 2.0 Hz, 1H),
6.83 (d, J=8.4 Hz, 1H), 2.98 (s, 2H) 2.48 (q, J=6.8 Hz,
4H), 0.92 (t, J=7.2 Hz, 6H). 13C NMR (100 MHz, DMSO-
d6) δ 169.65, 168.91, 139.54, 133.89, 133.72, 132.55,
131.70, 131.22, 130.40, 130.08, 129.21, 127.55, 127.49,
127.04, 125.96, 124.69, 122.70, 121.45, 115.63, 110.29.
57.82, 48.25 (2C), 12.44 (2C). HRMS (Q-TOF): calculated
for C25H25N3O2 [M]: 399.1947. Found [M+H]+: 400.2020.
(E)‑3‑cyclohexyl‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑oxoin‑
dolin‑5‑yl)propanamide (16l) Following general procedure
B, starting from intermediate (11l) and 1-naphthaldehyde
(8h) to aford compound 16l as an E isomer, yellow solid
1
(yield 35.0%), melting point 212 °C. H NMR (400 MHz,
DMSO-d6) δ 10.58 (s, 1H), 9.53 (s, 1H), 8.07 (m, 3H),
7.96 (m, 1H), 7.88 (d, J=7.2 Hz, 1H), 7.63 (m, 3H), 7.53
(d, J=1.6 Hz, 1H), 7.43 (dd, J=8.4, 1.6 Hz, 1H), 6.82 (d,
J=8.4 Hz, 1H), 2.14 (t, J=7.6 Hz, 2H), 1.61 (m, 5H), 1.37
(q, J=6.8 Hz, 2H), 1.13 (m, 4H), 0.82 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 171.37, 168.93, 139.16, 133.73
(2C), 133.64, 131.69, 131.31, 130.42, 130.05, 129.20,
127.55, 127.44, 127.06, 126.10, 124.63, 122.28, 121.40,
115.13, 110.30, 37.25, 34.28, 33.05, 33.03 (2C), 26.57,
26.22 (2C). HRMS (Q-TOF): calculated for C28H28N2O2
[M]: 424.2151. Found [M+H]+: 425.2239.
(E)‑3‑(diethylamino)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑
2‑oxoindolin‑5‑yl)propanamide (16p) Following general
procedure B, starting from intermediate (11p) and 1-naph-
thaldehyde (8h) to aford compound 16p as an E isomer,
orange solid (yield 21.5%), melting point 115 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.93 (s, 1H), 8.07
(m, 3H), 7.95 (m, 1H), 7.85 (d, J=7.2 Hz, 1H), 7.63 (m,
3H), 7.49 (dd, J=8.4, 2.0 Hz, 1H), 7.36 (d, J=1.6 Hz, 1H),
6.82 (d, J=8.4 Hz, 1H), 2.59 (t, J=6.8 Hz, 2H), 2.39 (q,
J=7.2 Hz, 4H), 2.25 (t, J=6.8 Hz, 2H), 0.86 (t, J=7.2 Hz,
6H). 13C NMR (100 MHz, DMSO-d6) δ 170.32, 168.87,
139.15, 133.85, 133.74, 133.55, 131.79, 131.20, 130.36,
130.16, 129.20, 127.54, 127.29, 127.06, 126.06, 124.66,
122.04, 121.49, 114.84, 110.36, 48.74, 46.40 (2C), 34.22,
12.22 (2C). HRMS (Q-TOF): calculated for C26H27N3O2
[M]: 413.2103. Found [M+H]+: 414.2174.
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑2‑(pyrrolidin‑1‑yl) acetamide (16m) Following
general procedure B, starting from intermediate (11m) and
1-naphthaldehyde (8h) to aford compound 16m as an E iso-
1
mer, orange solid (yield 29.1%), melting point 127 °C. H
NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.39 (s, 1H),
8.07 (m, 3H), 7.96 (m, 1H), 7.88 (d, J=6.8 Hz, 1H), 7.63
(m, 3H), 7.55 (d, J=1.2 Hz, 1H), 7.40 (dd, J=8.4, 2.0 Hz,
1H), 6.82 (d, J=8.4 Hz, 1H), 3.08 (s, 2H), 2.47 (m, 4H),
1.68 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 168.94,
168.58, 139.46, 133.80, 133.72, 132.90, 131.67, 131.26,
130.42, 130.04, 129.21, 127.55, 127.52, 127.04, 126.06,
124.67, 122.77, 121.37, 115.69, 110.25, 59.74, 54.05 (2C),
23.85 (2C). HRMS (Q-TOF): calculated for C25H23N3O2
[M]: 397.1790. Found [M+H]+: 398.1753.
(E)‑3‑(naphthalen‑1‑ylmethylene)‑2‑oxo‑N‑(2‑(piperidin‑
1‑yl)ethyl)indoline‑5‑carboxamide (16q) Following gen-
eral procedure B, starting from intermediate (11q) and
1-naphthaldehyde (8h) to aford compound 16q as an E
isomer, yellow solid (yield 21.0%), melting point 134 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.17 (s,
1H), 8.09 (m, 2H), 8.02 (m, 1H), 7.95 (m, 1H), 7.90 (d,
J = 7.2 Hz, 1H), 7.65 (m, 5H), 6.92 (d, J = 8.0 Hz, 1H),
3.21 (q, J = 6.4 Hz, 2H), 2.30 (m, 6H), 1.43 (m, 4H),
1.34 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 169.04,
166.20, 145.71, 134.80, 133.75, 131.72, 131.25, 130.67,
129.54, 129.37, 129.27, 128.33, 127.64, 127.56, 127.12,
(E)‑N‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑3‑(pyrrolidin‑1‑yl)propanamide (16n) Following
general procedure B, starting from intermediate (11n) and
1-naphthaldehyde (8h) to aford compound 16n as an E
isomer, orange solid (yield 22.3%), melting point 140 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.81 (s,
1H), 8.07 (m, 3H), 7.96 (m, 1H), 7.87 (d, J=6.8 Hz, 1H),
7.63 (m, 3H), 7.46 (m, 2H), 6.82 (d, J=8.4 Hz, 1H), 2.67 (t,
1 3

Molecular Diversity
125.92, 124.71, 122.69, 121.34, 109.74, 58.12, 54.48
(2C), 37.30, 26.00 (2C), 24.46. HRMS (Q-TOF): calcu-
lated for C27H27N3O2 [M]: 425.2103. Found [M + H]+:
426.2178.
Cell proliferation assay
Cell viability assays were performed using the CCK-8 kit
according to the manufacturer’s instructions. Briefy, A
variety of human cell lines were seeded in a 96-well plate
at 2000 cells per well, then they were treated with gradient
concentrations of compounds for 48 h. Each well was incu-
bated with 10 μL CCK-8 (MedChemExpress, Monmouth
Junction, NJ, USA) for 2 h in 37 °C cell culture incuba-
tor, then the absorbance was measured at a wavelength of
450nm by SpectraMAX M5 microplate spectrophotometer
(Molecular Devices, Sunnyvale, CA, USA). The inhibition
rates of diferent concentrations of compounds were calcu-
lated and the IC50 values were calculated by GraphPad Prism
7 software (San Diego, USA) using XY modeling.
(E)‑3‑(naphthalen‑1‑ylmethylene)‑5‑((3‑(piperidin‑1‑yl)
propyl)amino)indolin‑2‑one (16r) Following general pro-
cedure B, starting from intermediate (11r) and 1-naph-
thaldehyde (8h) to aford compound 16r as an E iso-
mer, dark red solid (yield 23.8%), melting point 98 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.05
(m, 2H), 7.98 (s, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.82 (d,
J = 6.8 Hz, 1H), 7.61 (m, 3H), 6.63 (d, J = 8.0 Hz, 1H),
6.41 (d, J = 8.0 Hz, 1H), 6.21 (s, 1H), 5.21 (brs, 1H), 2.60
(t, J = 6.0 Hz, 2H), 2.15 (m, 6H), 1.40 (m, 8H). 13C NMR
(100 MHz, DMSO-d6) δ 168.61, 144.38, 133.79, 133.69,
132.57, 132.40, 131.42, 131.06, 129.93, 129.14, 127.42,
127.05, 126.96, 125.83, 124.87, 122.08, 114.53, 110.88,
107.71, 57.15, 54.42 (2C), 42.66, 25.97 (2C), 25.85,
24.52. HRMS (Q-TOF): calculated for C27H29N3O [M]:
411.2311. Found [M + H]+: 412.2386.
Acute toxicity study
Seven-week-old male and female Balb/c mice (4 male and
4 female mice) were orally administered 9h or 16s once in a
single day at a dose of 500 mg/kg each time, while another
panel of control rats (3 male and 3 female mice) received the
same volume of vehicle. The condition of the experimental
mice was monitored daily for 15 days. Mortality, clinical
signs, and gross fndings were observed. Then, mice were
sacrifced and gross histological examinations of the major
organs were performed. The blood of each mice was col-
lected for hematological evaluation. Hematological analy-
sis was performed using ADVIA2120 (Siemens, Germany),
while biochemical parameters were measured using COBAS
Integra 400 Plus (Roche, Switzerland).
(E)‑1‑(3‑(naphthalen‑1‑ylmethylene)‑2‑ oxoindo‑
lin‑5‑yl)‑3‑(2‑(piperidin‑1‑yl)ethyl)urea (16s) Following
general procedure B, starting from intermediate (11s) and
1-naphthaldehyde (8h) to aford compound 16s as an E iso-
mer, orange solid (yield 26.2%), melting point 207 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.23 (s, 1H),
8.07 (m, 3H), 7.95 (m, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.63
(m, 3H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H), 7.17 (d, J = 1.6 Hz,
1H), 6.75 (d, J = 8.4 Hz, 1H), 5.78 (t, J = 5.2 Hz, 1H), 3.07
(q, J = 6.0 Hz, 2H), 2.27 (m, 6H), 1.46 (m, 4H), 1.36 (m,
2H). 13C NMR (100 MHz, DMSO-d6) δ 168.84, 155.65,
137.86, 134.87, 133.72, 133.40, 131.86, 131.25, 130.33
(2C), 129.20, 127.55, 127.35, 127.08, 126.08, 124.67,
121.51, 121.01, 113.90, 110.38, 58.59, 54.44 (2C),
36.82, 25.96 (2C), 24.54. HRMS (Q-TOF): calculated for
C27H28N4O2 [M]: 440.2212. Found [M + H]+: 441.2271.
In silico docking
In silico docking was performed through Maestro 1.15
(Schrodinger). Ligands were frstly generated in ChemDraw
followed by generation of energy minimized 3D structures
using LigPrep module of Maestro 1.15. Protein was prepared
by Protein Preparation Wizard of Maestro 1.15 following
general procedure. In silico docking was accomplished using
Glide XP (extra precision) of Ligand Docking module of
Maestro 1.15.
Cell culture and reagents
A549 and H1299 (human non-small lung carcinoma cell
lines), HCT116 and DLD1 (human colorectal carcinoma
cell lines), and LO2 (human hepatic cell lines) were pur-
chased from American Type Culture Collection (ATCC).
Cells were maintained in RPMI-1640 media or DMEM
media (Gibco) supplemented with 10% fetal bovine serum
(Hyclone), 100 U ml−1 penicillin (Sigma-Aldrich, USA)
and 100 μg ml−1 streptomycin (Sigma-Aldrich, USA) at
37 °C in a humidifed atmosphere containing 5% CO2. All
tumor cell lines were maintained according to the ATCC
procedures.
Acknowledgements This work was fnancially supported by grants
from the National Natural Science Foundation of China (Grant
Nos. 81473253, 81602956 and 81973368), National Major Pro-
gram of China during the 13th Five-Year Plan Period (Grant No.
2018ZX09721001-001-001).
Compliance with ethical standards
Conflict of interest The authors declare no confict of interest.
1 3

Molecular Diversity
8. Herrero A, Crespo P (2016) Tumors topple when ERKs uncouple.
Mol Cell Oncol 3:e1091875
References
9. Chaikuad A, Tacconi E, Zimmer J, Liang Y, Gray NS, Tarsounas
M, Knapp S (2014) A unique inhibitor binding site in ERK1/2 is
associated with slow binding kinetics. Nat Chem Biol 10:853–860
10. Casar B, Pinto A, Crespo P (2008) Essential role of ERK dimers in
the activation of cytoplasmic but not nuclear substrates by ERK-
scafold complexes. Mol Cell 31:708–721
1. Arozarena I, Calvo F, Crespo P (2011) Ras, an actor on many
stages: posttranslational modifcations, localization, and site-
specifed events. Genes Cancer 2:182–194
2. Herrero A, Pinto A, Colón-Bolea P, Casar B, Jones M, Agudo-
Ibáñez L, Vidal R, Tenbaum SP, Nuciforo P, Valdizán EM, Hor-
vath Z, Orf L, Pineda-Lucena A, Bony E, Keri G, Rivas G, Pazos
A, Gozalbes R, Palmer HG, Hurlstone A, Crespo P (2015) Small
molecule inhibition of ERK dimerization prevents tumorigenesis
by RAS-ERK pathway oncogenes. Cancer Cell 28:170–182
3. Wimmer R, Baccarini M (2010) Partner exchange: protein–protein
interactions in the Raf pathway. Trends Biochem Sci 35:660–668
4. Ward RA, Bethel P, Cook C, Davies E, Debreczeni JE, Fairley G,
Feron L, Flemington V, Graham MA, Greenwood R, Grifn N,
Hanson L, Hopcroft P, Howard TD, Hudson J, James M, Jones
CD, Jones CR, Lamont S, Lewis R, Lindsay N, Roberts K, Simp-
son I, St-Gallay S, Swallow S, Tang J, Tonge M, Wang Z, Zhai B
(2017) Structure-guided discovery of potent and selective inhibi-
tors of ERK1/2 from a modestly active and promiscuous chemical
start point. J Med Chem 60:3438–3450
11. Pinto A, Crespo P (2010) Analysis of ERKs’ dimerization by elec-
trophoresis. Methods Mol Biol 661:335–342
12. Casar B, Pinto A, Crespo P (2009) ERK dimers and scafold pro-
teins: unexpected partners for a forgotten (cytoplasmic) task. Cell
Cycle 8:1007–1013
13. Julie LW, Yu-Chi J, Andrei VK, Ewen G, Derk B, Elizabeth JG,
Cobb MH (2006) Characterization of mitogen-activated protein
kinase (MAPK) dimers. Biochemistry 45:13175–13182
14. Bertram JC, Andrei K, Melanie HC, Elizabeth JG (1997) Activa-
tion mechanism of the MAP Kinase ERK2 by dual phosphoryla-
tion. Cell 90:859–869
15. Bernhard CL, Peter DM, Hampton ES, Robert W, Romain S, Yann
W, Gregory PR, Stefan JR, Elena BP (2017) Structure-guided
strategy for the development of potent bivalent ERK inhibitors.
ACS Med Chem Lett 8:726–731
5. Horgan AM, Stork PJ (2003) Examining the mechanism of Erk
nuclear translocation using green fuorescent protein. Exp Cell
Res 285:208–220
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jurisdictional claims in published maps and institutional afliations.
6. Lidke DS, Huang F, Post JN, Rieger B, Wilsbacher J, Thomas
JL, Pouyssegur J, Jovin TM, Lenormand P (2010) ERK nuclear
translocation is dimerization-independent but controlled by the
rate of phosphorylation. J Biol Chem 285:3092–3102
7. Radhakrishnan K, Edwards J, Lidke DS, Jovin TM, Wilson BS,
Oliver JM (2009) Sensitivity analysis predicts that the ERK-
pMEK interaction regulates ERK. IET Syst Biol 3:329–341
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