Spirotriazoline oxindoles: A novel chemical scaffold with in vitro anticancer properties

Article abstract of DOI:10.1016/j.ejmech.2017.09.037

The design and synthesis of a library of twenty-six spirotriazoline oxindoles and their in vitro evaluation as potential anticancer agents is reported. The antiproliferative activity of the synthesized compounds was assessed against four different cancer cell lines (HCT-116 p53 ^(+/+), HCT-116 p53 ^(?/?), MCF-7, and MDA-MB-231). Four spirotriazoline oxindoles showed selectivity against the four cancer cell lines tested over the non-cancer derived HEK 293T cell line. To characterize the molecular mechanisms involved in compound antitumoral activity, two spirotriazoline oxindoles were selected for further studies. Both compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase and upregulated p53 steady-state levels, while decreasing its main inhibitor MDM2, in HCT-116 cells. Importantly, cytotoxic effects induced by spirotriazoline oxindoles occurred in cancer cells without eliciting cell death in non-malignant CCD-18Co human colon fibroblasts. In addition, four spirotriazoline oxindoles showed selectivity against the triple-negative breast cancer cell line MDA-MB-231 with IC₅₀ values of 3.5–6.7 μM. These results highlight the anticancer potential of spirotriazoline oxindoles, especially when dealing with aggressive and challenging triple-negative breast cancer.

Full text of DOI:10.1016/j.ejmech.2017.09.037

European Journal of Medicinal Chemistry 140 (2017) 494e509
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Spirotriazoline oxindoles: A novel chemical scaffold with in vitro
anticancer properties
Carlos J.A. Ribeiro, Rute C. Nunes, Joana D. Amaral, Lídia M. Gonçalves,
Cecília M.P. Rodrigues, Rui Moreira, Maria M.M. Santos*
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of a library of twenty-six spirotriazoline oxindoles and their in vitro evaluation
as potential anticancer agents is reported. The antiproliferative activity of the synthesized compounds
was assessed against four different cancer cell lines (HCT-116 p53 (þ/þ), HCT-116 p53 (ꢀ/ꢀ), MCF-7, and
MDA-MB-231). Four spirotriazoline oxindoles showed selectivity against the four cancer cell lines tested
over the non-cancer derived HEK 293T cell line. To characterize the molecular mechanisms involved in
compound antitumoral activity, two spirotriazoline oxindoles were selected for further studies. Both
compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase and upregulated p53
steady-state levels, while decreasing its main inhibitor MDM2, in HCT-116 cells. Importantly, cytotoxic
effects induced by spirotriazoline oxindoles occurred in cancer cells without eliciting cell death in non-
malignant CCD-18Co human colon fibroblasts. In addition, four spirotriazoline oxindoles showed
Received 1 August 2017
Received in revised form
7 September 2017
Accepted 19 September 2017
Available online 21 September 2017
Keywords:
Spirooxindole
Triazoline
Cytotoxicity
Anti-cancer agents
Breast cancer
selectivity against the triple-negative breast cancer cell line MDA-MB-231 with IC50 values of 3.5e6.7 mM.
These results highlight the anticancer potential of spirotriazoline oxindoles, especially when dealing
with aggressive and challenging triple-negative breast cancer.
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
the identification of novel targets, especially for TNBC patients [3].
Heterocycles possessing a spirooxindole framework are found in
It is well established that cancer represents a major public
health issue worldwide. In 2012, it was estimated 14.1 million new
cancer cases and 8.2 million deaths [1], and the World Health Or-
ganization estimates a rise of 13.1 million cancer deaths until 2030.
Specifically, breast cancer is the most common cancer in woman,
and is the leading cause of cancer death in women aged 20e59
years in the USA [2]. In particular, a specific tumor subtype, known
as triple-negative breast cancer (TNBC) is characterized as a more
aggressive type of cancer with poorer prognostic. TNBCs are char-
acterized by the lack of expression of estrogen and progesterone
receptors, and absence of human epidermal growth factor receptor
2 overexpression, representing the hardest breast cancer to treat
[3]. Although early detection and efficient therapies contributed to
a decrease of cancer mortality in developed countries, overcoming
drug intrinsic and acquired resistance represents a major challenge
[4,5] and imposes a constant development of new molecules and
many natural products and medicinal agents with diverse biolog-
ical activities [6e8]. The attractiveness of this type of scaffold in
organic and medicinal chemistry is evident by the increasing
number of publications on this topic over the last 10 years. This can
be ascribed, at least partially, to the fact that the central spiro car-
bon atom imposes a conformational restriction to the structure that
can be beneficial for ligand-target binding, and thus potentially
promoting an increase in potency and/or specificity [7].
Furthermore, the reduced molecular flexibility displayed by the
spirooxindole scaffold can also potentially lead to better pharma-
cokinetic properties [9]. Antiproliferative activity against cancer
cell lines is one of the biological effects reported for spirooxindoles
(Fig. 1) [10]. Specifically, CFI-400945 is a Polo-like kinase 4 (PLK4)
inhibitor [11], compound (ꢀ)-1 interferes with microtubule poly-
merization and arrests mitosis [12], and compounds MI-77301 [13]
and SM13 [14] modulate p53 activity.
We have recently developed several small molecules showing
potential anticancer activity [15e23], with a focus on novel five-
membered ring spirooxindoles that act as p53 modulators
(Fig. 2). Initial studies explored the spiroisoxazoline oxindole
* Corresponding author.
E-mail address: mariasantos@ff.ulisboa.pt (M.M.M. Santos).
https://doi.org/10.1016/j.ejmech.2017.09.037
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
495
Fig. 1. Spirooxindoles with biological activity.
Fig. 2. Optimization strategy towards spirotriazoline oxindoles 5.
scaffold 2 [21]. Then, the spirooxadiazoline oxindole scaffold 3 was
obtained by replacing the chiral isoxazoline C40 for a N40 atom.
Further optimization led to a spirooxadiazoline oxindole 15.6-fold
more potent in HCT-116 colorectal cancer cells than the most
active spiroisoxazoline oxindole previously developed by us [15].
We also explored the addition of a fourth lipophilic moiety to the
main scaffold, by changing the isoxazoline oxygen by a N-phenyl
group (spiropyrazoline oxindole scaffold 4) [20,23]. These com-
pounds were tested in two breast adenocarcinoma cell lines, MCF-7
and MDA-MB-231. One compound, spiropyrazoline oxindole 4a,
was found to be at least 13.7-fold more potent against MCF-7 cells
over MDA-MB-231 cells (TNBC cell line), and the non-cancer
derived human embryonic kidney HEK 293T cell line (Fig. 3).
Interestingly, spiropyrazoline oxindole 4b showed approxi-
mately the same potency against both breast cancer cell lines, but
maintained selectivity (>4.5-fold) over the HEK 293T cell line. This
observation suggests that it would be possible to obtain differential
selectivity in breast cancer cell lines depending on the position of
the halogen in the oxindole moiety.
Since both strategies gave rise to improved and/or more selec-
tive compounds, we decided to apply and explore simultaneously
both optimization strategies: replacing the chiral isoxazoline C40

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C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
2. Results and discussion
2.1. Synthesis of compounds
Spirotriazoline oxindoles 5a-z were synthesized by 1,3-dipolar
cycloaddition between 3-arylimino indolin-2-ones 6 and nitrile
imines (Scheme 1, Table 1). The 1,3-dipoles were generated in situ
by dehydrohalogenation of hydrazonoyl chlorides 7, mediated by
triethylamine (60e95% yield) [28,29]. 3-Arylimino indolin-2-ones 6
were synthesized by reacting indoline-2,3-diones (8) with different
anilines (9), in the presence of acetic acid (68e93% yield) [15]. The
hydrazonoyl chlorides 7 were synthesized by reacting N-chlor-
osuccinimide-dimethyl sulphide complex prepared in situ, with the
appropriate N-arylhydrazone (10) at ꢀ78 ꢁC (62e88% yield) [20].
Hydrazones (10) were prepared by reacting benzaldehyde de-
rivatives (11) with phenylhydrazines (12) in aqueous ethanol 20%
(81e99% yield) [30].
Fig. 3. Chemical structure of spiropyrazoline oxindoles 4a and 4b.
The spiropyrazoline equivalent (compound 4c) of the most
potent compound in MDA-MB-231 breast cancer cell line (com-
pound 5i) was also synthesized (Scheme 2). Spiropyrazoline oxin-
dole 4c was obtained in 65% yield by reacting 3-methylene
indoline-2-one 13 with hydrazonoyl chloride 7a (Scheme 2).
Compound 13 was synthesized by aldolic condensation between 5-
bromoindolin-2-one (14) and 3-chlorobenzaldehyde [21], and the
former by reduction of 5-bromoindolin-2,3-dione (8a) in the
presence of TiCl4/Zn [31], as described in literature.
All compounds gave spectroscopic data in agreement with their
structures. For all spirotriazolines, the regioisomer formed was the
spiro[indoline-3,30-triazoline]-2-one as shown by the chemical
shift of the spiro carbon signal at 87.88e89.83 ppm, in line with
reported data [28]. The regioisomer formed for the spiropyrazoline
oxindole 4c was established to be the spiro[indoline-3,30-pyrazo-
line]-2-one [20,32]: the chemical shift of the spiro carbon appears
at 77.22 ppm, the H-40 at 5.45 ppm, and C-40 at 62.34 ppm. The
for a N40 atom, and changing the isoxazoline oxygen by a N-phenyl
group (Fig. 2). As a result, a series of spirotriazoline oxindoles 5 was
synthesized and their anticancer activity was evaluated. These new
five-membered ring spirooxindoles resulted from the juxtaposition
of oxindole and 1,2,4-triazole moieties, two scaffolds that are
described to have anticancer activity [24e27]. We started by per-
forming a screening in breast cancer cell lines MCF-7 and MDA-MB-
231. For the most active compounds we evaluated their activity in
non-cancer HEK 293T cells. To amplify their anticancer scope, and
compare with our previous results obtained with related five-
membered ring spirooxindoles, we further assessed their anti-
proliferative activity in two additional cancer cell lines: the HCT116
human colorectal carcinoma isogenic pair, with and without p53.
The mechanism of action of the most promising compounds was
further evaluated.
Scheme 1. Synthesis of spirotriazoline oxindoles 5a-z: (a) aqueous ethanol 20%, r.t., 2e3 h, 81e99%; (b) NCS, S(CH3)2, 0 ꢁC, 15 min; ꢀ78 ꢁC, 1 h, then allowed to warm up to r.t.,
62e88%.; (c) CH3COOH, EtOH, reflux, 3e24 h, 68e93%; (d) Et3N, DCM, r.t., 24 h, 60e95%.

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497
Table 1
In vitro antiproliferative activities of spirotriazoline oxindoles 5a-z toward breast cancer cell lines and non-tumor cell line.
Compound
R1
R2
R3
R4
Breast cancer cell lines
MCF-7 MDA-MB-231
Non-cancer cell line
HEK 293T
SIb,d
SIc,d
IC50 IC50
Ma Ma
,
m
,
m
IC50
, m
Ma
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
H
H
H
5-Cl
5-Cl
5-Br
5-Br
5-Br
5-Br
7-Cl
7-Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-Cl
4-Cl
H
H
H
3-Cl
3-Cl
4-Cl
4-OMe
H
H
H
3-Cl
3-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
36.2 3.3
24.1 4.8
13.7 0.4
19.5 0.7
19.1 0.8
9.8 1.5
23.0 1.7
16.8 1.0
21.4 2.2
11.0 2.4
10.8 0.9
21.8 1.6
13.1 2.4
10.9 2.0
28.4 5.0
14.2 2.4
10.1 0.5
13.1 0.5
8.0 1.4
23.4 4.3
10.7 1.0
12.5 1.1
32.3 4.0
11.7 0.6
8.9 1.9
6.7 0.4
5.2 1.0
3.5 1.0
4.6 0.3
8.9 0.6
23.7 4.5
11.6 1.3
8.2 0.8
24.8 0.8
15.7 2.4
9.0 1.2
14.5 0.5
10.2 1.3
14.6 2.1
14.1 1.1
11.7 1.7
10.7 3.1
15.0 4.4
7.6 2.4
7.0 1.9
ND
>100
25.7 5.6
ND
32.6 9.3
>100
56.3 11.3
21.6 6.8
21.6 6.8
16.6 3.7
>100
4-Cl
3-Cl
3-Cl,4-F
H
3-Cl
H
4-Cl
3-Cl
3-Cl, 4-F
H
2.3
>4.1
>10.2
2.4
3.4
3.2
6.1
2.4
>9.3
H
ND
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
20.5 5.3
22.7 5.3
ND
H
H
H
H
2.1
2-Cl
3-Cl
4-Cl
2-Cl
H
15.4 2.6
15.8 4.4
35.0 12.2
12.5 1.1
20.3 6.5
25.9 5.8
20.9 3.0
16.9 3.4
16.2 2.4
21.4 7.4
9.0 1.7
H
2.7
2.3
5s
5t
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
H
H
10.1 0.9
12.3 1.5
10.2 0.8
9.8 0.9
11.4 0.9
9.5 3.1
5u
5v
5w
5x
5y
5z
2-Cl
3-Cl
4-Cl
2-Cl
3-Cl
6.9 0.7
ND: not determined.
a
IC50 determined by the MTT method after 72 h. Each value is the mean (IC50 SD) of three independent experiments.
Selectivity index against MDA-MB-231 expressed by the ratio MDA-MB-231 IC50/MCF-7 IC50.
Selectivity index towards cancer cell lines over HEK 293T, expressed by the ratio HEK 293T IC50/highest IC50 obtained in breast cancer cell lines.
b
c
d
Selectivity is depicted when SI > 2.
Scheme 2. Synthesis of spiropyrazoline oxindole 4c: (a) TiCl4, Zn, THF, 65%; (b) 3-chlorobenzaldehyde, piperidine, EtOH, reflux, 5 h, 94%; (c) 7a, Et3N, DCM, r.t., 24 h, 75%.
relative stereochemistry of spirooxindole 4c was assigned by
comparison with reported spiropyrazoline oxindole X-ray crystal-
lography structure [33].
(CRC) is one of the most commonly diagnosed cancers and presents
the highest cause of cancer deaths [1], the scope of anticancer ac-
tivity of all spirotriazoline oxindoles with a IC50 lower than 15 mM in
MCF-7 cells was further expanded to p53 wild-type and null
isogenic (p53 (þ/þ) and p53 (ꢀ/ꢀ)) HCT-116 colorectal cancer cell line.
Specifically, we were interested in evaluating if these compounds
could have an effect mediated by a p53-dependent pathway. These
compounds were also tested in the non-cancer derived HEK 293T to
assess their cytotoxicity to normal cells. The IC50 values obtained in
the five cell lines are presented on Tables 1 and 2.
2.2. Biological evaluation
2.2.1. Structure-activity relationship studies (SAR)
The SAR study for spirotriazoline oxindoles 5 was performed
primarily in human breast cancer cell lines (MCF-7 and MDA-MB-
231) for better comparison with the structurally similar spiropyr-
azoline oxindoles 4 previously reported. Since colorectal cancer
We started our study by synthesizing and evaluating

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- all spirotriazoline oxindoles were more active against the MCF-
7 cell line, with the exception of compound 5g, containing a
spirotriazoline oxindoles 5e, 5g, and 5k-l with the same sub-
stituents of spiropyrazoline oxindoles 4a and 4b (chlorine and
bromine at position 5 or 7 of the oxindole and no substituents in
the phenyl rings), and the non-halogenated counterpart 5a. In this
initial study, the comparison between antiproliferative activities
with their spiropyrazoline oxindole counterparts (5a, 5e, 5g and 5k
versus 4a-b, and 4d-e) revealed that in breast cancer cell lines
(Table 3):
bromine atom at C-5 indole (IC50
¼
23.0 mM vs. 4a,
IC50 ¼ 7.3 M). However, spirotriazoline oxindole 5g was at least
m
14.9-fold more potent against MDA-MB-231 cell line, revealing
that by altering C40 for N40 it is possible to change selectivity
between breast cancer cell lines, going from a compound at least
13.7-fold more selective towards MCF-7 cells (4e) to a derivative
3.4-fold more selective toward MDA-MB-231 cells (5g).
Table 2
In vitro antiproliferative activities of spirotriazoline oxindoles 5a-z toward colorectal cancer cell lines and non-cancer cell line.
Compound
R1
R2
R3
R4
Colorectal cancer cell lines
Non-cancer cell line
HEK 293T
SIc,d
HCT-116 p53(þ/þ)
HCT-116 p53(ꢀ/ꢀ)
IC50
,
m
Ma
IC50
,
m
Ma
IC50
, m
Mb
5a
H
H
H
H
ND
ND
ND
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
H
H
H
4-Cl
3-Cl
3-Cl,4-F
H
3-Cl
H
4-Cl
3-Cl
3-Cl, 4-F
H
H
H
H
H
H
H
H
H
H
H
H
3-Cl
4-Cl
H
H
H
3-Cl
3-Cl
4-Cl
4-OMe
H
H
H
3-Cl
3-Cl
e
H
H
H
H
H
H
H
H
H
H
H
H
15.0 1.5
19.9 1.4
ND
18.5 1.2
22.1 2.0
ND
>100
25.7 5.6
ND
32.6 9.3
>100
56.3 11.3
21.6 6.8
21.6 6.8
16.6 3.7
>100
>4.1
5-Cl
5-Cl
5-Br
5-Br
5-Br
5-Br
7-Cl
7-Br
H
H
H
H
H
21.6 0.6
16.3 0.2
22.2 0.5
16.3 1.2
15.7 1.4
13.5 1.0
23.7 0.6
ND
22.1 3.2
17.9 0.6
22.5 4.4
18.2 0.9
17.8 2.0
15.9 1.3
20.1 1.7
ND
>5.6
2.5
>4.2
H
ND
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
e
17.4 2.4
16.8 2.2
ND
16.1 1.6
18.5 1.6
ND
20.5 5.3
22.7 5.3
ND
H
2-Cl
3-Cl
4-Cl
2-Cl
H
19.8 1.4
18.5 0.9
25.3 1.3
12.6 1.2
10.8 1.2
22.2 1.4
21.5 2.1
12.0 1.1
15.0 1.3
13.9 1.1
10.2 1.7
4.0 1.2
18.5 0.9
18.7 0.5
34.6 2.9
13.0 1.4
10.8 0.9
25.8 1.2
31.6 1.5
13.1 1.4
17.2 1.8
17.7 1.3
9.0 0.8
47.8 1.9
15.4 2.6
15.8 4.4
35.0 12.2
12.5 1.1
20.3 6.5
25.9 5.8
20.9 3.0
16.9 3.4
16.2 2.4
21.4 7.4
9.0 1.7
e
H
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
5-Br
e
H
H
2-Cl
3-Cl
4-Cl
2-Cl
3-Cl
e
5y
5z
Nutlin-3a
e
ND: not determined.
a
IC50 determined by the MTS method after 72 h. Each value is the mean (IC50 SD) of three independent experiments performed in duplicate.
IC50 determined by the MTT method after 72 h. Each value is the mean (IC50 SD) of three independent experiments.
Selectivity index towards cancer cell lines over HEK 293T, expressed by the ratio HEK 293T IC50/highest IC50 obtained in cancer cell lines.
Selectivity is depicted when SI > 2.
b
c
d
Table 3
In vitro antiproliferative activities of spiropyrazoline oxindoles 4 vs spirotriazoline oxindoles 5.
R1
R2
R3
R4
Compd
MCF-7
IC50
MDA-MB-231
IC50
Compd
MCF-7
IC50
MDA-MB-231
IC50, mM
,
m
M
,
m
M
,
m
M
5-Br
7-Cl
5-Br
H
H
H
3-Cl
H
H
H
H
H
H
H
H
H
H
H
H
4a
4b
4c
4d
4e
7.3 1.4
22.4 3.5
10.0 0.7
>100
>100
5g
5k
5i
5a
5e
23.0 1.7
10.8 0.9
21.4 2.2
36.2 3.3
19.1 0.8
6.7 0.4
8.9 0.6
3.5 1.0
23.4 4.3
11.7 0.6
16.9 1.2
12.6 0.3
ND
5-Cl
37.7 14.1
ND
IC50 determined by the MTT method after 72 h. Each value is the mean (IC50 SD) of three independent experiments.
ND: not determined.

C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
499
- the non-halogenated spirotriazoline oxindole (5a) was at least
2.8-fold more potent against the MCF-7 cell line than the spi-
The spiropyrazoline oxindole 4c, with the same substitution
pattern as the most potent compound in MDA-MB-231 cancer cell
line (compound 5i), was found to be 3.6-fold less active in MDA-
MB-231 cells, and 2.2-fold more potent in MCF-7 cells than com-
pound 5i. This compound lost selectivity among breast cancer cell
lines, and the non-cancer cell line HEK 293T (HEK 293T
ropyrazoline oxindole (4d) counterpart (5a, IC50 ¼ 36.2
mM vs.
4d, IC50 > 100 M).
m
- 5-chlorooxindole spirotriazoline oxindole (5e) was 2.0-fold
more potent in the MCF-7 cell line than its spiropyrazoline
(4e) counterpart (5e, IC50 ¼ 19.1
m
M vs. 4e, IC50 ¼ 37.7
mM).
IC50 ¼ 17.5
mM).
- 7-chlorooxindole spirotriazoline oxindole (5k) was also 2-fold
more potent against both breast cancer cell lines (MCF-7:
2.2.2. Study of cell death mechanism
IC50 ¼ 10.8
m
M
vs. 4b, IC50
¼
22.4
mM).
mM; MDA-MB-231:
To further explore the molecular mechanisms underlying
compound-mediated loss of cell viability, we selected compounds
with different and promising antiproliferative profiles: (a) spiro-
triazoline oxindole 5f, the most selective compound against all four
cancer cell lines over HEK 293T cells; (b) spirotriazoline oxindole
5g, the most selective derivative toward MDA-MB-231 breast can-
cer cell line; and (c) spirotriazoline oxindole 5y, the most active
compound in the HCT116 p53(þ/þ) colon cancer cell line with at
least a 1.3-fold more potency over HCT116 p53(ꢀ/ꢀ) cells to assess
the involvement of p53-MDM2 interaction, as observed with our
previous spirooxindoles.
We evaluated apoptosis and cell cycle progression after treat-
ment of cells with spirotriazoline oxindoles 5f and 5y, by flow
cytometry analysis, using the HCT-116 p53(þ/þ) cancer cell line. At
the IC50 concentration, both compounds were able to induce
apoptosis in HCT-116 p53(þ/þ) cells, as observed by the significant
increase of 3.1-fold (p < 0.01) and 2.8-fold (p < 0.05), respectively, in
the percentage of cells in early apoptosis (Fig. 4A). Furthermore, a
robust dose-dependent response was observed when a 2xIC50
concentration was used. In addition, after 24 h of exposure, com-
pound 5f induced a G0/G1 phase arrest, as detected by a 50% in-
crease of cells in this phase, while promoting a decrease of
approximately 10% in the G2/M phase (Fig. 4B). Spirotriazoline
oxindole 5y was also able to promote a 22% decrease of cells in G2/
M phase.
After 48 h, approximately 54% of cells treated with both com-
pounds 5f and 5y were in the G0/G1 phase, whereas only 42% of the
control cells progressed into this phase. Moreover, compounds 5f
and 5y decreased the percentage of cells in G2/M phase in about
15% and 10%, respectively.
Regarding the mode of action of compound 5g in MDA-MB-231
breast cancer cells, we detected at the IC50 concentration a 2.5-fold
increase in caspase-3 and -7 activity, comparing with DMSO
(vehicle control) and Doxorubicin (positive control) (Fig. 5), indic-
ative of apoptosis. However, the presence of 5g did not affect MDA-
MB-231 cell cycle progression (data not shown).
Spirotriazoline oxindole 5y only displayed a 1.5-fold selectivity
index between HCT-116 p53(þ/þ) colon cancer cells and HEK 293T
cells. Therefore, to reassure their non-cytotoxic effect to non-cancer
derived cells at tested concentrations (IC50), we further evaluated
their growth inhibition potential in CCD-18Co human normal colon
fibroblasts using the IC50 and IC80 equitoxic concentrations, previ-
ously determined for HCT-116 p53(þ/þ) cells. As observed in Fig. 6,
treatment with compounds 5f and 5y at the IC50 concentration did
not affect CCD-18Co cell viability. When we used higher concen-
trations, we could detect a reduction of approximately 40% in cell
viability, which is still far below the values obtained for the HCT-116
p53(þ/þ) cell line. The absence of cytotoxic effects induced by
compounds 5f and 5y in human normal colon fibroblasts is clearly
an advantage for using these compounds as potential therapeutic
agents.
IC50 ¼ 8.9 M vs. 4b, IC50 ¼ 16.9
m
In the spirotriazoline series, a 2.0-fold decrease in potency in
both breast cancer cells lines was observed when 7-chlorine (5k)
was changed to 7-bromine (MCF-7: 5l, IC50 ¼ 21.8
IC50 ¼ 10.8 M; MDA-MB-231: 5l, IC50 ¼ 23.7
IC50 ¼ 8.9 M).
The remarkable selectivity of compound 5g towards the TNBC
cell line MDA-MB-231 (IC50 of 6.7 M) spurred the synthesis of new
mM vs. 5k,
m
mM vs. 5k,
m
m
derivatives with 5-bromoxindole moiety. We started by probing
ring A at meta and para position (5h-j). In general, any additional
halogen to one or both positions tested led to an activity
improvement in MCF-7 cells, reaching a 2.1-fold increase in po-
tency for compound 5j. Interestingly, comparing these 5-
bromooxindole derivatives to their non-halogenated oxindole
counterparts (5b-d) revealed that their activities were fairly similar
(less than 1.8-fold difference) in MCF-7 cells. However, in the MDA-
MB-231 cell line, 5-bromooxindole derivatives were substantially
more active, with derivative 5j (R2 ¼ 3-Cl, 4-F) showing a 7.0-fold
increase in potency (5j, IC50 ¼ 4.6
m
M vs. 5d, IC50 ¼ 32.3
mM). The
most active spirotriazoline oxindole in MDA-MB-231 cells was
compound 5i (R1 ¼ 5-Br and R2 ¼ 3-Cl) with a IC50 of 3.5
mM,
representing a 1.9-fold activity improvement and better selectivity
between breast cancer cell lines in comparison to the non-
halogenated ring A counterpart 5g. Furthermore, maintaining the
same substituent in ring A (R2 ¼ 3-Cl), but changing the halogen at
R1 from 5-bromo to 5-chloro, the selectivity between breast cancer
cell lines was lost (5f vs. 5i).
Then we decided to investigate different substitutions at rings B
and C (spirotriazoline oxindoles 5m-z, Table 1). Two compounds
were selected as starting points for this endeavor: the most active
compound in the MDA-MB-231 cells (compound 5i), and the non-
toxic derivative (HEK-293T IC50 > 100 mM) with selectivity between
the two breast cancer cell lines (compound 5b). Except for com-
pound 5o, introducing chlorine atoms in different positions in one
or both rings doubled the potency in MCF-7 cells (compounds 5m-
n, 5p-t, and 5v-z). The most active spirotriazoline oxindole tested in
MCF-7 cells was compound 5z (MCF-7 IC50 ¼ 6.9
mM). However,
these new substitutions did not improve activity in the MDA-MB-
231 cell line. In fact, for all 5-bromooxindole derivatives at least a 2-
fold decrease in activity was observed. The same pattern of
response was also achieved for compound 5u (R3 ¼ 4-OMe).
Interestingly, three compounds still showed 2.0-fold higher selec-
tivity towards both breast cancer cell lines over HEK 293K cells,
making a total of seven selective derivatives (5b, 5f-g, 5k, 5n, 5r,
5y). In addition, derivative 5r also showed a 2.0-fold selectivity over
both HCT116 cell lines.
More importantly, from this study performed in four cancer cell
lines and HEK 293T, we were able to achieve four derivatives (5g-j)
selective against MDA-MB-231 cells (ꢂ2.4-fold) over the other four
cell lines. In addition, four derivatives (5b, 5f-g, 5k) showed
selectivity towards all four cancer cell lines (ꢂ2.5-fold) over the
non-cancer derived cell line.
Since our previous spirooxindoles studied were able to disrupt
the interaction between p53 and MDM2 [15,21], we also explored
the effect of spirotriazoline oxindole 5y on this interaction. By

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Fig. 4. Compounds 5f and 5y induce apoptosis and cell cycle arrest in the HCT-116 p53(þ/þ) cell line. A. Apoptosis was investigated by flow cytometry using 7-aminoactinomycin D
(7-AAD) and Annexin-V-FITC. Cells were treated with either DMSO or tested compounds at IC50 or 2-fold IC50 and incubated for 72 h. Data represents percentage of apoptotic
cells SD of three independent experiments. B. Cellular DNA was stained with propidium iodide (PI), and flow cytometry analysis was performed to determine cell cycle dis-
tribution. Cells were treated with either DMSO or tested compounds at IC50 and incubated for 24 and 48 h. Following flow cytometry analysis, frequencies of cells in each phase of
the cell cycle were calculated using Mod Fit LT 4.1 software. Histograms show one representative example from at least two independent experiments (top). Results are expressed in
the graph bar as means SD of at least two independent experiments (bottom).

C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
501
3. Conclusion
Twenty-six compounds were synthesized with different sub-
stituents attached to the spirotriazoline oxindole scaffold. The
antiproliferative activity of the synthesized compounds was
assessed in four different cancer cell lines, and in the non-cancer
derived HEK 293T cell line. Interestingly, the replacement of a
pyrazoline ring by a triazoline ring in the spirooxindole core,
changed the antiproliferative profile of spirotriazoline oxindoles 5
throughout the five cell lines tested. Specifically, we were able to
achieve four spirotriazoline oxindoles (compounds 5g-j) selective
against MDA-MB-231 cells (ꢂ2.4-fold) over the other four cell lines.
In addition, four spirotriazoline oxindoles (compounds 5b, 5f-g, 5k)
showed selectivity towards all four cancer cell lines (ꢂ2.5-fold)
over the non-cancer derived cell line HEK 293T, with compound 5f
achieving a selectivity higher than 5.6-fold (highest cancer cell line
Fig. 5. - Compound 5g induces caspase-3/-7 activity in MDA-MB-231 cells. Caspase-3
and -7 activities were measured using Caspase-Glo 3/7 luminescent assay (Promega).
MDA-MB-231 cells were treated with DMSO, Doxorubicin or 5g at IC50 and incubated
for 48 h. Data represents mean SEM of two independent experiments.
IC50 ¼ 17.9
mM vs. HEK 293T IC50 > 100 mM). The spiropyrazoline
oxindole 4c, with the same substitution pattern as the most potent
Fig. 6. Compounds 5f and 5y are non-cytotoxic to CCD-18Co cell line at their HCT-116
p53(þ/þ) IC50. Cell viability in CCD-18Co fibroblasts was determined by the MTS method
after 72 h for compounds 5f and 5y at their IC50 and IC80 in HCT116 p53(þ/þ). Data
represent mean SEM of two independent experiments.
Fig. 7. Compound 5y decreases p53eMDM2 interaction evaluated by the BiFC assay.
HCT-116 p53(ꢀ/ꢀ) cells were co-transfected with V1-p53/MDM2-V2 BiFC combination
plasmids for 24 h. Vehicle, nutlin-3a (5 and 10
m
M) and compound 5y (5 and 10
mM)
were included in the culture medium
4
h after transfection. V1-p53/MDM2-V2
complementation was evaluated by flow cytometry. Results are expressed as the
percentage (%) of fluorescence normalized to vehicle control DMSO (100% fluorescence
intensity). Data represent mean SEM of three independent experiments. *p < 0.05
from control with DMSO.
applying a Venus-based bimolecular fluorescence complementa-
tion system methodology (BiFC) [34], we observed that compound
5y was able to decrease the p53eMDM2 interaction at 10
mM
(Fig. 7). Importantly, this effect may contribute for both apoptotic
and cell cycle arrest outcomes observed for this compound.
Additionally, compound 5y ability to disrupt p53-MDM2 bind-
ing was further corroborated by the significant decrease of MDM2
protein expression levels accompanied by an increase of p53
(Fig. 8).
Fig. 8. Effect of compounds 5f and 5y in p53 and MDM2 protein levels. Total proteins
were isolated from HCT-116 p53(þ/þ) cells following incubation with compounds 5f and
5y at equitoxic IC50 concentration, or DMSO (vehicle control), for 72 h. Ponceau S
staining was used as loading control. Data represent mean SEM of two independent
experiments.

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C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
compound in MDA-MB-231 cancer cell line (compounds 5i), was
found to be 3.6-fold less active in MDA-MD-231 cell line, and 2.2-
fold more potent in MCF-7 cell line, while losing selectivity
among all cancer cell lines, and HEK 293T cells.
reported before (adapted from Ref. [30]).
4.1.1.1. 1-Benzylidene-2-(2-chlorophenyl)hydrazine (10e). Light pink
solid (2.4 g, 92%). 1H NMR (300 MHz, CDCl3)
d (ppm): 8.08 (br s, 1H,
The mechanism of action of spirotryazoline oxindoles as po-
tential anti-cancer agents acting by a p53-dependent pathway was
studied in more detail. Compounds 5f and 5y were not cytotoxic in
normal cells, being selective for cancer cells over normal cells,
while inducing apoptosis and cell cycle arrest in G0/G1 phase in the
HCT-116 colorectal cancer cell line. Additionally, compounds 5f and
5y slightly induced p53 and decreased MDM2 expression levels.
These changes in p53 and MDM2 expression levels, together with
the results obtained in the BiFC assay strongly suggest the ability of
spirotryazoline oxindoles to modulate p53-MDM2 interaction.
Furthermore, four spirotriazoline oxindoles derivatives (5g-j,
NH), 7.84 (s, 1H), 7.73e7.66 (m, 2H), 7.64 (dd, J ¼ 8.2, 1.4 Hz, 1H),
7.44e7.31 (m, 3H), 7.31e7.21 (m, 2H), 6.87e6.75 (m, 1H); 13C NMR
(75 MHz, CDCl3)
d (ppm): 140.68 (Cq), 139.64 (CH), 135.05 (Cq),
129.23(CH), 129.00 (CH), 128.80 (2CH), 128.09 (CH), 126.55 (2CH),
120.14 (CH), 117.01 (Cq), 114.33 (CH).
4.1.1.2. 1-Benzylidene-2-(2-chlorophenyl)hydrazine
Off-white solid (2.5 g, 84% yield). 1H NMR (300 MHz, CDCl3)
(ppm): 7.70e7.62 (m, 3H), 7.59 (br s, 1H, NH), 7.45e7.28 (m, 3H),
7.23e7.11 (m, 2H), 6.91 (dd, J ¼ 8.2, 1.2 Hz, 1H), 6.84 (dd, J ¼ 7.9,
1.0 Hz, 1H); 13C NMR (75 MHz, CDCl3)
(ppm): 145.86 (Cq), 138.48
(10f).
d
d
IC50 ¼ 3.5e6.7
m
M) were selective against the TNBC cell line MDA-
(CH), 135.28 (Cq), 134.98 (Cq), 130.39 (CH), 128.93 (CH), 128.79
(2CH), 126.48 (2CH), 120.03 (CH), 112.85 (CH), 111.00 (CH).
MB-231 (SI > 2.4-fold) over the other four cell lines, with com-
pound 5g exhibiting 8.4-fold selectivity against MDA-MB-231 over
HEK 293T cells. Moreover, exposure of MDA-MB-231 cells to 5g
resulted in activation of effector caspases-3 and -7, thus suggesting
that apoptosis is, at least in part, involved as the death mechanism.
These four compounds have the common feature of containing a
bromine at position 5 of the oxindole, and unsubstituted phenyl
rings B and C. We are currently investigating in more detail the
selectivity of these compounds against the more challenging TNBC
cell line.
4.1.1.3. 1-(3-chlorobenzylidene)-2-(2-chlorophenyl)hydrazine (10h).
White solid (2.7 g, 90% yield). 1H NMR (300 MHz, CDCl3)
d (ppm):
8.15 (br s, 1H, NH), 7.76 (s, 1H), 7.71 (s, 1H), 7.63 (dd, J ¼ 8.2, 1.4 Hz,
1H), 7.53e7.47 (m, 1H), 7.35e7.22 (m, 4H), 6.87e6.77 (m, 1H); 13C
NMR (75 MHz, CDCl3) d (ppm): 140.30 (Cq),137.74 (CH),136.91 (Cq),
134.83 (Cq), 129.99 (CH), 129.26 (CH), 128.77 (CH), 128.12 (CH),
126.01 (CH), 124.80 (CH), 120.51 (CH), 117.12 (Cq), 114.40 (CH).
4.1.1.4. 1-(3-chlorobenzylidene)-2-(3-chlorophenyl)hydrazine (10i).
4. Experimental section
Light pink solid (2.6 g, 88% yield). 1H NMR (300 MHz, CDCl3)
d
(ppm): 7.74 (br s, 1H, NH), 7.66 (s, 1H), 7.63 (s, 1H), 7.53e7.47 (m,
4.1. Chemistry: general conditions
1H), 7.32e7.23 (m, 2H), 7.22e7.14 (m, 2H), 6.93 (ddd, J ¼ 8.3, 2.1,
0.9 Hz, 1H), 6.85 (ddd, J ¼ 7.9, 2.0, 0.9 Hz, 1H); 13C NMR (75 MHz,
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification, with exception of
NEt3 and reaction solvents, which were dried prior to their use.
Melting points were determined using a Kofler camera Bock
monoscope M and are uncorrected. The infrared spectra were
collected on a Shimadzu FTIR Affinity-1 spectrophotometer. Merck
Silica Gel 60 F254 plates were used for analytical TLC; flash column
chromatography was performed on Merck Silica Gel (200e400
mesh) and Combi-Flash Rf from Teledyne ISCO (columns RediSep
Rf, silica). Preparative TLC was performed on Merck Silica Gel 60
GF254 over glass plates with 1 mm thickness. 1H and 13C NMR
spectra were recorded on a Bruker 400 Ultra-Shield at 400 MHz (1H
NMR) and 100 MHz (13C NMR), and on a Bruker 300 Avance at
300 MHz (1H NMR) and 75 MHz (13C NMR). Data are reported as
CDCl3) d (ppm): 145.48 (Cq), 136.87 (Cq), 136.62 (CH), 135.34 (Cq),
134.85 (Cq), 130.45 (CH), 130.02 (CH), 128.74 (CH), 126.06 (CH),
124.66 (CH), 120.44 (CH), 112.97 (CH), 111.11 (CH).
4.1.2. General procedure for the synthesis of hydrazonoyl chlorides
To NCS (3.0 equiv) in CH2Cl2 (3.5 mL/mmol of hydrazone) at 0 ꢁC
was added methyl sulfide (6.0 equiv) over 5 min. After stirring for
15 min, the reaction was further cooled to ꢀ78 ꢁC. Then the cor-
responding hydrazone 10 (1.0 equiv) dissolved in CH2Cl2 (1 mL/
mmol of hydrazone) was added. The reaction was stirred at ꢀ78 ꢁC
for 1 h, and then slowly allowed to warm to room temperature over
3 h. The reaction was quenched by addition of cold water. The
organic layer was then washed with brine (1x), saturated sodium
sulfite aqueous solution (2x), and water. The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated to afford
the corresponding hydrazonoyl chloride (adapted from Ref. [32]).
Spectroscopic characteristics of hydrazonoyl chlorides 7a [38], 7b-
d [32], 7f [32], and 7g [39] have been reported before.
follows: chemical shift (d), multiplicity (s: singlet, d: doublet, dd:
doublet of doublet; t: triplet, m: multiplet, br: broad), coupling
constants (J) in Hertz and integration. 1H and 13C chemical shifts are
expressed in ppm using the solvent as internal reference. All
compounds tested showed appropriate purity by elemental anal-
ysis (C, H, and N), performed in a Flash 2000 CHNS-O analyzer
(ThermoScientific, UK) at Liquid Chromatography and Mass Spec-
trometry Laboratory, Faculty of Pharmacy of Universidade de
Lisboa.
4.1.2.1. N'-(2-chlorophenyl)benzohydrazonoyl
chloride
(7e).
Pink solid (1.9 g, 84% yield). 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.57
(br s, 1H, NH), 7.98e7.91 (m, 2H), 7.60 (dd, J ¼ 8.2, 1.5 Hz, 1H),
7.47e7.37 (m, 3H), 7.33 (dd, J ¼ 8.0, 1.4 Hz, 1H), 7.30e7.22 (m, 1H),
6.87 (ddd, J ¼ 7.9, 7.5,1.5 Hz,1H); 13C NMR (75 MHz, CDCl3)
d (ppm):
4.1.1. Synthesis of hydrazones
139.52 (Cq), 134.31 (Cq), 129.71 (CH), 129.42 (CH), 128.59 (2CH),
128.12 (CH), 127.37 (Cq), 126.74 (2CH), 121.23 (CH), 118.14 (Cq),
114.75 (CH).
A mixture of phenylhydrazine derivative (1.0 equiv), and benz-
aldehyde derivative (1.2 equiv) in aqueous ethanol 20% (2 mL/mmol
of phenylhydrazine) was stirred at room temperature in the dark
for 2e3 h. The precipitate formed was filtered and washed with
aqueous ethanol 20%. For compounds 10f-g, and 10i, the starting
phenylhydrazine was purchased in its chloridrate form, and
therefore triethylamine (1.0 equiv) was added 15 min before adding
the corresponding benzaldehyde. Spectroscopic characteristics of
hydrazones 10a [35], 10b [36], 10c-d [35], and 10g [37], have been
4.1.2.2. 3-Chloro-N'-(2-chlorophenyl)benzohydrazonoyl
(7h). Dark pink-reddish solid (1.6 g, 73% yield). 1H NMR (300 MHz,
CDCl3) (ppm): 8.59 (br s,1H, NH), 7.94e7.92 (m,1H), 7.85e7.79 (m,
1H), 7.59 (dd, J ¼ 8.2, 1.5 Hz, 1H), 7.37e7.24 (m, 4H), 6.93e6.85 (m,
1H); 13C NMR (75 MHz, CDCl3)
(ppm): 139.17 (Cq), 136.00 (Cq),
134.71 (Cq), 129.79 (CH), 129.59 (CH), 129.47 (CH), 128.17 (CH),
chloride
d
d

C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
503
126.55 (CH), 125.70 (Cq), 124.78 (CH), 121.61 (CH), 118.29 (Cq),
114.83 (CH).
solid (308.0 mg, 87% yield). 1H NMR (400 MHz, CDCl3) major (E)
isomer
(d, J ¼ 8.1 Hz, 1H), 7.30e7.24 (m, 1H), 7.00 (d, J ¼ 7.8 Hz, 2H), 6.72 (t,
J ¼ 8.0 Hz, 1H), 6.57 (d, J ¼ 7.8 Hz, 1H).
d
(ppm): 8.25 (br s, 1H, NH), 7.44 (t, J ¼ 7.8 Hz, 2H, HAr), 7.32
4.1.2.3. 3-Chloro-N'-(3-chlorophenyl)benzohydrazonoyl chloride (7i).
Dark brown-reddish solid (1.6 mg, 73% yield). 1H NMR (300 MHz,
CDCl3)
d
(ppm): 8.08 (br s,1H, NH), 7.91e7.88 (m, 1H), 7.83e7.77 (m,
4.1.3.8. 7-Bromo-3-(phenylimino)indolin-2-one (6l).
1H), 7.38e7.34 (m, 2H), 7.24e7.19 (m, 2H), 7.01 (ddd, J ¼ 8.3, 2.2,
Synthesized according to 6k. Yellow solid (245.0 mg, 80% yield). 1H
0.9 Hz, 1H), 6.93 (ddd, J ¼ 8.0, 1.9, 0.9 Hz, 1H); 13C NMR (75 MHz,
NMR (400 MHz, CDCl3) major (E) isomer
d (ppm): 8.04 (br s, 1H,
CDCl3)
d
(ppm): 144.20 (Cq), 135.92 (Cq), 135.44 (Cq), 134.72 (Cq),
NH), 7.48e7.41 (m, 3H), 7.30e7.24 (m, 1H), 7.00 (d, J ¼ 8.0 Hz, 2H),
130.56 (CH), 129.80 (CH), 129.57 (CH), 126.52 (CH), 124.71 (CH),
124.41 (Cq), 121.54 (CH), 113.74 (CH), 111.85 (CH).
6.67 (t, J ¼ 7.7 Hz, 1H), 6.61 (d, J ¼ 7.6 Hz, 1H).
4.1.4. Synthesis of 5-bromo-3-(3-chlorobenzylidene)indolin-2-one
4.1.3. Synthesis of 3-imino-indoline-2-ones
(13)
A mixture of indoline-2,3-dione derivative 8 (1.0 equiv), aniline
derivative 9 (1.0 equiv), and acetic acid (0.1 equiv) in ethanol
(4.0 mL/mmol of indoline-2,3-dione derivative) was heated at
reflux for 3e5 h, under nitrogen atmosphere. If after 5 h the reac-
tion was not completed an additional 0.5 equiv of aniline derivative
9 was added and refluxed to maximum reaction duration of 24 h.
The reaction mixture was allowed to cool to room temperature, and
the residue was filtrated. The solid obtained was washed with cold
ethanol (adapted from Ref. [40]). Spectroscopic characteristics of 3-
imino-indoline-2-ones 6a [41,42], 6b [43], 6c [44], and 6d [45] have
been reported before.
A mixture of 5-bromoindolin-2,3-dione (1.0 equiv), 3-
chlorobenzaldehyde (1.2 equiv) and piperidine (0.1 equiv) in
ethanol was refluxed for 8 h, under nitrogen atmosphere. After the
reaction was complete, the solvent was removed under reduced
pressure and the residue was purified by flash chromatography on
silica gel using as eluent a gradient from n-hexane/EtOAc (4:1) to n-
hexane/EtOAc (2:1) to afford the final product, as a mixture of E/Z
isomers (adapted from Ref. [46])
4.1.4.1. Compound 13. Yellow solid (369.2 mg, 94% yield). 1H NMR
(300 MHz, CDCl3) major (E) isomer
d (ppm): 7.84 (br s, 1H), 7.79 (s,
1H), 7.69 (d, J ¼ 1.8 Hz, 1H), 7.61 (d, J ¼ 0.9 Hz, 1H), 7.56e7.51 (m,
1H), 7.48e7.44 (m, 2H), 7.38 (dd, J ¼ 8.3, 1.8 Hz, 1H), 6.79 (d,
J ¼ 8.3 Hz, 1H).
4.1.3.1. 5-Chloro-3-(phenylimino)indolin-2-one (6e). Dark yellow
solid (1.2 g, 84% yield). 1H NMR (400 MHz, CDCl3) major (E) isomer
d
(ppm): 9.18 (br s, 1H, NH), 7.47 (t, J ¼ 7.8 Hz, 2H), 7.33e7.27 (m,
2H), 7.02 (d, J ¼ 7.8 Hz, 2H), 6.88 (d, J ¼ 8.3 Hz, 1H), 6.64 (d,
J ¼ 1.4 Hz, 1H).
4.1.5. General procedure for the synthesis of 20,40-dihydrospiro
[indoline-3,3'-[1,2,4]triazol]-2-ones
Triethylamine (2.0 equiv) was added dropwise to a mixture of 3-
imino indolin-2-one derivative 6 (1.0 equiv), and hydrazonoyl
chloride derivative 7 (2.0 equiv) in dry DCM (1 mL/0.1 mmol of 3-
iminoidolin-2-one) under nitrogen atmosphere. The reaction was
stirred at room temperature for 24 h. The mixture was then washed
with brine (2x) and the aqueous phase extracted with DCM. The
combined organic extracts were dried over anhydrous Na2SO4 and
the solvent was removed under reduce pressure. The residue was
purified by flash chromatography on silica gel using as eluent a
gradient of n-hexane/EtOAc (95:5) to n-hexane/EtOAc (75:25),
preparative thin layer chromatography (PTLC) on silica gel using as
eluent n-hexane:EtOAc (2:1), and recrystallized from diethyl ether/
n-hexane to afford the final product.
4.1.3.2. 5-Chloro-3-((3-chlorophenyl)imino)indolin-2-one
Dark orange solid (1.1 g, 68% yield). 1H NMR (400 MHz, CDCl3)
major (E) isomer
(6f).
d
(ppm): 8.43 (br s, 1H, NH), 7.40 (t, J ¼ 7.9 Hz, 1H),
7.33 (dd, J ¼ 8.4, 1.6 Hz, 1H), 7.28 (d, J ¼ 7.8 Hz, 1H), 7.04 (s, 1H),
6.91e6.86 (m, 2H), 6.67 (s, 1H).
4.1.3.3. 5-Bromo-3-(phenylimino)indolin-2-one (6g). Yellow solid
(1.1 g, 80% yield).1H NMR (400 MHz, CDCl3) major (E) isomer
d
(ppm): 8.44 (br s, 1H, NH), 7.50e7.42 (m, 3H), 7.30 (t, J ¼ 7.6 Hz,
1H), 7.02 (d, J ¼ 7.5 Hz, 2H), 6.81 (d, J ¼ 8.4 Hz, 1H), 6.78 (s, 1H).
4.1.3.4. 5-Bromo-3-((3-chlorophenyl)imino)indolin-2-one
(6h).
Dark orange solid (698.3 mg, 93% yield). 1H NMR (400 MHz, CDCl3)
major (E) isomer
d
(ppm): 8.19 (br s, 1H, NH), 7.48 (d, J ¼ 8.1 Hz, 1H),
4.1.5.1. 20,40,50-triphenyl-20,40-dihydrospiro[indoline-3,3'-[1,2,4]tri-
azol]-2-one (5a). Synthesized according to the general procedure,
and starting with 6a and 7a, this compound was obtained as a
yellow solid (151.2 mg, 81% yield). Mp: 203e204 ꢁC (Lit. 202 ꢁC
[28]); IR (KBr, selected peaks): 3245 (NH), 1735 (C¼O), 1594 (C¼N),
7.40 (t, J ¼ 7.9 Hz, 1H), 7.30e7.25 (m, 1H), 7.04 (s, 1H), 6.89 (d,
J ¼ 7.6 Hz, 1H), 6.86e6.79 (m, 2H).
4.1.3.5. 5-Bromo-3-((4-chlorophenyl)imino)indolin-2-one
Orange solid (637.2 mg, 86% yield). 1H NMR (400 MHz, CDCl3 major
(E) isomer (ppm): 8.07 (br s, 1H, NH), 7.50e7.40 (m, 3H),
(6i).
1493, 1472, 1385, 1320, 1203, 751, 694 cmꢀ1 1H NMR (400 MHz,
;
d
Acetone-d6)
d
(ppm): 7.55 (d, J ¼ 7.4 Hz, 1H), 7.48 (d, J ¼ 7.0 Hz, 2H),
7.05e6.96 (m, 2H), 6.93 (s, 1H), 6.86e6.78 (m, 1H).
7.38e7.27 (m, 4H), 7.18e7.03 (m, 6H), 6.97e6.89 (m, 3H), 6.87e6.79
(m, 2H), 6.73 (t, J ¼ 7.3 Hz, 1H); 13C NMR (100 MHz, Acetone-d6)
4.1.3.6. 5-bromo-3-((3-chloro-4-fluorophenyl)imino)indolin-2-one
(6j). Orange solid (641.3 mg, 82% yield). 1H NMR (400 MHz, CDCl3)
major (E) isomer
7.29e7.23 (m, 1H), 7.16e7.10 (m, 1H), 6.96e6.88 (m, 2H), 6.84 (d,
J ¼ 8.2 Hz, 1H).
d (ppm): 173.49 (Cq), 148.51 (Cq), 144.43 (Cq), 143.06 (Cq), 139.18,
(Cq) 132.31 (CH), 130.07 (CH), 129.67 (2CH), 129.61 (2CH), 129.03
(2CH), 128.88 (Cq), 128.62 (2CH), 128.61 (2CH), 127.72 (CH), 127.34
(Cq), 127.26 (CH), 123.99 (CH), 120.33 (CH), 114.54 (2CH), 111.73
(CH), 88.46 (Cspiro). Anal. Calcd for C27H20N4O$0.25H2O: C 77.03, H
4.92, N 13.31, found: C 76.63, H 4.99, N 13.05.
d
(ppm): 8.34 (br s,1H, NH), 7.50 (d, J ¼ 8.4 Hz,1H),
4.1.3.7. 7-Chloro-3-(phenylimino)indolin-2-one (6k).
Synthesized according to the general procedure. However, since
compound 6k did not precipitate after cooling to room tempera-
ture, the solvent was removed under reduced pressure and the
residue was purified by flash chromatography on silica gel using as
eluent a gradient from 100% DCM to DCM:EtOAc (90:10). Yellow
4.1.5.2. 4'-(4-chlorophenyl)-20,50-diphenyl-20,40-dihydrospiro[indo-
line-3,3'-[1,2,4] triazol]-2-one (5b). Synthesized according to the
general procedure, and starting with 6c and 7a, this compound was
obtained as a yellow solid (146.9 mg, 84% yield). Mp: 185e187 ꢁC. IR
(KBr, selected peaks): 3243 (NH), 1738 (C¼O), 1597 (C¼N), 1491,

504
C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
1471, 1385, 1320, 1200, 745, 691 cmꢀ1; 1H NMR (400 MHz, Acetone-
d6)
4.1.5.6. 5-Chloro-4'-(3-chlorophenyl)-20,50-diphenyl-20,40-dihy-
drospiro[indoline-3,3'-[1,2,4]triazol]-2-one (5f). Synthesized ac-
cording to the general procedure, and starting with 6f and 7a, this
compound was obtained as a yellow solid (148.4 mg, 89% yield).
Mp: 172e173 ꢁC. IR (KBr, selected peaks): 3252 (NH), 1741 (C¼O),
d
(ppm): 9.68 (br s, 1H, NH), 7.56 (d, J ¼ 7.4 Hz, 1H), 7.49 (d,
J ¼ 8.0 Hz, 2H), 7.40e7.30 (m, 4H), 7.18 (d, J ¼ 8.6 Hz, 2H), 7.13e7.07
(m, 3H), 6.97e6.90 (m, 3H), 6.82 (d, J ¼ 8.6 Hz, 2H), 6.74 (t,
J ¼ 7.3 Hz, 1H); 13C NMR (100 MHz, Acetone-d6)
d (ppm): 173.21
(Cq), 148.15 (Cq), 144.30 (Cq), 142.98 (Cq), 138.14 (Cq), 132.73 (Cq),
132.51 (CH), 130.26 (CH), 130.04 (2CH), 129.76 (2CH), 129.65 (2CH),
129.18 (2CH), 128.61 (2CH), 128.52 (Cq), 127.26 (CH), 127.06 (Cq),
124.16 (CH), 120.55 (CH), 114.62 (2CH), 111.88 (CH), 88.42 (Cspiro).
Anal. Calcd for C27H19ClN4O$0.65H2O: C 70.09, H 4.43, N 12.11,
found: C 69.67, H 4.27, N 11.69.
1590 (C¼N), 1495, 1477, 1385, 1323, 1199, 745, 689 cmꢀ1
;
1H NMR
(400 MHz, Acetone-d6)
d (ppm): 9.86 (br s, 1H, NH), 7.65 (d,
J ¼ 1.7 Hz, 1H), 7.56e7.48 (m, 2H), 7.44e7.32 (m, 4H), 7.25e7.16 (m,
2H), 7.13 (t, J ¼ 8.0 Hz, 2H), 7.01 (d, J ¼ 8.2 Hz,1H), 6.94 (d, J ¼ 8.0 Hz,
2H), 6.89 (s, 1H), 6.86e6.81 (m, 1H), 6.78 (t, J ¼ 7.3 Hz, 1H); 13C NMR
(100 MHz, Acetone-d6)
d (ppm): 173.05 (Cq), 148.01 (Cq), 144.04
(Cq), 141.73 (Cq), 140.48 (Cq), 134.62 (Cq), 132.54 (CH), 131.18 (CH),
130.43 (CH), 129.80 (2CH), 129.22 (2CH), 129.03 (Cq), 128.79 (Cq),
128.72 (2CH), 128.30 (Cq), 128.14 (CH), 127.91 (CH), 127.39 (CH),
126.96 (CH), 120.97 (CH), 114.77 (2CH), 113.40 (CH), 88.28 (Cspiro).
Anal. Calcd for C27H18Cl2N4O$0.5H2O: C 65.59, H 3.88, N 11.34,
found: C 65.19, H 4.23, N 11.31.
4.1.5.3. 4'-(3-chlorophenyl)-20,50-diphenyl-20,40-dihydrospiro[indo-
line-3,3'-[1,2,4] triazol]-2-one (5c). Synthesized according to the
general procedure, and starting with 6b and 7a, this compound was
obtained as a yellow solid (158.2 mg, 90% yield). Mp: 208e209 C; IR
(KBr, selected peaks): 3255 (NH), 1735 (C¼O), 1591 (C¼N), 1494,
1470, 1384, 1320, 1198, 753, 689 cmꢀ1; 1H NMR (400 MHz, Acetone-
4.1.5.7. 5-Bromo-20,40,50-triphenyl-20,40-dihydrospiro[indoline-3,3'-
[1,2,4]triazol]-2-one (5g). Synthesized according to the general
procedure, and starting with 6g and 7a, this compound was ob-
tained as a yellow solid (155.7 mg, 95% yield). Mp: 212e214 ꢁC. IR
(KBr, selected peaks): 3211 (NH), 1741 (C¼O), 1595 (C¼N), 1493,
1473, 1385, 1320, 1200, 766, 693 cmꢀ1; 1H NMR (400 MHz, Acetone-
d6)
d
(ppm): 9.72 (br s, 1H, NH), 7.57 (d, J ¼ 7.3 Hz, 1H), 7.51 (d,
J ¼ 8.0 Hz, 2H), 7.43e7.32 (m, 4H), 7.20e7.14 (m, 2H), 7.13e7.08 (m,
3H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.94 (d, J ¼ 7.9 Hz, 2H), 6.81 (s, 1H),
6.80e6.71 (m, 2H); 13C NMR (100 MHz, Acetone-d6)
d (ppm): 173.11
(Cq), 147.91 (Cq), 144.25 (Cq), 142.92 (Cq), 140.71 (Cq), 134.49 (Cq),
132.59 (CH), 131.02 (CH), 130.34 (CH), 129.67 (2CH), 129.23 (2CH),
128.62 (2CH), 128.51 (Cq), 127.97 (CH), 127.61 (CH), 127.28 (CH),
126.90 (Cq), 126.86 (CH), 124.20 (CH), 120.67 (CH), 114.73 (2CH),
111.87 (CH), 88.36 (Cspiro). Anal. Calcd for C27H19ClN4O$0.6H2O: C
70.23, H 4.42, N 12.14, found: C 69.96, H 4.32, N 11.92.
d6)
d
(ppm): 9.74 (br s, 1H, NH), 7.75 (s, 1H), 7.52 (d, J ¼ 8.6 Hz, 1H),
7.48 (d, J ¼ 7.6 Hz, 2H), 7.38e7.27 (m, 3H), 7.20e7.10 (m, 5H),
6.96e6.85 (m, 5H), 6.76 (t, J ¼ 7.3 Hz, 1H); 13C NMR (100 MHz,
Acetone-d6)
d (ppm): 173.13 (Cq), 148.59 (Cq), 144.18 (Cq), 142.19
(Cq), 138.92 (Cq), 135.18 (CH), 130.18 (CH), 130.12 (CH), 129.83
(2CH),129.76 (2CH),129.56 (Cq),129.04 (2CH), 128.70 (4CH),128.62
(Cq), 128.01 (CH), 120.65 (CH), 116.04 (Cq), 114.56 (2CH), 113.68
(CH), 88.32 (Cspiro). Anal. Calcd for C27H19BrN4O: C 65.46, H 3.87, N
11.31, found: C 65.31, H 3.97, N 11.05.
4.1.5.4. 4'-(3-chloro-4-fluorophenyl)-20,50-diphenyl-20,40-dihy-
drospiro[indoline-3,3'-[1,2,4]triazol]-2-one (5d). Synthesized ac-
cording to the general procedure, and starting with 6d and 7a, this
compound was obtained as a yellow solid (119.5 mg, 70% yield).
Mp: 203e205 ꢁC; IR (KBr, selected peaks): 3246 (NH), 1731 (C¼O),
4.1.5.8. 5-Bromo-4'-(4-chlorophenyl)-20,50-diphenyl-20,40-dihy-
drospiro[indoline-3,3'-[1,2,4]triazol]-2-one (5h). Synthesized ac-
cording to the general procedure, and starting with 6i and 7a, this
compound was obtained as a yellow solid (146.1 mg, 93% yield).
Mp: 140e142 ꢁC; IR (KBr, selected peaks): 3186, 1730, 1598, 1490,
1597 (C¼N), 1493, 1470, 1385, 1325, 1203, 746, 690 cmꢀ1
;
1H NMR
(400 MHz, Acetone.d6)
d (ppm): 9.72 (br s, 1H, NH), 7.61 (d,
J ¼ 7.3 Hz, 1H), 7.52 (d, J ¼ 8.0 Hz, 2H), 7.44e7.32 (m, 4H), 7.17e7.07
(m, 4H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.96e6.90 (d, J ¼ 8.0 Hz, 3H),
6.87e6.80 (m, 1H), 6.76 (t, J ¼ 7.3 Hz, 1H); 13C NMR (100 MHz,
1473, 1385, 1307, 1205, 747, 690 cmꢀ1 1H NMR (400 MHz, CDCl3)
;
d
(ppm): 8.09 (br s, 1H, NH), 7.70 (s, 1H), 7.50e7.41 (m, 3H),
Acetone-d6)
d
(ppm): 173.13 (Cq), 157.26 (d, JFC ¼ 247.0 Hz, Cq),
7.37e7.22 (m, 3H), 7.12 (t, J ¼ 7.7 Hz, 2H), 7.05 (d, J ¼ 8.4 Hz, 2H),
147.92 (Cq), 144.26 (Cq), 142.98 (Cq), 136.29 (d, JFCCCC ¼ 3.0 Hz, Cq),
132.67 (CH), 130.59 (CH), 130.41 (CH), 129.69 (2CH), 129.37 (d,
JFCCC ¼ 8.0 Hz, CH) 129.28 (2CH), 128.67 (2CH), 128.27 (Cq), 127.33
(CH), 126.83 (Cq), 124.27 (CH), 121.02 (d, JFCC ¼ 19.0 Hz, Cq), 120.71
(CH), 117.66 (d, JFCC ¼ 22.0 Hz, CH), 114.68 (2CH), 111.96 (CH), 88.52
(Cspiro). Anal. Calcd for C27H18ClFN4O$0.3H2O: C 68.37, H 3.96, N
11.81, found: C 68.75, H 3.62, N 11.42.
6.90 (d, J ¼ 8.0 Hz, 2H), 6.83 (t, J ¼ 7.4 Hz, 1H), 6.69 (d, J ¼ 8.4 Hz,
2H), 6.64 (d, J ¼ 8.3 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d (ppm):
173.21 (Cq), 147.96 (Cq), 143.10 (Cq), 139.72 (Cq), 136.41 (Cq), 134.63
(CH), 133.04 (Cq), 129.78 (CH), 129.58 (CH), 129.42 (2CH), 129.29
(2CH),129.05 (Cq),128.83 (2CH),128.49 (2CH),128.03 (2CH),126.91
(Cq), 120.99 (CH), 116.85 (Cq), 114.49 (2CH), 113.07 (CH), 88.09
(Cspiro). Anal. Calcd for C27H18BrClN4O$0.05H2O: C 61.10, H 3.44, N
10.56, found: C 60.71, H 3.50, N 10.23.
4.1.5.5. 5-Chloro-20,40,50-triphenyl-20,40-dihydrospiro[indoline-3,3'-
[1,2,4]triazol]-2-one (5e). Synthesized according to the general
procedure, and starting with 6e and 7a, this compound was ob-
tained as a yellow solid (153.3 mg, 87% yield). Mp: 218e220 ꢁC; IR
(KBr, selected peaks): 3244 (NH), 1742 (C¼O), 1595 (C¼N), 1493,
1476, 1384, 1320, 1201, 764, 693 cmꢀ1; 1H NMR (400 MHz, Acetone-
4.1.5.9. 5-Bromo-4'-(3-chlorophenyl)-20,50-diphenyl-20,40-dihy-
drospiro[indoline-3,3'-[1,2,4]triazol]-2-one (5i). Synthesized ac-
cording to the general procedure, and starting with 6h and 7a, this
compound was obtained as a yellow solid (144.8 mg, 92% yield).
Mp: 143e145 ꢁC; IR (KBr, selected peaks): 328 (NH), 1738 (C¼O),
d6)
d
(ppm): 9.74 (br s, 1H, NH), 7.64 (s, 1H), 7.50 (d, J ¼ 7.7 Hz, 2H),
1597 (C¼N), 1493, 1385, 1325, 1201, 745, 687 cmꢀ1
;
1H NMR
7.42e7.29 (m, 4H), 7.21e7.11 (m, 5H), 6.99e6.93 (m, 3H), 6.91 (d,
(400 MHz, Acetone-d6) d (ppm): 9.88 (br s, 1H, NH), 7.78 (d,
J ¼ 7.2 Hz, 2H), 6.78 (t, J ¼ 7.2 Hz, 1H); 13C NMR (100 MHz, Acetone-
J ¼ 1.7 Hz, 1H), 7.56 (dd, J ¼ 8.4, 1.7 Hz, 1H), 7.52 (d, J ¼ 8.2 Hz, 2H),
7.42e7.33 (m, 3H), 7.24e7.17 (m, 2H), 7.14 (t, J ¼ 7.8 Hz, 2H), 6.97 (d,
J ¼ 8.4 Hz, 1H), 6.93 (d, J ¼ 7.8 Hz, 2H), 6.89 (s, 1H), 6.86e6.81 (m,
d6)
d (ppm): 173.33 (Cq), 148.59 (Cq), 144.20 (Cq), 141.76 (Cq),
138.93 (Cq), 132.27 (CH), 130.18 (CH), 129.82 (2CH), 129.74 (2CH),
129.20 (Cq), 129.04 (2CH), 128.82 (Cq), 128.69 (4CH), 128.63 (Cq),
127.99 (CH), 127.33 (CH), 120.65 (CH), 114.58 (2CH), 113.22 (CH),
88.38 (Cspiro). Anal. Calcd for C27H19ClN4O$0.75H2O: C 69.69, H
4.47, N 12.04, found: C 69.82, H 4.46, N 12.07.
1H), 6.78 (t, J ¼ 7.6 Hz, 1H); 13C NMR (75 MHz, Acetone-d6)
d (ppm):
172.90 (Cq),148.04 (Cq),144.04 (Cq),142.19 (Cq),140.49 (Cq),135.47
(CH), 134.64 (Cq), 131.20 (CH), 130.45 (CH), 130.21 (CH), 129.82
(2CH), 129.24 (2CH), 129.15 (Cq), 128.74 (2CH), 128.31 (Cq), 128.18

C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
505
(Cq), 127.94 (CH), 126.98 (CH), 120.98 (CH), 116.24 (Cq), 114.77
(2CH), 113.85 (CH), 88.23 (Cspiro). Anal. Calcd for
7.61e7.54 (m, 2H), 7.44e7.31 (m, 4H), 7.24e7.16 (m, 2H), 7.15e7.05
(m, 3H), 6.98e6.91 (m, 3H), 6.89e6.82 (m, 2H), 6.81e6.73 (m, 1H);
C
27H18BrClN4O$0.15H2O: C 60.89, H 3.47, N 10.52, Found: C 60.76, H
13C NMR (75 MHz, Acetone-d6)
d (ppm): 173.08 (Cq), 146.87 (Cq),
3.87, N 10.19.
144.01 (Cq),142.93 (Cq),137.75 (Cq),134.59 (Cq),133.01 (Cq),132.61
(CH), 130.92 (CH), 130.49 (Cq), 130.11 (CH), 130.02 (2CH), 129.91
(2CH), 129.70 (2CH), 128.23 (CH), 127.33 (CH), 126.89 (CH), 126.75
(Cq), 124.20 (CH), 120.87 (CH), 114.72 (2CH), 111.93, 88.60 (Cspiro).
Anal. Calcd for C21H14ClN3O2$0.9H2O: C 64.65, H 3.99, N 11.17,
found: C 64.43, H 3.83, N 11.47.
4.1.5.10. 5-Bromo-4'-(3-chloro-4-fluorophenyl)-20,50-diphenyl-20,40-
dihydrospiro
[indoline-3,3'-[1,2,4]triazol]-2-one
(5j).
Synthesized according to the general procedure, and starting with
6j and 7a, this compound was obtained as a yellow solid (138.9 mg,
90% yield). MP: 135e137 ꢁC; IR (KBr, selected peaks): 3242 (NH),
1735 (C¼O), 1597 (C¼N), 1492, 1473, 1384, 1261, 1194, 764,
4.1.5.14. 40,50-bis(4-chlorophenyl)-20-phenyl-20,40-dihydrospiro[indo-
line-3,3'-[1,2,4] triazol]-2-one (5n). Synthesized according to the
general procedure, and starting with 6c and 7c, this compound was
obtained as a yellow solid (163.5 mg, 86% yield). Mp: 155e157 ꢁC; IR
(KBr, selected peaks): 3205 (NH), 1731 (C¼O), 1600 (C¼N), 1492,
1474, 1319, 1203, 1014, 835, 746 cmꢀ1; 1H NMR (300 MHz, Acetone-
691 cmꢀ1; 1H NMR (400 MHz, Acetone-d6)
d (ppm): 9.75 (br s, 1H,
NH), 7.82 (s, 1H), 7.56 (d, J ¼ 8.4 Hz, 1H), 7.52 (d, J ¼ 7.2 Hz, 2H),
7.42e7.32 (m, 3H), 7.20e7.10 (m, 3H), 7.03 (dd, J ¼ 6.5, 2.2 Hz, 1H),
6.96 (d, J ¼ 8.4 Hz, 1H), 6.95e6.88 (m, 3H), 6.78 (t, J ¼ 7.3 Hz, 1H);
13C NMR (100 MHz, Acetone-d6)
d (ppm): 172.84 (Cq), 157.42 (d,
JFC ¼ 247.0 Hz, Cq), 148.03 (Cq), 144.02 (Cq), 142.14 (Cq), 136.02 (d,
JFCCCC ¼ 4.0 Hz, Cq), 135.51 (CH), 130.83 (CH), 130.49 (CH), 130.22
(CH), 129.82 (2CH), 129.48 (d, JFCCC ¼ 7.0 Hz, CH), 129.27 (2CH),
129.04 (Cq), 128.78 (2CH), 128.04 (Cq), 121.17 (d, JFCC ¼ 18.0 Hz, Cq),
120.99 (CH), 117.82 (d, JFCC ¼ 23.0 Hz, CH), 116.29 (Cq), 114.70 (2CH),
113.91 (CH), 88.35 (Cspiro). Anal. Calcd for C27H18BrClFN4O$0.1H2O:
C 59.00, H 3.16, N 10.20, Found: C 58.67, H 3.52, N 10.17.
d6)
d
(ppm): 9.68 (br s, 1H, NH), 7.56 (d, J ¼ 7.4 Hz, 1H), 7.53e7.46
(m, 2H), 7.43e7.34 (m, 3H), 7.24e7.17 (m, 2H), 7.15e7.05 (m, 3H),
7.00e6.89 (m, 3H), 6.88e6.80 (m, 2H), 6.79e6.71 (m, 1H); 13C NMR
(75 MHz, Acetone-d6) d (ppm): 173.13 (Cq), 147.20 (Cq), 144.15 (Cq),
143.02 (Cq), 137.89 (Cq), 135.58 (Cq), 132.96 (Cq), 132.61 (CH),
130.17 (2CH), 130.04 (2CH), 129.91 (2CH), 129.70 (2CH), 129.41
(2CH), 127.32 (CH and Cq), 126.90 (Cq), 124.20 (CH), 120.78 (CH),
114.70 (2CH), 111.94 (CH), 88.56 (Cspiro). Anal. Calcd for
4.1.5.11. 7-Chloro-20,40,50-triphenyl-20,40-dihydrospiro[indoline-3,3'-
[1,2,4]triazol]-2-one (5k). Synthesized according to the general
procedure, and starting with 6k and 7a, this compound was ob-
tained as a yellow solid (159.6 mg, 91% yield). Mp: 186e188 ꢁC; IR
(KBr, selected peaks): 3059 (NH), 1750 (C¼O), 1594 (C¼N), 1494,
C21H14ClN3O2$0.75H2O: C 65.00, H 3.95, N 11.23, found: C 64.64, H
3.90, N 11.50.
4.1.5.15. 2'-(2-chlorophenyl)-4'-(4-chlorophenyl)-50-phenyl-20,40-
1475, 1337, 1177, 1154, 749, 694 cmꢀ1 1H NMR (400 MHz, CDCl3)
;
dihydrospiro
[indoline-3,3'-[1,2,4]triazol]-2-one
(5o).
d
(ppm): 7.53e7.44 (m, 3H), 7.32 (d, J ¼ 8.2 Hz, 1H), 7.29 (d,
Synthesized according to the general procedure, and starting with
6c and 7e, this compound was obtained as a white solid (146.7 mg,
76% yield). Mp: 212e214 ꢁC; IR (KBr, selected peaks): 3261 (NH),
1739 (C¼O), 1616 (C¼N), 1492, 1472, 1384, 1202, 756, 697 cmꢀ1; 1H
J ¼ 7.2 Hz, 1H), 7.27e7.21 (m, 2H), 7.19e7.02 (m, 6H), 6.91 (d,
J ¼ 8.0 Hz, 2H), 6.82 (t, J ¼ 7.3 Hz, 1H), 6.80e6.72 (m, 2H); 13C NMR
(100 MHz, CDCl3)
d (ppm): 172.37 (Cq), 148.38 (Cq), 143.34 (Cq),
138.47 (Cq), 137.86 (Cq), 131.27 (CH), 129.48 (CH), 129.20 (2CH),
129.17 (2CH), 128.87 (Cq), 128.27 (2CH), 128.07 (2CH), 127.77 (2CH),
127.38 (Cq), 127.32 (CH), 125.02 (CH), 124.74 (CH), 120.86 (CH),
116.08 (Cq), 114.80 (2CH), 88.97 (Cspiro). Anal. Calcd for
NMR (300 MHz, Acetone-d6)
d (ppm): 9.31 (br s, 1H, NH), 7.70 (dd,
J ¼ 8.1, 1.5 Hz, 1H), 7.58e7.50 (m, 2H), 7.45e7.32 (m, 3H), 7.29 (ddd,
J ¼ 8.1, 7.3, 1.5 Hz, 1H) 7.21e7.08 (m, 4H), 7.04 (ddd, J ¼ 8.0, 7.3,
1.5 Hz, 1H), 6.95 (d, J ¼ 7.0 Hz, 1H), 6.84e6.69 (m, 4H); 13C NMR
C
27H19ClN4O$0.65H2O: C 69.69, H 4.47, N 12.04, found: C 69.37, H
(75 MHz, Acetone-d6) d (ppm): 173.99 (Cq),152.73 (Cq),144.20 (Cq),
4.31, N 11.73.
144.15 (Cq), 138.61 (Cq), 132.44 (Cq), 132.03 (CH), 130.59 (CH),
130.28 (CH), 129.93 (2CH), 129.65 (2CH), 129.65 (Cq), 129.21 (2CH),
129.00 (2CH), 128.72 (Cq), 128.48 (CH), 127.82 (2CH), 126.76 (CH),
124.92 (Cq), 122.59 (CH), 111.27 (CH), 89.65 (Cspiro). Anal. Calcd for
4.1.5.12. 7-Bromo-20,40,50-triphenyl-20,40-dihydrospiro[indoline-3,3'-
[1,2,4]triazol]-2-one (5l). Synthesized according to the general
procedure, and starting with 6l and 7a, this compound was ob-
tained as a yellow solid (154.1 mg, 94% yield). Mp: 220e222 ꢁC; IR
(KBr, selected peaks): 3057 (NH), 1741 (C¼O), 1594 (C¼N), 1494,
1472, 1385, 1337, 1178, 761, 694 cmꢀ1; 1H NMR (400 MHz, Acetone-
C27H18Cl2N4O$0.3H2O: C 66.07, H 3.83, N 11.42, found: C 65.77, H
3.81, N 11.17.
d6)
d
(ppm): 9.86 (br s, 1H, NH), 7.58 (d, J ¼ 7.4 Hz, 1H), 7.55 (d,
4.1.5.16. 2'-(3-chlorophenyl)-4'-(4-chlorophenyl)-50-phenyl-20,40-
J ¼ 8.0 Hz, 1H), 7.47 (d, J ¼ 7.1 Hz, 2H), 7.38e7.28 (m, 3H), 7.20e7.09
(m, 5H), 7.06 (t, J ¼ 7.7 Hz, 1H), 6.92 (d, J ¼ 8.0 Hz, 2H), 6.87e6.81
(m, 2H), 6.76 (t, J ¼ 7.3 Hz, 1H); 13C NMR (100 MHz, Acetone-d6)
dihydrospiro
[indoline-3,3'-[1,2,4]triazol]-2-one
(5p).
Synthesized according to the general procedure, and starting with
6c and 7f, this compound was obtained as a yellow solid (172.0 mg,
91% yield). Mp: 120e122 ꢁC; IR (KBr, selected peaks): 3255 (NH),
1735 (C¼O), 1591 (C¼N), 1491, 1474, 1385, 1324, 1196, 1092,
d
(ppm): 173.40 (Cq), 148.62 (Cq), 144.22 (Cq), 142.26 (Cq), 138.87
(Cq), 135.10 (CH), 130.21 (CH), 129.84 (2CH), 129.76 (2CH), 129.25
(Cq), 129.07 (2CH), 128.70 (2CH), 128.64 (2CH), 128.59 (Cq), 128.07
(CH), 126.36 (CH), 125.56 (CH), 120.73 (CH), 114.70 (2CH), 104.43
(Cq), 89.29 (Cspiro). Anal. Calcd for C27H19BrN4O: C 65.46, H 3.87, N
11.31, found: C 65.36, H 3.99, N 11.18.
754 cmꢀ1 1H NMR (300 MHz, Acetone-d6)
; d (ppm): 9.79 (br s, 1,
NH), 7.59 (dd, J ¼ 7.6, 0.7 Hz, 1H), 7.55e7.47 (m, 2H), 7.45e7.31 (m,
4H), 7.23e7.17 (m, 2H), 7.12 (td, J ¼ 7.6, 1.0 Hz, 1H), 7.11e7.04 (m,
2H), 6.98 (d, J ¼ 7.7 Hz, 1H), 6.87e6.80 (m, 2H), 6.75 (ddd, J ¼ 8.0,
2.0, 0.9 Hz, 1H), 6.67 (ddd, J ¼ 8.4, 2.3, 0.9 Hz, 1H); 13C NMR
4.1.5.13. 5'-(3-chlorophenyl)-4'-(4-chlorophenyl)-20-phenyl-20,40-
(75 MHz, Acetone-d6) d (ppm): 172.86 (Cq),149.09 (Cq),145.34 (Cq),
dihydrospiro
[indoline-3,3'-[1,2,4]triazol]-2-one
(5m).
143.01 (Cq), 137.78 (Cq), 135.19 (Cq), 133.08 (Cq), 132.87 (CH), 131.14
(CH), 130.61 (CH), 130.22 (2CH), 129.90 (2CH), 129.28 (2CH), 128.83
(2CH), 128.16 (Cq), 127.36 (CH), 126.33 (Cq), 124.40 (CH), 119.99
(CH), 114.39 (CH), 112.27 (CH), 112.06 (CH), 88.12 (Cspiro). Anal.
Calcd for C27H18Cl2N4O$0.4H2O: C 65.83, H 3.85, N 11.38, found: C
65.51, H 3.76, N 11.12.
Synthesized according to the general procedure, and starting with
6c and 7b, this compound was obtained as a yellow solid (169.0 mg,
89% yield). Mp: 98e100 ꢁC. IR (KBr, selected peaks): 3244 (NH),
1733 (C¼O),1593 (C¼N),1491,1471,1318,1197,1093, 746, 688 cmꢀ1
;
1H NMR (300 MHz, Acetone-d6)
d (ppm): 9.72 (br s, 1H, NH),

506
C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
4.1.5.17. 20,40-bis(4-chlorophenyl)-50-phenyl-20,40-dihydrospiro[indo-
line-3,3'-[1,2,4] triazol]-2-one (5q). Synthesized according to the
general procedure, and starting with 6c and 7g, this compound was
obtained as a yellow solid (159.3 mg, 84% yield). MP: 130e132 ꢁC; IR
(KBr, selected peaks): 3208 (NH), 1731 (C¼O), 1595 (C¼N), 1490,
1385, 1320, 1194, 1014, 820, 746 cmꢀ1; 1H NMR (300 MHz, Acetone-
(Cq), 142.27 (Cq), 140.24 (Cq), 135.74 (Cq), 135.52 (CH), 134.76 (Cq),
131.32 (CH), 130.30 (2CH), 130.23 (CH), 129.85 (2CH), 129.44 (2CH),
128.98 (Cq), 128.13 (2CH), 127.13 (Cq), 127.03 (CH), 121.15 (CH),
116.21 (Cq),114.82 (2CH),113.91 (CH), 88.37 (Cspiro). Anal. Calcd for
C27H17BrCl2N4O: C 57.47, H 3.04, N 9.93, Found: C 57.87, H 2.84, N
9.77.
d6)
d
(ppm): 9.68 (br s, 1H, NH), 7.57 (d, J ¼ 7.4 Hz, 1H), 7.53e7.46
(m, 2H), 7.42e7.30 (m, 4H), 7.22e7.16 (m, 2H), 7.16e7.07 (m, 3H),
4.1.5.21. 5-Bromo-4'-(3-chlorophenyl)-5'-(4-methoxyphenyl)-20-
phenyl-20,40-dihydro spiro [indoline-3,3'-[1,2,4]triazol]-2-one (5u).
Synthesized according to the general procedure, and starting with
6j and 7d, this compound was obtained as a yellow solid (50.2 mg,
60% yield). Mp: 123e125 ꢁC; IR (KBr, selected peaks): 3259 (NH),
1746 (C¼O), 1590 (C¼N), 1498, 1475, 1385, 1255, 1177, 1032,
6.96 (d, J ¼ 7.8 Hz, 1H), 6.94e6.86 (m, 2H), 6.86e6.78 (m, 2H); 13C
NMR (75 MHz, Acetone-d6) d (ppm): 172.93 (Cq),148.85 (Cq),143.19
(Cq), 143.03 (Cq), 137.95 (Cq), 132.98 (Cq), 132.75 (CH), 130.50 (CH),
130.15 (2CH), 129.86 (2CH), 129.56 (2CH), 129.25 (2CH), 128.76
(2CH), 128.32 (Cq), 127.35 (CH), 126.56 (Cq), 124.87 (Cq), 124.32
(CH), 116.05 (2CH), 112.07 (CH), 88.41 (Cspiro). Anal. Calcd for
689 cmꢀ1; 1H NMR (300 MHz, Acetone-d6)
d (ppm): 9.86 (br s, 1H,
C
21H14ClN3O2: C 66.81, H 3.75, N 11.55, found: C 66.60, H 3.76, N
NH), 7.75 (d, J ¼ 2.0 Hz, 1H), 7.54 (dd, J ¼ 8.3, 2.0 Hz, 1H), 7.47e7.41
(m, 2H), 7.25e7.17 (m, 2H), 7.16e7.09 (m, 2H), 6.96 (d, J ¼ 8.3 Hz,
1H), 6.93e6.87 (m, 5H), 6.85e6.80 (m, 1H), 6.76 (t, J ¼ 7.3 Hz, 1H),
11.36.
4.1.5.18. 2'-(2-chlorophenyl)-5'-(3-chlorophenyl)-4'-(4-
chlorophenyl)-20,40-dihydrospiro [indoline-3,3'-[1,2,4]triazol]-2-one
(5r). Synthesized according to the general procedure, and start-
ing with 6c and 7h, this compound was obtained as a white solid
(173.8 mg, 86% yield). Mp: 244e245 ꢁC. IR (KBr, selected peaks):
3284 (NH), 1745 (C¼O), 1605 (C¼N), 1492, 1473, 1427, 1375, 1195,
3.80 (s, 3H); 13C NMR (75 MHz, Acetone-d6)
d (ppm): 173.00 (Cq),
161.68 (Cq), 147.92 (Cq), 144.21 (Cq), 142.16 (Cq), 140.64 (Cq), 135.38
(CH), 134.60 (Cq), 131.16 (CH), 130.31 (2CH), 130.16 (CH), 129.78
(2CH), 129.25 (Cq), 128.27 (CH), 127.89 (CH), 127.10 (CH), 120.74
(CH), 120.38 (Cq), 116.19 (Cq), 114.68 (2CH), 114.64 (2CH), 113.80
(CH), 88.10
28H20BrClN4O2$0.3H2O: C 59.49, H 3.68, N 9.91, found: C 59.17, H
3.34, N 10.08.
(Cspiro),
55.67 (CH3).
Anal. Calcd
for
753, 688 cmꢀ1. 1H NMR (300 MHz, Acetone-d6)
d
(ppm): 9.37 (br s,
C
1H, NH), 7.70 (dd, J ¼ 8.1, 1.5 Hz, 1H), 7.65e7.60 (m, 1H), 7.46e7.33
(m, 3H), 7.29 (ddd, J ¼ 8.1, 7.2, 1.6 Hz, 1H), 7.20e7.11 (m, 4H), 7.05
(ddd, J ¼ 8.0, 7.2, 1.6 Hz, 1H), 6.97 (dd, J ¼ 7.5, 1.5 Hz, 1H), 6.88e6.81
(m, 2H), 6.80e6.70 (m, 2H); 13C NMR (75 MHz, Acetone-d6)
4.1.5.22. 5-Bromo-2'-(2-chlorophenyl)-4'-(3-chlorophenyl)-50-
phenyl-20,40-dihydrospiro [indoline-3,3'-[1,2,4]triazol]-2-one (5v).
Synthesized according to the general procedure, and starting with
6j and 7e, this compound was obtained as a white solid (105.2 mg,
63% yield). Mp: 244e246 ꢁC; IR (KBr, selected peaks): 3168 (NH),
d
(ppm): 173.93 (Cq), 151.35 (Cq), 144.14 (Cq), 143.77 (Cq), 138.27
(Cq), 134.65 (Cq), 132.71 (Cq), 132.15 (CH), 130.98 (CH), 130.74 (Cq),
130.48 (CH), 130.34 (CH), 129.91 (2CH), 129.82 (2CH), 129.70 (Cq),
128.69 (CH), 128.57 (CH), 127.97 (CH), 127.88 (CH), 127.35 (CH),
127.01 (CH), 124.55 (Cq), 122.63 (CH), 111.30 (CH), 89.83 (Cspiro).
Anal. Calcd for C27H17Cl3N4O: C 62.38, H 3.30, N 10.78, Found: C
62.78, H 3.41, N 10.65.
1747 (C¼O), 1588 (C¼N), 1474, 1384, 1196, 828, 761, 690 cmꢀ1
;
1H
NMR (300 MHz, Acetone-d6)
d (ppm): 9.53 (br s, 1H, NH), 7.77 (dd,
J ¼ 8.1, 1.5 Hz, 1H), 7.59e7.54 (m, 2H), 7.46e7.30 (m, 5H), 7.22e7.06
(m, 5H), 6.90e6.86 (m, 1H), 6.85e6.81 (m, 1H), 6.77 (d, J ¼ 8.3 Hz,
1H); 13C NMR (75 MHz, Acetone-d6)
d (ppm): 173.60 (Cq), 152.48
4.1.5.19. 5-Bromo-40,50-bis(3-chlorophenyl)-20-phenyl-20,40-dihy-
drospiro[indoline-3,3'-[1,2,4]triazol]-2-one (5s). Synthesized ac-
cording to the general procedure, and starting with 6j and 7b, this
compound was obtained as a yellow solid (142.5 mg, 85% yield).
Mp: 104e106 ꢁC; IR (KBr, selected peaks): 3251 (NH), 1738 (C¼O),
(Cq), 143.75 (Cq), 143.26 (Cq), 140.86 (Cq), 134.91 (CH), 134.57 (Cq),
131.40 (CH), 131.06 (CH), 130.75 (CH), 130.38 (CH), 129.59 (Cq),
129.24 (2CH), 129.11 (2CH), 128.44 (Cq), 128.31 (CH), 128.10 (CH),
127.96 (CH), 127.63 (CH), 127.26 (CH), 126.99 (Cq), 126.75 (CH),
114.64 (Cq), 113.10 (CH), 89.48 (Cspiro). Anal. Calcd for
1590 (C¼N), 1474, 1434, 1384, 1314, 1197, 748, 689 cmꢀ1
;
1H NMR
C27H17BrCl2N4O$0.55H2O: C 56.48, H 3.18, N 9.76, found: C 56.21, H
3.32, N 9.37.
(300 MHz, Acetone-d6)
d
(ppm): 9.91 (br s, 1H, NH), 7.81 (d,
J ¼ 2.0 Hz, 1H), 7.61 (d, J ¼ 0.9 Hz, 1H), 7.55 (dd, J ¼ 8.3, 2.0 Hz, 1H),
7.45e7.34 (m, 3H), 7.26e7.20 (m, 2H), 7.15 (t, J ¼ 8.0 Hz, 2H),
7.00e6.91 (m, 4H), 6.90e6.85 (m, 1H), 6.80 (t, J ¼ 7.3 Hz, 1H); 13C
4.1.5.23. 5-Bromo-20,40-bis(3-chlorophenyl)-50-phenyl-20,40-dihy-
drospiro[indoline-3,3'-[1,2,4]triazol]-2-one (5w). Synthesized ac-
cording to the general procedure, and starting with 6j and 7f, this
compound was obtained as a yellow solid (147.4 mg, 88% yield).
Mp: 118e120 ꢁC; IR (KBr, selected peaks): 3245 (NH), 1735 (C¼O),
NMR (75 MHz, Acetone-d6)
d (ppm): 172.79 (Cq), 146.77 (Cq),
143.77 (Cq), 142.16 (Cq), 140.13 (Cq), 135.56 (CH), 134.79 (Cq),
134.64 (Cq), 131.36 (CH), 130.97 (CH), 130.32 (2CH), 130.30 (Cq),
129.87 (2CH), 128.82 (Cq), 128.42 (CH), 128.22 (CH), 128.19 (CH),
127.06 (CH), 127.04 (CH), 121.27 (CH), 116.29 (Cq), 114.87 (2CH),
113.88 (CH), 88.42 (Cspiro). Anal. Calcd for C27H17BrCl2N4O: C 57.47,
H 3.04, N 9.93, Found: C 57.80, H 3.23, N 9.72.
1589 (C¼N), 1474, 1384, 1328, 1197, 773, 691 cmꢀ1
;
1H NMR
(300 MHz, Acetone-d6)
d
(ppm): 7.83 (d, J ¼ 2.0 Hz, 1H), 7.57 (dd,
J ¼ 8.4, 2.0 Hz, 1H), 7.55e7.51 (m, 2H), 7.43e7.35 (m, 3H), 7.23e7.19
(m, 2H), 7.14e7.06 (m, 2H), 6.99 (d, J ¼ 8.4 Hz, 1H), 6.90 (dd, J ¼ 2.4,
1.3 Hz, 1H), 6.88e6.82 (m, 1H), 6.78 (ddd, J ¼ 7.9, 2.0, 0.6 Hz, 1H),
6.65 (ddd, J ¼ 8.3, 2.2, 0.6 Hz, 1H); 13C NMR (75 MHz, Acetone-d6)
4.1.5.20. 5-Bromo-4'-(3-chlorophenyl)-5'-(4-chlorophenyl)-20-
phenyl-20,40-dihydrospiro [indoline-3,3'-[1,2,4]triazol]-2-one (5t).
Synthesized according to the general procedure, and starting with
6j and 7c, this compound was obtained as a yellow solid (138.6 mg,
82% yield). Mp: 116e118 ꢁC; IR (KBr, selected peaks): 3251 (NH),
1735 (C¼O), 1590 (C¼N), 1491, 1474, 1384, 1315, 1092, 821,
d
(ppm): 172.54 (Cq), 148.91 (Cq), 145.04 (Cq), 142.19 (Cq), 140.09
(Cq), 135.78 (CH), 135.31 (Cq), 134.72 (Cq), 131.30 (2CH), 130.75
(CH), 130.30 (CH), 129.29 (2CH), 128.92 (2CH), 128.39 (Cq), 128.34
(CH), 128.22 (CH), 127.91 (Cq), 127.11 (CH), 120.38 (CH), 116.44 (Cq),
114.56 (CH), 114.00 (CH), 112.25 (CH), 87.88 (Cspiro). Anal. Calcd for
689 cmꢀ1; 1H NMR (300 MHz, Acetone-d6)
d
(ppm): 9.89 (br s, 1H,
C27H17BrCl2N4O$0.85H2O: C 55.95, H 3.26, N 9.67, found: C 55.61, H
NH), 7.78 (d, J ¼ 2.0 Hz, 1H), 7.58e7.50 (m, 3H), 7.41 (d, J ¼ 8.6 Hz,
3.13, N 9.39.
2H), 7.25e7.19 (m, 2H), 7.15 (t, J ¼ 8.0 Hz, 2H), 7.00e6.92 (m, 3H),
6.91 (d, J ¼ 1.0 Hz, 1H) 6.88e6.83 (m, 1H), 6.80 (t, J ¼ 7.3 Hz, 1H); 13
C
4.1.5.24. 5-Bromo-4'-(3-chlorophenyl)-2'-(4-chlorophenyl)-50-
phenyl-20,40-dihydrospiro [indoline-3,3'-[1,2,4]triazol]-2-one (5x).
NMR (75 MHz, Acetone-d6)
d
(ppm): 172.86 (Cq),147.07 (Cq),143.86

C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
507
Synthesized according to the general procedure, and starting with
6j and 7g, this compound was obtained as a yellow solid (149.4 mg,
89% yield). Mp: 203e205 ꢁC; IR (KBr, selected peaks): 3085 (NH),
1746 (C¼O), 1590 (C¼N), 1491, 1475, 1385, 1199, 776, 694 cmꢀ1; 1H
residue was purified by flash chromatography on silica gel using as
eluent a gradient of 100% n-hexane to n-hexane/EtOAc (60:40), and
recrystallized from diethyl ether to afford the desired compound.
NMR (300 MHz, Acetone-d6)
d
(ppm): 7.79 (d, J ¼ 2.0 Hz, 1H),
4.1.6.1. Compound 4c. White solid (59.3 mg, 75%). Mp: 185e187 ꢁC;
7.58e7.49 (m, 3H), 7.42e7.31 (m, 3H), 7.24e7.17 (m, 2H), 7.15 (d,
J ¼ 9.0 Hz, 2H), 6.96 (d, J ¼ 8.2 Hz, 1H), 6.92 (d, J ¼ 9.0 Hz, 2H), 6.89
(dd, J ¼ 2.3, 1.2 Hz, 1H), 6.87e6.80 (m, 1H); 13C NMR (75 MHz,
IR (KBr, selected peaks): 3215 (NH), 1726, 1705, 1596, 1492, 1473,
1384,1194, 751, 689 cmꢀ1; 1H NMR (400 MHz, Acetone-d6)
d (ppm):
9.92 (br s, 1H, NH), 7.81e7.71 (m, 2H), 7.40e7.26 (m, 6H), 7.18e7.07
(m, 4H), 6.98e6.91 (m, 3H), 6.86e6.78 (m, 1H), 6.54 (d, J ¼ 2.0 Hz,
Acetone-d6)
d (ppm): 172.60 (Cq), 148.66 (Cq), 142.87 (Cq), 142.27
(Cq), 140.27 (Cq), 135.65 (CH), 134.70 (Cq), 131.25 (CH), 130.64 (CH),
130.23 (CH), 129.71 (2CH), 129.27 (2CH), 128.85 (2CH), 128.62 (Cq),
128.26 (CH), 128.09 (CH), 128.08 (Cq), 127.02 (CH), 125.23 (Cq),
116.32 (Cq), 116.14 (2CH), 114.02 (CH), 88.18 (Cspiro). Anal. Calcd for
1H), 5.45 (s, 1H, H-40); 13C NMR (100 MHz, Acetone-d6)
d (ppm):
176.76 (Cq),149.40 (Cq),145.06 (Cq),141.67 (Cq),138.24 (Cq),135.00
(Cq), 133.32 (CH), 132.37 (Cq), 131.17 (CH), 130.05 (CH), 129.89 (CH),
129.84 (CH), 129.74 (2CH), 129.44 (2CH), 129.04 (CH), 128.65 (CH),
128.40 (Cq), 127.55 (2CH), 121.80 (CH), 115.90 (2CH), 114.67 (Cq),
112.95 (CH), 77.22 (Cspiro), 62.34 (CH). Anal. Calcd for
C
27H17BrCl2N4O$0.15H2O: C 57.19, H 3.08, N 9.88, found: C 56.86, H
3.27, N 9.70.
C
28H19BrClN3O$0.2H2O: C 63.16, H 3.68, N 7.89, found: C 62.76, H
4.1.5.25. 5-Bromo-2'-(2-chlorophenyl)-40,50-bis(3-chlorophenyl)-
4.07, N 7.99.
20,40-dihydrospiro
[indoline-3,3'-[1,2,4]triazol]-2-one
(5y).
Synthesized according to the general procedure, and starting with
6j and 7h, this compound was obtained as a white solid (143.1 mg,
80% yield). Mp: 239e240 ꢁC; IR (KBr, selected peaks): 3242 (NH),
1747 (C¼O), 1589 (C¼N), 1474, 1431, 1384, 1195, 1077, 764,
4.2. Biology: general conditions
4.2.1. Cell culture
HEK 293T (ATCC CRL-11268), MCF-7 (ATCC HTB-22TM) and
MDA-MB-231 (ATCC HTB-26TM) cells were cultured in RPMI 1640
culture medium supplemented with 10% fetal bovine serum, 100
units of penicillin G (sodium salt), 100 mg of streptomycin sulfate
695 cmꢀ1; 1H NMR (300 MHz, Acetone-d6)
d (ppm): 9.57 (br s, 1H,
NH), 7.77 (dd, J ¼ 8.1, 1.5 Hz, 1H), 7.67e7.64 (m, 1H), 7.48e7.30 (m,
5H), 7.24 (d, J ¼ 2.0 Hz, 1H), 7.23e7.14 (m, 3H), 7.11 (ddd, J ¼ 8.0, 7.3,
1.6 Hz,1H), 6.96e6.92 (m,1H), 6.91e6.86 (m,1H), 6.77 (d, J ¼ 8.3 Hz,
and 2 mM L-glutamine (Sigma-Aldrich, St Louis, MO, USA). HCT116
1H); 13C NMR (75 MHz, Acetone-d6)
d
(ppm): 173.54 (Cq), 151.10
cells were grown in McCoy's 5A supplemented with 10% fetal
bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA,
USA) and 1% penicillin/streptomycin solution (Sigma-Aldrich). Cells
were maintained at 37 ꢁC in a humidified atmosphere of 5% CO2.
CCD18-Co cells were grown in DMEM culture medium supple-
mented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin/
streptomycin solution (Sigma-Aldrich), 1% Glutamax (Gibco), 1%
Non-Essential Amino Acids Solution (NEAA) (Gibco) and 0.025%
(Cq), 143.44 (Cq), 143.19 (Cq), 140.53 (Cq), 135.03 (CH), 134.74 (Cq),
134.67 (Cq), 131.53 (CH), 131.24 (CH), 131.00 (CH), 130.65 (CH),
130.49 (Cq), 130.44 (CH), 129.64 (Cq), 128.86 (CH), 128.48 (CH),
128.11 (CH), 128.01 (CH), 127.93 (CH), 127.48 (2CH), 126.82 (CH),
126.67 (Cq), 114.70 (Cq), 113.12 (CH), 89.69 (Cspiro). Anal. Calcd for
C
27H16BrCl3N4O: C 54.16, H 2.70, N 9.36, found: C 54.54, H 2.80, N
9.29.
TNF-a Human.
4.1.5.26. 5-Bromo-20,40,50-tris(3-chlorophenyl)-20,40-dihydrospiro
[indoline-3,3'-[1,2,4] triazol]-2-one (5z). Synthesized according to
the general procedure, and starting with 6j and 7i, this compound
was obtained as a yellow solid (156.3 mg, 88% yield). Mp:
113e115 ꢁC; IR (KBr, selected peaks): 3240 (NH), 1740 (C¼O), 1589
(C¼N), 1475, 1430, 1385, 1326, 1195, 768, 691 cmꢀ1.1H NMR
4.2.2. Cell treatment
Compound stock solutions were prepared in sterile DMSO. Prior
to all treatments, cells were allowed to adhere for 24 h and then
exposed to test compounds diluted in culture medium for the
indicated time. All experiments were performed in parallel with
DMSO vehicle control. The final DMSO concentration did not
exceed 0.8% (v/v).
(300 MHz, Acetone-d6)
d (ppm): 10.02 (br s, 1H, NH), 7.87 (d,
J ¼ 2.0 Hz, 1H), 7.64e7.60 (m, 1H), 7.58 (dd, J ¼ 8.3, 2.0 Hz, 1H),
7.46e7.33 (m, 3H), 7.27e7.22 (m, 2H), 7.16e7.08 (m, 2H), 6.99 (d,
J ¼ 8.3 Hz, 1H), 6.96 (dd, J ¼ 2.5, 1.3 Hz, 1H), 6.92e6.87 (m, 1H), 6.81
4.2.3. In vitro antiproliferative assays
(ddd, J ¼ 8.0, 2.0, 0.8 Hz, 1H), 6.68 (ddd, J ¼ 8.4, 2.3, 0.8 Hz, 1H); 13
C
Cells were seeded in 96-well plates at 2 ꢃ 105 cells/mL (MCF-7,
NMR (75 MHz, Acetone-d6)
d
(ppm): 172.38 (Cq), 147.63 (Cq),
MDA-MB-231, HEK 293T),
1
ꢃ
105 cells/mL (HCT116), and
144.79 (Cq), 142.08 (Cq), 139.70 (Cq), 135.87 (CH), 135.34 (Cq),
134.86 (Cq), 134.68 (Cq), 131.43 (CH), 131.34 (CH), 131.01 (CH),
130.61 (CH), 130.41 (CH), 129.90 (Cq), 128.60 (CH), 128.49 (CH),
128.33 (CH), 128.07 (Cq), 127.26 (CH), 127.15 (CH), 120.69 (CH),
116.50 (Cq), 114.65 (CH), 114.00 (CH), 112.39 (CH), 88.05 (Cspiro).
Anal. Calcd for C27H16BrCl3N4O: C 54.16, H 2.70, N 9.36, found: C
54.51, H 2.79, N 9.22.
4 ꢃ 103 cells/well (CCD18-co human normal colon fibroblasts). The
cellular growth inhibitory activity was evaluated in five cell lines.
Antiproliferative assays in MCF-7, MDA-MB-231, and HEK 293T
were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) [47]. Each compound concentration
and DMSO was tested in triplicate in a single experiment which was
repeated at least 3 times. Cell viability was assessed 72 h after
compound incubation. Briefly, the medium was removed and
replaced with fresh medium, and the MTT dye solution was added
to each well (5 mg/mL in 10 mM phosphate buffer solution at pH
7.4), and after 3 h of incubations the media was removed and
intracellular formazan crystals were solubilized and extracted with
DMSO. Absorbance was measured at 570 nm, after 15 min at room
temperature using a microplate reader (FLUOstar Omega, BMG
Labtech, Germany), and the percentage of viable cells was deter-
mined for each compound concentration as described previously
[48]. For cytotoxicity assays in HCT116 cells, each compound con-
centration and DMSO was tested in duplicate in a single experiment
4.1.6. Synthesis of 5-bromo-4'-(3-chlorophenyl)-20,50-diphenyl-
20,40-dihydrospiro [indoline-3,30-pyrazol]-2-one (4c)
Triethylamine (2.0 equiv, 0.3 mmol, 42 mL) was added dropwise
to a mixture of 3-methylene indolin-2-one 13 (1.0 equiv, 0.15 mmol,
50.0 mg), and hydrazonoyl chloride 7a (2.0 equiv, 0.3 mmol,
69.2 mg) in dry DCM (1.5 mL) under nitrogen atmosphere. The
reaction was stirred at room temperature for 5 h. The mixture was
then washed with brine (2x) and the aqueous phase extracted with
DCM. The combined organic extracts were dried over anhydrous
Na2SO4 and the solvent was removed under reduce pressure. The

508
C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
which was repeated at least 3 times. Cell viability was assessed 72 h
after compound incubation by using the CellTiter96® AQueous
Non-Radioactive Cell Proliferation Assay (Promega Corporation,
Madison, WI, USA), according to the manufacturer's protocol. The
absorbance was measured at 490 nm using Bio-Rad microplate
reader Model 680 (Bio-Rad, Hercules, CA, USA). Nutlin-3a was used
as positive control. Additionally, for non-cytotoxic potential assays
in CCD18-Co cells, each compound concentration and DMSO was
tested in triplicate in a single experiment which was repeated at
least 2 times. Cell viability was assessed as described for the
HCT116 cell line. The concentrations of the compounds that
inhibited cell growth by 50 (IC50) and 80% (IC80) were determined
by non-linear regression using GraphPad PRISM software.
concentration was determined by the colorimetric Bradford
method using the Bio-Rad Protein Assay reagent (Bio-Rad), ac-
cording to the manufacturer's instructions. BSA (Sigma-Aldrich)
was used as standard, and absorbance measurements were per-
formed at 595 nm using GloMax-Multi þ Detection System
(Promega).
4.2.7. Caspase-3 and -7 activity assay
Caspases-3 and -7 activity was measured using the Caspase-Glo
3/7 Assay (Promega). Fifteen
mg of total protein extracts from MDA-
MB-231 cells in a total volume of 50
m
L were mixed with 50 L of
m
caspase-Glo 3/7 reagent with orbital shaking for 30 s. Subsequently,
the mixture was incubated at room temperature for 30 min. The
luminescence signal was measured using the GloMax-
Multi þ Detection System (Promega). Data were analyzed using
Excel and GraphPad PRISM software. Doxorubicin was used as
positive control.
4.2.4. Apoptosis evaluation
Apoptosis was quantified using the Guava Nexin Reagent kit
(Merck Millipore). The Nexin assay uses two distinct dyes, Annexin
V to detect phosphatidylserine (PS) on the external membrane of
apoptotic cells, and the cell impermeant dye, 7-AAD, as an indicator
of membrane structural integrity. For this purpose, HCT-116 p53(þ/
þ) cells were plated in 24-well plates at 5 ꢃ 104 cells/well. Twenty-
four hours after plating, cells were exposed to: 1) compounds in
test at IC50 concentration; 2) compounds in test at 2-fold IC50
concentration, and 3) DMSO (control group), for 72 h. After that, the
culture medium was collected and cells detached with Accutase.
Cells were collected and centrifuged at 500 g for 5 min at 4 ꢁC. The
4.2.8. Western blot and densitometric analysis
Steady-state protein levels were determined by Western blot
analysis. Briefly, total protein extracts were separated on 8% and
14% (w/v) sodium dodecyl sulfate (SDS) - polyacrylamide gel elec-
trophoresis and transferred onto nitrocellulose membranes using
the Trans-blot Turbo Transfer System (Biorad). Uniform protein
loading and transfer was confirmed by transient staining with 0.2%
Ponceau S (Merck, Darmstadt, Germany). Next, nonspecific binding
sites were blocked with a 5% milk solution in Tris-buffered saline
(TBS) for 1 h. Membranes were then incubated overnight at 4 ꢁC
with mouse monoclonal anti-p53 (Pab-240, sc-99, 1:200) and anti-
Mdm2 (SMP-14, sc-965, 1:200) antibodies, (Santa Cruz Biotech-
nology, Santa Cruz, CA). After washing, membranes were incubated
with anti-mouse secondary antibody conjugated with horseradish
peroxidase (Bio-Rad) for 2 h at room temperature. The immuno-
reactive complexes were visualized by chemiluminescence with
Immobilon™ Western (Milipore) or SuperSignal West Femto sub-
strate (Thermo Fisher Scientific, Waltham, MA, USA). Ponceau S was
used as loading control. Densitometric analysis was performed
with the Image Lab software Version 5.1 Beta (Bio-Rad).
cell pellet was resuspended in PBS/2% FBS. Subsequently, 50
mL of
cell suspension were mixed with 50 L of Guava Nexin reagent and
m
incubated for 20 min, at room temperature in the absence of light.
Following the staining procedure, sample acquisition and data
analysis of at least 5000 events per sample were performed using
the Guava easyCyte™ Flow Cytometer (Merck Millipore) and Nexin
software module.
4.2.5. Cell cycle distribution analysis
The effects of compounds on cell cycle progression were
determined using a standard propidium iodide (PI) staining pro-
cedure followed by flow cytometry analysis. PI is a fluorescent
intercalating agent that has high affinity to nucleic acids.
HCT116 p53(þ/þ)cells were plated in 6-well plates at
1.5 ꢃ 105 cells/well. Twenty-four hours after plating, cells were
treated with the compounds in test at the IC50 concentration, or
DMSO, for additional 24 or 48 h. After that, cells were detached and
collected by centrifugation at 800g for 5 min, at 4 ꢁC. Cell pellets
were resuspended in cold PBS and added an equal volume of 80%
ice-cold ethanol (ꢀ20 ꢁC) drop by drop, while vortexing gently.
Samples were stored at ꢀ4 ꢁC until data acquisition. For cell cycle
analysis, cells were centrifuged again at 850 g for 5 min, at 4 ꢁC, and
4.2.9. Venus-based bimolecular fluorescence complementation
(BiFC) assay
To evaluate p53-MDM2 protein-protein interaction, HCT116
p53(ꢀ/ꢀ) cells were co-transfected using 1
mg of each BiFC pair
plasmid (V1-p53/MDM2-V2) and Lipofectamine 2000 (Invitrogen,
Thermo Fisher Scientific, Waltham, MA, USA), following the man-
ufacturer's instructions. Four to 6 h after transfection, the medium
was replaced with fresh medium, and compound 5y and nutlin-3a
cell pellets were resuspended in 25
mg/mL PI (Fluka, Sigma-Aldrich)
were added to a final concentration of 5 and 10
mM. The same
and 50 g/mL RNase A (Sigma-Aldrich) and further incubated for
m
concentrations of DMSO were tested as control. Cells were washed
twice with Ca2þ- and Mg2þ-free phosphate buffered saline (PBS)
(Invitrogen), treated with Accutase (Gibco) and harvested in culture
medium. Cell suspensions were centrifuged, supernatants dis-
carded, and cell pellets resuspended in PBS supplemented with 2%
FBS [34]. Fluorescence was measured using Guava® easyCyte™
Flow Cytometer (Merck Millipore).
30 min. Sample acquisition and data analysis were performed using
the Guava easyCyte™ Flow Cytometer (Merck Millipore) and Guava
analysis software, with acquisition of at least 10000 events per
sample.
4.2.6. Total protein extraction and quantification
HCT-116 p53(þ/þ) cells were plated in 60 mm dishes at
8 ꢃ 105 cells/dish. Twenty four hours after plating, cells were
exposed to compounds in test at the IC50 concentration, or vehicle
control (DMSO), for 72 h. After that, floating and adherent cells
were collected directly in lysis buffer (1% NP-40, 20 mM Tris-HCl pH
7.4, 150 mM NaCl, 5 mM EDTA, 10% Glycerol, 1 mM dithiothreitol,
and 1X proteases and phosphatases inhibitors), followed by soni-
cation and centrifugation at 3200g for 10 min at 4 ꢁC. Total protein
extracts were recovered and stored at ꢀ80 ꢁC. Protein
Acknowledgements
This study was supported by FCT by research project UID/DTP/
04138/2013, and by fellowships: SFRH/BD/69258/2010 (C. J. A. R.)
and SFRH/BPD/100961/2014 (J. D. A.). M. M. M. Santos would like to
acknowledge FCT, “Programa Operacional Potencial Humano” and
the European Social Fund for the IF Program (IF/00732/2013).

C.J.A. Ribeiro et al. / European Journal of Medicinal Chemistry 140 (2017) 494e509
509
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