In?vitro targeting of colon cancer cells using spiropyrazoline oxindoles

Article abstract of DOI:10.1016/j.ejmech.2017.07.057

We report on the synthesis and biological evaluation of a library of twenty-three spiropyrazoline oxindoles. The antiproliferative activity of the chemical library was evaluated in HCT-116 p53^(+/+) human colon cancer cell line with eight deriva

Full text of DOI:10.1016/j.ejmech.2017.07.057

Accepted Manuscript
In vitro targeting of colon cancer cells using spiropyrazoline oxindoles
Rute C. Nunes, Carlos J.A. Ribeiro, Ângelo Monteiro, Cecília M.P. Rodrigues, Joana
D. Amaral, Maria M.M. Santos
PII:
S0223-5234(17)30579-2
10.1016/j.ejmech.2017.07.057
EJMECH 9620
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 April 2017
Revised Date: 23 June 2017
Accepted Date: 23 July 2017
Please cite this article as: R.C. Nunes, C.J.A. Ribeiro, Â. Monteiro, Cecí.M.P. Rodrigues, J.D. Amaral,
M.M.M. Santos, In vitro targeting of colon cancer cells using spiropyrazoline oxindoles, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.057.
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ACCEPTED MANUSCRIPT
In vitro targeting of colon cancer cells using spiropyrazoline oxindoles
Rute C. Nunes, Carlos J. A. Ribeiro, Ângelo Monteiro, Cecília M. P. Rodrigues, Joana
D. Amaral*, Maria M. M. Santos*
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade
de Lisboa. Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal

ACCEPTED MANUSCRIPT
In vitro targeting of colon cancer cells using spiropyrazoline oxindoles
Rute C. Nunes, Carlos J. A. Ribeiro, Ângelo Monteiro, Cecília M. P. Rodrigues, Joana
D. Amaral*, Maria M. M. Santos*
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade
de Lisboa. Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
*Corresponding authors
Maria M. M. Santos/ Joana D. Amaral
Research Institute for Medicines, iMed.ULisboa
Faculty of Pharmacy, Universidade de Lisboa
Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
Phone: (+) 351 21 794 6400; Fax: (+) 351 21 794 6470
E-mail: mariasantos@ff.ulisboa.pt/ jamaral@ff.ulisboa.pt
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Abstract
We report on the synthesis and biological evaluation of a library of twenty-three
spiropyrazoline oxindoles. The antiproliferative activity of the chemical library was
evaluated in HCT-116 p53(+/+) human colon cancer cell line with eight derivatives
displaying good activities (IC50˂15µM). To characterize the molecular mechanisms
involved in compound antitumoral activity, two spiropyrazoline 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. Importantly, cytotoxic effects induced by spiropyrazolines oxindoles
occurred in cancer cells without eliciting cell death in non-malignant CCD-18Co human
colon fibroblasts. Additionally, we demonstrated that the combination of
spiropyrazoline oxindole 2e with sub-toxic concentrations of the chemotherapeutic
agent 5-fluorouracil (5-FU) exerted a synergistic inhibitory effect on HCT-116 colon
cancer cell proliferation. Collectively, our results show the potential of spiropyrazoline
oxindoles for development of novel anticancer agents.
Keywords: spirooxindoles; pyrazoline; cytotoxicity; anticancer agents; colorectal
cancer.
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1. Introduction
Cancer is a multifaceted and multifactorial disease comprised of complex genetic and
epigenetic aberrations that destabilize the normal balance of cell homeostasis [1].
Colorectal cancer (CRC) is among the five most diagnosed cancers and it is also the
main contributor for mortality and health loss [2,3]. The most often used drugs for CRC
include 5-fluorouracil (5-FU), irinotecan, oxaliplatin, and trifluridine combined with
tipiracil. Frequently, two or more of these drugs are combined to achieve greater
efficacy.
Over the years, several studies have shown the importance of the p53 tumor
suppressor protein in cellular homeostasis. This transcription factor is found inactivated
in approximately 50% of all human cancers, either by mutation or deletion of its gene,
while in the other cases the p53 pathway is partially abrogated by inactivation of other
signaling components [4]. The role of p53 is well established in the development of
several types of malignant diseases, such as in CRC [3]. In tumors expressing wild-type
p53, the inactivation of p53 is frequently observed by reversible inhibition, which
results from the overexpression of p53 main negative regulators, MDM2 and MDMX.
Several studies have shown that p53 interaction with MDM2 and MDMX involves three
main hydrophobic amino acids (Phe19, Trp23 and Leu26) in the p53 protein.
The p53 reactivation can be facilitated by inhibition of its negative regulators. In
recent years, several families of compounds have been designed and developed as
modulators of p53, such as spirooxindoles compounds [5,6]. This family of compounds
is frequently reported as a core structure of a variety of medicinal agents and natural
products used in several therapeutic applications, such as in anticancer therapy [7].
However, the development of a p53 reactivating treatment through inhibition of p53–
MDM interaction is only just beginning, with few candidates entering in clinical trials
(e.g. compound SAR405838) [8]. In this regard, the discovery of more potent and
selective small molecule inhibitors of p53–MDM interaction is still an unmet need.
In an attempt to find novel anti-cancer agents, over the last years, we have been
developing novel spirooxindoles with different spiro five-membered rings [7,9–11]
(Figure 1). More specifically, we have developed a family of spiroisoxazoline oxindoles
1, in which the most potent derivative showed an IC50 of 26 µM in HCT-116 human
colon carcinoma cell line [9]. By changing the isoxazoline oxygen to a N-phenyl group,
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spiropyrazoline oxindoles 2 were obtained, presenting good antiproliferative activity in
human breast cancer cell lines [10]. Here, we report on the synthesis of novel
spiropyrazoline oxindole derivatives and the evaluation of biological effects in human
colon carcinoma cells.
Figure 1 - Spiroisoxazoline oxindoles 1 and spiropyrazoline oxindoles 2 chemical structures.
2. Results and discussion
2.1. Synthesis of a library of spiropyrazoline oxindoles and structure-activity
relationship studies (SAR)
Previous studies with spirooxindoles have shown that the presence of a halogen
in the oxindole moiety can sometimes increase the antiproliferative activity and the
selectivity profile in cancer cell lines [9–13]. Therefore, we have synthesized
spiropyrazoline oxindoles with different halogens at the oxindole moiety and different
substituents attached to the pyrazoline ring. To validate the importance of having a
halogen in the oxindole moiety, derivatives lacking this substituent were also
synthesized. Moreover, different substituents in the pyrazoline ring were evaluated,
such as aromatic groups (with both electron-withdrawing and electron-donating groups)
and the alquilic group t-butil. Specifically, spiropyrazoline oxindoles 2a-x were
obtained by 1,3-dipolar cycloaddition reaction between 2-indolinones 3 and
hydrazonoyl chlorides 4 (Scheme 1). Compounds 3a-h were synthesized by aldolic
condensation reaction of substituted 2-indolinones with different aromatic aldehydes in
the presence of piperidine, as reported previously [10,14–17]. Additionally, 2-
indolinone 3e was synthesized by reaction of 5-bromo isatin in the presence of
hydrazine monohydrate and ethylene glycerol, under reflux [18]. The relative
configuration of the final products was established by NMR comparison with other
spiropyrazoline oxindoles described in the literature, with published X-ray
crystallography structure [19,20].
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Scheme 1 – General scheme for the synthesis of spiropyrazoline oxindoles 2a-x.
The antiproliferative potential of spiropyrazoline oxindoles derivatives 2a-x (Figure 2)
was first evaluated in human colon carcinoma HCT-116 cells expressing wild-type p53
(HCT-116 p53(+/+)) to compare the activity of this chemical family with previously
developed spirooxindoles, which presented good activities in this cancer cell line
[11,21]. The results showed that the most active compounds were 2b, 2e, 2g, 2i, 2j, 2l,
2m and 2q (Table 1). The structure-activity relationship studies for this family of
compounds indicated that by changing a tert-butyl by a p-methoxy group at the R3
group (compound 2s versus compound 2l) leads to increased activity in HCT-116 cells.
Changing the phenyl group by a p-chloro phenyl group, at the R4 group, in most cases
(compound 2v versus compound 2o and compound 2d versus compound 2h) resulted in
decreasing activities, with IC50 values higher than 20 µM. Quite interestingly,
spiropyrazoline oxindole derivatives 2 were more active than the corresponding
spiroisoxazoline oxindoles 1 counterparts. In particular, the presence of a N-phenyl
group (compounds 2c, 2f and 2l) in the five-membered ring led to an increase in
potency, representing a more than 2-fold increase compared to the equivalent
spiroisoxazoline oxindoles (compounds 1a, 1b and 1c) (Table 2) [9]. This reassures the
importance of including an extra aryl group to improve the activity of the
spiropyrazoline oxindoles.
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Ph
Ph
Ph
Ph
N
N
N
N
Ph
Ph
Ph
Ph
NPh
NPh
NPh
NPh
Cl
O
O
O
O
N
N
N
N
Cl
H
H
H
H
Cl
2a
2b
2c
2d
Ph
Ph
Ph
Ph
N
Cl
N
N
N
Ph
Ph
Ph
Ph
Cl
N
NPh
NPh
O
NPh
O
Br
F
O
O
N
N
N
N
Br
Cl
H
H
H
H
2e
2f
2g
2h
MeO
MeO
MeO
MeO
MeO
N
N
N
N
N
Ph
Ph
Ph
Cl
Ph
Cl
NPh
O
NPh
O
NPh
O
NPh
NPh
Cl
O
O
N
H
N
N
N
N
Br
Cl
H
H
H
H
2i
2j
2k
2l
2m
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
N
N
N
N
N
N
Ph
Ph
Ph
Ph
Cl
Ph
Ph
NPh
O
NPh
O
NPh
NPh
O
NPh
O
NPh
Cl
Br
O
O
N
H
N
N
N
N
N
Cl
Br
H
H
H
H
H
2p
2n
2o
2q
2r
2s
t-Bu
t-Bu
t-Bu
t-Bu
N
Cl
N
Cl
N
Cl
N
Ph
Ph
Ph
N
N
N
Cl
Cl
NPh
O
Cl
O
O
O
N
H
N
N
N
H
H
H
Cl
2t
2u
2v
2x
Figure 2 - Spirooxindoles screened in this study.
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Table 1 - In vitro antiproliferative activities of spiropyrazoline oxindoles 2a-x.
HCT-116 p53(+/+)
Compound
R1
R2
R3
R4
[a]
IC50, µM
2a
2b
2c
2d
2e
2f
H
5-Cl
6-Cl
7-Cl
5-Br
6-Br
6-Cl, 5-F
7-Cl
H
Ph
Ph
Ph
Ph
Ph
Ph
˃20
11.0±0.3
16.7±0.9
16.6±1.5
13.1±1.0
15.9±0.6
12.4±0.7
˃20
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
2g
2h
2i
Ph
Ph
Ph
Ph
Ph
Ph-o-Cl
Ph
Ph
Ph-p-O-CH3
Ph-p-O-CH3
Ph-p-O-CH3
Ph-p-O-CH3
Ph-p-O-CH3
t-Bu
11.7±0.3
10.6±1.5
15.6±0.9
12.8±0.7
10.9±0.8
˃20
2j
5-Cl
7-Cl
6-Br
6-Cl
H
Ph
Ph
2k
2l
Ph
Ph
Ph
Ph
2m
2n
2o
2p
2q
2r
2s
Ph-m-Cl
Ph
Ph
Ph
5-Cl
6-Cl
7-Cl
5-Br
6-Br
6-Cl
H
Ph
t-Bu
Ph
17.0±1.3
˃20
Ph
t-Bu
Ph
Ph
t-Bu
Ph
13.3±1.4
˃20
Ph
t-Bu
Ph
Ph
t-Bu
Ph
>20
2t
Ph-m-Cl
Ph
t-Bu
Ph
18.4±1.8
˃20
2u
2v
2x
t-Bu
Ph-o-Cl
Ph-o-Cl
Ph-o-Cl
5-Cl
7-Cl
Ph
t-Bu
˃20
Ph
t-Bu
17.3±0.2
[a] IC50 determined by the MTS method after 72 h compound incubation. Each value is the mean (IC50
SD) of three independent experiments performed in duplicate.
±
Table 2 – IC50 values obtained for spiroisoxazoline oxindoles 1 and the corresponding counterparts
spiropyrazoline oxindoles 2.
HCT-116 p53 (+/+)
Compound
R1
R2
R3
R4
IC50, µM
16.7±0.9
39.8±1.1
15.9±0.6
35.0±1.1
12.8±0.7
33.4±1.1
2c
1a
2f
6-Cl
6-Cl
6-Br
6-Br
6-Br
6-Br
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
-
Ph
Ph
Ph
-
1b
2l
Ph
Ph-p-O-CH3
Ph-p-O-CH3
Ph
-
1c
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2.2. 2e and 2m inhibit p53-MDM2 interaction
To better characterize the molecular mechanisms underlying the antiproliferative
activity of this chemical library, we selected two of the active compounds (compounds
2e and 2m) for further analysis. Firstly, we investigated if these spirooxindole
derivatives could inhibit p53 interaction with its inhibitor MDM2 as described for other
five-membered ring spirooxindoles [22,23] (e.g. spirooxindole MI-77301). For that, we
used a simple assay that allows the direct visualization and quantification of p53-
MDM2 interaction in live cells [9,24]. The system relies on a Venus-based bimolecular
fluorescence complementation (BiFC) assay, where p53 and MDM2 are fused to non-
fluorescent fragments of Venus fluorescent reporter protein (V1 and V2). When both
proteins interact, the non-fluorescent halves get close enough to fold together and
reconstitute the functional fluorophore. Fluorescence is therefore proportional to the
amount of p53-MDM2 dimers generated, and can be analyzed by flow cytometry. As
shown in Figure 3, exposure of HCT-116 p53(-/-) cells co-transfected with V1-p53 and
MDM2-V2 to compounds 2e and 2m at 20µM concentration resulted in a mild but
significant decrease of BiFC signal (p < 0.05) corresponding to disruption of the p53-
MDM2 interaction. We obtained similar results when using nutlin-3a at half the
concentration as positive control.
100
DMSO
Nutlin-3a
2e
*
*
*
**
80
60
40
20
0
**
2m
5
5
10 20
5
10 20
5
10 20
DMSO
Concentration (µM)
Figure 3 - Compounds 2e and 2m inhibit p53-MDM2 interaction. HCT-116 p53(–/–) cells were co-
transfected with the V1-p53/MDM2-V2 BiFC constructs for 24 h. Vehicle, nutlin-3a and compounds 2e
and 2m 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 are mean ± SD of three independent
experiments. *p<0.05 and **p<0.01 versus DMSO control.
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Next, the evaluation of the steady-state levels of p53 and MDM2 was made by
(+/+)
Western blot. As depicted in Figure 4, incubation of HCT-116 p53
cells with
compounds 2e and 2m at the IC50 concentration induced slight variations in p53 and
MDM2 total levels, with a tendency to increase p53 and decrease MDM2 levels.
Figure 4 – Effect of compounds 2e and 2m in p53 and MDM2 protein levels. Total proteins were isolated
from HCT-116 p53(+/+) cells following incubation with compounds 2e and 2m at IC50 concentration, or
DMSO (vehicle control), for 72 h. Blots were normalized to endogenous β-actin. Data represent mean ±
SEM of two independent experiments.
To confirm the contribution of p53 activation to compound activity in HCT-116
p53(+/+) cells, 2e and 2m were tested in HCT-116 cells in which p53 has been knocked
out (HCT-116 p53(-/-)). None of the compounds was selective for HCT-116 p53(+/+) over
HCT-116 p53(-/-) cells (2e IC50=14.2±0.8 µM; 2m IC50=11.6±0.8 µM), revealing that
other mechanisms, besides p53-dependent effects, may contribute for compounds
antiproliferative activity. This result was further supported by evaluating the binding of
spiropyrazoline oxindole 2e with MDM2 using a fluorescence polarization (FP)
competitive binding assay. Compound 2e was not able to compete with the fluorescent
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probe molecule, which binds to MDM2 potently, up to 400 µM. This contrasts with the
reported MDM2 inhibitor Nutlin-3a developed by Roche.
2.3. 2e and 2m induce cell death and cell cycle arrest
The levels of general cell death were evaluated by measuring the enzymatic
activity of lactate dehydrogenase (LDH) released from damaged cells. Surprisingly, the
results revealed that incubation of HCT-116 p53(+/+) cells with compounds 2e and 2m at
the IC50 concentration for 72 hours, did not induce the release of LDH (Figure 5).
However, when both compounds were used at twice of the IC50 concentration the LDH
activity was approximately 1.5-fold higher comparing to DMSO controls, suggesting
that membrane permeabilization and leakage of cytosolic cellular contents, such as
LDH, may be a late-stage event in the cell death cascade. In contrast, mitochondria
dysfunction, an organelle partially involved in MTS metabolism, may represent an
initial event.
3
IC50
2xIC50
*
2
**
1
0
DMSO
2e
2m
Figure 5 - Compounds 2e and 2m induce cell death in HCT-116 p53(+/+) cells. General cell death was
evaluated by the LDH release assay following 72 h of incubation with compounds 2e and 2m at equitoxic
(IC50 and 2xIC50) concentration, or DMSO (vehicle control). Data are mean ± SEM of three independent
experiments. *p<0.05, **p<0.01 versus respective DMSO control.
The cell death by apoptosis was assessed by performing a double staining method
with Annexin V-FITC and 7-Aminoactinomycin D (7-AAD) followed by flow
cytometry analysis. As illustrated in Figure 6, exposure of HCT-116 p53 (+/+) cells to
compounds 2e and 2m at the IC50 concentration, resulted in the induction of apoptosis,
as revealed by a 10% increase of cells undergoing early phases of apoptosis. When cells
were exposed to the same compounds at twice of the IC50 concentration, the percentage
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of cells in early apoptosis increased, especially with compound 2m. Moreover, a
massive increase in cells undergoing secondary apoptosis was detected. Since, cell
death by apoptosis appears to contribute only in part to compound cytotoxic effects, the
association between 2e and 2m-induced growth inhibition and the regulation of cell
cycle progression was explored. HCT-116 p53(+/+) cells exposed to compounds 2e and
2m at the respective IC50 concentrations were collected and stained with propidium
iodide (PI) to determine cellular DNA content by flow cytometry. The percentage of
cells in each phase of the cell cycle was analyzed after 24 and 48 hours of compound
incubation. As observed in Figure 7, compound 2m induced a significant accumulation
of cells in the G0/G1 phase after 24 h, with a concomitant decrease in the G2/M phase,
comparing to control (p < 0.01). In respect to the S phase, no significant changes were
observed.
Figure 6 – Compounds 2e and 2m induce apoptosis in HCT-116 p53(+/+). Evaluation of apoptosis by flow
cytometry using Guava Nexin assay, following cell incubation with 2e and 2m at equitoxic concentrations
(IC50 and 2xIC50), or DMSO (vehicle control), for 72 h. Representative flow cytometry profiles (left) and
respective quantification (right). Data are mean ± SD of three independent experiments. **p<0.01;
***p<0.001 versus DMSO control.
When analysing the DNA content after 48 hours of compound exposure (Figure 7),
we observed that treatment with 2e resulted in accumulation of cells in the G0/G1
phase, representing a total of 62% compared to 42% of cells in the control. Compound
2e also had an impact in the G2/M phase with a 3-fold reduction in the percentage of
cells (p < 0.01). Collectively, these data indicate that compounds 2e and 2m induce cell
cycle arrest in G0/G1 in a time-dependent manner, with 2m acting earlier than 2e. The
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arrest of cells in the G0/G1 phase may allow the repair of damaged DNA, and/or inhibit
cell growth [25,26]. These results suggest that both induction of apoptosis and cell cycle
arrest are important mechanisms underlying 2e and 2m cytotoxic effects in HCT-116
p53(+/+) cells.
Figure 7 - Effects of compounds 2e and 2m on cell cycle progression. HCT-116 p53(+/+) were treated with
compounds 2e and 2m at equitoxic (IC50) concentration, or DMSO (vehicle control) for 24 h and 48 h.
Cellular DNA was stained with PI, and flow cytometry analysis was performed to determine cell cycle
distribution. 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 three
independent experiments (top). Results are expressed in the graph bar as means ± SD of three
independent experiments (bottom). **p<0.01 versus DMSO control.
2.4. 2e and 2m are not toxic in human normal colon fibroblasts
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Finally, compound cytotoxicity was evaluated in CCD-18Co fibroblasts, a human
normal colon cell line. Notably, exposure of CCD-18Co cells to compounds 2e and 2m
at IC50 concentration (previously determined for HCT-116 p53(+/+)) did not affect
viability (Figure 8). When compounds were incubated at IC80 concentration, cell
viability decreased in approximately 20%, suggesting that these molecules may serve as
promising molecules for further optimization and development of new anti-tumoral
agents with reduced off-target effects.
IC50
IC80
100
80
60
40
20
0
**
DMSO
2e
2m
Figure 8- Effect of compounds 2e and 2m in a non-tumoral cell line. CCD-18Co human normal colon
fibroblasts were incubated with compounds 2e and 2m at equitoxic (IC50 and IC80) concentrations as
determined for HCT-116 p53(+/+) cells, or vehicle (DMSO), for 72 h. Cell viability was assessed using the
MTS metabolism assay. Data are mean ± SEM of three independent experiments. **p<0.01 versus
DMSO control.
2.5. Synergistic antiproliferative effect of compound 2e and 5-fluorouracil
Drug combination is widely used to achieve treatment efficacy of several types of
tumors. The main goal is to achieve a synergistic therapeutic effect accompanied by a
decrease of toxicity, and diminished drug resistance [27,28]. Taking this into account,
we analysed the potential of compound 2e to increase HCT-116 p53 (+/+) cell sensitivity
to conventional chemotherapeutic 5-fluorouracil. 5-FU acts as a thymidylate synthase
inhibitor (blocking DNA replication) [29,30], while the compounds in test are capable
of inducing apoptosis and cell cycle arrest. First, we determined the IC50 value of 5-FU
in HCT-116 p53 (+/+) cells under the same conditions as we did for compounds in test
(data not shown). After that, cells were exposed to a fixed concentration of 5-FU
combined with increasing concentrations of compound 2e. Interestingly, combination of
5-FU at IC10 concentration with 2e at IC20 concentration resulted in the highest
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synergistic effect (CDI<1) (Figure 9). When compound 2e was used at IC10
concentration, a mild synergism was still observed, but the effect was lost when used at
IC50 concentration (CDI>1) (Figure 9). Overall, the results suggest that combining 5-FU
at IC10 concentration and compound 2e at concentrations below the IC50 lead to better
results and possibly less side effects.
2e IC10
2e IC20
2e IC50
†
100
50
0
1.0
0.8
0.6
0.4
0.2
0.0
*
±
§
±
no addition
5-FU IC10
5-FU IC10
Figure 9 - Coefficient drug interaction (CDI) analysis of compound 2e with 5-fluorouracil (5-FU). HCT-
116 cells were exposed to compounds for 72h and cell viability was evaluated using the MTS metabolism
assay. CDI value threshold = 1. 2e [IC10]=9.5µM; 2e [IC20]=10.7 µM; 2e [IC50]=13.0 µM and 5-FU
[IC10]=1.7 µM. Data are mean ± SEM of five independent experiments. §p<0.005 versus 2e IC10; †p<0.05
versus 2e alone; *p<0.01 versus 2e alone; ±p<0.005 versus respective 2e IC10.
3. Conclusions
A library of twenty-three spiropyrazoline oxindoles was designed and synthesized by
1,3-dipolar cycloaddition reaction. The compounds were screened for the first time in
HCT-116 human colon carcinoma cells expressing wild-type p53 and eight
spiropyrazoline oxindoles (compounds 2b, 2e, 2g, 2i, 2j, 2l, 2m and 2q) displayed
activities bellow 15 µM. From these, two derivatives were selected to further
characterize the molecular mechanisms involved in compound cytotoxicity.
Importantly, a dose dependent increase of general cell death and apoptosis was found in
HCT-116 cells after treatment with compounds 2e and 2m. These compounds also
induced time-dependent arrest of cells in G0/G1 phase of cell cycle. Interestingly,
compounds 2e and 2m showed to partially inhibit p53-MDM2 protein-protein
interaction in live cells using a bimolecular fluorescence complementation (BiFC)
assay. However, evaluation of these compounds in HCT-116 cells lacking p53 revealed
that spiropyrazoline oxindoles activity also rely on other pathways independent from
p53 and MDM2. Finally, compounds 2e and 2m were non-cytotoxic to human normal
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colon fibroblasts for the tested concentrations. Interestingly, combined therapy studies
demonstrated
a
synergistic effect of spiropyrazoline oxindoles with the
chemotherapeutic agent 5-FU, which further highlights the potential of this chemical
family as anticancer agents.
4. Experimental Section
4.1. Chemistry: General conditions
All chemical and solvents were obtained from commercial suppliers and were used
without further purification. When used as reaction solvents, CH2Cl2 was dried over
CaCl2 and distilled; THF was distilled from sodiumbenzophenone system. Et3N was
dried over KOH, distilled and stored with molecular sieves. Compounds 2a-2f, 2j-2l,
2p, 2q and 2s were synthesized according to the method described in the literature [10].
Compounds 4b, 4d, 4e were synthesized according to the method described in the
literature [31] and compounds 4a, 4c were synthesized as described in the literature
[32,33].
Thin layer chromatography was performed using Merck Silica Gel 60 F254
aluminium plates and visualized by UV light. Flash column chromatography was
performed on Merck Silica Gel (200-400 mesh ASTM). Preparative TLC was
1
13
performed on Merck Silica Gel 60 GF254 over glass plates. H and C NMR spectra
were recorded on a Bruker 400 Ultra-Shield at 400 MHz (1H NMR) and 101 MHz (13C
NMR) or on a Bruker 300 Ultra-Shield at 300 MHz (1H NMR) and 75 MHz (13C NMR).
1H and 13C chemical shifts (δ) are expressed in parts per million (ppm) using the solvent
1
as internal reference, and proton coupling constants (J) in hertz (Hz). H spectral data
are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; dd, doublet of doublets; dt, doublet of triplets, and br, broadened),
coupling constant, and integration. The infrared spectra were collected on a Shimadzu
FTIR Affinity-1 spectrophotometer. The spectra were determined using KBr disks.
Only the most significant absorption bands are reported. All compounds tested showed
appropriate purity by elemental analysis (C, H, and N), performed in a Flash 2000
CHNS-O analyzer (ThermoScientific, UK) at Liquid Chromatography and Mass
Spectrometry Laboratory, Faculty of Pharmacy of Universidade de Lisboa.
15

ACCEPTED MANUSCRIPT
4.1.1. General Procedure for the Synthesis of 2-Indolinones
A reaction mixture of the proper substituted indolin-2-ones 6, aldehyde (1.2 eq.), and
piperidine (0.1 eq.) in ethanol (1 mL/0.5 mmol oxindole) was refluxed for 3-5 h, under
nitrogen atmosphere. After, the mixture was cooled and put on ice, where the product
precipitated. The product was filtered in vacuum with cold ethanol [10,14–17]. Finally,
the product was placed in the vacuum desiccators to give the expected compound. The
indolin-2-ones 3e was generated through substituted isatin in the presence of hydrazine
monohydrate and ethylene glycerol, under reflux [18].
4.1.2. General Procedure for the Synthesis of 2',4'-Dihydrospiro[Indoline-3,3'-
[Pyrazol]-2-ones(2)
Triethylamine/N,N-Diisopropylethylamine was added dropwise to a mixture of
indolin-2-one 3 derivative and hydrazonyl chloride 4 derivative in dry CH2Cl2 under
nitrogen atmosphere. The reaction mixture was quenched with water (H2O). The phases
were separated and the aqueous phase was extracted with Ethyl Acetate (3xEtOAc). The
combined organic extracts were dried over anhydrous Na2SO4 and the product was dried
on the rotary evaporator. The residue was purified by flash chromatography on silica to
afford the final spiropyrazoline oxindole compound 2 [10].
6-chloro-5-fluoro-2',4',5'-triphenyl-2',4'-dihydrospiro[indoline-3,3'-pyrazol]-2-one
(2g). Following the general procedure, to a solution of 3g (0.0175 g, 0.06 mmol, 1 eq)
in CH2Cl2 (2 ml) was added 4a (0.04 g, 0.16 mmol, 2.5 eq) and triethylamine (0.023 ml,
0.15 mmol, 2.5 eq). Reaction time: 46 h. The compound was purified by flash
chromatography (eluent: EtOAc/n-Hexane 1:3) to afford compound 2g as a white solid
1
(0.01 g, 0.03 mmol, 40.0 %); IR (KBr): 3419 (NH), 1730 (C=O), 1597 (C=N) cm-1; H
NMR (300 MHz, CDCl3) δ 8.03 (s, 1H, NH), 7.70-7.60 (m, 2H, ArH), 7.35 – 7.05 (m,
8H, ArH), 7.00 – 6.80 (m, 6H, ArH), 6.17 (d, J = 8.0 Hz, 1H, ArH), 5.14 (s, 1H, H-5´);
13C NMR (75 MHz, CDCl3) δ 178.47 (C=O), 154.21 (d, J = 243.0 Hz), 149.23 (C=N),
144.07 (Cq), 136.44 (Cq), 133.97 (CH), 131.37 (Cq), 129.26 (CH), 129.16 (CH), 129.06
(CH), 128.69 (CH), 128.62 (CH), 126.99 (CH), 125.80 (d, J = 7.2 Hz), 122.09 (d, J =
19.5 Hz), 121.69 (CH), 115.48 (CH), 115.17 (d, J = 25.5 Hz), 112.52 (CH), 77.00
16

ACCEPTED MANUSCRIPT
(Cspiro), 62.81 (CH-5’); LC-MS m/z calcd for C28H19ClFN3O: 467.1, Found: 468.3
[M+H]+.
7-chloro-2'-(2-chlorophenyl)-4',5'-diphenyl-2',4'-dihydrospiro[indoline-3,3'-
pyrazol]-2-one (2h). Following the general procedure, to a solution of 3d (0.05 g, 0.20
mmol, 1 eq) in CH2Cl2 (1.5 ml) was added 4b (0.08 g, 0.29mmol, 1.5 eq) and N,N-
Diisopropylethylamine (0.10 mL, 0.59 mmol, 3 eq.). Reaction time: 23 h. The
compound was purified by flash chromatography (eluent: EtOAc/n-Hexane 1:4) to
afford compound 2h as a white solid (0.09 g, 0.18 mmol, 93.9 %); IR (KBr): 3412
(NH), 1720 (C=O), 1620 (C=N) cm-1; 1H NMR (300 MHz, CDCl3) δ 7.79 (s, 1H, NH),
7.71 – 7.63 (m, 2H, ArH), 7.44 (dd, J = 8.4, 1.3 Hz, 1H, ArH), 7.30-7.25 (m, 3H, ArH),
7.23 – 7.13 (m, 5H, ArH), 7.10 – 6.95 (m, 4H, ArH), 6.34 (t, J = 7.9 Hz, 1H, ArH), 5.89
(d, J = 7.5 Hz, 1H, ArH), 5.07 (s, 1H, H-5´); 13C NMR (75 MHz, CDCl3) δ 177.61
(C=O), 151.84 (C=N), 141.18 (Cq), 138.50 (Cq), 134.80 (Cq), 131.51 (Cq), 130.37
(CH), 129.72 (Cq), 129.22 (CH), 129.11 (CH), 128.95 (CH), 128.55 (CH), 128.24 (CH),
127.27 (CH), 127.12 (CH), 126.55 (CH), 126.00 (CH), 125.88 (CH), 125.50 (Cq),
122.42 (CH), 114.65 (Cq), 78.98 (Cspiro), 62.59 (CH-5’); Anal. Calc. for
C28H19Cl2N3O: C 69.42, H 3.96, N 8.68, Found: C 69.04, H 3.78, N 9.30.
5'-(4-methoxyphenyl)-2',4'-diphenyl-2',4'-dihydrospiro[indoline-3,3'-pyrazol]-2-
one (2i). Following the general procedure, to a solution of 3a (0.075 g, 0.34 mmol, 1
eq) in CH2Cl2 (2 ml) was added 4c (0.13 g, 0.51mmol, 1.5 eq) and triethylamine (0.14
ml, 1.02 mmol, 3 eq). Reaction time: 17 h. The compound was purified by flash
chromatography (eluent: EtOAc/n-Hexane 1:3) to afford compound 2i as a white solid
1
(0.1 g, 0.3 mmol, 73.5 %); IR (KBr): 3362 (NH), 1741 (C=O), 1600 (C=N) cm-1; H
NMR (400 MHz, CDCl3) δ 8.63 (s, 1H, NH), 7.62 (d, J = 8.0 Hz, 2H, ArH), 7.16 – 7.00
(m, 6H, ArH), 7.00-6.90 (m, 4H, ArH), 6.85-6.78 (m, 3H, ArH), 6.74 (d, J = 8.0 Hz,
1H, ArH), 6.57 (t, J = 8.0 Hz, 1H, ArH), 6.36 (d, J = 8.0 Hz, 1H, ArH), 5.13 (s, 1H, H-
13
4´), 3.78 (s, 3H, CH3); C NMR (101 MHz, CDCl3) δ 178.52 (C=O), 160.08 (C=N),
148.81 (Cq), 144.48 (Cq), 139.89 (Cq), 134.75 (Cq), 129.31 (CH), 129.21 (CH), 128.91
(CH), 128.53 (CH), 128.36 (CH), 127.88 (CH), 126.47 (CH), 125.72 (Cq), 124.41 (Cq),
122.36 (CH), 120.78 (CH), 115.25 (CH), 113.86 (CH), 110.44 (CH), 77.23 (Cspiro),
62.78 (CH-4’), 55.26 (O-CH3); Anal. Calc. for C29H23N3O2: C 78.18, H 5.20, N 9.43,
Found: C 77.96, H 5.19, N 9.34.
17

ACCEPTED MANUSCRIPT
6-chloro-4'-(3-chlorophenyl)-5'-(4-methoxyphenyl)-2'-phenyl-2',4'-
dihydrospiro[indoline-3,3'-pyrazol]-2-one (2m). Following the general procedure, to a
solution of 3h (0.075 g, 0.26 mmol, 1 eq) in CH2Cl2 (2 ml) was added 4c (0.14 g, 0.52
mmol, 2 eq) and triethylamine (0.11 ml, 0.78 mmol, 3 eq). Reaction time: 22 h. The
compound was purified by flash chromatography (eluent: EtOAc/n-Hexane 1:3) and
then recrystallized in diethyl ether to afford compound 2m as a white solid (0.13 g, 0.25
1
mmol, 97.8 %); IR (KBr): 3406 (NH), 1703 (C=O), 1597 (C=N) cm-1; H NMR (400
MHz, CDCl3) δ 8.81 (s, 1H, NH), 7.59 (d, J = 8.8 Hz, 2H, ArH), 7.17 (d, J = 8.0 Hz,
1H, ArH), 7.11 (t, J = 8.0 Hz, 3H, ArH), 6.97 (s, 1H, ArH), 6.90 (d, J = 8.0 Hz, 2H,
ArH), 6.87-6.80 (m, 4H, ArH), 6.78 (d, J = 1.5 Hz, 1H, ArH), 6.62 (dd, J = 8.0, 1.7 Hz,
13
1H, ArH), 6.28 (d, J = 8.0 Hz, 1H, ArH), 5.02 (s, 1H, H-4´), 3.79 (s, 3H, CH3); C
NMR (101 MHz, CDCl3) δ 178.29 (C=O), 160.49 (C=N), 148.59 (Cq), 144.34 (Cq),
141.13 (Cq), 136.73 (Cq), 135.65 (Cq), 134.87 (Cq), 130.18 (CH), 129.18 (CH), 128.58
(CH), 128.46 (CH), 127.48 (Cq), 127.29 (CH), 123.88 (Cq), 122.77 (CH), 121.51 (CH),
115.59 (CH), 114.17 (CH), 111.62 (CH), 76.66 (Cspiro), 62.20 (CH-4´), 55.43 (O-
CH3); Anal. Calcd for C29H21Cl2N3O2: C 67.71, H 4.11, N 8.17, Found: C 67.19, H
4.15, N 7.98.
5'-(tert-butyl)-2',4'-diphenyl-2',4'-dihydrospiro[indoline-3,3'-pyrazol]-2-one (2n).
Following the general procedure, to a solution of 3a (0.05 g, 0.26 mmol, 1 eq) in
CH2Cl2 (2 ml) was added 4d (0.1 g, 0.47 mmol, 2 eq) and triethylamine (0.11 ml, 0.78
mmol, 3 eq). Reaction time: 5h30min. The compound was purified by flash
chromatography (eluent: EtOAc/n-Hexane 1:3) and then recrystallized in
dichloromethane:n-hexane to afford compound 2n as a white solid (0.07 g, 0.16 mmol,
1
72.7 %); IR (KBr): 3236 (NH), 1709 (C=O), 1599 (C=N) cm-1; H NMR (300 MHz,
CDCl3) δ 7.65 (s, 1H, NH), 7.35 (m, 2H, ArH), 7.16 -6.99 (m, 5H, ArH), 6.83 (dd, J =
8.8, 1.0 Hz, 3H, ArH), 6.80 – 6.74 (m, 2H, ArH), 6.57 (td, J = 7.6, 1.0 Hz, 1H, ArH),
6.24 (d, J = 7.2 Hz, 1H, ArH), 4.46 (s, 1H, H-4´), 1.19 (s, 9H, C(CH3)3); 13C NMR (75
MHz, CDCl3) δ 178.37 (C=O), 161.70 (C=N), 145.79 (Cq), 140.40 (Cq), 135.29 (Cq),
129.28 (CH), 128.90 (CH), 128.12 (CH), 126.41 (CH), 125.74 (Cq), 122.24 (CH),
120.92 (CH), 115.96 (CH), 110.46 (CH), 77.28 (Cspiro), 62.32 (CH-4’), 34.87
(C(CH3)3), 29.49 (C(CH3)3); Anal. Calc. for C26H25N3O.0.05H2O: C 78.77, H 6.40, N
10.60, Found: C 77.70, H 6.47, N 10.18.
18

ACCEPTED MANUSCRIPT
5'-(tert-butyl)-5-chloro-2',4'-diphenyl-2',4'-dihydrospiro[indoline-3,3'-pyrazol]-2-
one (2o). Following the general procedure, to a solution of 3b (0.05 g, 0.23 mmol, 1 eq)
in CH2Cl2 (2 ml) was added 4d (0.09 g, 0.47 mmol, 2 eq) and triethylamine (0.09 ml,
0.68 mmol, 3 eq). Reaction time: 4h50min. The compound was purified by flash
chromatography (eluent: EtOAc/n-Hexane 1:2) to afford compound 2o as a white solid
1
(0.08 g, 0.19 mmol, 80.2 %); IR (KBr): 3254 (NH), 1718 (C=O), 1691 (C=N) cm-1; H
NMR (300 MHz, CDCl3) δ 7.62 (s, 1H, NH), 7.47 – 7.34 (m, 2H, ArH), 7.31 (d, J = 7.8
Hz, 1H, ArH), 7.20 (m, 1H, ArH), 7.12 – 7.02 (m, 3H, ArH), 6.85-6.78 (m, 4H, ArH),
6.69 (d, J = 8.3 Hz, 1H, ArH), 6.15 (d, J = 2.1 Hz, 1H, ArH), 4.46 (s, 1H, H-4´), 1.19 (s,
9H, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ 177.60 (C=O), 161.51 (C=N), 145.42
(Cq), 138.52 (Cq), 134.53 (Cq), 129.09 (CH), 128.87 (CH), 128.39 (CH), 127.71 (Cq),
127.46 (Cq), 126.64 (CH), 121.13 (CH), 115.87 (CH), 111.06 (CH), 77.16 (Cspiro),
62.39 (CH-4´), 34.74 (C(CH3)3), 29.33 (C(CH3)3); Anal. Calc. for
C26H24ClN3O.0.05H2O: C 72.45, H 5.67, N 9.68, Found: C 72.47, H 5.74, N 9.52.
5-bromo-5'-(tert-butyl)-2',4'-diphenyl-2',4'-dihydrospiro[indoline-3,3'-pyrazol]-2-
one (2r). Following the general procedure, to a solution of 3e (0.05 g, 0.18 mmol, 1 eq)
in CH2Cl2 (2 ml) was added 4d (0.09 g, 0.43 mmol, 2.5 eq) and triethylamine (0.06 ml,
0.43 mmol, 2.5 eq). Reaction time: 5h30min. The compound was purified by flash
chromatography (eluent: EtOAc/n-Hexane 1:1) to afford compound 2r as a white solid
1
(0.03 g, 0.06 mmol, 41.5 %); IR (KBr): 3254 (NH), 1726 (C=O), 1695 (C=N) cm-1; H
NMR (300 MHz, CDCl3) δ 7.69 (s, 1H, NH), 7.48-7.27 (m, 3H, ArH), 7.23 (dd, J = 9.0,
2.0 Hz, 2H, ArH), 7.10-7.02 (m, 2H, ArH), 6.86 – 6.80 (m, 3H, ArH), 6.64 (d, J = 8.3
Hz, 2H, ArH), 6.27 (d, J = 1.9 Hz, 1H, ArH), 4.45 (s, 1H, H-4´), 1.19 (s, 9H, C(CH3)3);
13C NMR (75 MHz, CDCl3) δ 177.17 (C=O), 161.68 (C=N), 145.62 (Cq), 139.06 (Cq),
134.71 (Cq), 132.11 (CH), 129.73 (CH), 129.04 (CH), 128.59 (CH), 127.97 (Cq),
121.42 (CH), 116.21 (CH), 115.20 (Cq), 111.45 (CH), 77.36 (Cspiro), 62.63 (CH-4´),
34.91 (C(CH3)3), 29.50 (C(CH3)3); LC-MS m/z calcd for C26H24BrN3O: 475.1, Found:
476.3 [M+H]+.
5'-(tert-butyl)-6-chloro-4'-(3-chlorophenyl)-2'-phenyl-2',4'-dihydrospiro[indoline-
3,3'-pyrazol]-2-one (2t). Following the general procedure, to a solution of 3h (0.075 g,
0.26 mmol, 1 eq) in CH2Cl2 (2 ml) was added 4d (0.11 g, 0.52 mmol, 2 eq) and
triethylamine (0.11 ml, 0.78 mmol, 3 eq). Reaction time: 18h30min. The compound was
purified by flash chromatography (eluent: EtOAc/n-Hexane 1:3) and then recrystallized
19

ACCEPTED MANUSCRIPT
in diethyl ether to afford compound 2t as a white solid (0.10 g, 0.23 mmol, 82.7 %); IR
1
(KBr): 3419 (NH), 1720 (C=O), 1616 (C=N) cm-1, H NMR (400 MHz, CDCl3) δ 8.40
(s, 1H, NH), 7.31 (s, 1H, ArH), 7.28 – 7.20 (m, 2H, ArH), 7.07 (t, J = 8.0 Hz, 2H, ArH),
6.90-6.75 (m, 4H, ArH), 6.73 – 6.50 (m, 2H, ArH), 6.18 (s, 1H, ArH), 4.40 (s, 1H, H-
4´), 1.19 (s, 9H, C(CH3)3); 13C NMR (101 MHz, CDCl3) δ 177.60 (C=O), 161.34
(C=N), 145.48 (Cq), 141.42 (Cq), 137.17 (Cq), 135.52 (Cq), 134.81 (Cq), 129.72 (CH),
129.04 (CH), 128.62 (CH), 127.27 (CH), 123.88 (Cq), 122.62 (CH), 121.67 (CH),
116.41 (CH), 111.30 (CH), 76.97 (Cspiro), 61.75 (CH-4´), 34.96 (C(CH3)3), 29.47
(C(CH3)3); Anal. Calcd for C26H23Cl2N3O.0.05H2O: C 67.25, H 4.99, N 9.05, Found: C
66.61, H 5.14, N 8.82.
5'-(tert-butyl)-2'-(2-chlorophenyl)-4'-phenyl-2',4'-dihydrospiro[indoline-3,3'-
pyrazol]-2-one (2u). Following the general procedure, to a solution of 3a (0.05 g, 0.26
mmol, 1 eq) in CH2Cl2 (2 ml) was added 4e (0.08 g, 0.33 mmol, 1.5 eq) and
triethylamine (0.115 mL, 0.083 mmol, 3 eq.). Reaction time: 26h50min. The compound
was purified by flash chromatography (eluent: EtOAc/n-Hexane 1:4) to afford
compound 2u as a white solid (0.06 g, 0.14 mmol, 61.8 %); IR (KBr): 3419 (NH), 1716
1
(C=O), 1618 (C=N) cm-1; H NMR (300 MHz, CDCl3) δ 7.91 (s, 1H, NH), 7.33 – 7.20
(m, 4H, ArH), 7.18-7.05 (m, 3H, ArH), 6.99 (t, J = 7.8 Hz, 1H, ArH), 6.93 (t, J = 7.8
Hz, 1H, ArH), 6.67 (d, J = 7.8 Hz, 1H, ArH), 6.38 (t, J = 7.8 Hz, 1H, ArH), 5.89 (d, J =
7.6 Hz, 1H, ArH), 4.40 (s, 1H, H-4´), 1.20 (s, 9H, C(CH3)3); 13C NMR (75 MHz,
CDCl3) δ 178.55 (C=O), 164.14 (C=N), 141.65 (Cq), 140.88 (Cq), 135.54 (Cq), 130.27
(CH), 129.85 (Cq), 129.17 (CH), 128.06 (CH), 127.60 (CH), 127.03 (CH), 125.72 (CH),
124.79 (CH), 123.70 (Cq), 121.52 (CH), 109.49 (CH), 77.95 (Cspiro), 61.79 (CH-4´),
34.93 (C(CH3)3), 29.51 (C(CH3)3); LC-MS m/z calcd for C26H23ClN3O: 429.2, Found:
430.3 [M+H]+.
5'-(tert-butyl)-5-chloro-2'-(2-chlorophenyl)-4'-phenyl-2',4'-dihydrospiro[indoline-
3,3'-pyrazol]-2-one (2v). Following the general procedure, to a solution of 3b (0.05 g,
0.02 mmol, 1 eq) in CH2Cl2 (2 ml) was added 4e (0.12 g, 0.41 mmol, 3 eq) and N,N-
Diisopropylethylamine (0.118 mL, 0.068 mmol, 3 eq.). Reaction time: 21 h. The
compound was purified by flash chromatography (eluent: EtOAc/n-Hexane 1:4) to
afford compound 2v as a white solid (0.03 g, 0.05 mmol, 27.5 %); IR (KBr): 3383
(NH), 1722 (C=O), 1620 (C=N) cm-1; 1H NMR (300 MHz, CDCl3) δ 8.56 (s, 1H, NH),
7.57 – 7.38 (m, 2H, ArH), 7.33 – 7.22 (m, 3H, ArH), 7.20 – 7.06 (m, 3H, ArH), 6.95 (d,
20

ACCEPTED MANUSCRIPT
J = 5.5 Hz, 2H, ArH), 6.61 (d, J = 7.1 Hz, 1H, ArH), 5.78 (s, 1H, ArH), 4.39 (s, 1H, H-
4´), 1.20 (s, 9H, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ 178.34 (C=O), 164.16 (C=N),
141.42 (Cq), 139.48 (Cq), 134.91 (Cq), 130.39 (CH), 129.81 (Cq), 129.10 (CH), 128.42
(CH), 127.91 (CH), 127.16 (CH), 126.93 (CH), 126.11 (CH), 125.45 (Cq), 124.95 (CH),
110.47 (CH), 78.18 (Cspiro), 61.93 (CH-4´), 34.94 (C(CH3)3), 29.48 (C(CH3)3); LC-MS
m/z calcd for C26H23Cl2N3O: 463.1, Found: 464.3 [M+H]+.
5'-(tert-butyl)-7-chloro-2'-(2-chlorophenyl)-4'-phenyl-2',4'-dihydrospiro[indoline-
3,3'-pyrazol]-2-one (2x). Following the general procedure, to a solution of 3d (0.07 g,
0.27 mmol, 1 eq) in CH2Cl2 (2 ml) was added 4e (0.11 g, 0.45 mmol, 1.5 eq) and N,N-
Diisopropylethylamine (0.13 mL, 0.82 mmol, 3 eq.). Reaction time: 22h20min. The
compound was purified by flash chromatography (eluent: EtOAc/n-Hexane 1:4) to
afford compound 2x as a white solid (0.09 g, 0.2 mmol, 70.8 %); IR (KBr): 3407 (NH),
1
1712 (C=O), 1620 (C=N) cm-1; H NMR (300 MHz, CDCl3) δ 7.51 (s, 1H, NH), 7.35
(dd, J = 8.6, 1.6 Hz, 1H, ArH), 7.30-7.22 (m, 3H, ArH), 7.20 – 7.10 (m, 3H, ArH), 7.01
– 6.93 (m, 2H, ArH), 6.68 (s, 1H, ArH), 6.40 – 6.29 (m, 1H, ArH), 5.83 (d, J = 7.6 Hz,
1H, ArH), 4.42 (s, 1H, H-4´), 1.19 (s, 9H, C(CH3)3); 13C NMR (75 MHz, CDCl3) δ
177.63 (C=O), 164.32 (C=N), 141.50 (Cq), 138.70 (Cq), 135.14 (Cq), 130.26 (CH),
130.06 (Cq), 129.14 (CH), 128.24 (CH), 127.19 (CH), 126.22 (CH), 125.94 (CH),
125.53 (CH), 125.18 (Cq), 122.22 (CH), 114.54 (Cq), 78.97 (Cspiro), 61.97 (CH-4´),
34.92 (C(CH3)3), 29.46 (C(CH3)3); Anal. Calc. for C26H23Cl2N3O.0.05H2O: C 67.24, H
5.00, N 9.05, Found: C 69.62, H 5.12, N 9.19.
4.2. Biological Effects: General Conditions
4.2.1 Cell Culture
HCT-116 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, St Louis, MO, USA). CCD-
18Co cells were grown in DMEM culture medium supplemented with 10% FBS
(Gibco), 1% penicillin/streptomycin solution (Sigma-Aldrich), 1% Glutamax
(Gibco), 1% Non-Essential Amino Acids Solution (NEAA) (Gibco) and 0.025%
TNF-α Human. Cultures were maintained at 37ºC in a humidified atmosphere of
5% CO2. HCT-116 cells were seeded in 96-well plates at 1x104 cells/well for
21

ACCEPTED MANUSCRIPT
dose-response curves, cell viability, and cell death assays. Additionally, HCT-116
p53(+/+) cells were seeded in 60-mm dishes at 8x105 cells/dish for Western blot
analysis, in 24-well plates at 5x104 cells/well for evaluation of apoptosis by flow
cytometry, and in 6-well plates at 1.5×105 cells/well for cell cycle analysis.
Finally, CCD-18Co colon fibroblasts cells were seeded in 96-well plates at 4x103
cells/well for cell viability assays.
4.2.2. Cell Treatment
Stock solutions of the spiropyrazoline oxindoles 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 mentioned time. To plot dose-response
curves, cells were exposed to 0.5-50 µM test compounds. All experiments were
performed in parallel with DMSO vehicle control. The final DMSO concentration did
not exceed 0.8% (v/v).
4.2.3. Viability assays
Cell viability was assessed 72 h after compound incubation using the
CellTiter96® AQueous Non-Radioactive Cell Proliferation Assay (Promega
Corporation, Madison, WI, USA), according to the manufacturer’s instructions.
This colorimetric assay is based on the bio-reduction of 3-(4,5-dimethylthiazo-2-
yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt
(MTS) to formazan by dehydrogenase enzymes found within metabolically active
cells. The amount of water soluble formazan product can be measured by the
amount of 490 nm absorbance, correlating with the number of living cells in
culture. For this purpose, changes in absorbance were assessed using a GloMax®
Multi Detection System (Sunnyvale, CA, USA). For dose-response experiments,
best-fit IC50 and IC80 values from at least three independent experiments were
calculated using GraphPad Prism software (version 5.00; San Diego, CA, USA),
using the log (inhibitor) vs response (variable slope) function.
4.2.4. Venus-based bimolecular fluorescence complementation (BiFC) assay
To evaluate p53-MDM2 protein-protein interaction, HCT-116 p53(-/-) cells were co-
transfected using 1 µg of each BiFC pair plasmid (V1-p53/MDM2-V2) and
Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions. Four to six
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h after transfection, the medium was replaced with fresh medium, and the compounds in
test were added to a final concentration of 5, 10 and 20 µM for additional 20 h. Nutlin-
3a at the same concentrations was used as positive control. Equal amounts of vehicle
(DMSO) were used as control. Cells were washed twice with Ca2+ and Mg2+ free PBS
(Invitrogen Corp.), treated with accutase and harvested with culture medium. Cell
suspensions were centrifuged, supernatants discarded, and cell pellets resuspended in
PBS. Fluorescence was measured using a Guava easyCyte™ Flow Cytometer (Merck
Millipore) [9,24].
4.2.5. Total protein extraction
HCT-116 p53(+/+) cells were exposed to the compounds in test at the IC50 and 2xIC50
concentration, or vehicle control (DMSO), for 72 h. After that, floating and adherent
cells were collected directly in nonyl phenoxypolyethoxylethanol (NP-40) lysis buffer
(1% NP-40, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% Glycerol, 1
mM dithiothreitol (DTT), and 1X proteases and phosphatases inhibitors), followed by
sonication and centrifugation at 3200 g for 10 min at 4°C. Total protein extracts were
recovered and stored at - 80 ºC.
Protein concentration was determined by the colorimetric Bradford method using the
Bio-Rad Protein Assay reagent (Bio-Rad), according to the manufacturer’s instructions.
BSA (Sigma-Aldrich) was used as standard, and absorbance measurements were
performed at 595 nm using GloMax-Multi+ Detection System (Promega).
4.2.6. Western blot analysis and densitometric analysis
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 electrophoresis (PAGE) 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 hour. Membranes were then incubated
overnight at 4⁰C with a mouse monoclonal anti-p53
(Pab-240, sc-99, 1:200) and anti-
Mdm2 SMP-14, sc-965, 1:200), (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or
(
a rabbit polyclonal anti-β-actin (AC-15; 1:8000; Sigma-Aldrich). Membranes were then
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washed three times with TBS containing 0.2% Tween 20 (TBS-T), and incubated with
anti-rabbit or anti-mouse secondary antibodies conjugated with horseradish peroxidase
(Bio-Rad) for 2 h at room temperature. After rinsing three times with TBS-T, the
immunoreactive complexes were visualized by chemiluminescence with ImmobilonTM
Western (Millipore) or SuperSignal West Femto substrate (Thermo Fisher Scientific,
Inc.). Ponceau was used as loading control. Densitometric analysis was performed with
the Image Lab software Version 5.1 Beta (Bio-Rad).
4.2.7. Competitive FP binding assay
The binding affinity of spiropyrazoline oxindole was evaluated by a competitive
fluorescence polarization-based (FP-based) binding assay, using a recombinant human
His-tagged MDM2 protein (residues 1-118) and a FAM tagged p53-based peptide as the
fluorescent probe. The design of the fluorescent probe was based upon a previously
reported high affinity p53-based peptidomimetic compound (5-FAM-βAla-βAla-Phe-
Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2) [34]. The affinity of this probe
compound to MDM2 was determined to be 1.4 ± 0.3 nM by preliminary protein
saturation experiments. Mixtures of 5 µl of the tested compound with different
concentrations in DMSO and 120 µl of preincubated protein/fluorescent probe complex
with fixed concentrations in the assay buffer (100mM potassium phosphate, pH 7.5, 100
µg/ml bovine γ-globulin, 0.02% sodium azide, with 0.01% Triton X-100) were added
into assay plates and incubated at room temperature for 30 minutes with gentle shaking.
Final concentrations of the protein and fluorescent probe in the competitive assays were
10 and 1 nM, respectively, and final DMSO concentration was 4%. Negative controls
containing protein/fluorescent probe complex only (equivalent to 0% inhibition), and
positive controls containing free fluorescent probe only (equivalent to 100% inhibition),
were included in each assay plate and utilized to calculate inhibition rates. Fluorescence
polarization (mP) values were measured using the Infinite M-1000 plate reader (Tecan
U.S., Research Triangle Park, NC) in Microfluor 1 96-well, black, round-bottom plates
(Thermo Scientific) at an excitation wavelength of 485 nm and an emission wavelength
of 530 nm.
4.2.8. General cell death assay
General cell death was evaluated using the lactate dehydrogenase (LDH)
Cytotoxicity Detection KitPLUS (Roche Diagnostics GmbH, Manheim, Germany),
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according to the manufacturer’s instructions. The LDH assay measures membrane
integrity as a function of the amount of cytoplasmic LDH released into the medium that
can be quantified by a coupled enzymatic reaction. In the first step, LDH catalyzes the
conversion of lactate to pyruvate via reduction of NAD+ to NADH. In the second step,
diaphorase uses NADH to reduce a tetrazolium salt (INT) to a red formazan product.
Thus, the level of formazan is directly proportional to the amount of LDH released,
which is indicative of cytotoxicity. Hence, 50 µL of supernatant from treated cells was
transferred into a new 96-well plate and incubated with 50 µL of assay substrate for 10
to 30 min, at room temperature, protected from light. Absorbance readings were
measured at 490 nm, with 620 nm reference wavelength using a Bio-Rad Model 680
microplate reader. The percentage of LDH release was normalized with DMSO
(control).
4.2.9. 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(+/+) cells
were exposed to: 1) compounds in test at IC50 concentration; 2) compounds in test at
2xIC50 concentration, and 3) DMSO (control), 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 minutes at 4ºC. The cell pellet was resuspended in PBS/2% FBS.
Subsequently, 50 µL of cell suspension were mixed with 50 µL of Guava Nexin reagent
and incubated for 20 minutes, 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 easyCyteTM Flow Cytometer (Merck
Millipore) and Nexin software module.
4.2.10. Cell cycle distribution analysis
The effects of compounds on cell cycle progression were determined using a
standard propidium iodide staining procedure followed by flow cytometry analysis.
Propidium iodide is a fluorescent intercalating agent that has high affinity to nucleic
acids. HCT-116 p53(+/+) cells were treated with the compounds in test at the IC50
concentration, or DMSO, for 24 or 48 h. After that, cells were detached with Tryple
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reagent and collected by centrifugation at 800 g 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 850g for 5 minutes,
at 4˚C, and cell pellets were resuspended in 25µg/mL propidium iodide (PI) (Fluka,
Sigma-Aldrich) and 50µg/mL RNase A (Sigma-Aldrich) and further incubated for 30
min. Sample acquisition and data analysis were performed using the Guava easyCyteTM
Flow Cytometer (Merck Millipore) and Guava analysis software, with the acquisition of
at least 10000 events per sample.
4.2.11. Combined Therapy Strategies
5-Fluorouracil was kindly provided by the hospital pharmacy of Hospital Santa
Maria in Lisboa. HCT-116 p53(+/+) cells were treated with compound at IC10, IC20 and
IC50 concentrations of 2e and with the same concentrations of 5-Fluorouracil. The effect
of combined treatments on cell growth was analyzed after 72 h incubation, using the
MTS assay. Data were analyzed using Excel and GraphPad PRISM software using the
following equation, where CDI is coefficient drug interaction: AB is the cell viability
ratio of combinatory strategy; A is the cell viability ratio of single agent A; and B is the
cell viability ratio of single agent B. Coefficient drug interaction (CDI) is a tool to
analyze interaction between drugs allowing distinguishing synergistic, additive and
antagonistic effects. For CDI values < 1, drugs have a synergistic effect; for CDI = 1,
drugs have an additive effect; and for CDI values > 1, drugs have an antagonistic effect
[27,35–44].
4.3. Statistical analysis
Data were analyzed statistically using the GraphPad software. Differences between
means were tested for significance using the Student's t-test (*p < 0.05; **p < 0.01;
***p < 0.001).
Acknowledgements
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This study was supported by Fundação para a Ciência e a Tecnologia, Portugal
(FCT) through grant UID/DTP/04138/2013 and fellowship 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).
This work was funded in part by iMed.ULisboa through the Young Investigator´s
Project for Collaborative Cross-disciplinary Studies 2015. The authors thank Dr.
Shaomeng Wang and Dr. Liu Liu for MDM2 binding testing.
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