Synthesis and evaluation of spiroisoxazoline oxindoles as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2013.10.048

Restoring p53 levels through disruption of p53-MDM2 interaction has been proved to be a valuable approach in fighting cancer. We herein report the synthesis and evaluation of eighteen spiroisoxazoline oxindoles derivatives as p53-MDM2 interaction inhibito

Full text of DOI:10.1016/j.bmc.2013.10.048

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Bioorganic & Medicinal Chemistry 22 (2014) 577–584
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of spiroisoxazoline oxindoles as anticancer
agents
⇑
Carlos J.A. Ribeiro, Joana D. Amaral, Cecília M.P. Rodrigues, Rui Moreira, Maria M.M. Santos
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, 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:
Restoring p53 levels through disruption of p53–MDM2 interaction has been proved to be a valuable
approach in fighting cancer. We herein report the synthesis and evaluation of eighteen spiroisoxazoline
oxindoles derivatives as p53–MDM2 interaction inhibitors. Seven compounds showed an antiprolifera-
tive profile superior to the p53–MDM2 interaction inhibitor nutlin-3, and induced cell death by apopto-
sis. Moreover, proof-of-concept was demonstrated by inhibition of the interaction between p53 and
MDM2 in a live-cell bimolecular fluorescence complementation assay.
Received 24 August 2013
Revised 21 October 2013
Accepted 29 October 2013
Available online 7 November 2013
Keywords:
Spiro compounds
Ó 2013 Elsevier Ltd. All rights reserved.
Protein–protein interactions
p53
MDM2
Cancer
1. Introduction
cis-imidazolines (e.g., nutlin-3a, Fig. 1),9 benzodiazepinediones
(e.g., TDP665759, Fig. 1),30 and spiro-oxindoles (e.g., MI-219,
Tumor suppressor p53 was discovered over 30 years ago and
since then it has emerged as a key protein in many biological pro-
cesses. p53 plays a pivotal role in the regulation of cell cycle, apop-
tosis, DNA repair, senescence and angiogenesis, and consequently
carcinogenesis.1,2 It is now believed that loss of p53 function oc-
curs in virtually all cancers. Indeed, in approximately 50% of hu-
man cancers, the TP53 gene is mutated or deleted. In tumors
with wild type p53, protein function is usually inactivated by over-
expression of negative regulators, primarily the murine double
minute-2 (MDM2), which form an autoregulatory feedback loop.3
Since increased levels of p53 inactivator MDM2 are present in
7% of all cancers, targeting the p53–MDM2 interaction to reactivate
p53 has emerged as a promising new cancer therapeutic strategy.4
Furthermore, studies using p53–MDM2 interaction inhibitors
showed that in normal cells the activation of p53 induces cell cycle
arrest but not cell death, revealing a selective apoptotic effect on
tumor cells.5
Fig. 1).31 Recently a new family containing a piperidone ring as
emerged as a promising new scaffold (e.g., AM-8553, Fig. 1).17 Each
Cl
O
NH2
Cl
Cl
Cl
O
NH
O
N
CH3
I
N
N
Cl
O
N
N
O
O
N
N
Nutlin-3a
TDP665759
In the bound conformation, the
a-helix of the transactivation
HO
H
N
O
domain of p53 projects residues Phe19, Trp23 and Leu26 into a
deep hydrophobic cleft in the MDM2 protein, representing the crit-
ical residues for binding between these two proteins to occur.6
In recent few years, several compounds families were designed
and developed as modulators of p53.7–29 Three classes of
p53–MDM2 interaction inhibitors are of particular importance:
O
OH
O
N
OH
NH
O
OH
F
Cl
N
H
Cl
Cl
MI-219
AM-8553
⇑
Corresponding author. Tel.: +351 217946400; fax: +351 217946470.
E-mail address: mariasantos@ff.ul.pt (M.M.M. Santos).
Figure 1. Representative p53–MDM2 interaction inhibitors.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.048

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
578
C. J. A. Ribeiro et al. / Bioorg. Med. Chem. 22 (2014) 577–584
Table 1
of them detains a unique, rigid heterocyclic scaffold, from which
three lipophilic groups are projected into p53 pocket in MDM2
mimicking the three pivotal Phe19, Trp23 and Leu26. All the inter-
actions are mostly hydrophobic, with potency increasing by intro-
duction of halide-substituted aromatic groups.32
In vitro antiproliferative activities in HepG2 cell line
R4
R3
However, the goal of developing a p53 reactivating cancer ther-
apy through inhibition of p53–MDM2 interaction is still in its in-
fancy, with only a few candidates entering clinical trials.33 In
spite of this, more potent and selective p53–MDM2 interaction
inhibitors are still largely required.
Therefore, we synthesized a library of novel spiroisoxazoline
oxindoles, to be evaluated as p53–MDM2 interaction inhibitors
in cell-based assays. The oxindole moiety would mimic the indole
side chain of Trp23 of p53 and the isoxazoline ring, would act as
the rigid heterocyclic scaffold. In addition, due to the fact that
mimicking p53 entails three hydrophobic moieties, we synthesized
18 derivatives with different aromatic side chains attached to the
isoxazoline moiety.
N
O
O
R2
N
R1
a
Compds
R1
R2
R3
R4
HepG2 GI50
>100
(lM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
p-NO2
p-OCH3
H
52.14 1.09
77.77 1.05
>100
OCH3
OCH3
OCH3
H
H
>100
>100
p-OCH3
H
p-OCH3
p-NO2
p-OCH3
H
p-OCH3
p-NO2
p-CH3
o-OCH3
p-OCH3
H
5-NO2
5-NO2
7-Cl
7-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Br
6-Br
83.59 1.04
53.10 1.14
55.48 1.11
55.79 1.11
37.07 1.08
38.14 1.08
33.84 1.11
29.11 1.09
35.70 1.07
>100
H
H
H
H
H
H
H
H
2. Results and discussion
Spiroisoxazoline oxindoles 1 (Scheme 1) were synthesized by
1,3-dipolar cycloaddition between 3-methylene indolin-2-ones 3
and nitrile oxides. Aldoximes 5 were synthesized from the corre-
sponding aldehyde 4 by reaction with hydroxylamine.34 Then,
aldoximes were reacted with NCS to form hydroximoyl chlorides.
With exception of compound 1e, the next step proceeded without
isolation of chlorooxime by addition of compound 3 to form the fi-
nal spirooxindole compound. In these cases, the 1,3-dipoles were
generated in situ through dehydrohalogenation of the chlorooxime
in the presence of triethylamine. Intermediates 3 were synthesized
by aldolic condensation of substituted indolin-2-ones with differ-
ent aromatic aldehydes in the presence of piperidine.35 Compound
1e was synthesized by reaction of the corresponding intermediate
3 with N-hydroxybenzimidoyl chloride in the presence of metallic
Zinc.36 The relative configuration was established by NMR compar-
isons with other spiroisoxazoline oxindoles, with published X-ray
crystallography structure.36,37 In this configuration the isoxazoline
proton (H-40) and the H-4 are more shielded in comparison to the
alternative configuration, as observed by Risitano.37 Furthermore,
we observed NOE effect between H-4 and the ortho-protons of
the aryl at C-40, revealing spatially proximity and corroborating
the established configuration.
OCH3
H
32.84 1.07
31.69 1.10
51.31 1.04
H
p-OCH3
Nutlin-3
a
GI50 determined by the MTS method. Each value is the mean (GI50 SEM) of
three independent experiments.
p53 (Table 1). Compound 1a with no substituents in the three
phenyl rings was not active. However, any substituent introduced
at position R2 and R4 gave rise to active compounds (1b–c, 1g–o,
and 1q–r). Introducing a methoxy group at position R3 abrogates
cytotoxicity, despite the pattern of substituents in other positions
(1d–f, and 1p).
The best compounds were obtained when a halogen was intro-
duced at position 6 (Cl or Br) of the oxindole moiety (1k–o, and
1q–r). In fact, a spiropyrrolidine oxindole derivative already pub-
lished with co-crystal structure bound to MDM238 revealed that
the oxindole moiety can perfectly mimic the indole side chain of
Trp23 of p53. Furthermore, the strength of this interaction is in-
creased by introducing a halogen at position 6 that can fill a small
pocket not occupied by indole, corroborating our findings. Differ-
ent substituents at R4 in para position (methoxy, nitro and methyl)
and ortho-methoxy are well tolerated, not affecting substantially
potency. The best activity was found for compound 1n
We started by evaluating the antiproliferative activity in a hu-
man hepatocellular carcinoma cell line (HepG2) with wild type
R3
R2
O
(GI50 = 29.1
To investigate if cytotoxicity is mediated by p53, we tested all
compounds more active than nutlin-3 (GI50 <50 M) in three other
lM).
a
N
R1
R2
O
l
N
cell lines with different p53 status: an isogenic pair of wild type
p53 and null human colorectal cancer cell lines [HCT116 p53(+/+)
and p53(ꢀ/ꢀ)]; and a p53 mutant human colorectal adenocarcinoma
cell line (SW620) (Table 2). All compounds showed only a marginal
increase in potency in cell lines harboring wild type p53, revealing
that p53 non-related effects are also contributing to cytotoxicity.
However, due to similar response profile obtained for nutlin-3 in
results assessed after 24 h, we further investigated if the com-
pounds can in fact inhibit p53–MDM2 interaction. By applying a
Venus-based bimolecular fluorescence complementation system
methodology (BiFC) developed by our group,39 we demonstrated
that compound 1k can inhibit p53–MDM2 interaction in the same
extent as nutlin-3 (Fig. 2). Similar results were also obtained for
compounds 1l and 1n (Supplementary data).
R1
2
3
R4
R3
OH
H
O
N
b
c
4'
H
R4
N
O
R4
4
O
R2
N
R1
4
5
1
Scheme 1. Synthesis of spiroisoxazoline oxindoles 1. Reagents and conditions:
(a) aromatic aldehydes, piperidine, EtOH, reflux, 76–99%; (b) NH2OH.HCl, Na2CO3,
H2O, reflux, 87–98%; (c) 1. NCS, piridine, CHCl3; 2. Compound 3, Et3N, CHCl3, reflux,
43–76%.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
C. J. A. Ribeiro et al. / Bioorg. Med. Chem. 22 (2014) 577–584
579
Table 2
In vitro antiproliferative activities
a
a
a
a
Compds
HepG2 GI50
(lM)
HCTp53(+/+) GI50
(l
M)
HCTp53(ꢀ/ꢀ) GI50
(
lM)
SW620 GI50 (lM)
1k
1l
1m
1n
1o
1q
1r
Nutlin-3
37.07 1.08
39.80 1.10
34.72 1.14
32.37 1.12
26.56 1.07
30.51 1.08
35.01 1.07
33.38 1.13
39.65 1.12
48.22 1.13
40.14 1.17
38.97 1.14
30.64 1.12
35.56 1.10
40.55 1.11
39.03 1.18
52.34 1.15
48.86 1.08
53.20 1.10
33.52 1.07
31.56 1.05
36.74 1.04
39.65 1.07
40.36 1.09
57.04 1.04
38.14 1.08
33.84 1.11
29.11 1.09
35.70 1.07
32.84 1.07
31.69 1.10
51.31 1.04
a
GI50 determined by MTS method. Each value is the mean (GI50 SEM) of three independent experiments.
Figure 2. Compound 1k decreases p53–MDM2 interaction by BiFC. HCT116 p53(ꢀ/ꢀ) cells were co-transfected with V1-p53/MDM2-V2 BiFC combination plasmids for 24 h.
Vehicle, nutlin-3 (50 M) and compound 1k (10 and 50 M) were included in the culture medium 4 h after transfection. Representative flow cytometry profiles of the
disruption of V1-p53/MDM2-V2 complementation (n = 3).
l
l
Figure 3. Compounds 1l and 1n induces caspase-3 activation and PARP cleavage. HCT116 p53(+/+) cells were incubated with vehicle or 25 and 50
lM of compounds 1l and 1n,
for 24 h. Representative immunoblots of caspase-3 processing (left) and PARP cleavage (right) analyzed in whole cell extracts. Blots were normalized to endogenous b-actin.
Data represent mean SEM of three independent experiments. ⁄p <0.01 and §p <0.05 from control.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
580
C. J. A. Ribeiro et al. / Bioorg. Med. Chem. 22 (2014) 577–584
Inhibition of p53–MDM2 interaction will restore p53 in cancer
(J) in Hertz and integration. 1H and 13C chemical shifts are ex-
cell lines leading to its activation, and consequently induce a
p53-mediated signaling pathway that will culminate in cell death
by apoptosis. The activation of caspase-3 upon cleavage by up-
stream proteases and subsequent cleavage of caspase-3 substrate
PARP are considered reliable markers of the apoptotic process. In
accordance, compounds 1l and 1n induced a significant dose-
dependent increase of cleaved PARP (p <0.01) and active caspase-
3 (p <0.01 and p <0.05 for compound 1l and 1n, respectively) as de-
tected by Western blotting (Fig. 3).
Chemical stability in pH 7.4 phosphate buffer and metabolic
stability in human plasma and rat liver microsomes at 37 °C were
evaluated for compounds 1l and 1r. Both compounds were stable
in phosphate buffer and plasma for the duration of the assays
(3 days). Compounds 1l and 1r exhibited degradation when incu-
bated in rat microsomes with NADPH regenerating system, with
half-lives of 3.15 0.07 h and 3.62 0.06 h, respectively, indicating
moderate susceptibility towards co-factor dependent microsomal
enzymes.
pressed in ppm using the solvent as internal reference.
4.1.1. General preparation of derivatives 1a-d and 1f–r
To a stirred solution of N-chlorosuccinimide (3 equiv) and pyr-
idine (0.3 equiv) in chloroform (2.5 mL/0.1 mmol of 3-methylene
indolin-2-one) was added the appropriate aldoxime (3 equiv).
The reaction mixture was stirred for 24 h at room temperature be-
fore addition of the appropriate 3-methylene indolin-2-one. The
mixture was then heated to 50 °C and triethylamine (3.5 equiv)
was added in a dropwise manner. After heating at refluxed for
26 h, the mixture was washed with brine (2ꢁ) and the aqueous
phase extracted with CH2Cl2 or EtOAc depending on the solubility
of the product. 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 sil-
ica gel using as eluent n-hexane/EtOAc (4:1) to EtOAc (100%) and
recrystallized from EtOAc/n-hexane to afford the final product
(adapted from Ref. 37).
4.1.1.1.
(1a)25
30,40-Diphenyl-spiro[indoline-3,50-isoxazoline]-2-one
Obtained as a white solid (16.4 mg, 0.0482 mmol,
3. Conclusion
.
53%). 1H NMR (400 MHz, CDCl3) d (ppm): 7.95 (br s, 1H), 7.69–
7.65 (m, 2H), 7.43–7.27 (m, 3H), 7.28–7.23 (m, 3H, partially ob-
scured by CHCl3 signal), 7.12 (td, J = 7.8, 1.2 Hz, 1H), 7.09–7.05
(m, 2H), 6.80 (d, J = 7.8 Hz, 1H), 6.63 (t, J = 7.8 Hz, 1H), 6.25 (d,
J = 7.8 Hz, 1H), 5.08 (s, 1H); 13C NMR (100 MHz, CDCl3) d (ppm):
177.08, 159.19, 141.62, 133.68, 130.65, 130.45, 129.15, 129.09,
128.82, 128.58, 128.55, 127.92, 127.40, 123.03, 122.72, 110.21,
89.05, 60.41. Anal. Calcd for C22H16N2O2ꢂ0.5H2O: C, 75.63; H,
4.91; N, 8.02. Found: C, 75.36; H, 4.91; N, 7.82.
Eighteen spiroisoxazoline oxindole derivatives were synthe-
sized with different substituents attached to the three phenyl rings
to probe their capacity of inhibiting the p53–MDM2 interaction.
Screening the compounds in a HepG2 cell line revealed that com-
pounds with chloro or bromo at position 6 of the oxindole aromatic
ring were more active than nutlin-3. The profile of inhibitory activ-
ity in cell lines with different p53 status was comparable to that of
nutlin-3, revealing also p53-independent effects. In addition, com-
pound 1k showed inhibition of p53–MDM2 interaction in a cell-
based bimolecular fluorescence complementation assay. Moreover,
compounds 1l and 1r showed good stability in 7.4 phosphate buf-
fer and plasma, and moderate susceptibility towards NADPH-
dependent rat microsomal enzymes.
4.1.1.2. 30-(4-Nitrophenyl)-40-phenyl-spiro[indoline-3,50-isoxaz-
oline]-2-one (1b).
0.256 mmol, 57%). Mp 274–276 °C; IR (KBr): 3198, 1712, 1522,
1337 cmꢀ1 1H NMR (400 MHz, acetone-d6) d (ppm): 9.58 (br s,
Obtained as a light yellow solid (98.6 mg,
;
In conclusion, besides the potential use of the active compounds
as molecular probes and possible anticancer agents, compound 1n
represent a useful lead compound for the development of more po-
tent and selective p53–MDM2 interaction inhibitors.
1H), 8.25 (br d, J = 8.8 Hz, 2H), 7.98 (br d, J = 8.8 Hz, 2H), 7.39–
7.29 (m, 3H), 7.27–7.21 (m, 2H), 7.10 (t, J = 7.7 Hz, 1H), 6.91 (d,
J = 7.7 Hz, 1H), 6.63 (t, J = 7.7 Hz, 1H), 6.29 (d, J = 7.7 Hz, 1H), 5.46
(s, 1H); 13C NMR (100 MHz, acetone-d6) d (ppm): 176.54, 158.99,
149.54, 144.31, 135.92, 134.48, 131.69, 130.12, 129.97, 129.45,
129.43, 127.88, 124.86, 123.47, 122.66, 111.02, 90.52, 59.63.
4. Experimental
4.1. Chemistry
4.1.1.3.
isoxazoline]-2-one (1c).
(117.0 mg, 0.316 mmol, 70%). Mp 267–269 °C; IR (KBr): 3198,
1712, 1522, 1337 cmꢀ1 1H NMR (400 MHz, acetone-d6) d (ppm):
30-(4-Methoxyphenyl)-40-phenyl-spiro[indoline-3,50-
Obtained as white solid
a
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. 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 and the UV spectra on a Shima-
dzu UV–Vis Recording Spectrophotometer UV-160. Elemental
analysis (C, H, and N) were performed in a Flash 2000 CHNS-O ana-
lyzer (ThermoScientific, UK) at Liquid Chromatography and Mass
Spectrometry Laboratory, Faculty of Pharmacy of Lisbon University
and also in a LECO model CHNS-932 elemental analyzer at the Unit
Elemental Analysis, University of Santiago de Compostela, Spain.
The 7 most active compounds also showed purity P95% by analyt-
ical HPLC with absorbance at 260 nm. Merck Silica Gel 60 F254
plates were used for analytical TLC; flash column chromatography
was performed on Merck Silica Gel (200–400 mesh) and Combi-
Flash Rf from Teledyne ISCO (columns RediSep Rf, silica). 1H and
13C NMR spectra were recorded on a Bruker 400 Ultra-Shield at
400 MHz (1H NMR) and 100 MHz (13C NMR). Data are reported
as follows: chemical shift (d), multiplicity (s: singlet, d: doublet,
dd: doublet of doublet; ddd: doublet doublet of doublets, t: triplet,
td: triplet of doublets, m: multiplet, br: broad), coupling constants
;
9.51 (br s, 1H), 7.64 (br d, J = 8.9 Hz, 2H), 7.34–7.25 (m, 3H),
7.22–7.18 (m, 2H), 7.15 (t, J = 7.7 Hz, 1H), 6.91 (br d, J = 8.9 Hz,
2H), 6.88 (d, J = 7.7 Hz, 1H), 6.60 (t, J = 7.7 Hz, 1H), 6.24 (d,
J = 7.7 Hz, 1H), 5.27 (s, 1H), 3.79 (s, 3H); 13C NMR (100 MHz, ace-
tone-d6) d (ppm): 177.07, 162.07, 159.43, 144.14, 135.41, 131.21,
130.05, 129.96, 129.63, 128.96, 127.72, 124.18, 122.37, 122.28,
114.96, 110.71, 89.33, 60.49, 55.66; Anal. Calcd for C23H18N2O3-
ꢂ0.4H2O: C, 73.15; H, 5.03; N, 7.42. Found: C, 72.84; H, 4.90; N, 7.36.
4.1.1.4.
isoxazoline]-2-one (1d)25
40-(4-Methoxyphenyl)-30-phenyl-spiro[indoline-3,50-
Obtained as white solid
.
a
(35.2 mg, 0.0950, 57%). 1H NMR (400 MHz, CDCl3) d (ppm): 8.60
(br s, 1H), 7.70–7.65 (m, 2H, HAr), 7.38–7.29 (m, 3H), 7.10 (td,
J = 7.8, 1.2 Hz, 1H), 6.99 (br d, J = 8.4 Hz, 2H), 6.82–6.76 (m, 3H),
6.66 (td, J = 7.8, 0.8 Hz, 1H), 6.31 (d, J = 7.8 Hz, 1H), 5.03 (s, 1H),
3.76 (s, 3H); 13C NMR (100 MHz, CDCl3) d (ppm): 177.64, 159.61,
159.50, 141.84, 130.58, 130.38, 130.24, 128.79, 128.65, 127.91,
127.40, 125.59, 123.07, 122.72, 114.46, 110.46, 89.16, 59.69, 55.36.