Novel 3,3-disubstituted oxindole derivatives. Synthesis and evaluation of the anti-proliferative activity

Article abstract of DOI:10.1016/j.bmcl.2019.126845

3,3-Disubstituted oxindole derivatives bearing a nitrogen atom at the C-3 position have been synthesized starting from 3-alkyl oxindole through a metal free pathway. These derivatives have been tested in five human tumor cell lines (PC3, MCF7, SW620, MiaP

Full text of DOI:10.1016/j.bmcl.2019.126845

Journal Pre-proofs
Novel 3,3-disubstituted oxindole derivatives. Synthesis and evaluation of the
anti-proliferative activity
Michael S. Christodoulou, Ferdinando Nicoletti, Katia Mangano, Maria Assunta
Chiacchio, Giorgio Facchetti, Isabella Rimoldi, Egle M. Beccalli, Sabrina
Giofrè
PII:
S0960-894X(19)30819-4
DOI:
https://doi.org/10.1016/j.bmcl.2019.126845
BMCL 126845
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 September 2019
15 November 2019
16 November 2019
Please cite this article as: Christodoulou, M.S., Nicoletti, F., Mangano, K., Assunta Chiacchio, M., Facchetti, G.,
Rimoldi, I., Beccalli, E.M., Giofrè, S., Novel 3,3-disubstituted oxindole derivatives. Synthesis and evaluation of the
anti-proliferative activity, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126845
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Graphical Abstract
Novel 3,3-disubstituted oxindole derivatives.
Synthesis and evaluation of the anti-
proliferative activity
Leave this area blank for abstract info.
Michael S. Christodoulou, Ferdinando Nicoletti, Katia Mangano, Maria Assunta Chiacchio, Giorgio Facchetti,
Isabella Rimoldi, Egle M. Beccalli and Sabrina Giofrè

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Novel 3,3-disubstituted oxindole derivatives. Synthesis and evaluation of the
anti-proliferative activity
Michael S. Christodouloua , Ferdinando Nicolettib , Katia Manganob , Maria Assunta Chiacchioc , Giorgio
Facchettia , Isabella Rimoldia , Egle M. Beccallia, * and Sabrina Giofrèa
aDISFARM, Sezione di Chimica Generale e Organica “A. Marchesini” Università degli Studi di Milano, via Venezian 21, 20133 Milano, Italy.
bUniversity of Catania, BIOMETEC, Via Santa Sofia 97, 95123, Catania, Italy
cUniversity of Catania, Dipartimento di Scienze del Farmaco, Viale Andrea Doria, 6, 95125 Catania, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
3,3-Disubstituted oxindole derivatives bearing a nitrogen atom at the C-3 position have been
synthesized starting from 3-alkyl oxindole through a metal free pathway. These derivatives have
been tested in five human tumor cell lines (PC3, MCF7, SW620, MiaPaca2 and A375) and on
primary cells (PBMCs) from healthy donors providing compound 6d showing a strong
anticancer effect in all cancer lines on the low micromolar range.
2009 Elsevier Ltd. All rights reserved.
Keywords:
3,3-disubstituted oxindole
isocyanate
iminophosphorane
Anti-proliferative activity
Human cancer cell lines
The oxindole is a scaffold of a large family of natural and
unnatural compounds endowed with biological activities.1-5
Among them 3,3-disubstituted oxindole derivatives are
recognized as valuable compounds for drug discovery,6-9 and also
as key intermediates for the synthesis of many kinds of drug
candidates.7,10-15 In particular, oxindoles bearing an amino group
at the 3-position are belonging to a variety of pharmacological
active molecules, such as the gastrin/CCK-B receptor antagonist
AG-041R (1),16 the vasopressin VIb receptor antagonist SSR-
149415 (2)17-20 and the HIV protease inhibitors 3 (Figure 1).21
Herein, based on our experience in the functionalization of
oxindole derivatives32-34 and in the transformation of heterocyclic
rings,35-38 we describe the synthesis of a new class of 3,3-
disubstituted oxindoles, bearing a nitrogen substituent in C-3
exploiting a metal free pathway. Continuing our researches in the
synthesis of anticancer compounds,39-49 the aim was to investigate
the potential biological activity of the target compounds on
different cancer cell lines.
Starting from 3-alkyl oxindoles, the 3-bromo derivatives 4
were obtained in quantitative yield by reaction with bromine in
OH
dichloromethane at r.t., following
a procedure previously
described by our group.37 Subsequently, compounds 4 were
reacted with sodium azide to form the 3-azido derivatives 5,
which were then treated with triphenylphosphine to provide the
stable iminophosphoranes 6 (Scheme 1). The compounds 6a and
6c were quantitatively deprotected at the position 1 under basic
conditions to give the corresponding derivatives 6f and 6g
(Scheme 1). The synthesis of the target isocyanates 7 was
achieved by reacting the iminophosphoranes 6 with CO2, in a
close vessel, at 50 °C in THF. Compounds 7a, 7b, 7d and 7e
were obtained in moderate to good yields (55-70 %). Low
solubility of compounds 6f and 6g in THF, drove us to use as
solvents DMF for 6f and MeOH for 6g. In these solvents, the
reaction of 6f and 6g with CO2 provided the amine 8f and the
amide 9 correspondingly (Scheme 1). The iminophosphorane 6c
resulted unreactive under the standard conditions.
TolHN
HN
O
HO
NHTol
N
O
Cl
R
CONHMe2
O
N
O
O
O
N
N
O
OEt
OEt
S
N
O
H
O
3
O
1
2
Figure 1. 3,3-Disubstituted oxindole derivatives.
Due to the interest of this class of oxindole derivatives, a lot of
attention has been devoted to the development of new synthetic
strategies to access to oxindoles bearing a nitrogen atom at the C-
3 position.22-24 In this field, significant advances have been
achieved in the organocatalytic25-29 or metal-catalyzed30,31 α-
amination of 2-oxindoles.

R2
R2
R2
R2
R1
R1
R1
R1
Br
N=PPh3
R4
N3
b
d
a
N
O
N
O
N
O
N
O
R3
R3
R3
R3
4
6a-e
7 8 9
, ,
5a-e
1
2
3
1
2
3
4
4a
: R = H, R = Bn, R = Ac
6a, 6c
7a
7b
7d
7e
8f:
9:
: R = H, R = Bn, R = Ac, R = NCO
1
2
3
1
2
3
4
4b
4c
: R = H, R = CH2CO2Et, R = Ac
c
: R = H, R = CH2CO2Et, R = Ac, R = NCO
1
2
3
1
2
3
1
2
3
4
: R = H, R = C3H7, R = CO2Et
6f
: R = H, R = Bn, R = H
: R = Br, R = Bn, R = H, R = NCO
1
2
3
1
2
3
1
2
3
4
4d:
4e:
R = Br, R = Bn, R = H
6g
: R = H, R = C3H7, R = H
: R = Br, R = Bn, R = CO2Et, R = NCO
1
2
3
1
2
3
4
R = Br, R = Bn, R = CO2Et
R = H, R = Bn, R = H, R = NH2
1
2
3
4
R = H, R = C3H7, R = H, R = NHCO2Me
Scheme 1. Reagents and conditions: (a) NaN3, tetrahexylammonium iodide, DCM/H2O, r.t., overnight; (b) PPh3 DEE, r.t., 3 h; (c) NaOH, EtOH, r.t.,
overnight; (d) CO2, THF (DMF for 6f and MeOH for 6g), 50 ˚C (90 ˚C for 6f), overnight.
(Figures S3, S4) and with an IC50 of respectively 28 and 30 M
(Table 1).
Treatment of the azide 5d with a solution of HBr in AcOH,
allowed the formation of the amine 8d, which was subsequently
reacted with benzoyl chloride and diethyl chlorophosphate to
provide compounds 10a and 10b respectively (Scheme 2).
Table 1. Cell growth inhibition in the presence of examined
compounds
IC50 (µM)a
Bn
Bn
Bn
Cmpd
6d
PC3
8.63
MCF7 SW620
MiaPaca2 A375
PBMC
30.71
45.52
> 100
75.30
> 100
> 100
> 100
> 100
> 100
51.78
Br
Br
Br
NH2
R1
N3
a
b
< 6.25
14.38
30.44
17.68
> 50
< 6.25
> 50
> 50
9.81
< 6.25
47.01
> 50
9.04
> 50
> 50
6.41
> 50
> 50
> 50
> 50
> 50
> 50
N
O
N
O
N
O
H
H
H
7a
18.20
17.04
8.95
5d
8d
10
: R1 = NHCOPh
: R1 = NHPO(OEt)2
7b
10a
10b
7d
7.46
Scheme 2. Reagents and conditions: (a) HBr in AcOH, AcOH, 0 ˚C, 2 h; (b)
PhCOCl or ClP(=O)(OEt)2, Et3N, DCM, 25 °C, 3 h.
7e
32.51
> 50
> 50
N.D.b
> 50
> 50
> 50
> 50
37.05
> 50
> 50
27.70
> 50
8d
> 50
> 50
The synthesized compounds 6d, 7a-e, 8d, 8f, 9, 10a, 10b
having the 3-aminooxindole core were tested for their
antiproliferative activity on five human tumor cell lines, the PC3
(prostatic adenocarcinoma), the MCF7 (breast adenocarcinoma),
the SW620 (colorectal adenocarcinoma), the MiaPaca2
(pancreatic adenocarcinoma) and A375 (melanoma) cell lines at
50, 25, 12.5 and 6.25 M (Figures S1-S5) concentrations and
expressed as IC50 values (Table 1). Moreover, the toxicity of the
compounds on PBMCs (Peripheral Blood Mononuclear Cells)
from healthy donors was also tested at 100, 50, 25, 12.5 and 6.25
µM concentrations and expressed as IC50 values (Table 1, Figure
S6).
8f
> 50
43.09
N.D.b
> 50
9
> 50
10a
10b
> 50
> 50
30.21
aValues of at least two independent experiments.
b N.D. = Not Determined.
The evaluation test on the toxicity in the primary cells PBMC
showed that among the compounds that exerted an antitumor
activity, compound 6d showed an IC50 of 30.71 M but with an
anticancer activity at doses lower than 6.25 M (Table 1).
Compound 7d showed an IC50 on PBMCs higher than 50 µM and
compound 7a showed an IC50 of 45.5 µM (Table 1).
In a preliminary evaluation some of the tested compounds
showed a detectable activity (Table 1). In particular, the most
cytotoxic derivatives in all the considered tumor cell lines were
compound 6d, characterized by a N=PPh3 substituent linked to
the C-3 position and H in position 1 and compound 7d
characterized by a NCO substituent linked to the C-3 position
and H in position 1 (Scheme 1). They showed an IC50 on the low
micromolar range of respectively 6-10 and 7-20 M in all the
considered cancer cell lines inducing a 100% of mortality at
almost all the tested doses (Figures S1-S5).
In the present work, we have evaluated the potential anti-
tumor effects of several compounds on a panel of stabilized
human cancer cell lines. Among the compounds tested, ten of
them exerted anti-cancer activity, although at high concentrations
(in the µM range) and not in all the tested cell lines. However,
when evaluating the toxicity in primary cells, we observed that
none of the compounds was toxic at the concentrations effective
on cancer cells.
Other compounds such as 7a, 7b and 7e exhibited cytotoxic
dose dependent effects only on prostatic cancer cell line PC3
(Figure S1) and 7a and 7b also on breast cancer cell line MCF7
(Figure S2) at an IC50 lower than 50 M (Table 1). Compounds
8d, 8f, 9 and 10a exerted toxic effects on cancer cell lines
(Figures S1-S5) but only at high concentrations with an IC50
higher than 50 M (Table 1). Compound 10b showed an
antitumoral efficacy in adenocarcinoma cancer cell SW620 and
pancreatic cell line MiaPaca2 in a dose dependent manner
The most promising compounds that we believe deserve further
testing are the 7d and 6d. The first compound exhibits an
important anti-cancer effect with toxicity in PBMCs at the dose
of 50 and 100 M. Compound 6d showed to be toxic on PBMCs
cells already at the dose of 25 µM but it showed a strong
anticancer effect at doses lower than 6.25 µM.

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Declaration of interests
☒ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: