Synthesis of 4-substituted-3-Hydroxyquinolin-2(1H)-ones with anticancer activity

Article abstract of DOI:10.1016/j.tet.2020.130983

Herein we show that the 3-hydroxyquinolin-2(1H)-one (3HQ) core is a suitable platform to develop new compounds with anticancer activity against MCF-7 (IC₅₀ up to 4.82 μM) and NCI–H460 (IC₅₀ up to 1.8 μM) cancer cell lines. The ring-expansion reaction of isatins with diazo esters catalysed by di-rhodium (II) complexes proved to be a simple and effective strategy to synthesize 4-carboxylate-3HQs (yields up to 92%). 4-Carboxamide-3HQs were more efficiently prepared using NHS-diazoacetate in yields up to 88%. This innovative methodology enabled the construction of “peptidic-like” 3HQs, with several amino acid substituents. Among this series, the L-leucine derivative induced the cell death of MCF-7 (IC₅₀ of 15.1 μM) and NCI–H460 (IC₅₀ of 2.7 μM) cancer cell lines without causing any appreciable cytotoxicity against the non-cancer cell model (CHOK1).

Full text of DOI:10.1016/j.tet.2020.130983

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Synthesis of 4-substituted-3-Hydroxyquinolin-2(1H)-ones with anticancer activity
Roberta Paterna, Rita Padanha, Roberto Russo, Raquel Frade, Hélio Faustino, Pedro
M.P. Gois
PII:
S0040-4020(20)30078-8
https://doi.org/10.1016/j.tet.2020.130983
TET 130983
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 18 November 2019
Revised Date: 20 January 2020
Accepted Date: 26 January 2020
Please cite this article as: Paterna R, Padanha R, Russo R, Frade R, Faustino Hé, Gois PMP, Synthesis
of 4-substituted-3-Hydroxyquinolin-2(1H)-ones with anticancer activity, Tetrahedron (2020), doi: https://
doi.org/10.1016/j.tet.2020.130983.
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Synthesis of 4-Substituted-3-Hydroxyquinolin-2(1H)-
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Roberta Paterna,a Rita Padanha,a Roberto Russo,a Raquel Frade,a Hélio Faustino,a and Pedro M. P. Goisa,
a Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003
Lisbon, Portugal
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1
Tetrahedron
journal homepage: www.elsevier.com
Synthesis of 4-Substituted-3-Hydroxyquinolin-2(1H)-ones with Anticancer Activity
Roberta Paterna,a Rita Padanha,a Roberto Russo,a Raquel Frade,a Hélio Faustino,a and Pedro M. P. Goisa,
a Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal
E-mail: pedrogois@ff.ulisboa.pt.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Available online
Herein we show that the 3-hydroxyquinolin-2(1H)-one (3HQ) core is a suitable platform to
develop new compounds with anticancer activity against MCF-7 (IC50 up to 4.82 µM) and NCI-
H460 (IC50 up to 1.8 µM) cancer cell lines. The ring-expansion reaction of isatins with diazo
esters catalysed by di-rhodium(II) complexes proved to be a simple and effective strategy to
synthesize 4-carboxylate-3HQs (yields up to 92%). 4-Carboxamide-3HQs were more efficiently
prepared using NHS-diazoacetate in yields up to 88%. This innovative methodology enabled the
construction of “peptidic-like” 3HQs, with several amino acid substituents. Among this series,
the L-leucine derivative induced the cell death of MCF-7 (IC50 of 15.1 µM) and NCI-H460 (IC50
of 2.7 µM) cancer cell lines without causing any appreciable cytotoxicity against the non-cancer
cell model (CHOK1).
Keywords:
Alkaloids
Ring-expansion
Diazo compounds
Anticancer compounds
Hydroxyquinolines
2009 Elsevier Ltd. All rights reserved
.
———
Corresponding author. e-mail: heliofaustino@ff.ulisboa.pt (H. Faustino), pedrogois@ff.ulisboa.pt (P. M. P. Gois).

2
Tetrahedron
1. Introduction
modification of this core, by either decarboxylation or N-
alkylation resulted in the loss of the observed antiproliferative
activity, as indicated by the results of cell viability obtained in
the presence of compounds 6, 7 and 8. Hence, we addressed the
influence of the substituent at the position 4 on the activity of the
heterocycle.
The 3-hydroxyquinolin-2(1H)-one (3HQ) core is an important
structural motif present in the natural products like viridicatin,
viridicatol and 3-O-methyl viridicatin.[1–8] These metabolites,
isolated from penicillium species, have been shown to inhibit the
replication of human immunodeficiency virus and to be
promising lead compounds for the development of new anti-
inflammatory agents.[9,10] Furthermore, this unique heterocycle
was recognized to be a valuable bioisoster for the carboxylic acid
function of α-amino acids. This has triggered the interest towards
the development of procedures for the synthesis of this class of
compounds.[11] Although less acidic (pKa of 8.7) than a
carboxylic acid,[12,13] a series of 3HQs were prepared at Pfizer
and shown to be potent inhibitors of the D-amino acid oxidase
activity, eliciting similar binding interactions with the enzyme
active site as the carboxylic acid containing inhibitors.[14] These
discoveries were not left unnoticed, and recently this
pharmacophore was found to bind to metal cofactors, by this way
inhibiting the influenza A endonuclease.[15]
Rather surprisingly, and despite the many possibilities offered
by the 3HQ core to develop new biologically active compounds,
the use of this scaffold in the construction of anti-proliferative
agents, remained basically unexplored. However, while
developing new inhibitors of the HIV-1 reverse transcriptase
associated RNase H activity, Bailly and co-workers observed that
a series of 4-substituted 3HQs were significantly cytotoxic
against non-cancer MT-4 cells, and this precluded their further
use as antiviral agents.[16] Based on this observation, we
envisioned that 4-carboxylate and 4-carboxamides substituted
3HQs could be further explored as a valuable platform to prepare
innovative anticancer agents.
Scheme 2. Synthesis of 4-carboxylate substituted 3HQs 1-8 based on an Eistert
ring-expansion reaction of isatins with diazo acetate (EDA) catalysed by
Rh2(OAc)4.
Table 1. Anti-proliferative evaluation of compounds 1-8
against MCF-7, NCI-H460 and HT-29 cancer cell lines.
C
OMPOUND
MCF-7
NA
NA
NCI-H460
NA
HT-29
87%
NA
NA
NA
74%
NA
NA
1
2
3
4
5
6
7
8
NA
NA
NA
52%
97%
NA
NA
86%
95%
NA
NA
NA
NA
NA
Percentage of cell-viability; NA – Non-active at the concentration of 20 µM
To study this, a series of diazo compounds were prepared and
used in the Eistert ring-expansion reaction of the 5-
trifluoromethoxy-isatin.[18] As shown in Scheme 3, this simple
protocol afforded 3HQ 9-13 in yields ranging from 71 to 86%.
Once prepared, the 3HQs were evaluated against the
aforementioned panel of cancer cell lines. As shown in Table 2,
the operated structural modification had a profound impact on the
heterocycles activity. Promisingly, introducing a benzyl ester on
compound 11 resulted in an increased activity against the three
cancer cell lines with an IC50 as low as 1.8 µM, against NCI-
H460 cells. Analogously, the 3HQ 13 featuring a slightly longer
alkylic chain also showed an IC50 of 2.1 µM against the same cell
line. However, the indiscriminate activity observed for these
molecules, suggested the possibility of these 3HQs being also
significantly toxic against non-cancer cell lines.
Scheme 1. The 3-hydroxyquinolin-2(1H)-one (3HQ) core present in the structure
of naturally occurring compounds, as a carboxylic acid bioisoster and as an
enzyme inhibitor.
2. Results and discussion
Aiming to test this hypothesis, we set out to prepare a small
library of 4-substituted-3HQs to evaluate their potential anti-
proliferative activity. As shown in Scheme 2, exploring our
previously described Eistert ring-expansion reaction of isatins
with diazo acetate (EDA) catalysed by Rh2(OAc)4,[17] 4-
carboxylate-3HQs 1-5 were synthesised in good to excellent
yields. Once prepared, this set of compounds was evaluated
against a panel of breast cancer cells (MCF-7), human non-small
lung cancer cells (NCI-H460) and human colorectal
adenocarcinoma cells (HT-29) (Scheme 2, Table 1). This assay
revealed that 3HQ 1-4 were non-active against the three cancer
lines tested, though the 6- trifluoromethoxy -4-ethylacetate-3HQ
5 was could reduce the viability of the NCI-H460 cells by 8% at
the concentration of 20 µM (Table 1). This result prompted us to
explore the derivatization of the 3HQ scaffold bearing a
trifluoromethoxy substituent on position 6. Structural
Table 2. Anti-proliferative evaluation of compounds 9-13
against MCF-7, NCI-H460 and HT-29 cancer cell lines.
C
OMPOUND
MCF-7
10.8±1.1
13.4±2.5
10.1±2.1
12.1±1.0
16.0±1.2
NCI-H460
10.4±1.9
6.0±1.0
1.8±1.2
7.3±1.2
HT-29
NA
NA
11.4±1.1
NA
NA
9
10
11
12
13
2.1±1.1

3
Determined IC50 of the compounds in MCF-7, NCIH460 and HT-29 cancer
cell lines after 48 hours of incubation; NA – Non-active at the concentration
of 20 µM
N-benzyl isatin as a model scaffold in order to avoid potential
cross reactivity of the exposed amide group. To this end, N-
benzyl isatin was reacted with NHS-diazo acetate under different
basic conditions (see ESI). This step proved to be very reversible,
though when using 20 mol% of TEA in EtOH, intermediate 14
precipitated from the reaction mixture, and was isolated in 88%
yield without the need of chromatographic purification. Diazo
intermediate 14 was then submitted to the ring expansion
reaction catalysed by 0.5 mol% of Rh2(OAc)4. The reaction
proceeded smoothly in DCM, and compound 15 was isolated by
filtration in 84% yield. Finally, benzylamine was added to 3HQ
15 with sodium carbonate, and this afforded the targeted 4-
carboxamide 3HQ 16 in a good yield. Notably, this method
proved to be compatible with more complex amines, and glycine
methyl ester and L-phenylalanine ethyl ester afforded the
peptidic-like[12] 4-carboxamides-3HQs 17 and 18 in 79% and
70% yields respectively.
Once demonstrated the feasibility of this synthetic scheme, the
same protocol was used to functionalize the 5-trifluoromethoxy -
isatin (Scheme 3, Table 1). As shown in Scheme 5, the presence
of an unprotected amide group was not detrimental for the
preparation of the diazo intermediate 19 that was obtained in
93%, neither to the ring expansion step, that afforded the 3HQ
heterocycle 20 almost quantitatively using 0.5 mol% of
Rh2(OAc)4. As expected, primary and secondary amines also
reacted smoothly with the 6-trifluoromethoxy -4-NHS-3HQ 20 to
yield the carboxamides 21-26 in yields up to 90%. Similarly,
protected amino acids also afforded the 4-carboxamides-3HQs
27-32 in good to excellent yields without any chromatographic
step.
Scheme 3. Synthesis of 4-carboxylate substituted 3HQs 9-13 based on an Eistert
ring-expansion reaction of isatins with diazo acetate (EDA) catalysed by di-
rhodium complexes.
To study this, compound 12 was evaluated against the non-
cancer Chinese hamster ovary cells (CHOK1) and, as expected, it
proved to be quite cytotoxic to this model (IC50 of 5.6±1.0 µM).
The incorporation of alkylic esters at position 4 of the scaffold
clearly induced a higher anti-proliferative effect against cancer
cells but regrettably, also a significant toxicity towards non-
cancer cell lines. Therefore, to improve these compounds toxicity
profile, we studied the anti-proliferative properties of 6-
trifluoromethoxy-4-carboxamide-3HQs. This study was initiated
with the synthesis of 4-carboxamides-3HQs following reported
protocols, in which the ethyl ester is typically hydrolysed to the
corresponding acid under basic conditions, and then coupled with
primary and secondary amides via an acid chloride intermediate.
Unfortunately, and despite our many attempts, when starting with
6-trifluoromethoxy-4-ethylacetate-3HQ 5, this simple protocol
invariably resulted in a decarboxylation process yielding
compound 6 (Scheme 2).
Scheme 4. Possible synthetic routes to prepare 4-carboxamides-3HQs.
Scheme 5. Synthesis of 4-carboxamide-3HQs based on Eistert ring-expansion
reaction of protected isatins with NHS-diazo acetate, followed by an amidation step.
Interestingly, the reported synthesis of 4-carboxamides-3HQs
starting from the corresponding carboxylic acid derivatives
proceeds with poor yields (<40%), indicating the need to improve
the synthetic methodologies to access these compounds.[16]
Based on this, we conceived that a more direct route to prepare 4-
carboxamide-3HQs would be to perform the Eistert ring-
expansion reaction with NHS-diazo acetate,[19,20] followed by a
simple amidation step (Scheme 4). We tested this idea adopting
Once established the preparation of 3HQ 21-32, the anti-
proliferative activity of these compounds was evaluated.
Observation of the results shown in Table 3 indicates that 4-
carboxamides-3HQs were shown to be less toxic against the non-
cancer cell model (CHOK1) than the 4-carboxylate-3HQs series.
For instance, benzylamide 22 was only slightly less potent

4
Tetrahedron
towards MCF-7 and NCI-H460 cancer cell lines than its benzyl result obtained with the 4-carboxylate-3HQ series (Table 2).
ester counterpart 11, while being clearly less toxic to CHOK1 Consistently with what observed for the previously tested
cells at a concentration of 20 µM than compound 11 (Table 3).
compounds, also this series of 3HQ derivatives did not elicit any
anti-proliferative activity on HT-29 cancer cells. Due to their
interesting activity both in NCI-H460 and MCF-7 cells, compound
13 and 32 were further tested for to their ability to induce cell death
in these cell lines as measured by Lactate dehydrogenase (LDH)
release, which is an indicator of plasma membrane damage.
Interestingly, exposure to compound 13 or 32, at IC50 and 2x IC50,
significantly increased general cell death in both cell lines,
confirming the anticancer potential of these compounds (Figure 1,
SI).
3. Conclusion
In this study, we address for the first time the cytotoxic potential
of 3HQs. The Eistert ring-expansion reaction of isatins with diazo
compounds catalysed by Rh2(OAc)4 was shown to be a versatile
methodology to prepare 3HQs. The direct addition of structurally
diverse diazo compounds to isatins enabled the construction of a
series of 4-carboxylate-3HQs (in yields up to 86%) which were
shown to present anti-proliferative activity against a panel of MCF-
7, NCI-H460 and HT-29 cancer cell lines. Regrettably, this series
of compounds also induced severe cytotoxicity against a model of
non-cancer cell lines, which motivated us to evaluate the 4-
carboxamide-3HQ counterparts. The troublesome preparation of
these compounds was simplified by performing a ring expansion
reaction of isatin derivatives with NHS-diazo acetate. This
methodology afforded the targeted 4-carboxamides-3HQs in yields
up to 90%, and this series of cytotoxic 3HQs were shown to have
an improved selectivity towards MCF-7 (3HQ 23, IC50 of 4.8 µM)
and NCI-H460 (3HQ 26, IC50 of 7.3 µM) cancer cell lines.
Scheme 6. Synthesis of 4-carboxamide-3HQs based on Eistert ring-expansion
reaction of 5- trifluoromethoxy -isatin with NHS-diazo acetate, followed by an
amidation step.
4. References and notes
[1]
K.G. Cunningham, G.G. Freeman, , Biochem. J. 53 (1953)
328–332. [2] A. Bracken, A. Pocker, H. Raistrick,
Biochem. J. 57 (1954) 587–595.
J.H. Birkinshaw, M. Luckner, Y.S. Mohammed, K. Mothes,
C.E. StickingsBiochem. J. 89 (1963) 196–202. [4] M.
Luckner, K. Mothes, Tetrahedron Lett. 3 (1962) 1035–1039.
M. Luckner, K. Mothes, No Title, Arch. Pharm. 18 (1963)
296.
This profile was even more pronounced in the case of amides 23
and 26 that showed a good selectivity towards the MCF-7 (IC50 of
4.8 µM) and NCI-H460 (IC50 of 7.3 µM) cancer cell lines
respectively (Table 3), with negligible toxicity towards the CHOK1
cells. The “peptidic-like” 4-carboxamides-3HQs 30-32 were also
active against the MCF-7 and NCI-H460 cell lines.
[3]
[5]
[6]
Table 3. Anti-proliferative evaluation of compounds 21-32
against MCF-7, NCI-H460 and CHOK1 cell lines.
M.Y. Wei, R.Y. Yang, C.L. Shao, C.Y. Wang, D.S. Deng,
Z.G. She, Y.C. Lin, 47 (2011) 322–325.
C
OMPOUND
MCF-7
NA
12.0±1.0
4.8±1.2
17.5±2.4
NA
NCI-H460
NA
9.5±1.2
NA
NA
NA
7.3±1.2
NA
NA
CHOK1
NA
NA
NA
NA
ND
NA
ND
NA
ND
NA
NA
NA
[7]
[8]
D. Austin, M. Myers, J. Chem. Soc. 1 (1964) 1197–1198.
I.Z. El Euch, M. Frese, N. Sewald, S. Smaoui, M. Shaaban,
L. Mellouli, Med. Chem. Res. 27 (2018) 1085–1092. [9]
N. Ribeiro, H. Tabaka, J. Peluso, L. Fetzer, C. Nebigil, S.
Dumont, C.D. Muller, L. Désaubry, Bioorg. Med. Chem.
21
22
23
24
25
26
27
28
29
30
31
32
Lett. 17 (2007) 5523–5524. [10]
A. Heguy, P. Cai,
NA
NA
P. Meyn, D. Houck, S. Russo, R. Michitsch, C. Pearce, B.
Katz, G. Bringmann, D. Feineis, D.L. Taylor, A.S. Tyms,
Antivir. Chem. Chemother. 9 (1998) 149–155.
Y. Tangella, K. Manasa, N. hari krishna, B. Sridhar, A.
Kamal, B. Nagendra Babu, Org. Lett. 20 (2018).
C.B.M. Poulie, L. Bunch, ChemMedChem. 8 (2013) 205–
215.
C. Ballatore, D.M. Huryn, A.B. Smith, ChemMedChem. 8
(2013) 385–395.
A.J. Duplantier, S.L. Becker, M.J. Bohanon, K.A.
Borzilleri, B.A. Chrunyk, J.T. Downs, L.Y. Hu, A. El-
12.6±1.1
NA
9.4±7.5
9.5±1.0
15.1±1.9
NA
8.4±1.7
11.4±1.1
2.7±1.4
[11]
[12]
[13]
[14]
Determined IC50 of the compounds in MCF-7 and NCIH460 cancer cell lines and
CHOK1 non-cancer cell model after 48 hours of incubation; NA – Non-active at
the concentration of 20 µM; ND – Not determined
In particular, compound 32 elicited an IC50 of 2.7 µM against the
NCI-H460 cells (Table 3), which compares well with the best

5
Kattan, L.C. James, S. Liu, J. Lu, N. Maklad, M.N.
Mansour, S. Mente, M.A. Piotrowski, S.M. Sakya, S.
Sheehan, S.J. Steyn, C.A. Strick, V.A. Williams, L. Zhang,
J. Med. Chem. 52 (2009) 3576–3585.
[15]
[16]
H.Y. Sagong, A. Parhi, J.D. Bauman, D. Patel, R.S.K.
Vijayan, K. Das, E. Arnold, E.J. Lavoie, 2 (2013) 8–11.
V. Suchaud, F. Bailly, C. Lion, E. Tramontano, F. Esposito,
A. Corona, F. Christ, Z. Debyser, P. Cotelle, Bioorg. Med.
Chem. Lett. 22 (2012) 3988–3992.
[17]
R. Paterna, V. André, M.T. Duarte, L.F. Veiros, N.R.
Candeias, P.M.P. Gois, European J. Org. Chem. (2013)
6280–6290.
[18]
[19]
[20]
N.R. Candeias, R. Paterna, P.M.P. Gois, Chem. Rev. 116
(2016) 2937–2981.
M.P. Doyle, A. V. Kalinin, J. Org. Chem. 61 (1996) 2179–
2184.
A. Ouihia, L. Rene, J. Guilhem, C. Pascard, B. Badet, J.
Org. Chem. 58 (1993) 1641–1642.
5. Acknowledgements
The authors would like to thank Fundação para a Ciência e
Tecnologia (SAICTPAC/0019/2015, SFRH/BD/78301/2011,
SFRH/BPD/102296/2014, PTDC/QEQ-QOR/1434/2014). This
work was also funded, in part, by iMed.ULisboa
(UID/DTP/04138/2019) from Fundação para a Ciência e a
Tecnologia (FCT), Portugal and FEDER (PTDC/QUI-
QOR/29967/2017).