Chalcones and Chalcone-mimetic Derivatives as Notch Inhibitors in a Model of T-cell Acute Lymphoblastic Leukemia

Article abstract of DOI:10.1021/acsmedchemlett.8b00608

Based on hit-likeness and chemical diversity, a number of chalcones and chalcone-mimetic compounds were selected as putative Notch inhibitors. The evaluation of the antiproliferative effect combined with the inhibition of Notch1 expression in KOPTK1 cell line identified compound 18, featuring a tetrahydronaphthalene-based scaffold, as a new promising Notch-blocking agent.

Full text of DOI:10.1021/acsmedchemlett.8b00608

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Letter
Chalcones and chalcone-mimetic derivatives as Notch
inhibitors in a model of T-cell acute lymphoblastic leukemia
Deborah Quaglio, Nadezda Zhdanovskaya, Gloria Tobajas, Viviana Cuartas, Silvia
Balducci, Michael S Christodoulou, Giancarlo Fabrizi, Marta Gargantilla, Eva-María
Priego, Álvaro Carmona Pestaña, Danielle Passarella, Isabella Screpanti, Bruno
Botta, Rocco Palermo, Mattia Mori, Francesca Ghirga, and Maria-Jesus Perez-Perez
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00608 • Publication Date (Web): 26 Feb 2019
Downloaded from http://pubs.acs.org on February 27, 2019
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Chalcones and chalcone-mimetic derivatives as Notch inhibitors in a
model of T-cell acute lymphoblastic leukemia
Deborah Quaglio,†‡ Nadezda Zhdanovskaya,ǂ‡ Gloria Tobajas,§ Viviana Cuartas,§ Silvia Balducci,†
Michael S. Christodoulou,ǁ Giancarlo Fabrizi,† Marta Gargantilla,§ Eva-María Priego,§ Álvaro Carmona
Pestaña,ǂ Daniele Passarella,┴ Isabella Screpanti,ǂ Bruno Botta,† Rocco Palermo,ǂ* Mattia Mori,#*
Francesca Ghirga,V* María-Jesús Pérez-Pérez§
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†Department of Chemistry and Technology of Drugs “department of excellence 2018-2022”, Sapienza Università di Roma,
P.le Aldo Moro 5, 00185 Rome, Italy
ǂ Department of Molecular Medicine, Sapienza University, 00161 Rome, Italy
§ Instituto de Química Médica-CSIC, Juan de la Cierva 3, 28006 Madrid , Spain
ǁ DISFARM, Sezione di Chimica Generale e Organica “A. Marchesini”, Università degli Studi di Milano, Via Venezian 21,
20133 Milano, Italy
┴ Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi, 19, 20133 Milano, Italy
# Department of Biotechnology, Chemistry and Pharmacy “department of excellence 2018-2022”, University of Siena, via
Aldo Moro 2, I-53100, Siena, Italy
V Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy
KEYWORDS: Chalcone, tetrahydronaphthalene scaffold, Notch inhibitors, T-ALL, cancer.
ABSTRACT: Based on hit-likeness and chemical diversity, a number of chalcones and chalcone-mimetic compounds were
selected as putative Notch inhibitors. The evaluation of the anti-proliferative effect combined with the inhibition of Notch1
expression in KOPTK1 cell line identified compound 18, featuring a tetrahydronaphthalene-based scaffold, as a new promising
Notch-blocking agent.
Notch signaling is an evolutionarily conserved cell
communication pathway regulating many biological processes
including stem cells self-renewal, cell differentiation,
proliferation and survival in different tissues during embryonic
and adult life.1,2,3 The mammalian Notch family includes four
trans-membrane receptors (namely, Notch1–4), and five trans-
membrane ligands (namely, Jagged-1 and -2, Delta-like-1, -3
and -4). Notch signaling is a short-range inter-cellular
communication system, wherein a membrane tethered Notch
the majority of T-ALL patients shows increased expression
and function of Notch3.6,7,8 Moreover, Notch signaling may
contribute to chemotherapy resistance, thus suggesting Notch
10
inhibition as a strategy in anticancer targeted therapy.9,
(
Notwithstanding, small molecules (e.g. GS inhibitors (GSI)
such as MK-0752, RO4929097 and PF-03084014) and large
molecules (e.g. Notch1-specific antibody OMP-52M51)
currently in clinical development are not effective in all
patients, and often cause severe side-effects that limit their
widespread therapeutic use and raise the need for novel Notch-
blocking agents.11, 12
ligand on the signal-sending cell interacts with
a
transmembrane Notch receptor on the juxtaposed signal-
receiving cell.4 This activation induces a series of proteolytic
events such as the S2 cleavage performed by the ADAM
metalloproteases, and the S3 cleavage by the gamma secretase
(GS) complex, which trigger the release of activated intra-
cellular domain of Notch and its translocation to the nucleus.5
The subsequent interaction with the DNA binding protein CSL
(CBF1/Suppressor of Hairless/LAG-1; also known as RBPJ)
and Mastermind like protein (MAML) promotes the
transcription of Notch downstream target genes.5 Aberrant
Notch activation due to either gene mutation or amplification,
or to post-translational modifications has been linked to the
onset of different solid and hematological cancers, including
T-cell acute lymphoblastic leukemia (T-ALL). 60% of T-ALL
cases are associated with activating NOTCH1 mutations and
Recently, an in house library of natural products and their
derivatives was used as a source of potential inhibitors of the
Notch signaling in T-ALL.13, 14 Eight representative molecules
of the library were selected through a cheminformatics
approach and tested in vitro. The chalcone scaffold emerged as
a promising tool to inhibit Notch signaling, and in particular
the synthesis of several chalcones combined with their
biological evaluation highlighted the chalcone 8 as the most
potent Notch blocking agent of the series, suggesting the
synergistic activity of 2’ and 4-hydroxy groups.13, 14 Herein,
we designed and synthesized a number of chalcones and
chalcone-mimetic compounds based on hit-likeness and
chemical diversity, and we evaluated their anti-proliferative
activity in KOPTK1 cells. Small molecules able to reduce cell
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growth by more than 30% were further evaluated as inhibitors
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of Notch1 by means of Western blotting assay.
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Chart 1. Chemical structures of chalcone-8 derivatives (1-7) and chalcone mimetics (9-18).
Results And Discussion. Synthesis of chalcones and
chalcone-mimetic derivatives. The general synthetic approach
to efficiently yield chalcone scaffold bearing substituents at
different positions (1-7) was based on the Claisen-Schmidt
condensation as previously reported (Chart 1).13,15 For the
synthesis of the chalcone-mimetics (9-18) (Chart 1), the
bicyclic acids (19a-c) were used to perform the Friedel-Crafts
acylation of some arenes (20a-d) by using two different
procedures (Scheme 1).
acid,16 the ketone 9 was obtained in 70% yield. When
resorcinol (20c) was used as the arene, compound 11 was
obtained in 67% yield, so that the heating conditions favor the
formation of the C-acylated product.17 Removal of the
methoxy groups in 9 by using BBr318 led to the formation of a
mixture of 12 and 13 in 39% and 48% yields, respectively.
The trimethoxy derivative 14 was obtained in low yield
probably due to steric hindrance. On the other hand, the direct
acylation reaction of the fluoroarene 20d with the acid 19a by
adapting previous reports using TFAA/TfOH,19, 20 allowed the
formation of the regioisomers 15 and 16, as shown in Scheme
1. Similarly, reaction of the 7-methoxychromane-3-carboxylic
acid 19b or 7-methoxy-1,2,3,4-tetrahydronaphthalene-2-
carboxylic acid 19c with 20a in the presence of TFAA/TfOH
afforded the aryl derivatives 17 and 18 in 38% and 73% yield,
respectively.
In all cases, compounds 9-18 were obtained as racemates.
Conformation of chalcone 8 and chalcone mimetics. E-
Chalcones can adopt s-cis or s-trans conformations.21 Non-
substituted chalcones at the α-position adopt, in general, a s-
cis conformation and this was confirmed for the chalcone 8 by
2D-NOESY experiments (Figure S1). The incorporation of a
bicyclic ring in the structure of the chalcone-mimetics
(exemplified by compound 12 in Figure 1), where the double
bond conjugated to the enone has been removed, is meant to
restrict the conformational freedom of these analogues. The
energy minimized conformers (see computational methods
S20) of the 12-R and 12-S enantiomers present in the racemic
mixture were constructed and superimposed with the X-Ray
crystal structure of a model s-cis chalcone (that is, benzylidene
acetophenone, CCDC 1185311).22 As shown in Figure 1, both
enantiomers are able to locate the OH-substituent on ring B
close to the 4-position of ring B in the s-cis chalcone. Thus,
the compounds were assayed as racemic mixtures.
Figure 1. Superposition of the crystal structure of benzylidene
acetophenone (green) with energy minimized conformers of 12-S
(magenta) and 12-R (yellow). Atom pairs used for the
superimposition are indicated with black arrows.
The ketone 9 was prepared by the reaction between the
activated form of 1,2,3,4-tetrahydronaphthalene-2-carboxylic
acid 19a with 1,3-dimethoxybenzene 20a in the presence of
BF3·OEt2. Since the catalyst induced the partial demethylation
of the methoxy group vicinal to the ketone, the reaction
afforded a mixture of 9 and 10 with 31% and 42% yields,
respectively. Otherwise, performing the reaction with triflic
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Scheme 1. Synthesis of the chalcone-mimetics (9-18). Reagents and conditions: a) (1) SOCl2, toluene, reflux, 3 h; (2) BF3·OEt2,
ClCH2CH2Cl anh., 60 ºC, 16 h; b) (1) SOCl2, toluene, reflux, 3 h; (2) TfOH, ClCH2CH2Cl anh., 60 ºC, 1 h; c) BBr3, anhydrous CH2Cl2, 4
°C, 48 h; (d) TFAA, TfOH, ClCH2CH2Cl anh, 15 min, rt.
Biological Results and Discussion. To evaluate the
effectiveness of chalcones 1-7 and chalcone-mimetics 9-18,
we investigated their growth-inhibitory potency in KOPTK1
T-ALL cell line. These cells are sensitive to Notch inhibition
by GSI and harbor activating Notch1 gene mutations.7As we
demonstrated previously, in this cell line the reference
compound 8 inhibited Notch1 signaling and cell growth in the
concentration range between 1 and 2.5 μM.13 Therefore, we
compared the effects of compounds 1-7 and 9-18 in KOPTK1
cells on the endogenous levels of the activated domain of
Notch1 (N1Val) by western blotting and on cell growth by
MTS assay treating the cells with 2.5 μM of each compound
for 36 h and taking 30% decrease of cell viability as a
threshold value for further screening (Figure 2a-b and S2).
Notably, the comparison of the viability data and the N1Val
levels after exposure to all chalcone derivatives confirmed a
remarkably positive correlation between N1Val protein levels
and the sensitivity of KOPTK1 cells to the bioactive
compounds 1, 5, 9, 12 and 18 indicating that their
antiproliferative effects were tightly associated with Notch
signaling inhibition (Figure 2c). Further proving Notch
inhibition, selected compounds decreased the N1Val protein
expression and the endogenous mRNA levels of the Notch
target gene DELTEX1 (DTX1). Interestingly, among them,
only compounds 1 and 18 showed Notch1-inhibitory effects
comparable to that of compound 8, whereas the Notch
inhibition exerted by 5, 9 and 12 was weaker than that of
compound 8 (Figure 2d and S3). In addition, we determined
the half-maximal growth-inhibitory concentration (IC50) in a
statistical analysis of single-point comparative treatment
showed no significant differences in the inhibition of cell
growth between compounds 8, 1, 9, and 18, which provided a
comparable effect at 2.5 μM concentration. The slightly higher
IC50 values of 5 and 12 are in line with their weaker effect on
Notch1 inhibition (Figure 2a, 2d and S3). Determination of the
IC50 value for compound 1 was not possible due to irregular
shape of its growth inhibition curve (Figure S4). Moreover,
consistent with the effect induced by chalcone 8 in G1-phase
cell cycle arrest and apoptosis,13 we found that growth
inhibition by 1 was accompanied by both increased levels of
the anti-proliferative factor p27kip and of the apoptotic marker
cleaved poly ADP-ribose polymerase (CL PARP). On the
contrary, the most promising chalcone-mimetics 9 and 18
failed to upregulate p27kip, and promoted the highest levels of
PARP cleavage among tested compounds (Figure 2f), whereas
the weakest Notch inhibitors 5 and 12 induced p27kip without
affecting PARP processing (Figure 2f). Finally, chalcone-
mimetics 9 and 18 affected cell proliferation also in GSI-resistant
JURKAT cell line23 and in immortalized non-tumorigenic human
keratinocyte HaCat cells in which Notch signaling acts as onco-
suppressor.24 These latter observations suggest different
mechanisms of action and/or off-target effects by chalcones and
chalcone-mimetics (Figure S5).
SAR of chalcones and chalcone-mimetic derivatives as
Notch inhibitors. Structure-Activity Relationships (SAR) of a
previous series of chalcone derivatives revealed how the 2’-
and 4-hydroxy groups are essential for both cell proliferation
and Notch inhibition.13 The biological results obtained with
the chalcones described in this work confirmed the previous
SAR.
dose-response
proliferation
assay
with
increasing
concentrations (namely, 0, 0.5, 1, 2.5, 5, 10, 20, 50 μM) of
selected compounds 1, 5, 9, 12 and 18. These molecules
inhibited leukemic cell growth to a similar extent each other,
with IC50 values comparable to that of the reference compound
8 and ranging from 0.7 to 3.8 µM (Figure 2e). Notably,
Accordingly, the effectiveness of the chalcones 1 and 5 on
growth inhibition of KOPTK1 T-ALL cell line highlighted the
pivotal role of 4-OH substitution to ring B (Figure 2a).
However, the western blotting assay on N1Val expression
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N1Val expression obtained with optical densitometry and cell
using chalcone 8 as the reference compound showed a
comparable inhibitory effect just for 1 (Figure 2b). These
results reinforced the previous evidences also for the ring A
showing how the lack of 4-OH substitution led to a weaker
inhibitory activity towards activated Notch1. The introduction
of 3-CH3 group at ring B did not influence the inhibition of
endogenous Notch signaling activity and cell growth.
Chalcone-mimetics 9, 12 and 18 proved to decrease the cell
viability of KOPTK1 T-ALL cells with IC50 values in the low-
up to sub-micromolar range (Figure 2c). While compound 12,
with a substitution pattern at rings A and B similar to that of
the reference chalcone 8 showed some Notch-inhibitory
effects, the best inhibition of Notch1 intracellular domain in
this series was obtained by compound 18 (Figure 2b). Thus, it
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viability in KOPTK1 treated with compounds 1–18 vs DMSO.
R=0.9535, R2=0.9092, p<0.0001. d) N1Val and β-actin protein
expression levels (lower panel) and Deltex1 (DTX1) relative gene
expression levels (upper panel) in KOPTK1 cells treated with 2.5
μM of compounds 8, 1, 5, 9, 12, and 18 or vehicle alone (DMSO)
for 36 h. Charts data represent mean values for three independent
experiments performed in triplicate ± SEM. (e) IC50 values of
compounds 8, 1, 5, 9, 12, and 18 determined in KOPTK1 after 36
h of incubation. Data represent mean values for three independent
experiments performed in triplicate ± S.D. (f) Protein expression
of p27kip, of the not processed PARP (FL PARP) and its cleaved
form (CL PARP). β-actin is used as loading control.
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Conclusion. Based on previous evidences and molecular
modeling studies, several chalcones 1-7 and chalcone
mimetics 9-18 were selected, synthesized and tested towards
anti-proliferative activity and Notch inhibition in KOPTK1 T-
ALL cells. Compounds 1 and 18 exhibited cell growth
reduction combined with inhibition of Notch1 intracellular
domain, mostly comparable to the reference compound 8.
SAR analysis of the chalcone series confirmed the key role
played by the 2’ and 4-OH groups on both anti-proliferative
and Notch1 inhibitory activity. However, the introduction of
bicyclic ring in the structure of the chalcone-mimetics,
promotes Notch inhibition through different biological
responses, thus suggesting that more in-deep investigations are
needed to further develop tetrahydronaphthalene derivatives as
Notch blocking agents.
is interesting to note that compounds incorporating
a
tetrahydronaphthalene scaffold are also able to exert inhibition
of the Notch pathway, although with some differences
compared to chalcones.
ASSOCIATED CONTENT
Supporting Information
Experimental details for synthetic procedures, associated chemical
data for compounds 1-18, computational methods and biological
assays. The Supporting Information is available free of charge on
the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
* (MM) Phone +390577234256 E-mail: m.mattia79@gmail.com.
* (FG) Phone +390649693318. E-mail: francesca.ghirga@iit.it.
* (RP) Phone +390649255142. Email:
rocco.palermo@uniroma1.it
Present Addresses
Viviana Cuartas Present address: Grupo de Investigación de
Compuestos Heterocíclicos, Departamento de Química,
Universidad del Valle, A.A. 25360 Cali, Colombia.
viviana.cuartas@correounivalle.edu.co. Nadezda Zhdanovskaya
Present address: Department of Physics, University of Rome
"Sapienza", Rome, Italy.
Figure 2. Anti-proliferative and molecular effects of chalcones
and chalcone-like derivatives in KOPTK1 T-ALL cells. (a)
Protein expression levels of activated domain of Notch1 (N1Val)
in KOPTK1 cells treated for 36 h with 2.5 μM of compounds 1–
18 or vehicle alone (DMSO) β-actin is used as loading control. (b)
Histogram shows the mean of relative cell viability of KOPTK1
cells treated with 2.5 μM of compounds 1–18 or DMSO for 36 h
measured with MTS assay ± SEM obtained from two independent
experiments performed in triplicate. ****P<0.0001, comparing
with DMSO. (c) Representative graph showing the results of
Pearson’s correlation test between the average values of relative
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the
manuscript. / ‡These authors contributed equally.
Funding Sources
This Work has been partially supported by a grant from
MINECO/FEDER SAF2015-64629-C2-1-R (to M-J. P.-P and E-
M. P). MIUR PNR 2015-2020 (ARS01_00432) (to I.S.) Sapienza
University 2016 Project number RG116154E2C7A6FB (to I.S.).
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linfoblastica acuta
PCT/IB2017/058204 2017. Deposito 20-12-2017.
a
cellule T.
Domanda internazionale nr.
Notes
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The authors declare no competing financial interest.
(15) Moriello, A. S.; Luongo, L.; Guida, F.; Christodoulou, M. S.;
Perdicchia, D.; Maione, S.; Passarella, D.; Marzo, V. D.; De
Petrocellis, L. Chalcone Derivatives Activate and Desensitize the
Transient Receptor Potential Ankyrin 1 Cation Channel, Subfamily A,
Member 1 TRPA1 Ion Channel: Structure-Activity Relationships in
vitro and Anti-Nociceptive and Anti-inflammatory Activity in vivo.
CNS Neurol. Disord. Drug Targets 2016, 15, 987–994.
(16) Bueno, O.; Tobajas, G.; Quesada, E.; Estévez-Gallego, J.;
Noppen, S.; Camarasa, M.-J.; Díaz, J.-F.; Liekens, S.; Priego, E.-M.;
Pérez-Pérez, M.-J. Conformational mimetics of the α-methyl chalcone
TUB091 binding tubulin: Design, synthesis and antiproliferative
activity. Eur. J. Med. Chem. 2018, 148, 337-348.
ACKNOWLEDGMENT
The authors acknowledge networking contribution by the COST
Action CM1407 “Challenging organic syntheses inspired by
nature - from natural products chemistry to drug discovery”. V.C.
thanks the Universidad del Valle (CIAM-2017) and the Science,
Technology and Innovation Fund-General Royalties System
(FCTeI-SGR) under contract BPIN 2013000100007 for
a
9
predoctoral fellowship. This work has been supported by Italian
Ministry of Education, University and Research - Dipartimenti di
Eccellenza - L. 232/2016.
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(17) Adogla, E. A.; Janser, R. F. J.; Fairbanks, S. S.; Vortolomei,
C. M.; Meka, R. K.; Janser, I. Selective methoxy ether cleavage of
2,6-dimethoxyphenol followed by a selective acylation. Tetrahedron
Lett. 2012, 53, 11-14.
(18) McOmie, J. F. W.; Watts, M. L.; West, D. E. Demethylation
of aryl methyl ethers by boron tribromide. Tetrahedron 1968, 24,
2289-2292.
ABBREVIATIONS
ADAM,
a disintegrin and metalloproteinase; GS, gamma
secretase; GSI, gamma secretases inhibitor; CSL, CBF-
1/SuH/Lag-1; MAML, mastermind-like; T-ALL, T-cell acute
lymphoblastic leukemia.
(19) Tachrim, Z.; Wang, L.; Murai, Y.; Yoshida, T.; Kurokawa,
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Graphical abstract
76x55mm (300 x 300 DPI)
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