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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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Page 1 of 7 ACS Medicinal Chemistry Letters

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7 Chalcones and chalcone-mimetic derivatives as Notch inhibitors in a
8 model of T-cell acute lymphoblastic leukemia
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10 Deborah Quaglio,†‡ Nadezda Zhdanovskaya,ǂ‡ Gloria Tobajas,§ Viviana Cuartas,§ Silvia Balducci,†
11 Michael S. Christodoulou,ǁ Giancarlo Fabrizi,† Marta Gargantilla,§ Eva-María Priego,§ Álvaro Carmona
12
13
Pestaña,ǂ Daniele Passarella,┴ Isabella Screpanti,ǂ Bruno Botta,† Rocco Palermo,ǂ* Mattia Mori,#*
14 Francesca Ghirga,V* María-Jesús Pérez-Pérez§
15 †Department of Chemistry and Technology of Drugs “department of excellence 2018-2022”, Sapienza Università di Roma,
16 P.le Aldo Moro 5, 00185 Rome, Italy
17 ǂ Department of Molecular Medicine, Sapienza University, 00161 Rome, Italy
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§ Instituto de Química Médica-CSIC, Juan de la Cierva 3, 28006 Madrid , Spain
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20 ǁ DISFARM, Sezione di Chimica Generale e Organica “A. Marchesini”, Università degli Studi di Milano, Via Venezian 21,

21 20133 Milano, Italy

22 ┴ Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi, 19, 20133 Milano, Italy

23 # Department of Biotechnology, Chemistry and Pharmacy “department of excellence 2018-2022”, University of Siena, via
24 Aldo Moro 2, I-53100, Siena, Italy
25 V Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy
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27 KEYWORDS: Chalcone, tetrahydronaphthalene scaffold, Notch inhibitors, T-ALL, cancer.
28
29 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
30
expression in KOPTK1 cell line identified compound 18, featuring a tetrahydronaphthalene-based scaffold, as a new promising
31 Notch-blocking agent.
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35 Notch signaling is an evolutionarily conserved cell the majority of T-ALL patients shows increased expression
36 communication pathway regulating many biological processes and function of Notch3.6,7,8 Moreover, Notch signaling may
37 including stem cells self-renewal, cell differentiation, contribute to chemotherapy resistance, thus suggesting Notch
38 proliferation and survival in different tissues during embryonic inhibition as a strategy in anticancer targeted therapy.9, 10 (
and adult life.1,2,3 The mammalian Notch family includes four Notwithstanding, small molecules (e.g. GS inhibitors (GSI)
39
trans-membrane receptors (namely, Notch1–4), and five trans- such as MK-0752, RO4929097 and PF-03084014) and large
40 membrane ligands (namely, Jagged-1 and -2, Delta-like-1, -3 molecules (e.g. Notch1-specific antibody OMP-52M51)
41 and -4). Notch signaling is a short-range inter-cellular currently in clinical development are not effective in all
42 communication system, wherein a membrane tethered Notch patients, and often cause severe side-effects that limit their
43 ligand on the signal-sending cell interacts with a widespread therapeutic use and raise the need for novel Notch-
44 transmembrane Notch receptor on the juxtaposed signal- blocking agents.11, 12
45 receiving cell.4 This activation induces a series of proteolytic
Recently, an in house library of natural products and their
46 events such as the S2 cleavage performed by the ADAM
derivatives was used as a source of potential inhibitors of the
47 metalloproteases, and the S3 cleavage by the gamma secretase
Notch signaling in T-ALL.13, 14 Eight representative molecules
48 (GS) complex, which trigger the release of activated intra-
of the library were selected through a cheminformatics
cellular domain of Notch and its translocation to the nucleus.5
49 approach and tested in vitro. The chalcone scaffold emerged as
The subsequent interaction with the DNA binding protein CSL
50 (CBF1/Suppressor of Hairless/LAG-1; also known as RBPJ)
a promising tool to inhibit Notch signaling, and in particular
51 the synthesis of several chalcones combined with their
and Mastermind like protein (MAML) promotes the
52 biological evaluation highlighted the chalcone 8 as the most
transcription of Notch downstream target genes.5 Aberrant
53 potent Notch blocking agent of the series, suggesting the
Notch activation due to either gene mutation or amplification,
synergistic activity of 2’ and 4-hydroxy groups.13, 14 Herein,
54 or to post-translational modifications has been linked to the
we designed and synthesized a number of chalcones and
55 onset of different solid and hematological cancers, including
chalcone-mimetic compounds based on hit-likeness and
56 T-cell acute lymphoblastic leukemia (T-ALL). 60% of T-ALL
chemical diversity, and we evaluated their anti-proliferative
57 cases are associated with activating NOTCH1 mutations and
activity in KOPTK1 cells. Small molecules able to reduce cell
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growth by more than 30% were further evaluated as inhibitors

1 of Notch1 by means of Western blotting assay.
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22 Chart 1. Chemical structures of chalcone-8 derivatives (1-7) and chalcone mimetics (9-18).
23 Results And Discussion. Synthesis of chalcones and acid,16 the ketone 9 was obtained in 70% yield. When
24 chalcone-mimetic derivatives. The general synthetic approach resorcinol (20c) was used as the arene, compound 11 was
25 to efficiently yield chalcone scaffold bearing substituents at obtained in 67% yield, so that the heating conditions favor the
26 different positions (1-7) was based on the Claisen-Schmidt formation of the C-acylated product.17 Removal of the
27 condensation as previously reported (Chart 1).13,15 For the methoxy groups in 9 by using BBr318 led to the formation of a
28 synthesis of the chalcone-mimetics (9-18) (Chart 1), the mixture of 12 and 13 in 39% and 48% yields, respectively.
29 bicyclic acids (19a-c) were used to perform the Friedel-Crafts The trimethoxy derivative 14 was obtained in low yield
30 acylation of some arenes (20a-d) by using two different probably due to steric hindrance. On the other hand, the direct
procedures (Scheme 1). acylation reaction of the fluoroarene 20d with the acid 19a by
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adapting previous reports using TFAA/TfOH,19, 20 allowed the
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formation of the regioisomers 15 and 16, as shown in Scheme
33 1. Similarly, reaction of the 7-methoxychromane-3-carboxylic
34 acid 19b or 7-methoxy-1,2,3,4-tetrahydronaphthalene-2-
35 carboxylic acid 19c with 20a in the presence of TFAA/TfOH
36 afforded the aryl derivatives 17 and 18 in 38% and 73% yield,
37 respectively.
38 In all cases, compounds 9-18 were obtained as racemates.
39 Conformation of chalcone 8 and chalcone mimetics. E-
40 Chalcones can adopt s-cis or s-trans conformations.21 Non-
41 substituted chalcones at the α-position adopt, in general, a s-
42 cis conformation and this was confirmed for the chalcone 8 by
43 2D-NOESY experiments (Figure S1). The incorporation of a
44 bicyclic ring in the structure of the chalcone-mimetics
45 (exemplified by compound 12 in Figure 1), where the double
46 bond conjugated to the enone has been removed, is meant to
restrict the conformational freedom of these analogues. The
47
Figure 1. Superposition of the crystal structure of benzylidene energy minimized conformers (see computational methods
48 S20) of the 12-R and 12-S enantiomers present in the racemic
acetophenone (green) with energy minimized conformers of 12-S
49 (magenta) and 12-R (yellow). Atom pairs used for the mixture were constructed and superimposed with the X-Ray
50 superimposition are indicated with black arrows. crystal structure of a model s-cis chalcone (that is, benzylidene
51 acetophenone, CCDC 1185311).22 As shown in Figure 1, both
52 The ketone 9 was prepared by the reaction between the enantiomers are able to locate the OH-substituent on ring B
activated form of 1,2,3,4-tetrahydronaphthalene-2-carboxylic
53 close to the 4-position of ring B in the s-cis chalcone. Thus,
acid 19a with 1,3-dimethoxybenzene 20a in the presence of the compounds were assayed as racemic mixtures.
54 BF3·OEt2. Since the catalyst induced the partial demethylation
55 of the methoxy group vicinal to the ketone, the reaction
56 afforded a mixture of 9 and 10 with 31% and 42% yields,
57 respectively. Otherwise, performing the reaction with triflic
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23 Scheme 1. Synthesis of the chalcone-mimetics (9-18). Reagents and conditions: a) (1) SOCl2, toluene, reflux, 3 h; (2) BF3·OEt2,
24 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
25 °C, 48 h; (d) TFAA, TfOH, ClCH2CH2Cl anh, 15 min, rt.
26 Biological Results and Discussion. To evaluate the statistical analysis of single-point comparative treatment
27 effectiveness of chalcones 1-7 and chalcone-mimetics 9-18, showed no significant differences in the inhibition of cell
28 we investigated their growth-inhibitory potency in KOPTK1 growth between compounds 8, 1, 9, and 18, which provided a
29 T-ALL cell line. These cells are sensitive to Notch inhibition comparable effect at 2.5 μM concentration. The slightly higher
30 by GSI and harbor activating Notch1 gene mutations.7As we IC50 values of 5 and 12 are in line with their weaker effect on
demonstrated previously, in this cell line the reference Notch1 inhibition (Figure 2a, 2d and S3). Determination of the
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compound 8 inhibited Notch1 signaling and cell growth in the IC50 value for compound 1 was not possible due to irregular
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concentration range between 1 and 2.5 μM.13 Therefore, we shape of its growth inhibition curve (Figure S4). Moreover,
33 compared the effects of compounds 1-7 and 9-18 in KOPTK1 consistent with the effect induced by chalcone 8 in G1-phase
34 cells on the endogenous levels of the activated domain of cell cycle arrest and apoptosis,13 we found that growth
35 Notch1 (N1Val) by western blotting and on cell growth by inhibition by 1 was accompanied by both increased levels of
36 MTS assay treating the cells with 2.5 μM of each compound the anti-proliferative factor p27kip and of the apoptotic marker
37 for 36 h and taking 30% decrease of cell viability as a cleaved poly ADP-ribose polymerase (CL PARP). On the
38 threshold value for further screening (Figure 2a-b and S2). contrary, the most promising chalcone-mimetics 9 and 18
39 Notably, the comparison of the viability data and the N1Val failed to upregulate p27kip, and promoted the highest levels of
40 levels after exposure to all chalcone derivatives confirmed a PARP cleavage among tested compounds (Figure 2f), whereas
41 remarkably positive correlation between N1Val protein levels the weakest Notch inhibitors 5 and 12 induced p27kip without
and the sensitivity of KOPTK1 cells to the bioactive affecting PARP processing (Figure 2f). Finally, chalcone-
42 mimetics 9 and 18 affected cell proliferation also in GSI-resistant
compounds 1, 5, 9, 12 and 18 indicating that their
43 antiproliferative effects were tightly associated with Notch JURKAT cell line23 and in immortalized non-tumorigenic human
44 signaling inhibition (Figure 2c). Further proving Notch keratinocyte HaCat cells in which Notch signaling acts as onco-
45 inhibition, selected compounds decreased the N1Val protein suppressor.24 These latter observations suggest different
46 expression and the endogenous mRNA levels of the Notch mechanisms of action and/or off-target effects by chalcones and
47 target gene DELTEX1 (DTX1). Interestingly, among them, chalcone-mimetics (Figure S5).
48 only compounds 1 and 18 showed Notch1-inhibitory effects SAR of chalcones and chalcone-mimetic derivatives as
49 comparable to that of compound 8, whereas the Notch Notch inhibitors. Structure-Activity Relationships (SAR) of a
50 inhibition exerted by 5, 9 and 12 was weaker than that of previous series of chalcone derivatives revealed how the 2’-
51 compound 8 (Figure 2d and S3). In addition, we determined and 4-hydroxy groups are essential for both cell proliferation
the half-maximal growth-inhibitory concentration (IC50) in a and Notch inhibition.13 The biological results obtained with
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dose-response proliferation assay with increasing the chalcones described in this work confirmed the previous
53 concentrations (namely, 0, 0.5, 1, 2.5, 5, 10, 20, 50 μM) of SAR.
54 selected compounds 1, 5, 9, 12 and 18. These molecules Accordingly, the effectiveness of the chalcones 1 and 5 on
55 inhibited leukemic cell growth to a similar extent each other, growth inhibition of KOPTK1 T-ALL cell line highlighted the
56 with IC50 values comparable to that of the reference compound pivotal role of 4-OH substitution to ring B (Figure 2a).
57 8 and ranging from 0.7 to 3.8 µM (Figure 2e). Notably, However, the western blotting assay on N1Val expression
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using chalcone 8 as the reference compound showed a N1Val expression obtained with optical densitometry and cell
1 comparable inhibitory effect just for 1 (Figure 2b). These viability in KOPTK1 treated with compounds 1–18 vs DMSO.
2 results reinforced the previous evidences also for the ring A R=0.9535, R2=0.9092, p<0.0001. d) N1Val and β-actin protein
3 showing how the lack of 4-OH substitution led to a weaker expression levels (lower panel) and Deltex1 (DTX1) relative gene
inhibitory activity towards activated Notch1. The introduction expression levels (upper panel) in KOPTK1 cells treated with 2.5
4
of 3-CH3 group at ring B did not influence the inhibition of μM of compounds 8, 1, 5, 9, 12, and 18 or vehicle alone (DMSO)
5 for 36 h. Charts data represent mean values for three independent
6 endogenous Notch signaling activity and cell growth.
Chalcone-mimetics 9, 12 and 18 proved to decrease the cell experiments performed in triplicate ± SEM. (e) IC50 values of
7 compounds 8, 1, 5, 9, 12, and 18 determined in KOPTK1 after 36
viability of KOPTK1 T-ALL cells with IC50 values in the low-
8 up to sub-micromolar range (Figure 2c). While compound 12,
h of incubation. Data represent mean values for three independent
9 experiments performed in triplicate ± S.D. (f) Protein expression
with a substitution pattern at rings A and B similar to that of of p27kip, of the not processed PARP (FL PARP) and its cleaved
10 the reference chalcone 8 showed some Notch-inhibitory form (CL PARP). β-actin is used as loading control.
11 effects, the best inhibition of Notch1 intracellular domain in
12 this series was obtained by compound 18 (Figure 2b). Thus, it Conclusion. Based on previous evidences and molecular
13 is interesting to note that compounds incorporating a modeling studies, several chalcones 1-7 and chalcone
14 tetrahydronaphthalene scaffold are also able to exert inhibition mimetics 9-18 were selected, synthesized and tested towards
15 of the Notch pathway, although with some differences anti-proliferative activity and Notch inhibition in KOPTK1 T-
compared to chalcones. ALL cells. Compounds 1 and 18 exhibited cell growth
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reduction combined with inhibition of Notch1 intracellular
17 domain, mostly comparable to the reference compound 8.
18 SAR analysis of the chalcone series confirmed the key role
19 played by the 2’ and 4-OH groups on both anti-proliferative
20 and Notch1 inhibitory activity. However, the introduction of
21 bicyclic ring in the structure of the chalcone-mimetics,
22 promotes Notch inhibition through different biological
23 responses, thus suggesting that more in-deep investigations are
24 needed to further develop tetrahydronaphthalene derivatives as
25 Notch blocking agents.
26
27
ASSOCIATED CONTENT
28
29 Supporting Information
30 Experimental details for synthetic procedures, associated chemical
31 data for compounds 1-18, computational methods and biological
32 assays. The Supporting Information is available free of charge on
33 the ACS Publications website.
34 AUTHOR INFORMATION
35
36 Corresponding Author
37 * (MM) Phone +390577234256 E-mail: m.mattia79@gmail.com.
38 * (FG) Phone +390649693318. E-mail: francesca.ghirga@iit.it.
39 * (RP) Phone +390649255142. Email:
rocco.palermo@uniroma1.it
40
41 Present Addresses
42
Viviana Cuartas Present address: Grupo de Investigación de
43 Compuestos Heterocíclicos, Departamento de Química,
44 Universidad del Valle, A.A. 25360 Cali, Colombia.
45 viviana.cuartas@correounivalle.edu.co. Nadezda Zhdanovskaya
46 Present address: Department of Physics, University of Rome
47 "Sapienza", Rome, Italy.
48 Figure 2. Anti-proliferative and molecular effects of chalcones
and chalcone-like derivatives in KOPTK1 T-ALL cells. (a) Author Contributions
49
Protein expression levels of activated domain of Notch1 (N1Val) The manuscript was written through contributions of all authors. /
50 in KOPTK1 cells treated for 36 h with 2.5 μM of compounds 1– All authors have given approval to the final version of the
51 18 or vehicle alone (DMSO) β-actin is used as loading control. (b) manuscript. / ‡These authors contributed equally.
52 Histogram shows the mean of relative cell viability of KOPTK1
53 cells treated with 2.5 μM of compounds 1–18 or DMSO for 36 h Funding Sources
54 measured with MTS assay ± SEM obtained from two independent This Work has been partially supported by a grant from
55 experiments performed in triplicate. ****P<0.0001, comparing MINECO/FEDER SAF2015-64629-C2-1-R (to M-J. P.-P and E-
with DMSO. (c) Representative graph showing the results of M. P). MIUR PNR 2015-2020 (ARS01_00432) (to I.S.) Sapienza
56
Pearson’s correlation test between the average values of relative University 2016 Project number RG116154E2C7A6FB (to I.S.).
57
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Notes linfoblastica acuta a cellule T. Domanda internazionale nr.

1 The authors declare no competing financial interest. PCT/IB2017/058204 2017. Deposito 20-12-2017.
2 (15) Moriello, A. S.; Luongo, L.; Guida, F.; Christodoulou, M. S.;
ACKNOWLEDGMENT Perdicchia, D.; Maione, S.; Passarella, D.; Marzo, V. D.; De
3 Petrocellis, L. Chalcone Derivatives Activate and Desensitize the
4 The authors acknowledge networking contribution by the COST Transient Receptor Potential Ankyrin 1 Cation Channel, Subfamily A,
5 Action CM1407 “Challenging organic syntheses inspired by Member 1 TRPA1 Ion Channel: Structure-Activity Relationships in
6 nature - from natural products chemistry to drug discovery”. V.C. vitro and Anti-Nociceptive and Anti-inflammatory Activity in vivo.
thanks the Universidad del Valle (CIAM-2017) and the Science, CNS Neurol. Disord. Drug Targets 2016, 15, 987–994.
7 Technology and Innovation Fund-General Royalties System (16) Bueno, O.; Tobajas, G.; Quesada, E.; Estévez-Gallego, J.;
8 (FCTeI-SGR) under contract BPIN 2013000100007 for a Noppen, S.; Camarasa, M.-J.; Díaz, J.-F.; Liekens, S.; Priego, E.-M.;
9 predoctoral fellowship. This work has been supported by Italian Pérez-Pérez, M.-J. Conformational mimetics of the α-methyl chalcone
10 Ministry of Education, University and Research - Dipartimenti di TUB091 binding tubulin: Design, synthesis and antiproliferative
11 Eccellenza - L. 232/2016. activity. Eur. J. Med. Chem. 2018, 148, 337-348.
(17) Adogla, E. A.; Janser, R. F. J.; Fairbanks, S. S.; Vortolomei,
12 C. M.; Meka, R. K.; Janser, I. Selective methoxy ether cleavage of
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15 1/SuH/Lag-1; MAML, mastermind-like; T-ALL, T-cell acute of aryl methyl ethers by boron tribromide. Tetrahedron 1968, 24,
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30 Graphical abstract
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32 76x55mm (300 x 300 DPI)
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60 ACS Paragon Plus Environment