# Rational Design and Synthesis of Highly Stable Haloflavanone DNA Methyltransferase Inhibitors Inducing Tumor Suppressor Gene Re-expression in Cancer Cells

Francesco Calzaferri, Hiba Daher, Julie Gilbert, Yinshan Yang, Marine Tauziet, Corinne Jallet, Yannick Bessin, Arie van der Lee, Paola Barbara Arimondo, Isabelle Krimm, et al.

► To cite this version:

Francesco Calzaferri, Hiba Daher, Julie Gilbert, Yinshan Yang, Marine Tauziet, et al. Rational Design and Synthesis of Highly Stable Haloflavanone DNA Methyltransferase Inhibitors Inducing Tumor Suppressor Gene Re-expression in Cancer Cells. Journal of Medicinal Chemistry, 2025, 68 (11), pp.10704-10721. 10.1021/acs.jmedchem.4c02075. pasteur-05248866

HAL Id: pasteur-05248866

https://pasteur.hal.science/pasteur-05248866v1

Submitted on 7 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Rational design and synthesis of highly stable haloflavanone DNMT inhibitors inducing tumour suppressor gene re-expression in cancer cells

Francesco Calzaferri,<sup>1*</sup> Hiba Daher,<sup>2</sup> Julie Gilbert,<sup>1</sup> Yinshan Yang,<sup>3</sup> Marine Tauziet,<sup>2</sup> Corinne Jallet,<sup>4</sup>

Yannick Bessin,<sup>1</sup> Arie van der Lee,<sup>5</sup> Paola Barbara Arimondo,<sup>4</sup> Isabelle Krimm,<sup>3,6</sup> Eric Julien,<sup>2,7</sup> Marie

Lopez<sup>1*</sup>

<sup>1</sup> Institut des Biomolécules Max Mousseron (IBMM), UMR5247 CNRS-UM-ENSCM, 1919 route de

Mende 34090 Montpellier cedex 5, France

<sup>2</sup> Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM-UM-ICM, 08 avenue des

Apothicaires 34298 Montpellier cedex 5, France

<sup>3</sup>Centre de Biologie Structurale (CBS), CNRS-INSERM-UM, 34090 Montpellier, France

<sup>4</sup> Epigenetic Chemical Biology, Department of Structural Biology and Chemistry, Institut Pasteur,

Université Paris Cité, CNRS UMR 3523 Chem4Life, F-75015, Paris, France.

<sup>5</sup> Institut Européen des Membranes (IEM), UMR 5635 CNRS-UM, 300 avenue du Prof. E. Jeanbrau,

34296 Montpellier cedex 5, France

<sup>6</sup> Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard,

Centre de recherche en cancérologie de Lyon, 69373 Lyon, France

<sup>7</sup> Centre National de la Recherche Scientifique (CNRS), F-34293, Montpellier, France

*Corresponding authors' emails: francesco.calzaferri@cnrs.fr, marie.lopez@cnrs.fr

Abstract

DNA methylation is an epigenetic modification involved in cancer. The clinically approved nucleoside DNA methyltransferase (DNMT) inhibitors 5-azacytidine and 5-aza-2'-deoxycytidine lack selectivity and stability, resulting in high toxicity. Previously, we discovered 3-halo-3-nitroflavanones as non-nucleoside DNMT inhibitors. Here, we designed and synthesised a new series of 2-substituted haloflavanones to increase compound chemical stability. Moreover, replacement of the nitro by an additional halogen enhanced compound potency. Indeed, compound **34b** (anti-3-bromo-3-chloro-2-methoxyflavanone) exhibited submicromolar DNMT3A inhibitory activity, upregulated the expression of DNMT-targeted genes, and impaired cell proliferation. Importantly, **34b** triggered a critical cell-cycle arrest in G1/S transition, notably in p53-depleted HCT-116 colorectal cancer cells, which paves the way for novel therapeutic opportunities. **34b** competes for the same DNMT catalytic pocket as confirmed by STD-NMR, but assuming different docking poses as shown by computational studies. Overall, the high stability and activity of **34b** make it a promising DNMT inhibitor for anticancer research and therapy.

Introduction

DNA methylation is an epigenetic modification responsible for gene expression regulation.¹ In humans

DNA methylation is catalysed by three DNA methyltransferases (DNMTs) named DNMT1,

DNMT3A, and DNMT3B. While DNMT3A and 3B play a major role in DNA *de novo* methylation,

DNMT1 is considered as the main enzyme responsible for the maintenance of DNA methylation
during cell proliferation. Additionally, the non-catalytically active DNMT3L was identified as DNMT3A and 3B regulatory protein.¹ DNMTs catalyse the methylation on cytosines of DNA that are followed by a guanine (CpG). Frequent repetition of CpG in the genome is commonly defined as CpG islands and are present in gene promoters. In several cancers, abnormal DNA methylation of tumour suppressor gene (TSG) promoters are associated with transcriptional repression, which contributes to cancer initiation and progression.²,³ This was notably shown with the transcriptional repression of *p15*, *p16*, and *MPPED2* TSGs mainly caused by improper DNA methylation in colorectal cancer.⁴,⁵ Besides, DNA hypermethylation of several genes was identified for the diagnosis and prognosis of cancer, such as for *PRAC2* and *TDRD10* in breast cancer,⁶ and was responsible for cancer resistance to conventional therapy, as found in multiple myeloma.⁷,⁸

DNMT inhibitors (DNMTi) have been employed to revert DNA hypermethylation in cancer. Particularly, nucleoside DNMTi, i.e. 5-azacytidine (Vidaza®) and 5-aza-2'-deoxycytidine (5azadC, Dacogen™), have been approved by the FDA and EMA for the treatment of myelodysplastic syndromes and acute myeloid leukaemia, validating the efficacy of DNMTi for anticancer therapy.⁹,¹⁰ However, as nucleoside analogues, these compounds are chemically unstable and their mode of action, which implies their incorporation into DNA, results in toxic *in-target* side effects. Altogether, this explains the low therapeutic window of approved DNMTi and why the search for non-nucleoside DNMTi has always been of extensive interest. In the last decade, a rich library of bisubstrate compounds with different selectivity for DNMT1 and DNMT3A have been developed (compound 70, Figure 1),¹¹ as well as DNMT1-selective dicarbazole DC_517¹² and pyridine-based GSK3685032,¹³ and DNMT3A protein-protein interaction disruptors (Figure 1).¹⁴ Among these non-nucleoside derivatives that do not present either strong activity or optimal physicochemical properties, our group developed 3-halo-3-nitroflavanones, starting from 3-chloro-3-nitroflavanones previously discovered by high-throughput screening as DNMTi.¹⁵,¹⁶ However, these optimised compounds did not reach submicromolar activity and remain globally unstable in aqueous media.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

DC_517

<figure><img src="image_4.png" /></figure>

GSK3685032

<figure><img src="image_5.png" /></figure>

DNMT3A PPI disruptor

<figure><img src="image_6.png" /></figure>

MLo1502

<figure><img src="image_7.png" /></figure>

MLo1506

<figure><img src="image_8.png" /></figure>

MLo1507

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**Figure 1.** Selection of previously described DNMTi. PPI: protein-protein interaction.

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Here, we present our new generation of flavanones as DNMTi, which present a substitution at position 2 that greatly increases compound chemical stability in water. We also show that the replacement of the nitro group at position 3 from the original series with an additional halogen further increases their potency as DNMTi. Our work resulted in chemically stable DNMTi active in the nanomolar range and capable of inducing TSG re-expression and cell cycle arrest in colorectal cancer cells.

70

# Results and discussion

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## *Design*

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The previously discovered 3-halo-3-nitroflavanones DNMTi showed fast degradation in water at room temperature to generate 3-nitroflavone as the main product.<sup>15,16</sup> To increase the chemical stability of these derivatives, we envisaged to include an additional substituent at position 2 of the flavanone scaffold to avoid the presence of the hydrogen that proved to be responsible for flavanone degradation (Figure 2). The chosen substituents were the relatively small methyl and methoxy groups to avoid any negative impact on DNMT inhibitory activity.

78
Previous works

Ceccaldi et al. 2011

<figure><img src="image_9.png" /></figure>

Major degradation product

<figure><img src="image_10.png" /></figure>

Pechalrieu et al. 2020

<figure><img src="image_11.png" /></figure>

↑ Stability (t₁/₂ = 8 h)

= Activity (IC₅₀ = 4.7 ± 1.6 µM)

Serhouni et al. 2025

<figure><img src="image_12.png" /></figure>

Y = NH or S

= Stability

= Activity

This work

<figure><img src="image_13.png" /></figure>

X = Br or Cl

<figure><img src="image_14.png" /></figure>

↑ Stability
(t₁/₂ > 1 week)

↑ Activity
(IC₅₀ = 0.78 ± 0.14 µM)

**Figure 2.** Optimisation of flavanones as DNMTi. In this work a substituent was introduced at position 2 and the nitro group was replaced with an additional halogen to increase compound stability and activity, respectively.

*Synthesis of 3-halo-2-methyl-3-nitroflavanones*

For the synthesis of 2-methylflavanones we attempted the cyclisation in basic conditions of 2'-hydroxy-2-nitroacetophenone (1) and 4'-chloroacetophenone adapting the protocol of the reaction between 1 and benzaldehyde previously employed in our laboratory¹⁶ (Scheme 1A). However, no product was observed both in ethanol and acetonitrile as solvents. By increasing the temperature to 80 °C, degradation of 1 was observed after few hours. The desired flavanones were then obtained through the aldol condensation of 2'-hydroxyacetophenone and differently substituted acetophenones in acetonitrile and pyrrolidine at 80 °C as described by Rapposelli *et al.*¹⁷ (Scheme 1B). Globally, high conversions and yields were obtained when *meta*-substituted acetophenones were used. Yields decreased with substituents in *para* and even more in *ortho* position, above all with strong electron donating or withdrawing groups (compound **8, 10, 12** – Scheme 1). This suggests that both electronic and steric effects influence the condensation and cyclisation reactions.
A

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

1

B

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

2 R = 2'-Cl (33 %)

3 3'-Cl (97 %)

4 4'-Cl (37 %)

5 2'-Br (10 %)

6 3'-Br (93 %)

7 4'-Br (52 %)

8 2'-NO₂ (1 %)

9 3'-NO₂ (28 %)

10 4'-NO₂ (4 %)

11 3'-OMe (39 %)

12 4'-OMe (23 %)

13 R = 2'-Cl X = NO₂ (24 %)

14a-b 3'-Cl Br (27 %; 37 %)

15a-b 3'-Cl Cl (45 %; 47 %)

16a-b 4'-Cl Br (17 %; 20 %)

17 2'-Br NO₂ (25 %)

18 2'-Br Br (43 %)

19a-b 3'-Br Br (25 %; 22 %)

20a-b 4'-Br Br (13 %; 18 %)

21a-b 3'-NO₂ Br (53 %; 28 %)

22a-b 3'-OMe Br (52 %; 14 %)

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**Scheme 1.** A) The cyclisation in basic conditions of **1** with an acetophenone instead of a benzaldehyde as previously described¹⁶ did not result in the desired 3-nitroflavanone. B) Final optimised conditions for the synthesis of 3-halo-2-methyl-3-nitroflavanones. a) pyrrolidine, MeCN, 80°C, 1 week; b) HNO₃, AcOH, 0°C to rt; c) NBS or NCS, Na₂CO₃, THF, rt. When specified, final diastereomers were isolated and identified (**a** = syn- and **b** = anti-isomers). Yields are reported in brackets in the form “(yield **a**; yield **b**)”. 98 99 100 101 102 103

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2-Methylflavanones were then nitrated by HNO₃ in AcOH and halogenated by NBS or NCS in basic conditions, giving 3-halo-2-methyl-3-nitroflavanones **14-16** and **18-22** (**Scheme 1B**). Surprisingly, the nitration of *ortho*-haloflavanones **2** and **5** resulted in the double nitration at position 3, giving compounds **13** and **17**. This was confirmed by comparison of their ¹³C, HSQC, and HMBC NMR spectra with those of 3-halo-3-nitroflavanones: the former ones showed the C-3 signal at 117-118 ppm, the latter at 103-104 ppm. Additionally, HRMS spectra of compound **13** showed a characteristic isotopic distribution corresponding to a chlorine atom in the molecule (**Figure S1**). Finally, the structure of **17** was confirmed by X-ray diffraction (**Figure S2**). 105 106 107 108 109 110 111 112

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Most of 2-methylflavanones were obtained as mixture of diastereomers (named **a** and **b**) that we could separate and characterise. Both diastereomers mainly differ from the chemical shift of the methyl group in ¹H NMR: for **a**-isomers the methyl signal is at a higher chemical shift than that of **b**-isomers (2.11 ± 0.04 vs 2.01 ± 0.03 ppm). X-ray diffraction of compound **21a** (**Figure 3A**) indicated that **a**-isomers have a syn configuration, where the halogen and the phenyl are on the same side of the cycle. This is also in agreement with the configuration assigned to 3-halo-3-nitroaza- and thioflavanones by Serhouni *et al.*¹⁸ 113 114 115 116 117 118 119

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<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

**Figure 3.** Crystal structures of syn-3-bromo-2-methyl-2',3-dinitroflavanone **21a** (A), anti-3-bromo-2-methoxy-3-nitroflavanone **25** (B), and syn-3-bromo-3-chloro-2-methoxyflavanone **34a** (C). Data collected by X-ray diffraction. Structures are deposited on CCDC (**21a**: 2371610; **25**: 2371605; **34a**: 2371607).

*Synthesis of 3-halo-2-methoxy-3-nitroflavanones*

For the insertion of a methoxy group at position 2 of the flavanone scaffold, the simultaneous addition of the halogen and the methoxy to the flavone double-bond was first tested (**Scheme 2A**). The reaction of the commercial flavone with NBS in MeOH at rt, as described on chromanones by Merle and Descotes,¹⁹ provided the 3-bromo-2-methoxyflavanone (23) in high yield (89%). The same reaction starting from 3-nitroflavone (24), previously synthesised from 2'-hydroxy-2-nitroacetophenone (1) and triethyl orthobenzoate in acidic conditions, furnished 3-halo-2-methoxy-3-nitroflavanones **25** and **26**, using NBS or NCS, respectively, in MeOH at 50 °C overnight (**Scheme 2B**). The reaction was stereoselective, since only one diastereomer was isolated as result of an *anti*-addition. This was validated by x-ray diffraction of **25** (*Figure 3B*), which also confirmed the correct insertion of the bromine at position 3 and the methoxy at position 2.
<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

23 (89 %)

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

24 (27 %)

<figure><img src="image_32.png" /></figure>

25 X = Br (54 %)

26 Cl (20 %)

1

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**Scheme 2.** Synthetic route for 2-methoxy-3-bromoflavanone (A) and 2-methoxy-3-halo-3-nitroflavanones (B); a) NBS or NCS, MeOH, rt or 50 °C; b) triethyl orthobenzoate, H₂SO₄, 160 °C, 4.5 h.

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### Exploring the replacement of the nitro group at position 3

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The impact of the replacement of the nitro group at position 3, which has never been explored for the 3-halo-3-nitroflavanones activity as DNMTi, was then studied. First, we replaced the nitro group with a hydroxyl through the epoxidation of the flavone double bound (27, **Scheme 3A**) and its opening with methanol (28, **Scheme 3A**). However, this strategy allowed the insertion of a methoxide at position 2 but not any further functionalisation, e.g. halogenation at position 3, and was therefore abandoned. Subsequently, we replaced the nitro with a trifluoromethyl group through the one-pot addition of the -CF₃ group *via* vinyl triflate formation as described by Kawamoto *et al.*²⁰ The obtained 3-trifluoromethaneflavanone (30) was then halogenated, giving 3-bromo-3-trifluoromethaneflavanone **31** (**Scheme 3B**). Unfortunately, this strategy did not work on 2-methylflavanones and was not compatible with the synthesis of 2-methoxyflavanones.

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A

<figure><img src="image_33.png" /></figure>

B

<figure><img src="image_34.png" /></figure>

C

<figure><img src="image_35.png" /></figure>

**Scheme 3.** Synthetic route explored for the replacement of the nitro group at position 3 of the flavanone scaffold, affording 3-hydroxy-2-methoxyflavanone (A), 3-bromo-3-trifluoromethylflavanone (B), and 2-methoxy-3-bromo-3-haloflavanones (C); a) 1,1,1-trifluoroacetone, EDTA-Na₂, oxone, NaHCO₃, MeCN, 0 °C, 5 h; b) dry MeOH, Ar, rt, 3 h; c) 2,6-ditet-butylpyridine, Tf₂O, dry dichloroethane, Ar, rt, 2 h; d) BEt₃, dry dichloroethane, rt, overnight; e) NBS, NaOH, DMF, 75 °C, overnight; f) oxone, KBr, MeOH, rt, 40 h; g) NaOH, rt, 30 min; h) NBS or NCS, MeOH, 50 °C. Final diastereomers were isolated and characterised (a representing the *syn*-isomer, and b the *anti*-one).

Finally, we replaced the nitro group with an additional halogen. 3-Bromoflavone **32** was first prepared from commercial flavone as described by Peng *et al.*²¹ Afterwards, the halogen and the methoxide were inserted by alkene addition as described for 2-methoxy-3-nitroflavanones to afford compounds **33** (crystal structure shown in **Figure S3**) and **34** in good yields (**Scheme 3C**). The insertion of both halogens was confirmed by the isotopic distribution observed by HRMS (Figure SX). In this case, the alkene addition is not stereoselective, contrary to what was observed using 3-nitroflavone **24** as starting material (**Scheme 2B**). In fact, both diastereomers of **34** were isolated. **34a** is the *syn*-diastereomer obtained by a typical *anti*-addition mechanism, as confirmed by X-ray diffraction (**Figure 3C**). The isolation of **34b** suggests that the lack of a nitro group at position 3 induces the halonium ion intermediate to open, giving a carbocation at position 2 that is stabilised by resonance with the phenyl group, resulting in a *syn*-addition.

### Chemical stability assay

Compound stability was assessed in HEPES buffer by HPLC to test the hypothesis that 2-substituted flavanones are chemically more stable than previous described 3-halo-3-nitroflavanones. Interestingly, all the novel derivatives showed a much higher stability than reference compounds.¹⁶
3-Nitro-2-methylflavanones are significantly more stable than reference compound ML01506-07 (Figure 1) with half-life times ranging 1 to 5 days (Figure 4A).¹⁶ Syn- and anti-isomers (a vs b) have comparable stability in a week scale (Table S1). Notably, epimerisation of 3-halo-2-methyl-3-nitroflavanones was observed instantaneously when compounds were dissolved in DMSO (Figure S4). Epimerisation slowly occurs also in acetonitrile and in methanol at rt, but only after several days. At -20 °C epimerisation in MeOH was not observed even after more than 6 months. Therefore, stock solutions of compounds in MeOH were used for the stability assays. However, epimerisation kinetics and mechanism, which are beyond the scope of this paper, were not further investigated in this study, going beyond its scope.

Remarkably, 2-methoxyflavanones showed much higher stability with more than 75 % of remaining compound after one week (Figure 4B, Table S1). For this series, chlorinated derivatives with a nitro group at position 3 are more stable than the corresponding brominated ones (26 vs 25). This difference is not significant when the nitro is replaced by a bromine atom (33 vs 34). However, when the carbon at position 3 is not fully substituted like in 23, stability drastically decreased because of methanol elimination, giving 3-bromoflavone 32. Apart from this case, we were not able to determine the degradation products of the other compounds by MS analysis.

Overall, substitution at position 2 of the flavanone scaffold strongly increases the chemical stability of these compounds in HEPES buffer as hypothesised. Remarkably, methoxy-substitution leads to highly stable compounds with half-live time over one week.
<figure><img src="image_36.png" /><figcaption>**Figure 4.** Time-dependent chemical degradation of *syn*- and *anti*-2-methylflavanones (A) and 2-methoxyflavanones (B) respect to reference compounds **MLo1507**, **MLo1506** and **MLo1502** at rt in HEPES buffer (see experimental section for exact composition).¹⁶</figcaption></figure>

*DNMT inhibition assays*

2-Substituted flavanones were screened in our fluorescent-based DNMT3A assay¹⁶ to study the impact of substitution at position 2 on their inhibitory activity against DNMT3A. Compounds were first screened at two concentrations, 32 and 10 µM. Substitution at position 2 on 3-halo-3-nitroflavanones strongly decreased their DNMT3A inhibitory activity (Table S2). Similarly, the inhibitory activity is affected when the nitro group at position 3 is replaced by a trifluoromethyl group (30 and 31, Table 1). However, compounds bearing an additional halogen instead of the 3-nitro (33, 34a, 34b) resulted in potent DNMT3A inhibitors, despite the methoxy group at position 2 (Table 1).
<figure><img src="image_37.png" /></figure>

<table><thead><tr><th rowspan="2">Compound</th><th rowspan="2">X</th><th rowspan="2">Y</th><th rowspan="2">R</th><th colspan="2">DNMT3A-c % inhib.</th></tr><tr><th>32 µM</th><th>10 µM</th></tr></thead><tbody><tr><td>ML01502</td><td>Br</td><td>NO<sub>2</sub></td><td>H</td><td>95 ± 5</td><td>86 ± 14</td></tr><tr><td>23</td><td>Br</td><td>H</td><td>OMe</td><td>13</td><td>N/A</td></tr><tr><td>25</td><td>Br</td><td>NO<sub>2</sub></td><td>OMe</td><td>42 ± 21</td><td>15 ± 11</td></tr><tr><td>26</td><td>Cl</td><td>NO<sub>2</sub></td><td>OMe</td><td>4 ± 18</td><td>N/A</td></tr><tr><td>28</td><td>OH</td><td>H</td><td>OMe</td><td>N/A</td><td>N/A</td></tr><tr><td>30</td><td>CF<sub>3</sub></td><td>H</td><td>H</td><td>22 ± 8</td><td>7 ± 9</td></tr><tr><td>31</td><td>CF<sub>3</sub></td><td>Br</td><td>H</td><td>N/A</td><td>6 ± 11</td></tr><tr><td>33</td><td>Br</td><td>Br</td><td>OMe</td><td>93 ± 10</td><td>74 ± 8</td></tr><tr><td>34a</td><td>Br</td><td>Cl</td><td>OMe</td><td>62 ± 13</td><td>46 ± 14</td></tr><tr><td>34b</td><td>Cl</td><td>Br</td><td>OMe</td><td>91 ± 4</td><td>71 ± 0</td></tr></tbody></table>

**Table 1.** DNMT3A-c inhibition percentages for 2-methoxyflavanones and 3-modified flavanones at 32 and 10 µM. **ML01502** was used as reference compound.¹⁵ Values are represented as mean ± S.E.M. of duplicates of at least two independent experiments, except for compound **23**. N/A: not applicable.

Considering the potency of compounds **33**, **34a**, and **34b**, their IC₅₀ values were measured. **ML01502** and **ML01506** were chosen as reference compounds, being the former the 3-bromo-3-nitroflavanone with no substituents on the phenyl like our derivatives, and the latter the most active flavanone among the previously described analogues.¹⁶ Measured IC₅₀ are 0.80 ± 0.11, 1.11 ± 0.22, and 0.78 ± 0.14 µM, for **33**, **34a**, and **34b**, respectively (Figure 5). As far as we know, these compounds represent at the moment three of the four compounds ever discovered that are active at the nanomolar range as DNMT3A inhibitors, together with the bisubstrate-type analogue **70** described in 2017 by Halby *et al.* (Figure 1),¹¹ which presents solubility limitations. Moreover, since stability issues arose in enzyme inhibition assay conditions with the previous generation of flavanones, stability of compounds **33**, **34a**, and **34b** was assessed by HPLC measurements before and after incubation with DNMT3A-c at 37°C. We could not observe any degradation product, confirming the high stability of the novel compounds (Figure S4).
<figure><img src="image_38.png" /><figcaption>**Figure 5.** Concentration-response curves of 3,3-dihalo-2-methoxyflavanones on DNMT3A-c enzyme in our fluorescence-based *in vitro* assay. **MLo1502** and **MLo1506** were chosen as reference compounds (**Figure 1**).<sup>15</sup> Values are shown as mean ± S.E.M. of duplicated of two or more independent experiments.</figcaption></figure>

A selection of representative compounds of our library of flavanones bearing either a methyl or a methoxide at position 2 was screened at 32 µM on the recombinant DNMT1. In this case, no relevant inhibitory activity was detected for flavanones bearing a methoxide at position 2, except for **34b** that showed 47 ± 15 % inhibition value (**Figure S6**). Interestingly, 3-halo-2-methyl-3-nitroflavanones with a chlorine or nitro at meta position of the aryl group showed strong inhibition values ranging from 70 to 97 % (**Figure S6**).

*Cell viability assessment in colorectal cancer cells*

The cytotoxicity of novel 3,3-dihalo-2-methoxyflavanones **33**, **34a**, and **34b** was assessed in HCT-116 colorectal cancer cell line, where the impact of DNMT activities on the transcriptional repression of TSGs are well documented.<sup>4,5</sup> Exponentially growing cells were treated every day for 3 days with compounds at concentration ranging 0.1 to 30 µM (**Figure 6**). In these conditions, the compounds showed very similar cytotoxicity among themselves and reference compound **MLo1506** (**Figure 1**), whose cytotoxicity in HCT166 cells was previously described by Pechalrieu *et al*.<sup>16</sup> Cytotoxicity is in line with the one assessed in other proliferating cell lines (**Figure S7**).
<figure><img src="image_39.png" /><figcaption>**Figure 6.** Cell viability in HCT-116 colorectal cancer cells at different compound concentrations. Treatment was performed every day for 3 days. **MLo1506** was chosen as reference compound.¹⁶ Values are shown as mean ± S.E.M. of duplicated of three independent experiments.</figcaption></figure>

p15 and p16 tumour suppressor gene re-expression in colorectal cancer cells

To determine whether the treatment with compounds **33**, **34a**, and **34b** impacts on the expression of TSGs, normally repressed by DNMTs, we analysed by RT-qPCR experiments the mRNA levels of genes encoding for the cyclin-dependent kinases p15 and p16 in HCT-116 colorectal cancer cell line. It was previously shown that the DNMT-mediated methylation of the promoters of these two genes are hallmarks of colorectal cancer cells and that contribute to the process of tumorigenesis.²,⁴ Since the subsequent decrease in DNA methylation upon DNMT inhibition generally requires multiple rounds of DNA synthesis and cell replication, we examined by RT-qPCR after total RNA extraction the levels of *p15* and *p16* gene expression after 7 days of treatment, with cells treated at day 0, 2 and 5 at a concentration of 5 µM. Under these conditions, the treatment with the nucleoside DNMTi **5azadC** resulted in decreased levels of both *p15* and *p16*, probably due to its high toxicity and poor selectivity. Similarly, compound **MLo1506** has only a weak impact on the expression of these two genes. In contrast, 7 days treatment with compounds **34a** and **34b** significantly induced the up-regulation of *p15* expression by 1.44 ± 0.11 (72 %) and 1.76 ± 0.11 (88 %) folds, respectively, compared to non-treated cells (Figure 7). Compound **34b** also increased *p16* expression by 1.34 ± 0.07 (67%) folds. Data observed using this 7-day-long protocol, together with those obtained by shorter cell treatment (Figure S8), suggest that the effects of **34a** and **34b** on *p15* and *p16* gene expression are in line with an epigenetic cell reprogramming. All together, these results show that compounds **34a** and, to a limited extent, **34b** can improve the expression of genes normally inhibited by DNA methylation in HCT-116 cancer cells.
<figure><img src="image_40.png" /></figure>

**Figure 7.** Re-expression of *p15* and *p16* TSGs following a 7-day-long treatment of reference and test compounds. Drug treatment was accomplished at 5 µM at day 0, 2, and 5. Data were normalized to non-treated cell values (fold ratio = 1, dotted red line) and are shown as mean ± S.E.M. of duplicates of four independent experiments (three for **5azadC**). One sample t-test was performed comparing test compound fold ratio against 1. *p* < 0.05, **p** < 0.01

To assess whether **34b** also affects the expression of genes known to be inhibited by DNA methylation in other cancerous cell lines, we examined by RT-qPCR the mRNA levels of *SPDEF* and *KAI-1* genes in DU-145 prostate cancer cells and those of *p15* and *p21* genes in U2OS osteosarcoma cells after treatment with 5 µM of **5azadC**, **MLo1506**, or **34b** as described above in HCT116 cells.²²,²³ As shown in **Figure S9**, **34b** only induced a moderate increase in *SPDEF* and *p21* expression compared to **5azadC** treatment. In contrast, the treatment with the previous generation compound **MLo1506** reduced the expression of tested genes except for *p21* that was slightly increased (**Figure S9**). These findings suggest that the efficacy of the non-nucleoside DNMT inhibitor **34b** on DNMT-regulated genes varies by cell type and is most pronounced in HCT116 cells.

*Compound 34b only moderately modulate cell cycle in cancer cell lines*

To characterize the effects of compound **34b** on cell proliferation, HCT-116, DU-145, and U2OS cancer cell lines were treated with 5 µM of drug for 5 days. Cells were then pulse-labeled with BrdU to mark replicating cells, and the cell cycle profile was analyzed by measuring DNA content and BrdU incorporation via flow cytometry. **5azadC** and **MLo1506** were used as reference compounds.
While no significant cell cycle defects were observed following treatment with the previously described, less stable DNMT inhibitor **ML01506**, exposure to **5-azadC** markedly altered cell cycle progression in all three cell lines. Specifically, **5-azadC** treatment reduced the proportion of replicating cells (BrdU⁺ cells, **Figure 8**). This was accompanied by an increase in the percentage of cells with DNA content ≥ 4N in both DU-145 and U2OS cells (**Figure 8B-C**). However, HCT116 cells exhibited an accumulation in cells with DNA content ≤ 2N (**Figure 8A**). These cell-cycle alterations likely indicate the presence of DNA damage induced by **5azadC** treatment, as also evidenced by the appearance of defective replicating cells with reduced BrdU incorporation (**Figure 8**, **Figure S10**).

In comparison, treatment with compound **34b** primarily resulted in a mild but significant delay in the G1/S transition, as indicated by the increase in the proportion of cells with DNA content ≤ 2N and a slightly corresponding decrease in the percentage of replicating cells across all tested cell lines (**Figure 8**). Thus, despite its high potency in *in vitro* assays targeting DNMT3A-c activity, compound **34b** exhibited a comparatively weaker impact on cell cycle progression than **5azadC**. This difference may be attributed to the ability of **5azadC** to incorporate into DNA, which is responsible for its off-target effects. Another possible explanation is the lower activity of compound **34b** against DNMT1, which plays a critical role in restoring DNA methylation on newly synthesized DNA and maintaining genome stability during cell cycle.¹
<figure><img src="image_41.png" /><figcaption>**Figure 8.** Quantification of cell cycle flow cytometry analysis of HCT-116 (A), DU-145 (B), and U2OS (C) cancer cell lines following a 5-day-long treatment of 5 µM reference and test compounds. Data are shown as mean ± S.E.M. of two or three independent experiments. Cells were separated in diploid (2N), tetraploid (4N), and BrdU-positive and negative replicating cells. Unpaired t-test was performed comparing test compound cell % against DMSO one. \*p < 0.05 \*\*p < 0.01 \*\*\*p < 0.001</figcaption></figure>

*Compound 34b significantly alters cell cycle of p53-knockout colorectal cancer cells*

The tumour suppressor gene *p53* is mutated in a variety of cancers,²⁴ typically leading to the loss of its cancer-inhibitory function and promoting several oncogenic effects.²⁵ To mimic such predominant phenotype in cancer cells, we assessed the effects of compound **34b** in the expression of TSGs *p15*, *p16*, and *p21* in a p53-knockout (KO) HCT-116 cell line. **5azadC** was used as reference compounds. First, we observed that none of the test compounds was able to significantly increase *p21* expression, probably because of the deletion of p53 induced in our model (**Figure 9A**). Contrarily, **5azadC** could
330 induce both *p15* and *p16* re-expression (Figure 9A). Compound **34b** did not alter *p15* expression in 331 *p53*-KO cells, but successfully induced *p16* re-expression (Figure 9A), even more strongly than in 332 WT-HCT-116 cells (Figure 7).

333 Consistent with these results, flow cytometry-based cell cycle analysis of p53-KO HCT-116 cells 334 following treatment with test compounds also revealed notable effects. As shown in **Figure 9B**, both 335 **34b** and **5azadC** strongly impaired G1/S transition in absence of p53, as evidenced by an increased 336 percentage of cells with a DNA content ≤ 2N and a corresponding decrease in BrdU-positive cells 337 (Figure 9B). Notably, similarly to **5azadC**, **34b** treatment also led to a substantial population of 338 defective replicating cells with reduced BrdU incorporation compared to DMSO-treated p53-KO 339 controls (Figure 9B). These findings suggest that the novel non-nucleoside DNMTi **34b** holds 340 promise for treating p53-mutated cancers, expanding the therapeutic strategies available at the moment 341 targeting DNA methylation beyond the use of the chemically unstable and non-selective **5azadC**.²⁵
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**Figure 9.** TSG re-expression and cell cycle analysis of *p53*-KO HCT-116 cancer cells following a 5-day-long treatment of 5 µM reference and test compounds. A) Re-expression of *p21*, *p15*, and *p16* TSGs. Data were normalized to non-treated cell values (fold ratio = 1, dotted red line) and are shown as mean ± S.E.M. of duplicates of two independent experiments. B) Above, flow cytometry dot plot showing cell populations after treatment with DMSO as negative control or 5 µM of **5azadC** and **34b**. Below, quantification of cell cycle flow cytometry analysis. Data are shown as mean ± S.E.M. of two or three independent experiments. Cells were separated in diploid (2N), tetraploid (4N), and BrdU-positive or negative replicating cells. Unpaired t-test was performed comparing test compound cell % against DMSO one. \*p < 0.05 \*\*p < 0.01

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*Compound 34b and Mlo1507 compete for the same DNMT3A binding site*

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Saturation Transfer Difference (STD) experiment was conducted on compound **34b** in the presence of 5 µM DNMT3A-c to investigate its mode of interaction. STD signals of compound **34b** were detected, confirming that our compound interacts with the target protein (Figure 10A). To further characterise
the binding properties of **34b**, compound **MLo1507** (Figure 1), which has previously been shown to inhibit DNMT3A-c,<sup>16</sup> was tested for binding. The STD spectrum of **MLo1507** bound to DNMT3A-c is shown in **Figure 10B**. Subsequently, a competition experiment between **34b** and **MLo1507** was performed (Figure 10C). In the presence of a 5-fold excess of **MLo1507** (IC<sub>50</sub> = 3.2 ± 1.1 µM),<sup>16</sup> the binding of **34b** was reduced by approximately 50%, suggesting that **34b** is a reversible DNMTi and that both compounds, *i.e.* **34b** and **MLo1507**, compete for the same binding site. Nevertheless, these experiments do not give information about a possible reversible covalent nature of the interaction of 34 with the catalytic cysteine of DNMT, for instance, *via* transacetalation or addition on the carbonyl.

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**Figure 10.** STD-NMR spectra and competition experiments for compounds **34b** and **MLo1507**. A) 1D ¹H and STD-NMR spectra of 100 µM **34b** in the presence of 5 µM protein. (B) 1D ¹H and STD-NMR spectra of 300 µM **MLo1507** in the presence of 5 µM protein. (C) 1D ¹H and STD-NMR spectra of 50 µM **34b** bound to protein DNMT3A-c in the presence of 10 µM (black spectrum) and 300 µM (red spectrum) of **MLo1507**. STD signals of **34b** that do not overlap to those of **MLo1507** are highlighted with a black frame. (D) Zoom showing the NMR signals within the black frame, where the 1D ¹H and STD spectra of **34b** in the presence of 10 µM **MLo1507** (black spectra) and 300 µM **MLo1507** (red spectra) highlight the decrease in **34b** STD signals in the presence of an excess of **MLo1507**.

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*Identification of a new binding pose in the catalytic pocket of DNMT3A for 3,3-dihalo-2-methoxyflavanones*

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Molecular docking studies of compound **34a** and **34b** in the DNMT3A catalytic pocket were performed and results were compared to previously described 3-halo-3-nitroflavanones¹⁵ and aza/thioflavanones.¹⁸ Binding poses in the cofactor (S-adenosyl-L-methionine, SAM) pocket were found for both compounds and respective enantiomers (**Figure 11B-D**, **Table S3**). Interestingly, no poses were found at the interface of SAM and catalytic pockets, contrary to 3-chloro-3-nitroflavanones and 3-bromo-3-nitroazaflavanones (**Table S3**), as previously described.¹⁵ Instead, a novel binding pose within the catalytic pocket was identified with 3,3-dihalo-2-methoxyflavanones, which is closer to the catalytic cysteine (C706) involved in DNA cytosine methylation (**Figure 11A-C-E**). This could explain the increase of inhibitory activity of our novel ligands. The predicted binding pocket partially overlap with the binding pocket described for previous 3-halo-3-nitroflavanones,¹⁵,¹⁸ which would confirm the results obtained in the STD NMR competition assay between **34b** and **MLo1507**. The new binding pose identified for **34b** was also detected in preliminary data with 3-bromo-2-methyl-3-nitroflavanone, however the ligand was found farther away from C706 and with no convenient orientations to establish any H bonds (**Figure S11**). We think that this could be due to the presence of the nitro group, whose steric hindrance is not compatible with a relatively bulky substituent at position 2.

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<figure><img src="image_48.png" /><figcaption>**Figure 11.** Representation of docking poses of compound **34b** in DNMT3A-3L (PDB: 2QRV). A) 2S,3S-**34b** binding pose in the enzyme catalytic pocket; B) 2S,3S-**34b** binding pose in the SAM cofactor pocket, the same site described in Serhouni *et al*;¹⁸ C-D) representation of the interactions established by 2S,3S-**34b** in the catalytic pocket (C) and cofactor pocket (D). H-bonds are highlighted in cyan; E) 2R,3R-**34b** in the catalytic pocket is placed farther from C706 respect to its enantiomer 2S,3S. Panels A and B show the ligand represented as spheres and the enzyme surface in electrostatic colouring. Panels C, D, and E show the ligand as sticks and the enzyme structure as ribbons. Colour legend: carbons, grey (enzyme), goldenrod (2S,3S-**34b**), or gold (2R,3R-**34b**); nitrogen, blue; oxygen, red; sulphur, yellow; chlorine, green; bromine, dark red.</figcaption></figure>

We analysed the contribution of each enantiomer of **34a** and **34b** to the interactions in both the cofactor and catalytic pockets (Table S3). 2S-**34a** and 2S-**34b** shared the same pose in the SAM pocket with same energy scores (5.8 kcal/mol), as for 2R-**34a** and 2R-**34b** (5.9 vs 5.7 kcal/mol). This indicates that the different orientation of chlorine and bromine does not have an impact on the binding. In the catalytic pocket, 2R-**34a** and 2R-**34b** shared the same pose with similar energy scores (5.2 vs 4.9 kcal/mol), but no pose was found for 2S-**34a**, contrary to 2S-**34b** isomer (5.2 kcal/mol). This suggests that halogen orientation is relevant when carbon 2 has *S* configuration. Moreover, 2S-**34b** is oriented the closest to C706 (3.4 Å) respect to 2R-**34a** or 2R-**34b** (Figure 11E, Table S3), establishing H-bonds between C706 and the flavanone endocyclic oxygen (3.5 Å), and asparagine 707 (N707) and the flavanone carbonyl group (3.5 Å, Figure 11C). This might be in line with the more consistent activity of **34b** in cells, but it still needs to be confirmed. 2S-**34b** is the only isomer where these interactions were found. For other isomers several molecular orientations were identified (Figure S12), meaning
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that flavanones have a large degree of freedom in such pockets. Performing molecular dynamics on
such compounds would give more insights on this observation.

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Conclusions

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Through this research, we discovered 3,3-dihalo-2-methoxyflavanones as novel potent DNMTi with a submicromolar inhibitory activity and a high stability. We confirmed that the additional substituent at position 2 of previously described 3-halo-3-nitroflavanones¹⁶ greatly increases compound chemical stability in HEPES buffer, but decreases their activity as DNMTi. Notably, the substitution of the nitro at position 3 with an additional halogen led to a 10-fold increase of DNMT3A inhibitory activity even in the presence of a methoxide at position 2. This allows 3,3-dihalo-2-methoxyflavanones to have both high stability and potency. The *anti*-3-bromo-3-chloro-2-methoxyflavanone **34b** could also consistently increase the expression of the tumour suppressor genes *p15* and *p16* in HCT-116 colorectal cancer cells and moderately that of *SPEDF* and *p21* in DU-145 prostate cancer and U2OS osteosarcoma cells, respectively. Compound **34b** moderately arrested cell cycle in G1 phase in the above-mentioned cell lines, however such effect is greatly enhanced in p53-KO HCT-116 cells. These results suggest that **34b** could be an effective lead candidate for p53-mutated cancer and pave the way to novel anticancer therapeutic strategies.²⁵ Binding of **34b** in the DNMT3A-c pocket was further characterised by STD NMR as well as its competition with reference compounds **ML01507**, from the previous generation of 3-halo-3-nitroflavanones. Finally, docking studies suggest that **34b** establishes new interactions in the catalytic pocket respect to previous 3-halo-3-nitroflavanones and azaflavanones DNMTi.¹⁵ Altogether, this work reports the exploration of novel DNMTi from which **34b** stands out, to our knowledge, as the most potent non-nucleoside DNMT3A inhibitor described so far.

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**Experimental section**

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Chemical *materials and methods*

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All reagents and solvents were purchased from commercial suppliers (BLDpharm, Sigma-Aldrich, TCI, and ThermoFisher Scientific). All reactions were performed open on air, unless otherwise indicated, and monitored by TLC on silica gel 60 F<sub>254</sub> plates (Merck ref #1.05554). Purifications by flash chromatography were carried out on a Puriflash 430 instrument (Interchim) using 30 µm spheric or 40-60 µm irregular silica cartridges. Preparative and semi-preparative HPLC was performed on a Gilson® PLC 2250 Purification System equipped with a UV-Vis Glison® DAD detector and using a binary gradient with the following solutions (Solution A: 99.9% H<sub>2</sub>O, 0.1% TFA; Solution B: 99.9% MeCN, 0.1% TFA). The purification system was equipped with either a WATERS™ XSelect™ CSH C18 OBD Prep Column 186005493 (130 Å, 5 µm, 30 nm X 250 nm) or a Macherey-Nagel® VP HPLC Column 250/10 NUCLEODUR C18 Htec 762566.100 (110 Å, 7 µm, 10 nm X 250 nm). Compound purity and stability were measured by HPLC (Thermo Scientific, UltiMate3000) on C18 reversed-phase column (Thermo Scientific, Hypersil GOLD aQ, 2.1 x 50 mm, 1.9 µm). Elution was
performed using a binary gradient (solvent A: H₂O + 0.1% TFA; solvent B: MeCN + 0.1% TFA).
LCMS analyses were performed using electrospray in positive and negative modes on an instrument
equipped with a Shimadzu LC-20AD XR pump and an SPD-M20A diode array detector. A C18
reversed-phase column (Phenomenex, Kinetex polar C18, 3 x 750 mm, 2.6 µm, 100 Å) was used.
Elution was performed using a binary gradient (solvent A: H₂O + 0.1% formic acid; solvent B: MeCN
+ 0.1% formic acid). Final compounds were analysed by HRMS using a Bruker hybrid Q-TOF mass
spectrometer. When HRMS is not indicated for final compounds, no ionisation was detected by ESI,
ASAP, APCI, and MALDI. NMR spectra were recorded on a Bruker DRX-400 spectrometer at 400 or
600 MHz for ¹H and 101 MHz for ¹³C. 2D NMR experiments such as ¹H-¹H COSY, ¹H-¹³C HSQC,
¹H-¹³C HMBC experiments were performed to enable signal attributions or identify quaternary
carbons not visible in 1D ¹³C NMR. Chemical shifts (δ) are given in ppm, coupling constant (J) are
measured in Hz. Multiplicity is indicated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; dd,
doublet of doublets; ddd, doublet of doublet of doublets; m, multiplet. Purity was assessed by both
HPLC (UV detection at 214 and 254 nm) and NMR and compounds tested in the biological assays are
≥ 95% pure by HPLC analysis, otherwise differently indicated. C, H, N, and O values from elemental
analysis are indicated for certain final compounds. Melting points were obtained using a Buchi B-510
melting point apparatus. No Pan Assay Interference Compound (PAINS) was detected when searching
our flavanone derivatives on http://zinc15.docking.org/patterns/home.²⁶

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472 **Synthesis**

<figure><img src="image_49.png" /></figure>

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General procedure 1 – Aldol condensation and cyclisation procedure for the synthesis of 2-methyl
flavanones (2-12). In a reaction vial, 2'-hydroxy-acetophenone (1 equiv) was dissolved in acetonitrile
(0.7 M) and pyrrolidine (1 equiv) was added followed by differently substituted acetophenone (1
equiv). The reaction was sealed, heated to 80 °C and followed by HPLC for 7 days. The reaction
mixture was diluted with EtOAc and then washed twice with NaHCO₃ (sat), twice with HCl (1 M) and
once with brine. The organic layer was dried with Na₂SO₄ and then evaporated in vacuum after
filtration. The crude is then purified by flash chromatography (gradient from 0 to 50% DCM in
cyclohexane).

General procedure 2 – Nitration and halogenation procedure for the synthesis of 3-halo-2-methyl-3-nitro flavanones (13-22). 2-Methylflavanone (1 equiv) was dissolved in AcOH (0.5 M) in a reaction vial. HNO₃ (10 equiv) was added dropwise at 0 °C and the vial was firmly closed. Reaction was stirred at rt and monitored by TLC (9:1 cyclohexane/EtOAc) every 2 h until reaction is completed.

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Otherwise, additional 5 equiv of $HNO_3$ are added and the reaction is left overnight at rt. The crude mixture was poured into a beaker, neutralised with saturated $NaHCO_3$ until pH = 6-7, and extracted three times with DCM. The organic layers were combined, washed with brine, dried with $Na_2SO_4$, filtered, and evaporated in vacuum. The resulting yellow oil was directly transferred into a reaction vial and dissolved in THF (0.15 M). NBS or NCS (2 equiv) and $Na_2CO_3$ (2 equiv) were added and the mixture was stirred at rt until completion, typically between 3 and 6 h (TLC, 9:1 cyclohexane/EtOAc). Water was added to the crude, which was extracted three times with DCM. The organic layers were combined, washed with brine, dried over $Na_2SO_4$, filtered, and evaporated under vacuum. The obtained crude was purified by semipreparative HPLC to allow diastereomer separation.

**General procedure 3** – Alkene addition general procedure for the synthesis of 3-halo-2-methoxy-3-nitro and 3,3-dihalo-2-methoxy flavanones (23, 25-26, 33-34). Flavone or 3-substituted flavone (1 equiv) was dissolved in MeOH (0.15 M) and N-bromo- or N-chlorosuccinimide (NBS and NCS, respectively; 3 equiv) was added. The reaction was stirred at rt for flavone or 50 °C for 3-substituted flavone until completion as monitored by TLC (9:1 cyclohexane/EtOAc). After addition of EtOAc, the organic layer was washed with water and brine, and dried over $Na_2SO_4$, filtered, and evaporated in vacuum. The obtained white powder was purified by flash chromatography (gradient 0 to 30 % EtOAc in cyclohexane).

2-(2-chlorophenyl)-2-methylchroman-4-one (2). Compound 2 was obtained as a dark brown oil of 70 mg starting from 2'-chloroacetophenone (100 µL) and following the general procedure 1. Yield: 33 %. ¹H NMR (400 MHz, $CDCl_3$): $\delta$ (ppm) = 7.79 (ddd, $J$ = 7.8, 1.8, 0.5 Hz, 1H, H5), 7.52 (m, 2H, H7, H3'), 7.36 (m, 1H, H5'), 7.18 (m, 2H, H4', H6'), 7.12 (ddd, $J$ = 8.3, 1.1, 0.5 Hz, 1H, H8), 6.98 (ddd, $J$ = 7.8, 7.2, 1.1 Hz, 1H, H6), 3.61 (d, $J$ = 16.6 Hz, 1H, $CH_2$), 3.28 (d, $J$ = 16.6 Hz, 1H, $CH_2$), 1.94 (s, 3H, $CH_3$). ¹³C NMR (101 MHz, $CDCl_3$): $\delta$ (ppm) = 191.7 (C=O), 159.7 (C10), 139.3 (C1'), 136.4 (C7), 132.4 (C5'), 131.7 (C2'), 129.4 (C4' or C6'), 128.1 (C3'), 127.1 (C4' or C6'), 126.6 (C5), 121.4 (C6), 121.0 (C9), 118.1 (C8), 83.2 (C2), 47.2 ($CH_2$), 26.1 ($CH_3$).

2-(3-chlorophenyl)-2-methylchroman-4-one (3). Compound 3 was obtained as an amber oil of 205 mg starting from 3'-chloroacetophenone (100 µL) and following the general procedure 1. Yield: 97 %. ¹H NMR (400 MHz, $CDCl_3$): $\delta$ (ppm) = 7.77 (ddd, $J$ = 7.8, 1.8, 0.3 Hz, 1H, H5), 7.48 (ddd, $J$ = 8.3, 7.2, 1.8 Hz, 1H, H7), 7.43 (td, $J$ = 1.7, 0.6 Hz, 1H, H2'), 7.26 (m, 1H, H-Ar), 7.21 (m, 2H, H-Ar), 7.07 (ddd, $J$ = 8.3, 1.1, 0.5 Hz, 1H, H8), 6.95 (ddd, $J$ = 8.0, 7.2, 1.0 Hz, 1H, H6), 3.25 (d, $J$ = 16.5 Hz, 1H, $CH_2$), 3.07 (d, $J$ = 16.6 Hz, 1H, $CH_2$), 1.42 (s, 3H, $CH_3$). ¹³C NMR (101 MHz, $CDCl_3$): $\delta$ (ppm) = 191.2 (C=O), 159.7 (C10), 145.4 (C1'), 136.4 (C7), 134.8 (C3'), 130.0 (CH-Ar), 128.0 (CH-Ar), 126.7 (C5), 125.7 (C2'), 123.4 (CH-Ar), 121.4 (C6), 121.0 (C9), 118.4 (C8), 82.0 (C2), 48.0 ($CH_2$), 29.7 ($CH_3$).
2-(4-chlorophenyl)-2-methylchroman-4-one (4). Compound 4 was obtained as a brown oil of 78 mg starting from 4'-chloroacetophenone (101 µL) and following the general procedure 1. Yield: 37 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.76 (ddd, J = 7.8, 1.8, 0.5 Hz, 1H, H5), 7.47 (ddd, J = 8.3, 7.2, 1.8 Hz, 1H, H7), 7.37 - 7.32 (m, 2H, H3' and H5'), 7.29 - 7.24 (m, 2H, H2' and H6'), 7.05 (ddd, J = 8.3, 1.1, 0.5 Hz, 1H, H8), 6.95 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H, H6), 3.26 (d, J = 16.5 Hz, 1H, CH₂), 3.08 (d, J = 16.5 Hz, 1H, CH₂), 1.74 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 191.4 (C=O), 159.9 (C10), 141.7 (C1'), 136.4 (C7), 133.8 (C4'), 129.0 (C2' and C6'), 126.9 (C3'and C5'), 126.8 (C5), 121.4 (C6), 121.1 (C9), 118.4 (C8), 82.2, 48.1 (CH₂), 30.1 (CH₃).

2-(2-bromophenyl)-2-methylchroman-4-one (5). Compound 5 was obtained as a brown oil of 25 mg starting from 2'-bromoacetophenone (103 µL) and following the general procedure 1. Yield: 10 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.79 (dd, J = 7.9, 1.7 Hz, 1H, H5), 7.59 (dd, J = 7.9, 1.4 Hz, 1H, H3'), 7.55 - 7.46 (m, 2H, H7 and H6'), 7.24 (m, 1H, H4'), 7.15 - 7.06 (m, 2H, H6 and H8), 6.97 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H, H5'), 3.65 (d, J = 16.7 Hz, 1H, CH₂), 3.25 (d, J = 16.7 Hz, 1H, CH₂), 1.95 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 191.7 (C=O), 159.6 (C10), 140.5 (C1'), 136.4 (C7), 136.2 (C3'), 129.6 (C6), 128.4 (C6'), 127.6 (C4'), 126.6 (C5), 121.4 (C5'), 121.1 (C9), 120.6 (C2'), 118.5 (C8), 83.4 (C2), 47.4 (CH₂), 26.2 (CH₃).

2-(3-bromophenyl)-2-methylchroman-4-one (6). Compound 6 was obtained as an orange oil of 229 mg starting from 3'-bromoacetophenone (103 µL) and following the general procedure 1. Yield: 93 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.78 (ddd, J = 7.8, 1.8, 0.5 Hz, 1H, H5), 7.59 (td, J = 1.9, 0.4 Hz, 1H, H2'), 7.49 (ddd, J = 8.3, 7.2, 1.8 Hz, 1H, H7), 7.36 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H, H4'), 7.32 (ddd, J = 7.9, 1.8, 1.0 Hz, 1H, H6'), 7.17 (td, J = 7.9, 0.4 Hz, 1H, H5'), 7.07 (ddd, J = 8.3, 1.0, 0.5 Hz, 1H, H8), 6.96 (ddd, J = 7.8, 7.2, 1.1 Hz, 1H, H6), 3.25 (d, J = 16.5 Hz, 1H, CH₂), 3.07 (d, J = 16.5 Hz, 1H, CH₂), 1.73 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 191.2 (C=O), 159.8 (C10), 145.7 (C1'), 136.5 (CH), 131.0 (CH), 130.3 (CH), 128.6 (CH), 126.8 (CH), 123.9 (CH), 123.1 (C3'), 121.5 (CH), 121.0 (C9), 118.4 (CH-8), 82.0 (C2), 48.1 (CH₂), 29.7 (CH₃). MS (ESI⁺): m/z 357.9 [M+MeCN+H]⁺.

2-(4-bromophenyl)-2-methylchroman-4-one (7). Compound 7 was obtained as an orange oil of 123 mg starting from 4'-bromoacetophenone (147 mg) and following the general procedure 1. Yield: 52 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.79 (ddd, J = 7.8, 1.8, 0.5 Hz, 1H, H5), 7.50 (ddd, J = 8.3, 7.1, 1.7 Hz, 1H, H7), 7.45 (d, J = 8.7 Hz, 2H, H3'and H5'), 7.31 (d, J = 8.8 Hz, 2H, H2'and H6'), 7.08 (dd, J = 8.4, 1.1 Hz, 1H, H8), 6.98 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H, H6), 3.29 (d, J = 16.5 Hz, 1H, CH₂), 3.11 (d, J = 16.5 Hz, 1H, CH₂), 1.76 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 191.3 (C=O), 159.8 (C10), 142.2 (C1'), 136.4 (C7), 131.9 (C2' and C6'), 127.2 (C3' and C5'), 126.8 (C5), 121.9 (C), 121.4 (C6), 121.1 (C), 118.4 (C8), 82.2 (C2), 48.0 (CH₂), 30.0 (CH₃). MS (ESI⁺): m/z 358.0 [M+MeCN+H]⁺.
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2-methyl-2-(2-nitrophenyl)chroman-4-one (8). Compound **8** was obtained as dark brown oil of 3 mg starting from 2'-nitroacetophenone (103 µL) and following the general procedure 1. Yield: 1 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.17 (d, J = 8.9 Hz, 1H), 7.84 – 7.71 (m, 2H), 7.60 (d, J = 8.9 Hz, 1H), 7.55 – 7.46 (m, 2H), 7.16 – 7.07 (m, 1H), 7.02 – 6.95 (m, 1H), 3.30 (d, J = 16.6 Hz, 1H, CH₂), 3.16 (d, J = 16.6 Hz, 1H, CH₂), 1.79 (s, 3H, CH₃).

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2-methyl-2-(3-nitrophenyl)chroman-4-one (9). Compound **9** was obtained as a brown oil of 58 mg starting from 3'-nitroacetophenone (112 mg) and following the general procedure 1. Yield: 28 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.34 (ddd, J = 2.3, 1.9, 0.5 Hz, 1H, H2'), 8.11 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H, H4'), 7.77 (ddd, J = 7.8, 1.8, 0.5 Hz, 1H, H5), 7.74 (ddd, J = 7.8, 1.9, 1.0 Hz, 1H, H6'), 7.54 – 7.48 (m, 2H, H7 and H5'), 7.12 (ddd, J = 8.3, 1.1, 0.5 Hz, 1H, H8), 6.98 (ddd, J = 7.8, 7.2, 1.1 Hz, 1H, H6), 3.30 (d, J = 16.6 Hz, 1H, CH₂), 3.15 (d, J = 16.6 Hz, 1H, CH₂), 1.79 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 190.7 (C=O), 159.4 (C10), 148.7 (C3'), 145.7 (C1'), 136.7 (CH), 131.2 (CH), 129.9 (CH), 126.8 (CH), 122.9 (CH), 121.8 (CH), 120.9 (C9), 120.6 (CH), 118.5 (C8), 81.9 (C2), 48.0 (CH₂), 29.5 (CH₃).

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2-methyl-2-(4-nitrophenyl)chroman-4-one (10). Compound **10** was obtained as a brown oil of 9 mg starting from 4'-nitroacetophenone (112 mg) and following the general procedure 1. Yield: 4 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.17 (d, J = 9.1 Hz, 2H, H3' and H5'), 7.76 (ddd, J = 7.8, 1.8, 0.5 Hz, 1H, H5), 7.60 (d, J = 9.1 Hz, 2H, H2' and H6'), 7.51 (ddd, J = 8.3, 7.2, 1.8 Hz, 1H, H7), 7.10 (ddd, J = 8.4, 1.1, 0.5 Hz, 1H, H8), 6.98 (ddd, J = 7.8, 7.2, 1.1 Hz, 1H, H6), 3.30 (d, J = 16.6 Hz, 1H, CH₂), 3.16 (d, J = 16.5 Hz, 1H, CH₂), 1.78 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 190.7 (C=O), 159.6 (C10), 150.5 (C4'), 136.7 (C1'), 126.9 (C7), 126.4 (C2' and C6'), 124.1 (C3' and C5'), 121.89 (CH), 121.0 (CH), 118.4 (C8), 82.2 (C2), 48.0 (CH₂), 29.7 (CH₃).

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2-(3-methoxyphenyl)-2-methylchroman-4-one (11). Compound **11** was obtained as an orange oil of 77 mg starting from 3'-methoxyacetophenone (101 µL) and following the general procedure 1. Yield: 39 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.76 (dd, J = 7.8, 1.8 Hz, 1H, H5), 7.46 (ddd, J = 8.4, 7.2, 1.8 Hz, 1H, H7), 7.20 (td, J = 7.1, 0.7 Hz, 1H, H5'), 7.06 (dd, J = 8.3, 1.0 Hz, 1H, H8), 6.95 (m, 3H, H6, H2', and H6'), 6.75 (ddd, J = 8.4, 2.3, 1.4 Hz, 1H, H4'), 3.74 (s, 3H, OCH₃), 3.29 (d, J = 16.5 Hz, 1H, CH₂), 3.07 (d, J = 16.5 Hz, 1H, CH₂), 1.75 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 191.6 (C=O), 160.1 (C10 or C3'), 159.9 (C10 or C3'), 144.8 (C1'), 136.2 (C7), 129.7 (CH), 126.7 (CH), 121.2 (C9), 121.2 (CH), 118.4 (CH), 117.7 (CH), 112.8 (CH), 111.6 (C8), 82.5 (C2), 55.2 (OCH₃), 48.2 (CH₂), 30.0 (CH₃). MS (ESI⁺): *m/z* 310.0 [M+MeCN+H]⁺.

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2-(4-methoxyphenyl)-2-methylchroman-4-one (12). Compound **12** was obtained as a yellow oil of 48 mg starting from 4'-methoxyacetophenone (116 mg) and following the general procedure 1. Yield: 23 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.94 (d, J = 8.9 Hz, 2H, H2' and H6'), 7.75 (dd, J = 7.8, 1.8 Hz, 1H, H5), 7.45 (m, 1H, H7), 7.03 (dd, J = 8.3, 1.0 Hz, 1H, H8), 6.93 (m, 3H, H6, H3', and
590 H5'), 3.87 (s, 3H, OCH3), 3.29 (d, J = 16.5 Hz, 1H, CH2), 3.06 (d, J = 16.5 Hz, 1H, CH2), 2.55 (s, 3H, CH3). ¹³C NMR (101 MHz, CDCl3): δ (ppm) = 192.0 (C=O), 160.1 (C10), 159.1 (C4'), 136.2 (CH), 135.0 (C1'), 130.7 (CH), 126.7 (CH), 121.2 (C9), 121.1 (CH), 118.4 (CH), 114.0 (CH), 82.4 (C2), 55.6 (OCH3), 48.2 (CH2), 30.3 (CH3). MS (ESI⁺): m/z 310.0 [M+MeCN+H]⁺.
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2-(2-Chlorophenyl)-2-methyl-3,3-dinitrochroman-4-one (13). Compound 13 was obtained as a white powder of 21 mg starting from compound 2 (65 mg) and following general procedure 2. Purification was performed by flash chromatography (gradient from 0 to 40 % DCM in cyclohexane). Yield: 24 %. 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.99 (dd, J = 8, 1.6 Hz, 1H, H5), 7.64 (ddd, J = 8.4, 7.6, 2 Hz, 1H, H7), 7.42 (m, 2H, CH-Ar), 7.23 (m, 4H, CH-Ar) 2.43 (s, 3H, CH3). ¹³C NMR (101 MHz, CDCl3): δ (ppm) = 174.3 (C=O), 156.9 (C10), 138.3 (C7), 133.9 (C), 133.4 (CH-Ph), 132.0 (C), 130.8 (CH-Ph), 129.8 (CH-Ph), 128.4 (C5), 126.7 (CH-Ph), 124.3 (C6), 120.8 (C), 119.3 (C8), 117.9 (C3), 88.2 (C2), 27.0 (C), 21.8 (CH3). mp 108 °C (dec). HPLC-UV (λ= 254 nm), peak area 98 %. HRMS (ESIΓ) m/z [M - H]⁻ calcd for C₁₆H₁₀ClN₂O₆ 361.0233; found 361.0220.
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3-Bromo-2-(3-chlorophenyl)-2-methyl-3-nitrochroman-4-one (14a-b). Compounds 14a and 14b were achieved as white solids of 33 and 46 mg, respectively, starting from compound 3 (99 mg) and following general procedure 2. Yield: 64 % (14a: 27 %, 14b: 37 %). (14a) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.04 (dd, J = 7.9, 1.8 Hz, 1H, H5), 7.70 (ddd, J = 8.4, 7.3, 1.7 Hz, 1H, H7), 7.66 (td, J = 2, 0.5 Hz, 1H, H2'), 7.42 (ddd, J = 9.2, 2, 1.7 Hz, 1H, H4'), 7.35 (td, J = 7.9, 0.5 Hz, 1H, H5'), 7.22 (m, 3H, H6, H8, and H6'), 2.11 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 178.2 (C=O), 157.2 (C10), 138.3 (C7), 138.2 (C-Ar), 134.8 (C-Ar), 129.9 (C5'), 129.8 (C4'), 128.9 (C5), 127.1 (C2'), 124.1 (C6'), 123.6 (CH), 119.2 (CH), 117.4 (C-Ar), 105.2 (C3), 85.8 (C2), 22.7 (CH₃). mp 123-125 °C. HPLC-UV (λ= 254 nm), peak area 95 %. HRMS (ESI⁺) m/z [M + Na]⁺ calcd for C₁₆H₁₁BrClNNaO₄ 417.9452; found 417.9417. (14b) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.08 (dd, J = Hz, 1H, H5), 7.69 (m, 2H, H7 and H2'), 7.61 (ddd, J = Hz, 1H, H4' or H6'), 7.44 (ddd, J = Hz, 1H, H4' or H6'), 7.38 (t, J = Hz, 1H, H5'), 7.23 (H6), 7.15 (H8), 2.01 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): 177.1 (C=O), 157.8 (C10), 138.9 (C3'), 138.0 (C4' or C6'), 134.4 (C1'), 129.9 (C4' or C6'), 129.5 (C5'), 128.9 (C5), 127.2 (C2'), 125.0 (C4' or C6'), 123.6 (CH-6), 119.6 (C9), 119.2 (C8), 104.2 (C3), 86.9 (C2), 23.5 (CH₃). mp 136-137 °C. HPLC-UV (λ= 254 nm), peak area 96 %. HRMS (ESI⁺) m/z [M + Na]⁺ calcd for C₁₆H₁₁BrClNNaO₄ 417.9452; found 417.9451.
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3-chloro-2-(3-chlorophenyl)-2-methyl-3-nitrochroman-4-one (15a-b). Compounds 15a and 15b were achieved as white solids of 49 and 52 mg, respectively, starting from compound 3 (99 mg). Yield: 92 % (15a: 45 %, 15b: 47 %). (15a) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.05 (dd, J = 7.9 Hz, 1H, H5), 7.69 (m, 2H, H7 and H2'), 7.42 (ddd, J = 8.0, 2.0, 1.1 Hz, 1H, H4' or H6'), 7.36 (t, J = 7.9 Hz, 1H, H5'), 7.23 (m, 3H, H5, H6, and H4' or 6'), 2.11 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 178.5 (C=O), 156.9 (C10), 139.1 (C1'), 138.2 (C7), 134.7 (C3'), 129.8 (C4' or C6'), 129.76 (C5').
(C4' or C6'), 128.8 (C5), 126.9 (C2'), 123.9 (C5'), 123.6 (C6), 119.2 (C8), 117.3 (C9), 103.1 (C3), 85.7 (C2), 22.1 (CH₃). mp 120-121 °C. HPLC-UV (λ= 254 nm), peak area 93 %. HRMS (ASAP⁺) m/z [M + H]⁺ calcd for C₁₆H₁₁Cl₂NO₄ 351.0065; found 350.9615. **(15b)** ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.07 (ddd, *J* = 7.8, 1.7, 0.4 Hz, 1H, H5), 7.70 (ddd, *J* = 8.4, 7.3, 1.8 Hz, 1H, H7), 7.67 (td, *J* = 2.0, 0.4 Hz, 1H, H2'), 7.58 (ddd, *J* = 7.8, 1.9, 1.2 Hz, 1H, H6'), 7.44 (ddd, *J* = 8.0, 2.0, 1.2 Hz, 1H, H4'), 7.38 (td, *J* = 8.0, 0.4 Hz, 1H, H5'), 7.24 (ddd, *J* = 7.9, 7.3, 1.0 Hz, 1H, H6), 7.17 (ddd, *J* = 8.4, 1.0, 0.4 Hz, 1H, H8), 1.96 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 177.2 (C=O), 158.0 (C10), 138.4 (C1'), 138.2 (C7), 134.6 (C3'), 130.0 (C4'), 129.7 (C5'), 128.7 (C5), 126.9 (C2'), 124.8 (C6'), 123.6 (C6), 119.8 (C9), 119.4 (C8), 106.3 (C3), 86.6 (C2), 22.5 (CH₃). mp 154-156 °C (dec). HPLC-UV (λ= 254 nm), peak area 99 %. Anal. calcd for C₁₆H₁₁Cl₂NO₄: C, 54.57; H, 3.15; N, 3.98; O, 18.17; found: C, 54.345 ± 0.038; H, 3.391 ± 0.063; N, 3.697 ± 0.015; O, 18.787 ± 0.338.

**3-bromo-2-(4-chlorophenyl)-2-methyl-3-nitrochroman-4-one (16a-b).** Compounds **16a** and **16b** were achieved as white solids of 7 and 8 mg, respectively, starting from compound **4** (32 mg). Yield: 37 % (16a: 17 %, 16b: 20 %). (16a) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.05 (dd, *J* = 7.8, 1.7 Hz, 1H, H5), 7.69 (ddd, *J* = 8.4, 7.3, 1.8 Hz, 1H, H7), 7.46 (d, *J* = 9.0 Hz, 2H, H3' and H5'), 7.41 (d, *J* = 9.0 Hz, 2H, H2' and H6'), 7.23 (m, 1H, H6), 7.18 (m, 1H, H8), 2.10 (s, 3H, CH₃). ¹³C NMR (APT, 101 MHz, CDCl₃): δ (ppm) = 178.6 (C=O), 157.0 (C10), 138.1 (C7), 135.6 (C1'), 128.9 (C5), 128.7 (C2' and C6'), 127.6 (C3', C5', and C4'), 123.5 (C6), 119.1 (C8), 103.1 (C3), 85.9 (C2), 22.1 (CH₃). mp 128-129 °C. HPLC-UV (λ= 254 nm), peak area 96 %. HRMS (ESI⁺) m/z [M + Na]⁺ calcd for C₁₆H₁₁BrClNNaO₄ 417.9452; found 417.9450. (16b) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.08 (ddd, *J* = 7.9, 1.7, 0.4 Hz, 1H, H5), 7.69 (ddd, *J* = 8.4, 7.3, 1.8 Hz, 1H, H7), 7.64 (d, *J* = 9.0 Hz, 2H, H3' and H5'), 7.42 (d, *J* = 9.0 Hz, 2H, H2' and H6'), 7.24 (ddd, *J* = 7.9, 8.3, 1.0 Hz, 1H, H6), 7.14 (ddd, *J* = 8.3, 1.0, 0.5 Hz, 1H, H8), 2.01 (s, 3H, CH₃). ¹³C NMR (APT, 101 MHz, CDCl₃): δ (ppm) = 157.8 (C10), 138.0 (C7), 135.4 (C1'), 128.9 (C5), 128.5 (C2' and C6'), 128.2 (C3' and C5'), 123.6 (C6), 119.6 (C9), 119.2 (C8), 104.2 (C3), 87.1 (C2), 23.5 (CH₃). mp 151-152 °C. HPLC-UV (λ= 254 nm), peak area 95 %. HRMS (ESI⁺) m/z [M + Na]⁺ calcd for C₁₆H₁₁BrClNNaO₄ 417.9452; found 417.9453.

**2-(2-bromophenyl)-2-methyl-3,3-dinitrochroman-4-one (17) and 3-bromo-2-(2-bromophenyl)-2-methyl-3-nitrochroman-4-one (18).** Compounds **17** and **18** were achieved as yellow solids of 33 and 60 mg, respectively, starting from compound **5** (115 mg). Yield: 25 % and 43 %, respectively. (17) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.96 (dd, *J* = 7.8, 1.7 Hz, 1H, H5), 7.63 (m, 2H, H7 and H3'), 7.23 (m, 3H, H4', H5', and H6'), 7.16 (m, 2H, H6 and H8), 2.46 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 174.2 (C=O), 156.8 (C10), 138.1 (C7), 137.5 (C3'), 134.9 (C1'), 130.8 (C8), 130.0 (CH-Ar), 128.3 (C8), 127.1 (CH-Ar), 124.2 (C6), 121.3 (C9), 120.6 (C2'), 119.3 (CH-Ar), 117.9 (C3), 88.5 (C2), 22.4 (CH₃). mp 119-120 °C. HPLC-UV (λ= 254 nm), peak area 99 %. (18) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.06 (ddd, *J* = Hz, 1H, H5), 7.72 (dd, *J* = Hz, 1H), 7.65 (m, 2H), 7.35 (ddd, *J*
661 = Hz, 1H, H6), 7.19 (m, 3H), 2.27 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 177.1 (C=O), 156.9 (C10), 137.7 (C7), 136.8 (C3'), 134.9 (C1'), 131.1 (CH-Ar), 130.7 (CH-Ar), 128.6 (C5), 126.8 (CH-Ar), 123.1 (C6), 121.6 (C2'), 119.5 (C9), 118.9 (C8), 101.8 (C3), 88.3 (C2), 24.2 (CH₃). 662
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664 mp 106 °C. HPLC-UV (λ= 254 nm), peak area 86 %. HRMS (ESI⁻) *m/z* [M - H]⁻ calcd for C₁₆H₁₀Br₂NO₄ 437.8982; found 437.8968.
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666 3-bromo-2-(3-bromophenyl)-2-methyl-3-nitrochroman-4-one (19a-b). Compounds 19a and 19b were 667 achieved as white solids of 30 and 27 mg, respectively, starting from compound 5 (100 mg). Yield: 47 668 % (19a: 25 %, 19b: 22 %). (19a) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.06 (dd, J = 7.9, 1.6 Hz, 669 1H, H5), 7.83 (m, 1H, H2'), 7.70 (m, 1H, H7), 7.58 (m, 1H, H4'), 7.29 (m, 2H, H5'and H6'), 7.22 (m, 670 2H, H6 and H8), 2.11 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 178.6 (C=O), 156.9 671 (C10), 139.3 (C1'), 138.2 (C7), 132.7 (C6'), 130.1 (C5' or C4'), 129.8 (C2'), 128.9 (C5), 124.4 (C5' 672 or C4'), 123.6 (C6), 122.8 (C3'), 119.2 (C8), 117.4 (C9), 103.1 (C3), 85.6 (C2), 22.2 (CH₃). mp 133- 673 134 °C. HPLC-UV (λ= 254 nm), peak area 97 %. HRMS (ESI⁺) *m/z* [M + Na]⁺ calcd for C₁₆H₁₁Br₂NNaO₄ 461.8947; found 461.8959. (19b) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.08 (dd, J 674 = 7.9, 1.6 Hz, 1H, H5), 7.85 (t, J = 1.9 Hz, 1H, H2'), 7.68 (m, 2H, H7 and H4'), 7.59 (ddd, J = 8.0, 675 1.8, 0.9 Hz, 1H, H6'), 7.32 (t, J = 8.0 Hz, 1H, H5'), 7.23 (m, 1H, H6), 7.15 (d, J = 8.3 Hz, 1H, H8), 676 2.00 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 177.1 (C=O), 157.7 (C10), 139.1 (C1'), 677 138.0 (C7), 132.8 (C6'), 130.0 (C2'), 129.8 (C5'), 128.9 (C5), 125.5 (C4'), 123.6 (C6), 122.5 (C3'), 678 119.6 (C9), 119.2 (C8), 104.2 (C3), 86.8 (C2), 23.5 (CH₃). mp 139-142 °C. HPLC-UV (λ= 254 nm), 679 peak area 97 %. HRMS (ESI⁺) *m/z* [M + Na]⁺ calcd for C₁₆H₁₁Br₂NNaO₄ 461.8947; found 461.8944.
680

681 3-bromo-2-(4-bromophenyl)-2-methyl-3-nitrochroman-4-one (20a-b). Compounds 20a and 20b were 682 achieved as white solids of 18 and 25 mg, respectively, starting from compound 7 (115 mg). Yield: 31 683 % (20a: 13 %, 20b: 18 %). (20a) ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.98 (dd, J = 7.9, 1.5 Hz, 1H, 684 H5), 7.62 (m, 1H, H7), 7.50 (d, J = 8.7 Hz, 2H, H3' and H5'), 7.32 (d, J = 8.7 Hz, 2H, H2'and H6'), 685 7.14 (m, 2H, H6 and H8), 2.03 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 178.6 (C=O), 686 157.0 (C10), 138.1 (C7), 136.2 (C1'), 131.7 (C3' and C5'), 128.9 (C5), 127.9 (C2' and C6'), 123.5 687 (C6), 119.1 (C8), 117.4 (C9), 103.0 (C3), 85.9 (C2), 22.1 (CH₃). mp 124-125 °C. HPLC-UV (λ= 254 688 nm), peak area 99 %. HRMS (ESI⁺) *m/z* [M + K]⁺ calcd for C₁₆H₁₁Br₂KNO₄ 477.8686; found 689 477.8693. (20b) ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.08 (dd, J = 7.9, 1.6 Hz, 1H, H5), 7.68 (ddd, J 690 = 8.4, 7.3, 1.7 Hz, 1H, H7), 7.58 (s, 4H, CH-Ar), 7.23 (m, 1H, H6), 7.13 (dd, J = 8.3, 0.5 Hz, 1H, H8), 691 2.00 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 177.2 (C=O), 157.8 (C10), 138.0 (C7), 692 136.0 (C1'), 131.5 (C3' and C5'), 128.9 (C5), 128.5 (C2' and C6'), 124.3 (C4'), 123.6 (C6), 119.6 693 (C9), 119.2 (C8), 104.4 (C3), 87.1 (C2), 23.4 (CH₃). mp 151 °C. HPLC-UV (λ= 254 nm), peak area 96 694 % . HRMS (ESI⁺) *m/z* [M + Na]⁺ calcd for C₁₆H₁₁Br₂NNaO₄ 461.8947; found 461.8952.
695
695 3-bromo-2-methyl-3-nitro-2-(3-nitrophenyl)chroman-4-one (21a-b). Compounds 21a and 21b were achieved as white solids of 43 and 23 mg, respectively, starting from compound 9 (66 mg). Yield: 81 % (21a: 53 %, 21b: 28 %). Syn-(21a) The compound was recrystallized in CH₂Cl₂ by slow evaporation to obtain crystals for X-ray diffraction (Supporting information). ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.61 (t, J = Hz, 1H, H2'), 8.33 (ddd, J = Hz, 1H, H4'), 8.08 (dd, J = Hz, 1H, H5), 7.74 (ddd, J = Hz, 1H, H7), 7.65 (m, 2H, H5' and H6'), 7.27 (m, 2H, H6 and H8), 2.18 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 178.1 (C=O), 156.6 (C10), 148.3 (C3'), 139.3 (C1'), 138.4 (CH), 131.6 (CH), 129.7 (CH), 128.9 (CH), 124.6 (CH), 123.9 (CH), 122.2 (CH), 119.3 (CH), 117.2 (C9), 102.6 (C3), 85.4 (C2), 22.0 (CH₃). mp 136-137 °C. HPLC-UV (λ= 254 nm), peak area 98 %. HRMS (ESI⁺) m/z [M + Na]⁺ calcd for C₁₆H₁₁Br₂N₂NaO₆ 428.9693; found 428.9691. Anti-(21b) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.62 (t, 1H, H2') 8.34 (m, 1H, H8), 8.10 (m, 2H, H4' and H6'), 7.71 (m, 1H, H7), 7.65 (t, J = Hz, 1H, H5'), 7.28 (m, 1H, H6), 7.19 (dd, J = Hz, 1H, H8), 2.07 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 176.6 (C=O), 157.3 (C10), 148.1 (C3'), 139.2 (C1'), 138.2 (CH), 132.8 (CH), 129.4 (CH), 128.9 (CH), 124.6 (CH), 124.0 (CH), 122.3 (CH), 119.6 (C9), 119.2 (C8), 103.6 (C3), 86.7 (C2), 23.4 (CH₃). mp 159 °C. HPLC-UV (λ= 254 nm), peak area 92 %. HRMS (ESI⁺) m/z [M + Na]⁺ calcd for C₁₆H₁₁Br₂N₂NaO₆ 428.9693; found 428.9681.

711 3-bromo-2-(3-methoxyphenyl)-2-methyl-3-nitrochroman-4-one (22a-b). Compounds 22a and 22b were achieved as white solids of 56 and 15 mg, respectively, starting from compound 11 (86 mg). Yield: 66 % (22a: 52 %, 22b: 14 %). (22a) ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.05 (dd, J = 7.9, 1.7 Hz, 1H, H5), 7.68 (ddd, J = 8.4, 7.3, 1.7 Hz, 1H, H7), 7.34 (t, J = 8.1 Hz, 1H, H5'), 7.19 (m, 3H, H7, H2', and H4' or H6'), 6.99 (m, 2H, H8 and H4' or H6'), 3.85 (s, 3H, OCH₃), 2.11 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 178.9 (C=O), 159.5 (C), 157.2 (C), 138.7 (C), 138.0 (CH), 129.6 (CH), 128.8 (CH), 123.3 (CH), 119.2 (CH), 118.1 (CH), 117.5 (C), 114.4 (CH), 113.0 (CH), 86.1 (C2), 55.5 (OCH₃), 22.3 (CH₃). mp 133-134 °C. HPLC-UV (λ= 254 nm), peak area 99 %. HRMS (ESI⁺) m/z [M + Na]⁺ calcd for C₁₇H₁₄BrNNaO₅ 413.9948; found 413.9965. (22b) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.07 (dd, J = 7.9, 1.7 Hz, 1H, H5), 7.68 (ddd, J = 8.3, 7.2, 1.8 Hz, 1H, H7), 7.36 (t, J = 8.0 Hz, 1H, H5'), 7.29 (m, 1H, H4' or H6'), 7.24 (m, 2H, H2' and H6), 7.14 (dd, J = 8.3, 1.0 Hz, 1H, H4' or H6'), 6.98 (ddd, J = 8.2, 2.6, 1.0 Hz, 1H, H8), 3.84 (s, 3H, OCH₃), 2.02 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 177.5 (C=O), 159.4 (C), 158.2 (C), 138.3 (C), 137.9 (CH), 129.3 (CH), 128.8 (CH), 123.4 (CH), 119.6 (C), 119.2 (CH), 119.2 (CH), 114.6 (CH), 113.4 (CH), 104.8 (C3), 87.3 (C2), 55.5 (OCH₃), 23.7 (CH₃). mp 98-100 °C. HPLC-UV (λ= 254 nm), peak area 94 %. HRMS (ESI⁺) m/z [M + H]⁺ calcd for C₁₇H₁₅BrNO₅ 392.0128; found 392.0121.

727 3-bromo-2-methoxy-2-phenylchroman-4-one (23). Compounds 23 was achieved as a white solid of 80 mg, starting from flavone (60 mg) and following general procedure 3. Yield: 89 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.01 (dd, J = 7.8, 1.8 Hz, 1H, H5), 7.68 (m, 2H, H-Ph), 7.63 (ddd, J = 8.3, 7.2, 1.8 Hz, 1H, H7), 7.49 (m, 3H, H-Ph), 7.20 (m, 2H, H6 and H8), 4.42 (s, 1H, H3), 3.04 (s, 3H,
CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 186.5 (C=O), 155.7 (C10), 136.6 (C7), 135.7 (C-Ph), 129.7 (CH-Ph), 128.6 (2 CH-Ph), 127.9 (C5), 127.1 (2 CH-Ph), 122.9 (C8), 118.8 (C9), 118.1 (C6), 105.4 (C2), 52.2 (OCH₃), 50.2 (C3). mp 123-124 °C. HPLC-UV (λ= 214-254 nm), peak area 97 %. HRMS (ESI⁺) *m/z* [M + H]⁺ calcd for C₁₆H₁₄BrO₃ 333.0121; found 333.0139.

**3-nitro-2-phenyl-4H-chromen-4-one (24).** Compound **1** (990 mg, 1 equiv), previously synthesised as described in Pechalrieu *et al.*,¹⁶ is added to triethyl orthobenzoate (6.2 mL, 5 equiv), followed by H₂SO₄ (15 µL, 0.05 equiv). The mixture is heated to 160 °C for 7 h. The crude mixture was diluted in EtOAc, washed twice with HCl (1 M), water and brine, dried over Na₂SO₄, filtered, and finally evaporated under *vacuum*. The crude mixture was purified by column chromatography (gradient from 0 to 40 % of EtOAc in cyclohexane. A yellow powder was obtained, affording 400 mg of compound **24**. Yield: 27 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.34 (dd, *J* = 8.0, 1.7 Hz, 1H, H5), 7.81 (ddd, *J* = 8.5, 7.2, 1.7 Hz, 1H, H7), 7.76 (m, 2H, H-Ar), 7.62 (m, 2H, H-Ar), 7.55 (m, 3H, H-Ar).

**Anti-3-bromo-2-methoxy-3-nitro-2-phenylchroman-4-one (25).** Compounds **25** was achieved as a white solid of 40 mg, starting from compound **24** (50 mg) and following general procedure 3. Yield: 54 %. The compound was recrystallized in CH₂Cl₂ by slow evaporation to obtain crystals for X-ray diffraction (Supporting information). ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.10 (ddd, *J* = 7.9, 1.7, 0.3 Hz, 1H, H5), 7.70 (ddd, *J* = 8.3, 7.4, 1.8 Hz, 1H, H7), 7.61 (br, 2H, H-Ph), 7.50 (m, 3H, H-Ph), 7.28 (ddd, *J* = 7.9, 7.4, 1.0 Hz, 1H, H6), 7.21 (dd, *J* = 8.3, 0.6 Hz, 1H, H8), 3.15 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 179.0 (C=O), 154.5 (C), 137.6 (C7), 131.0 (CH-Ph), 130.8 (C), 128.7 (C5), 128.6 (2 CH-Ph), 128.3 (2 CH-Ph), 124.0 (C6), 118.6 (C8), 118.4 (C), 106.4 (C), 98.0 (C), 53.2 (CH₃). mp 161-163 °C. HPLC-UV (λ= 254 nm), peak area 96 %. HRMS (APCI⁺) *m/z* [M - H]⁻ calcd for C₁₆H₁₁BrNO₅ 375.99; found 375.6045.

**3-chloro-2-methoxy-3-nitro-2-phenylchroman-4-one (26).** Compounds **26** was achieved as a white solid of 5 mg, starting from compound **24** (20 mg) and following general procedure 3. Yield: 20 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.09 (ddd, *J* = 7.9, 1.7, 0.4 Hz, 1H, H5), 7.72 (ddd, *J* = 8.3, 7.3, 1.8 Hz, 1H, H7), 7.59 (br, 2H, H-Ph), 7.50 (m, 3H, H-Ph), 7.28 (ddd, *J* = 7.9, 7.4, 1.0 Hz, 1H, H6), 7.22 (ddd, *J* = 8.3, 1.0, 0.4 Hz, 1H, H8), 3.16 (s, 3H, CH₃). ¹³C NMR (APT, 101 MHz, CDCl₃): δ (ppm) = 137.8 (C7), 131.0 (CH-Ph), 128.8 (CH-Ph), 128.7 (CH-Ph and C5), 124.0 (C6), 118.7 (C8), 52.78 (CH₃). HPLC-UV (λ= 214-254 nm), peak area 99 %.

**1a-phenyl-1a,7a-dihydro-7H-oxireno[2,3-b]chromen-7-one (27).** Flavone (30 mg, 1 equiv) was dissolved in MeCN (1 mL). After addition of a solution 0.4 mM of EDTA disodium salt (670 µL), the reaction was cooled at 0 °C. 1,1,1-Trifluoroacetone (135 µL, 11.2 equiv) was picked with a syringe previously cooled in a freezer and injected into the reaction. A mixture of oxone® (207 mg, 5 equiv) and NaHCO₃ (88 mg, 7.8 equiv) was added portionwise throughout 1 h. The reaction was kept sealed and stirred at 0 °C for 2 h. The reaction did not reach completion even after the addition of 1 equiv of
766

oxone® and NaHCO₃ (TLC, 9:1 cyclohexane/EtOAc). Iced water was added to the mixture, which was then extracted three times with DCM, dried over Na₂SO₄, filtered, and evaporated in vacuum. Compound 27 was obtained as a white powder of 27 mg. Yield: 84 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.98 (ddd, J = 7.8, 1.8, 0.4 Hz, 1H, H5), 7.63 (m, 3H, H-Ar), 7.49 (m, 3H, H-Ar), 7.22 (m, 2H, H-Ar), 3.88 (s, 1H, CH₃). Compound characterisation in accordance with the literature.²⁷

767

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769

770

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777

778

779

780

781

3-hydroxy-2-methoxy-2-phenylchroman-4-one (28). Compound 27 (30 mg) was dissolved in dry MeOH (10 mL, 0.01 M). The solution was stirred at rt under Ar atmosphere for 5 h. Solvent was evaporated under vacuum and the crude was purified by flash chromatography (gradient from 0 to 100 % EtOAc in cyclohexane). Compound 28 was obtained as a white powder of 10 mg. Yield: 29 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.90 (dd, J = 7.8, 1.8 Hz, 1H, H5), 7.62 (m, 3H, H6, H-Ph), 7.43 (m, 3H, H-Ph), 7.19 (ddd, J = 8.4, 1.0, 0.4f Hz, 1H, H8), 7.12 (ddd, J = 7.8, 7.2, 1.0 Hz, 1H, H6), 4.37 (d, J = 4.5 Hz, 1H, H3), 3.17 (s, 3H, CH₃), 2.55 (d, J = 4.6 Hz, 1H, OH). ¹³C NMR (DEPT135, 101 MHz, CDCl₃): δ (ppm) = 136.6 (CH-Ar), 129.5 (CH-Ar), 128.6 (CH-Ar), 127.4 (2 CH-Ar), 122.3 (CH-Ar), 117.9 (CH-Ar), 75.0 (CH-Ar), 50.6 (OCH₃). mp 174 °C. HPLC-UV (λ= 214-254 nm), peak area > 99 %. HRMS (ESI⁺) m/z [M + H]⁺ calcd for C₁₆H₁₅O₄ 271.0965; found 271.0969. Characterisation in accordance with the literature for the acetylated form.²⁸

782

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784

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786

787

788

789

790

791

792

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796

797

2-phenyl-3-(trifluoromethyl)chroman-4-one (30). Commercial flavanone (250 mg, 1 equiv) was dissolved in dry 1,2-dichloroethane (4.5 mL) under Ar atmosphere. Trifluoromethanesulfonic anhydride (280 µL, 1.5 equiv) was added, followed by 2,6-di-tert-butylpyridine (480 µL, 2 equiv). The mixture was stirred 2 h at rt, then a solution 1 M of BEt₃ in hexane (555 µL) was added. After stirring overnight at rt, water was added, the crude was extracted three times with DCM, dried over Na₂SO₄, filtered, and evaporated in vacuum. After flash chromatography purification (gradient from 0 to 20 % EtOAc in cyclohexane), 48 mg of compound 30 were obtained, together with 192 mg of intermediate 29, which was recovered to obtain further 12 mg of compound 30 by repeating the BEt₃ addition and purification. Yield: 18 %. 2-phenyl-2H-chromen-4-yl trifluoromethanesulfonate (29) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.29 (m, 5H, H-Ph), 7.14 (m, 2H, H5 and H7), 6.85 (td, J = 7.6, 1.0 Hz, 1H, H6), 6.72 (dd, J = 8.2, 0.76 Hz, 1H, H8), 5.99 (d, J = 3.6, 1H), 5.72 (d, J = 3.6, 1H). (30) ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.87 (dd, J = 7.9, 1.6 Hz, 1H, H5), 7.55 (ddd, J = 8.7, 7.2, 1.8 Hz, 1H, H7), 7.34 (m, 5H, H-Ph), 7.05 (m, 2H, H6 and H8), 5.93 (d, J = 4.2 Hz, 1H, H2), 3.77 (dq, J = 9.1, 4.2 Hz, 1H, H3). mp 67 °C. HPLC-UV (λ= 214-254 nm), peak area 91 %. HRMS (ASAP⁺) m/z [M + H]⁺ calcd for C₁₆H₁₂O₂F₃ 293.0789; found 293.0810. Compound characterisation in accordance with the literature.²⁰

798

799

800

3-bromo-2-phenyl-3-(trifluoromethyl)chroman-4-one (31). Compound 30 (25 mg, 1 equiv) was dissolved in THF (570 µL). Na₂CO₃ (18 mg, 2 equiv) and NBS (31 mg, 2 equiv) were added, and the reaction was stirred over the weekend at rt. DCM was added and the mixture was washed twice with
water, once with brine, and then dried over Na₂SO₄, filtered, and evaporated in vacuum. After flash chromatography (gradient from 0 to 100 % EtOAc in cyclohexane), 22 mg of compound **31** was achieved as a white powder. Yield: 69 %. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.25 (dd, *J* = 8.0, 1.6 Hz, 1H, H5), 7.95 (m, 2H, H-Ph), 7.71 (ddd, *J* = 8.8, 7.2, 2.0 Hz, 1H, H7), 7.60 (dd, *J* = 8.8, 0.8 Hz, 1H, H8), 7.54 (m, 3H, H-Ph), 7.43 (ddd, *J* = 8.0, 7.2, 0.8 Hz, 1H, H6), 6.84 (s, 1H, CH). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 178.7 (C4), 163.6 (C1'), 156.4 (C10), 133.9 (C7), 131.9 (C3), 131.8 (C-Ph), 129.2 (2 CH-Ph), 126.5 (2 C-Ph), 125.9 (C5), 125.4 (C6), 124.1 (C9), 118.2 (C8), 107.7 (C2), 29.8 (CF₃). HPLC-UV (λ= 214-254 nm), peak area 98 %.

**3-bromo-2-phenyl-4H-chromen-4-one (32).** Flavone (100 mg, 1 equiv), oxone® (166 mg, 1.2 equiv) and KBr (65 mg, 1.2 equiv) were dissolved in MeOH (11 mL) and water (190 µL). The mixture was stirred at rt overnight. Oxone® (0.6 equiv) and KBr (0.6 equiv) were added one more time and the mixture was stirred until completion, as monitored by TLC (8:2 cyclohexane/EtOAc). NaOH (90 mg, 5 equiv) was added to the mixture, which was left stirring at rt for 3 h until completion (TLC). Water (50 mL) was added and the product was extracted three times with DCM. After drying over Na₂SO₄, filtration, and evaporation under vacuum, compound **32** was obtained without any further purification as a white powder of 137 mg. Quantitative yield. ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.30 (ddd, *J* = 8.0, 1.7, 0.4 Hz, 1H, H5), 7.86 (m, 2H, H-Ph), 7.72 (ddd, *J* = 8.7, 7.1, 1.7 Hz, 1H, H7), 7.51 (m, 5H, H-Ph, H6, H8). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 173.2 (C=O), 162.1 (C), 155.8 (C), 134.3 (CH), 133.0 (C), 131.3 (CH), 129.4 (2 CH), 128.4 (2 CH), 126.6 (CH), 125.9 (CH), 121.9 (C), 118.0 (CH), 109.4 (C). mp 105-107 °C. Compound characterisation in agreement with 3-bromo-4'-methoxyflavone described by Peng *et al.*²¹

**3,3'-dibromo-2-methoxy-2-phenylchroman-4-one (33).** Compound **33** was achieved as a white solid of 48 mg, starting from compound **32** (50 mg) and following general procedure 3. Yield: 70 %. The compound was recrystallized in CH₂Cl₂ by slow evaporation to obtain crystals for X-ray diffraction (Supporting information). ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.10 (dd, *J* = 7.8, 1.6 Hz, 1H, H5), 7.92 (m, 2H, H-Ph), 7.64 (ddd, *J* = 8.3, 7.3, 1.7 Hz, 1H, H7), 7.52 (m, 3H, H-Ph), 7.22 (ddd, *J* = 7.9, 7.4, 1.0 Hz, 1H, H6), 7.16 (dd, *J* = 8.3, 0.7 Hz, 1H, H8), 3.14 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ (ppm) = 180.8 (C=O), 154.6 (C10), 136.9 (C7), 132.5 (C-Ph), 130.4 (2 CH-Ph), 130.3 (CH-Ph), 129.1 (C5), 127.6 (2 CH-Ph), 123.4 (C6), 118.0 (C8), 117.4 (C9), 107.7 (C2), 69.7 (C3), 53.0 (CH₃). mp 161-162 °C. HPLC-UV (λ= 214-254 nm), peak area > 99 %. HRMS (ESI⁺) *m/z* [M + H]⁺ calcd for C₁₆H₁₃Br₂O₃ 410.9226; found 410.9231.

**3-bromo-3-chloro-2-methoxy-2-phenylchroman-4-one (34a-b).** Compound **34a** and **34b** were achieved as white solids of 4 and 9 mg, respectively, starting from compound **32** (50 mg) and following general procedure 3. Diastereomers were isolated by semipreparative HPLC purification. Yield: 69 % (34a: 7 %, **34b**: 15%, plus non-purified fraction of both compounds). Syn-(34a) The compound was
recrystallized in CH₂Cl₂ by slow evaporation to obtain crystals for X-ray diffraction (Supporting information). ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.08 (dd, *J* = 7.8, 1.6 Hz, 1H, H5), 7.87 (m, 2H, H-Ph), 7.65 (ddd, *J* = 8.4, 7.9, 1.7 Hz, 1H, H7), 7.52 (m, 3H, H-Ph), 7.22 (m, 1H, H6), 7.16 (d, *J* = 8.3, 0.4 Hz, 1H, H8), 3.16 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 180.4 (C=O), 154.9 (C10), 137.0 (C7), 132.1 (C-Ph), 130.3 (CH-Ph), 130.1 (2 CH-Ph), 129.1 (C5), 127.7 (2 CH-Ph), 123.4 (C6), 118.0 (C8), 117.4 (C9), 108.0 (C2), 79.8 (C3), 52.5 (CH₃). mp 158 °C. HPLC-UV (λ= 214-254 nm), peak area > 99%. *Anti-(34b)* ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.09 (dd, *J* = 7.9, 1.7 Hz, 1H, H5), 7.88 (m, 2H, H-Ph), 7.64 (ddd, *J* = 8.3, 7.3, 1.7 Hz, 1H, H7), 7.52 (m, 3H, H-Ph), 7.23 (m, 1H, H6), 7.16 (dd, *J* = 8.3, 2.5 Hz, 1H, H8), 3.13 (s, 3H, CH₃). ¹³C NMR (126 MHz, CDCl₃): δ (ppm) = 180.9 (C=O), 154.6 (C10), 136.9 (C7), 131.9 (C-Ph), 130.3 (CH-Ph), 130.1 (2 CH-Ph), 128.9 (C5), 127.8 (2 CH-Ph), 123.4 (C6), 118.1 (C8), 118.0 (C9), 107.9 (C2), 77.8 (C3), 52.9 (CH₃). HPLC-UV (λ= 214-254 nm), peak area > 99%. HRMS (ESI⁺) *m/z* [M + H]⁺ calcd for C₁₆H₁₃BrClO₃ 366.9731; found 366.9734.

### Stability assay

Test compounds, previously dissolved in DMSO or MeOH (when epimerisation was observed in DMSO) at 10 mM, were diluted in the same buffer used for methylation reaction in the DNMT assay (20 mM HEPES pH 7.2, 50 mM KCl, 1 mM EDTA) to reach a final concentration of 100 µM. Compound stability was measured by HPLC, following compound AUC % after 2, 4, 8, 12 and 24 h at 254 nm. At 24 h and higher time points, a volume of MeOH was added to the sample just before HPLC injection to avoid artifacts due to solubility issues. Data were plotted using GraphPad.

### DNMT3A-c inhibition assay

Compound inhibitory activities were determined with a fluorescence-based assay previously described.¹⁵ In brief, a double-strand DNA, comprising a unique CpG site overlaying an endonuclease restriction site for methylation-sensitive enzyme was used. This oligonucleotide comprises a fluorescent 6-FAM at one end and biotin on the other end allowing immobilisation into a 384-well plate (PerkinElmer) pre-coated with avidin. Compounds and SAM (20 µM) were added followed by recombinant DNMT3A-c to start the methylation reaction in methylation buffer (HEPES 20 mM, KCl 50 mM, EDTA 1 mM, BSA 0.1%) for 1 h 20 min at 37 °C. Wells were washed 3 times with PBS tween (0.05%) containing NaCl (0.5 M) and PBS tween (0.05%). HpyCH4IV restriction enzyme (New England, BioLabs) was incubated for 1 h at 37 °C. After washing, fluorescence was quantified on an SAFAS Xenius plate reader (SAFAS Monaco) and methylation activity was calculated using the following formula: (X<sub>meth</sub> - X<sub>restri</sub>)*100/(X<sub>DNA</sub> - X<sub>restri</sub>), where X<sub>meth</sub> are the fluorescence signals detected, while X<sub>restri</sub> and X<sub>DNA</sub> are those of restriction and fluorescent DNA controls, respectively. IC<sub>50</sub> values of active compounds (mean of at least 3 independent experiments) were calculated by nonlinear regression with sigmoidal dose-response (variable slope) tool of GraphPad Prism software.
871 *DNMT1 inhibition assay*

872 Full-length histidine-tagged human DNMT1 (182 kDa) was produced and purified as previously
873 described.²⁹ Compound activities were determined with a fluorescence-based assay.¹⁵ In brief, a
874 hemimethylated double-strand DNA comprising a unique CpG site overlaying an endonuclease
875 restriction site for hemimethylation-sensitive enzyme was used (Fam- 5' GCA TAT ATA TGA CGA
876 TmCG TGT AGG TCA CTA CCA GAC ATG CAC TG 3' / 5' Biot- CAG TGC ATG TCT GGT
877 AGT GAC CTA CAC GAT CGT CAT ATA TAT GC 3'). This oligonucleotide comprises a 6-FAM
878 at one end and biotin on the other end allowing immobilization into a 384-well plate (Perkin Elmer)
879 pre-coated with avidin. The methylation reaction is performed by adding human DNMT1 at the final
880 concentration of 200 nM in a total volume of 30 µL of methylation buffer (HEPES 20 mM, KCl 120
881 mM, MgCl₂ 3 mM, EDTA 1mM, BSA 0.1 mg/mL, Tween 0.005 %) in the presence of the chemical
882 compound to be tested at the desired concentration and the cofactor SAM at the final concentration of
883 20 µM. After incubation (1h at 37 °C), the plate is washed 3 times with a solution of Tween (0.05 %)
884 and NaCl (0.5 M) in PBS and 3 times with Tween (0.05%) in PBS. Then, the methylation sensitive
885 restriction enzyme Bsh1285I (Thermo Fisher Scientific) is added. After 1.5 h incubation at 60 °C the
886 plate is washed and the fluorescence measured with a PerkinElmer Envision Multilabel Plate Reader.
887 Data are normalized referring to the “restriction control” (wells coated with labelled duplex not treated
888 nor exposed to DNMT1 but only cleaved by the restriction enzyme) as the minimum of fluorescence
889 (0) and to the “DMSO control” as the maximum of DNMT1 activity (1) (total methylation). Data were
890 plotted and analysed by non-linear regression on GraphPad (GraphPad Software Inc).

891 *Cell culture*

892 Human colorectal carcinoma cell line HCT-116 was cultured in Glutamax-supplemented RPMI
893 (Gibco) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin (Pen Strep
894 10000 U/mL, Gibco). DU-145 and U2OS cell lines were cultured in DMEM-Glutamax (Gibco)
895 containing 10% FBS, 1% Penicillin/Streptomycin, and 1% Sodium Pyruvate (Pen Strep 10000 U/mL,
896 Gibco). Cells were maintained at 37 °C under 5% CO₂ and split every 2-3 days before complete
897 confluence. P53-KO HCT-116 cells were supplied by Dr. B. Vogelstein.³⁰ All cells were tested
898 negative for mycoplasma.

899 *Cell viability assay*

900 HCT-116 cells were plated in 96-well plates (2500 cells/well, 100 µL/well). The following day cell
901 medium was removed *in vacuum* and replaced with fresh medium containing 0.05% DMSO or test
902 compounds. This step was repeated after 24 and 48 h. After 72 h, 50 µL of resazulin (stock solution at
903 0.15 mg/mL, then diluted at 10% in RPMI) was added to each well and cells were incubated at 37 °C
904 for 2.5 h. Absorbance was measured by a plate reader. Cytotoxicity curves were represented and
905 analysed by GraphPad (GraphPad Software Inc).
RT-qPCR

HCT-116 cells were plated in 6-well plates (250000 cells/well, 2 mL/well). The following day, cells were treated with DMSO or test compounds (day 0). Cell treatment was repeated at day 2 and 5. At day 7, dead and living cells were collected and centrifugated at 1200 rpm for 5 min. The pellet was washed twice with PBS and the stocked at -80 °C. RNA was extracted using the Monarch® Total RNA Miniprep Kit (T2010S, New England BioLabs). RNA was quantified by nanodrop spectrophotometer (DeNovix DS-11 FX). Reverse transcription was performed with Superscript™ III reverse transcriptase (Invitrogen), according to the manufacturer's protocol. A 1/100 sample of this reaction was used for real-time qPCR amplification using ONEGreen® Fast qPCR premix (Ozyme), following the manufacturer's protocol. Primers are indicated in supporting information. Ct values of test samples were normalised to those of DMSO-treated samples. One-sample t-test was performed for each condition using GraphPad (GraphPad Software Inc).

Cell-Cycle analysis by Flow Cytometry

2 million cells were incubated with BrdU (50 µg/ml, Sigma-Aldrich) for 90 minutes before fixation in ice-cold 70% ethanol. After incubation for 30 minutes with solution containing 2N HCl and 0.5% Triton X-100 under gentle agitation, cells were washed with Borax buffer (pH8.5) and then sequentially incubated in blocking solution (PBS, 0,5% Tween 20,1% BSA) containing anti-BrdU primary antibody (1/100, BD Biosciences) and anti-mouse secondary antibody coupled to FITC (1/300, Sigma-Aldrich) for 2 hours each. Total DNA was labeled with 2 µg/ml of 7-AAD (7-amino-actinomycin D, Sigma-Aldrich) diluted in PBS containing 100 µg/ml of RNAse A (Sigma -Aldrich). FACS analysis was carried out with Gallios flow cytometer (Beckman Coulter) using the Kaluza Acquisition software (Beckman Coulter). Cell cycle profiles were analyzed using FlowJo software (BD Biosciences). Data were presented with mean ± S.E.M. from at least three independent experiments. Unpaired t-test was performed comparing test compound cell % against DMSO one using GraphPad (GraphPad Software Inc).

STD-NMR experiments

The NMR spectra were recorded on a Bruker AVANCE III 800 MHz spectrometer equipped with a cryogenic 5 mm Z-gradient probe head. STD-NMR experiments were carried out at 293K and 283K using the Bruker pulse sequence (stddiffesgp.3). The selective saturation (at 0.8 ppm) was achieved by cascades of 50 ms Gaussian-shaped pulse with a 4 us delay between successive pulses. Total saturation time for each STD measurement was 2 s with a recycling of 1.2 s. STD-NMR spectra were recorded with 4096 scans for compound 34b and 512 scans for MLo1507. The protein signals were suppressed by a trim pulse of 25 ms. % DMSO was kept at 5% in all experiments.
939 **Molecular docking**

940 Ligands were docked in the crystal structure of DNMT3A-3L (PDB: 2QRV).³¹ Molecules 3D-
941 conformations of all compounds were generated using MarvinSketch 24.1.2 (2024, Chemaxon -
942 http://www.chemaxon.com). Ligands docked in the catalytic-cofactor binding pocket of DNMT3A-3L
943 using a squared grid with side of 24 Å. The method was validated by obtaining the same poses for
944 SAM, Flv62 and Flv69 as described in the published crystal structure or by Ceccaldi et al. and
945 Serhouni et al.¹⁵,¹⁸ All structural files were prepared with AutodockTools. For each run, 9 docking
946 poses were generated with Autodock Vina,³² ranked by score, using an exhaustiveness of 64. All
947 docking figures were generated with UCSF Chimera.³³

948 **X-ray diffraction**

949 Crystal evaluation and data collection of all structures were performed at 173 K on a Bruker Venture
950 diffractometer with a Cu-Kα microsource and equipped with a Photon-II detector. The APEX3
951 program was used for data collection and SAINT, XPREP and SADABS for the integration of the
952 diffraction data using default parameters, for the empirical absorption correction using spherical
953 harmonics employing symmetry-equivalent and redundant data, and the correction for Lorentz and
954 polarization effects.³⁴ All six structures were solved using the *ab-initio* iterative charge flipping
955 method with parameters described by Van der Lee³⁵ using the *Superflip* program³⁶ and they were
956 refined using full-matrix least-squares procedures as implemented in CRYSTALS³⁷,³⁸ on all reflections
957 with *I* > -3σ(*I*). The H atoms were in general all located in a difference map but repositioned
958 geometrically. They were initially refined with soft restraints on the bond lengths and angles to
959 regularize their geometry (C---H in the range 0.93-0.98 Å) and Uiso(H) (in the range 1.2-1.5 times Ueq
960 of the parent atom). Hydrogen atoms were refined with riding constraints in all three structures.
961 Structural ORTEP-like drawings (**Figures XR1-6**) were prepared with the three-dimensional graphical
962 visualization program Mercury.³⁹ CCDC 2371609 (**17**), 2371608 (**19b**), 2371610 (**21a**), 2371605 (**25**),
963 2371606 (**33**), and 2371607 (**34a**) contain the supplementary crystallographic data for this paper.
964 These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via
965 www.ccdc.cam.ac.uk/structures.

966 **Supporting Information**

967 Supporting information includes full ¹H and ¹³C NMR spectra, primers used for RT-qPCR, molecular
968 SMILES, HPLC traces, and supplementary figures, tables, and x-ray diffraction data.

969 **Declaration of competing interest**

970 The authors do not have any conflicts of interest to declare.
971 **Author contribution**

972 F.C. and M.L. supervised the project. E.J. supervised all the in-cell experiments. F.C. designed the
973 experiments and performed synthesis, chemical characterisation, stability assays, and molecular
docking. F.C., H.D, M.T., and E.J. performed the in-cell experiments. I.K. and Y.Y. designed, carried
974 out, and analysed STD NMR data. J.G., C.J., and P.B.A. conducted DNMT1 assays. Y.B. produced
975 the DNMT3A-c. A.L. analysed X-ray diffraction data. F.C. and E.J. performed biological data
976 analysis. F.C., M.L., I.K., and E.J. wrote the manuscript. J.G. and A.L. contributed to manuscript
977 revision.
978

979 **Acknowledgements**

980 This work was supported by the CNRS through the Emergence 2021 grant awarded to M. Lopez and
981 the ARC Foundation (Fondation ARC pour la recherche contre le cancer), which funded F.
982 Calzaferri's postdoctoral fellowship. We would also like to thank the Analysis and Characterisation
983 Platform (PAC, UAR2041) from the University of Montpellier. We also thank the referees for all their
984 suggestions, which helped to improve the manuscript.

985 **Abbreviations**

986 BrdU, bromodeoxyuridine; DNMT, DNA methyltransferase; DNMT3A-c, recombinant catalytic
987 DNMT3A; DNMTi, DNMT inhibitor; EtOAc, ethyl acetate; MeCN, acetonitrile; MeOH, methanol;
sat, saturated; STD, saturation transfer difference; TSG, tumour suppressor gene.
988

989 **References**

990 (1) Mattei, A. L.; Bailly, N.; Meissner, A. DNA Methylation: A Historical Perspective. *Trends Genet.* **2022**, *38* (7), 676–707. https://doi.org/10.1016/j.tig.2022.03.010.

991
992 (2) Merlo, A.; Herman, J. G.; Mao, L.; Lee, D. J.; Gabrielson, E.; Burger, P. C.; Baylin, S. B.;
993 Sidransky, D. 5' CpG Island Methylation Is Associated with Transcriptional Silencing of the
994 Tumour Suppressor P16/CDKN2/MTS1 in Human Cancers. *Nat. Med.* **1995**, *1* (7), 686–692.
995 https://doi.org/10.1038/nm0795-686.

996 (3) Esteller, M. Epigenetics in Cancer. *N. Engl. J. Med.* **2008**, *358* (11), 1148–1159.
997 https://doi.org/10.1056/NEJMra072067.

998 (4) Ishiguro, A.; Takahata, T.; Saito, M.; Yoshiya, G.; Tamura, Y.; Sasaki, M.; Munakata, A.
999 Influence of Methylated P15 INK4b and P16 INK4a Genes on Clinicopathological Features in
1000 Colorectal Cancer. *J. Gastroenterol. Hepatol.* **2006**, *21* (8), 1334–1339.
1001 https://doi.org/10.1111/j.1440-1746.2006.04137.x.

1002 (5) Gu, S.; Lin, S.; Ye, D.; Qian, S.; Jiang, D.; Zhang, X.; Li, Q.; Yang, J.; Ying, X.; Li, Z.; Tang, M.;
1003 Wang, J.; Jin, M.; Chen, K. Genome-Wide Methylation Profiling Identified Novel Differentially
1004 Hypermethylated Biomarker MPPED2 in Colorectal Cancer. *Clin. Epigenetics* **2019**, *11* (1), 41.
1005 https://doi.org/10.1186/s13148-019-0628-y.

1006 (6) de Almeida, B. P.; Apolónio, J. D.; Binnie, A.; Castelo-Branco, P. Roadmap of DNA Methylation
1007 in Breast Cancer Identifies Novel Prognostic Biomarkers. *BMC Cancer* **2019**, *19* (1), 219.
1008 https://doi.org/10.1186/s12885-019-5403-0.

1009 (7) Viziteu, E.; Klein, B.; Basbous, J.; Lin, Y.-L.; Hirtz, C.; Gourzones, C.; Tiers, L.; Bruyer, A.;
1010 Vincent, L.; Grandmougin, C.; Seckinger, A.; Goldschmidt, H.; Constantinou, A.; Pasero, P.;
1011 Hose, D.; Moreaux, J. RECQ1 Helicase Is Involved in Replication Stress Survival and Drug
1012 Resistance in Multiple Myeloma. *Leukemia* **2017**, *31* (10), 2104–2113.
https://doi.org/10.1038/leu.2017.54.

1014 (8) Wu, D.; Miao, J.; Hu, J.; Li, F.; Gao, D.; Chen, H.; Feng, Y.; Shen, Y.; He, A. PSMB7 Is a Key Gene Involved in the Development of Multiple Myeloma and Resistance to Bortezomib. *Front. Oncol.* **2021**, *11*, 2916. https://doi.org/10.3389/fonc.2021.684232.

1017 (9) Sato, T.; Issa, J.-P. J.; Kropf, P. DNA Hypomethylating Drugs in Cancer Therapy. *Cold Spring Harb. Perspect. Med.* **2017**, *7* (5), a026948. https://doi.org/10.1101/cshperspect.a026948.

1019 (10) Sekeres, M. A.; Taylor, J. Diagnosis and Treatment of Myelodysplastic Syndromes: A Review. *JAMA* **2022**, *328* (9), 872–880. https://doi.org/10.1001/jama.2022.14578.

1021 (11) Halby, L.; Menon, Y.; Rilova, E.; Pechalrieu, D.; Masson, V.; Faux, C.; Bouhlel, M. A.; David-Cordonnier, M.-H.; Novosad, N.; Aussagues, Y.; Samson, A.; Lacroix, L.; Ausseil, F.; Fleury, L.; Guianvarc'h, D.; Ferroud, C.; Arimondo, P. B. Rational Design of Bisubstrate-Type Analogues as Inhibitors of DNA Methyltransferases in Cancer Cells. *J. Med. Chem.* **2017**, *60* (11), 4665–4679. https://doi.org/10.1021/acs.jmedchem.7b00176.

1026 (12) Chen, S.; Wang, Y.; Zhou, W.; Li, S.; Peng, J.; Shi, Z.; Hu, J.; Liu, Y.-C.; Ding, H.; Lin, Y.; Li, L.; Cheng, S.; Liu, J.; Lu, T.; Jiang, H.; Liu, B.; Zheng, M.; Luo, C. Identifying Novel Selective Non-Nucleoside DNA Methyltransferase 1 Inhibitors through Docking-Based Virtual Screening. *J. Med. Chem.* **2014**, *57* (21), 9028–9041. https://doi.org/10.1021/jm501134e.

1030 (13) Pappalardi, M. B.; Keenan, K.; Cockerill, M.; Kellner, W. A.; Stowell, A.; Sherk, C.; Wong, K.; Pathuri, S.; Briand, J.; Steidel, M.; Chapman, P.; Groy, A.; Wiseman, A. K.; McHugh, C. F.; Campobasso, N.; Graves, A. P.; Fairweather, E.; Werner, T.; Raoof, A.; Butlin, R. J.; Rueda, L.; Horton, J. R.; Fosbenner, D. T.; Zhang, C.; Handler, J. L.; Muliaditan, M.; Mebrahtu, M.; Jaworski, J.-P.; McNulty, D. E.; Burt, C.; Eberl, H. C.; Taylor, A. N.; Ho, T.; Merrihew, S.; Foley, S. W.; Rutkowska, A.; Li, M.; Romeril, S. P.; Goldberg, K.; Zhang, X.; Kershaw, C. S.; Bantscheff, M.; Jurewicz, A. J.; Minthorn, E.; Grandi, P.; Patel, M.; Benowitz, A. B.; Mohammad, H. P.; Gilmartin, A. G.; Prinjha, R. K.; Ogilvie, D.; Carpenter, C.; Heerding, D.; Baylin, S. B.; Jones, P. A.; Cheng, X.; King, B. W.; Luengo, J. I.; Jordan, A. M.; Waddell, I.; Kruger, R. G.; McCabe, M. T. Discovery of a First-in-Class Reversible DNMT1-Selective Inhibitor with Improved Tolerability and Efficacy in Acute Myeloid Leukemia. *Nat. Cancer* **2021**, *2* (10), 1002–1017.

1042 (14) Sandoval, J. E.; Ramabadran, R.; Stillson, N.; Sarah, L.; Fujimori, D. G.; Goodell, M. A.; Reich, N. First-in-Class Allosteric Inhibitors of DNMT3A Disrupt Protein–Protein Interactions and Induce Acute Myeloid Leukemia Cell Differentiation. *J. Med. Chem.* **2022**. https://doi.org/10.1021/acs.jmedchem.2c00725.

1046 (15) Ceccaldi, A.; Rajavelu, A.; Champion, C.; Rampon, C.; Jurkowska, R.; Jankevicius, G.; Sénamaud-Beaufort, C.; Ponger, L.; Gagey, N.; Ali, H. D.; Tost, J.; Vriz, S.; Ros, S.; Dauzonne, D.; Jeltsch, A.; Guianvarc'h, D.; Arimondo, P. B. C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development. *ChemBioChem* **2011**, *12* (9), 1337–1345. https://doi.org/10.1002/cbic.201100130.

1051 (16) Pechalrieu, D.; Dauzonne, D.; Arimondo, P. B.; Lopez, M. Synthesis of Novel 3-Halo-3-Nitroflavanones and Their Activities as DNA Methyltransferase Inhibitors in Cancer Cells. *Eur. J. Med. Chem.* **2020**, *186*, 111829. https://doi.org/10.1016/j.ejmech.2019.111829.

1054 (17) Rapposelli, S.; Settimo, F. D.; Digiacomo, M.; Motta, C. L.; Lapucci, A.; Sartini, S.; Vanni, M. Synthesis and Biological Evaluation of 2'-Oxo-2,3-Dihydro-3'H- Spiro[Chromene-4,5'-[1,3]Oxazolidin]-3'yl]Acetic Acid Derivatives as Aldose Reductase Inhibitors. *Arch. Pharm. (Weinheim)* **2011**, *344* (6), 372–385. https://doi.org/10.1002/ardp.201000302.

1058 (18) Serhouni, A.; Calzaferri, F.; Bessina, Y.; van der Lee, A.; Arimondo, P. B.; Louet, M.; Winum, J.-Y.; Lopez, M. 3-Halo-3-Nitro-Aza/Thioflavanones: DNMT Inhibitors with a Two-Site Binding Mode in the *h*DNMT3A Catalytic Pocket. *Bioorg. Med. Chem.* **2025**, *118*, 117988. https://doi.org/10.1016/j.bmc.2024.117988.

1062 (19) Merle, A.; Descotes, G. Synthèse et réactivité des bromhydrines dérivées de la chromone. *J. Heterocycl. Chem.* **1975**, *12* (5), 981–984. https://doi.org/10.1002/jhet.5570120532.

1064 (20) Kawamoto, T.; Sasaki, R.; Kamimura, A. Synthesis of α-Trifluoromethylated Ketones from Vinyl Triflates in the Absence of External Trifluoromethyl Sources. *Angew. Chem. Int. Ed.* **2017**, *56* (5), 1342–1345. https://doi.org/10.1002/anie.201608591.
1067 (21)Peng, T.; Wang, G.; Zhang, S.; Sun, Y.; Liu, S.; Wang, L. One-Pot Synthesis of 3-Haloflavones from Flavones Using Oxone® and Potassium Halide as a Halogenation Reagent. *Tetrahedron* **2020**, *61* (7), 151511. https://doi.org/10.1016/j.tetlet.2019.151511.

1068 (22)Vatanmakanian, M.; Steffan, J. J.; Koul, S.; Ochoa, A. C.; Chaturvedi, L. S.; Koul, H. K. Regulation of SPDEF Expression by DNA Methylation in Advanced Prostate Cancer. *Front. Endocrinol.* **2023**, *14*. https://doi.org/10.3389/fendo.2023.1156120.

1070 (23)Lee, J.; Lee, M.-S.; Jeoung, D.-I.; Kim, Y.-M.; Lee, H. Promoter CpG-Site Methylation of the KAI1 Metastasis Suppressor Gene Contributes to Its Epigenetic Repression in Prostate Cancer. *The Prostate* **2017**, *77* (4), 350–360. https://doi.org/10.1002/pros.23274.

1071 (24)Donehower, L. A.; Soussi, T.; Korkut, A.; Liu, Y.; Schultz, A.; Cardenas, M.; Li, X.; Babur, O.; Hsu, T.-K.; Lichtarge, O.; Weinstein, J. N.; Akbani, R.; Wheeler, D. A. Integrated Analysis of TP53 Gene and Pathway Alterations in The Cancer Genome Atlas. *Cell Rep.* **2019**, *28* (5), 1370–1384.e5. https://doi.org/10.1016/j.celrep.2019.07.001.

1072 (25)Joerger, A. C.; Fersht, A. R. The P53 Pathway: Origins, Inactivation in Cancer, and Emerging Therapeutic Approaches. *Annu. Rev. Biochem.* **2016**, *85*, 375–404. https://doi.org/10.1146/annurev-biochem-060815-014710.

1073 (26)Aldrich, C.; Bertozzi, C.; Georg, G. I.; Kiessling, L.; Lindsley, C.; Liotta, D.; Merz Jr., K. M.; Schepartz, A.; Wang, S. The Ecstasy and Agony of Assay Interference Compounds. *J. Med. Chem.* **2017**, *60* (6), 2165–2168. https://doi.org/10.1021/acs.jmedchem.7b00229.

1074 (27)Adam, W.; Golsch, D.; Hadjiarapoglou, L.; Patonay, T. Epoxidation of Flavones by Dimethyldioxirane. *J. Org. Chem.* **1991**, *56* (26), 7292–7297. https://doi.org/10.1021/jo00026a020.

1075 (28)Bernini, R.; Mincione, E.; Provenzano, G.; Fabrizi, G.; Tempesta, S.; Pasqualetti, M. Obtaining New Flavanones Exhibiting Antifungal Activities by Methyltrioxorhenium-Catalyzed Epoxidation–Methanolysis of Flavones. *Tetrahedron* **2008**, *64* (32), 7561–7566. https://doi.org/10.1016/j.tet.2008.05.101.

1076 (29)Lee, B. H.; Yegnasubramanian, S.; Lin, X.; Nelson, W. G. Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1 *. *J. Biol. Chem.* **2005**, *280* (49), 40749–40756. https://doi.org/10.1074/jbc.M505593200.

1077 (30)Bunz, F.; Dutriaux, A.; Lengauer, C.; Waldman, T.; Zhou, S.; Brown, J. P.; Sedivy, J. M.; Kinzler, K. W.; Vogelstein, B. Requirement for P53 and P21 to Sustain G2 Arrest after DNA Damage. *Science* **1998**, *282* (5393), 1497–1501. https://doi.org/10.1126/science.282.5393.1497.

1078 (31)Jia, D.; Jurkowska, R. Z.; Zhang, X.; Jeltsch, A.; Cheng, X. Structure of Dnmt3a Bound to Dnmt3L Suggests a Model for de Novo DNA Methylation. *Nature* **2007**, *449* (7159), 248–251. https://doi.org/10.1038/nature06146.

1079 (32)Trott, O.; Olson, A. J. AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. *J. Comput. Chem.* **2010**, *31* (2), 455–461. https://doi.org/10.1002/jcc.21334.

1080 (33)Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera—A Visualization System for Exploratory Research and Analysis. *J. Comput. Chem.* **2004**, *25* (13), 1605–1612. https://doi.org/10.1002/jcc.20084.

1081 (34)Bruker. *APEX3*, *SAINT*, *XPREP* and *SADABS*; Bruker AXS Inc., 2016.

1082 (35)Lee, A. van der. Charge Flipping for Routine Structure Solution. *J. Appl. Crystallogr.* **2013**, *46* (5), 1306–1315. https://doi.org/10.1107/S0021889813020049.

1083 (36)Palatinus, L.; Chapuis, G. SUPERFLIP – a Computer Program for the Solution of Crystal Structures by Charge Flipping in Arbitrary Dimensions. *J. Appl. Crystallogr.* **2007**, *40* (4), 786–790. https://doi.org/10.1107/S0021889807029238.

1084 (37)Betteridge, P. W.; Carruthers, J. R.; Cooper, R. I.; Prout, K.; Watkin, D. J. CRYSTALS Version 12: Software for Guided Crystal Structure Analysis. *J. Appl. Crystallogr.* **2003**, *36* (6), 1487–1487. https://doi.org/10.1107/S0021889803021800.

1085 (38)Cooper, R. I.; Thompson, A. L.; Watkin, D. J. CRYSTALS Enhancements: Dealing with Hydrogen Atoms in Refinement. *J. Appl. Crystallogr.* **2010**, *43* (5), 1100–1107. https://doi.org/10.1107/S0021889810025598.

1086 (39)Macrae, C. F.; Sovago, I.; Cottrell, S. J.; Galek, P. T. A.; McCabe, P.; Pidcock, E.; Platings, M.; Shields, G. P.; Stevens, J. S.; Towler, M.; Wood, P. A. Mercury 4.0: From Visualization to
1122 Analysis, Design and Prediction. *J. Appl. Crystallogr.* **2020**, *53* (1), 226–235.
1123 https://doi.org/10.1107/S1600576719014092.
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