# 3-Halo-3-nitro-aza/thioflavanones: DNMT inhibitors with a two-site binding mode in the hDNMT3A catalytic pocket

Alexandra Serhouni, Francesco Calzaferri, Yannick Bessina, Arie van Der Lee, Paola B Arimondo, Maxime Louet, Jean-Yves Winum, Marie Lopez

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Alexandra Serhouni, Francesco Calzaferri, Yannick Bessina, Arie van Der Lee, Paola B Arimondo, et al.. 3-Halo-3-nitro-aza/thioflavanones: DNMT inhibitors with a two-site binding mode in the hDNMT3A catalytic pocket. Bioorganic and Medicinal Chemistry, 2024, 118, pp.117988. 10.1016/j.bmc.2024.117988. hal-04817890

HAL Id: hal-04817890

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Submitted on 5 Aug 2025

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# 3-Halo-3-nitro-aza/thioflavanones: DNMT inhibitors with a two-site binding mode in the *h*DNMT3A catalytic pocket

Alexandra Serhouni<sup>a,1</sup>, Francesco Calzaferri<sup>a,1</sup>, Yannick Bessina<sup>a</sup>, Arie van der Lee<sup>b</sup>, Paola B. Arimondo<sup>c</sup>, Maxime Louet<sup>a</sup>, Jean-Yves Winum<sup>a</sup>, Marie Lopez<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**
DNA methylation
DNMT inhibitors
Epigenetics
Binding modes

## ABSTRACT

Flavonoid derivatives are natural product analogues that have shown great interest for therapeutic applications as modulators of DNA methylation. In this article we report new synthesis pathways to access ten novel flavonoid derivatives (i.e. 3-halo-3-nitro-aza/thioflavanones) to be used as potential DNA methyltransferase inhibitors. These compounds have a micromolar inhibition against human DNA methyltransferase 3A in our *in vitro* fluorescence-based assay. Importantly, a docking study of representative compounds of this series in the enzyme pocket highlights a mode of interaction in the catalytic pocket of *h*DNMT3A that has never been described.

## 1. Introduction

DNA methyltransferases (DNMTs) are epigenetic enzymes that catalyse DNA methylation at position 5 of a cytosine residue in a CpG context.<fnref n="1" /> As epigenetic modification, DNA methylation is involved in the control of gene expression. Besides, aberrant DNA methylation was shown in several pathological contexts including cancers.² Interestingly, two DNMT inhibitors (DNMTi), 5-azacytidine (5aza, Vidaza®) and 5-aza-2'-deoxycytidine (5azadC, Dacogen™), are used for leukaemia treatment, which validates the interest of DNMTs as therapeutic targets.³ However, as nucleoside analogues, 5aza and 5azadC show poor chemical stability and require incorporation in DNA for DNMT inhibition. This incorporation occurs without any DNA site or cell specificity, which causes important side-effects and therefore limits their administration at low doses and in combination. Considering these hurdles, there has been a strong interest in identifying new non-nucleoside DNMTi.⁴ Among non-nucleoside compounds affecting DNA methylation, several flavonoid derivatives were reported with anticancer properties (Fig. 1).⁵ For example, genistein affects chromatin configuration and DNA methylation,⁶ quercetin and EGCG and its alkylated analogues affect DNA methylation level together with histone acetylation level.⁷,⁸ In a previous work, our groups identified 3-chloro- and 3-bromo-3-nitroflavanones as potent DNMTi.⁹,¹⁰

3-Halo-3-nitroflavanones are potent DNMTi with micromolar active concentrations for DNMT inhibition activity and in leukaemia cell lines.⁹,¹⁰ One hurdle to further clinical development of these compound is their susceptibility to β-elimination leading to corresponding 3-nitroflavones, whose inhibition activity drastically decreases.⁹ Although 3-bromo-3-nitroflavanone showed a significant stability enhancement and good in-cell efficacy,¹⁰ this is not sufficient for further development. Considering the interesting properties of flavonoid derivatives to be used as therapeutic compounds,¹¹ the current study explored two new families of flavonoid derivatives, i.e. 3-halo-3-nitro-aza/thioflavanones, as DNMTi. We established synthesis pathways and obtained ten undescribed aza- and thioflavanones, which showed micromolar activity on purified DNMT3A-c. Importantly, a new mode of interaction in the catalytic pocket of the enzyme was identified by docking study, which opens new perspectives towards the development of DNMTi.

## 2. Results and discussion

### 2.1. Design

Although previous works extensively explored the impact of substitutions on phenyl rings A and B of the 3-halo-3-nitroflavanones on the DNMT inhibition activity,⁹,¹⁰ the influence of the heteroatom in the

<footnote><sup>a</sup> Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS-Université de Montpellier-ENSCM, 1919 route de Mende, 34296 Montpellier cedex 5, France</footnote>

<footnote><sup>b</sup> Institut Européen des Membranes (IEM), UMR 5635 CNRS-Université de Montpellier, 300 avenue du Prof. E. Jeanbrau, 34296 Montpellier cedex 5, France</footnote>

<footnote><sup>c</sup> Institut Pasteur, Université Paris Cité, CNRS UMR 3523 Chem4Life, Epigenetic Chemical Biology, Department of Structural Biology and Chemistry, 75015 Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: marie.lopez@cnrs.fr (M. Lopez).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Examples of previously described flavonoid derivatives reported to modulate DNA methylation and 3-halo-3-nitro-aza/thioflavanones, target compounds of this work.</figcaption></figure>

cycle C has never been studied. Previous docking studies showed an orientation of the oxygen atom nearby the catalytic pocket,⁹ which highlighted the possibility of additional interaction with the heteroatom. Besides, a change in the heteroatom will strongly impact the pKa of the proton H-2, which could lead to an improvement of the 3-halo-3-nitroflavonoid chemical stability, reducing the ability of flavanones to undergo β-elimination. Altogether this shows the interest of exploring the impact of modifying the heteroatom of 3-nitro-3-flavanones and designed compounds are represented Fig. 1.

## 2.2. Chemical synthesis

The first step of the synthesis of the target 3-halo-3-nitro-azaflavanones was based on acyl-amination reaction of anthranilate with nitroalkene described by Chaturvedi et al.,¹² which allows direct access to 3-nitro-azaflavanones (Scheme 1).

First, anthranilic acid was activated as the benzotriazole derivative 1, then acyl-amination reaction with appropriate nitrostyrene led to 3-nitro-azaflavanones 2–3. This reaction proceeded with moderate yield mainly due to the addition of benzotriazole moiety on the nitrostyrenes as secondary products. Importantly, this cyclisation mechanism led only to anti-diastereoisomers for compounds 2 and 3. These latter were characterised by NMR and LC-MS analysis and their structures were further confirmed by X-ray crystallography (Fig. 2).

Finally, halogenation reaction with either *N*-bromo- or *N*-chlorosuccinimide provided final compounds **NFlav2017–2019** (Scheme 1). Following halogenation reaction, only one diastereoisomer was identified but their characterisation did not enable us to determine which of the *syn*- and *anti*-isomer was obtained.

Regarding 3-bromo-3-nitro-thioflavanones, several strategies were

<figure><img src="image_6.png" /><figcaption>**Scheme 1.** Synthesis pathway of 3-halo-3-nitro-azaflavanones **NFlav2017–2019**. (a) BtH (0.85 equiv.), DCC (1.2 equiv.), CH₂Cl₂, rt, overnight, 48%; (b) nitrostyrene (1.2 equiv.), H₂O/ACN (1:1), 80 °C, 5 h, 36–52%; (c) NBS or NCS (1.6 equiv.), Na₂CO₃ (1.4 equiv.), rt, 5 min – 2 h, 14–45%.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Crystal structure of compounds 2 and 3.</figcaption></figure>

tested to prepare the thioflavanone scaffold, i.e. Newman-Kwart rearrangement,¹³ copper-catalysed domino reaction using xanthate as thiol surrogate¹⁴ and direct condensation reaction using 2'-mercaptoacetophenone¹⁵ (Scheme 2).

First, addition of *N*,*N*-dimethyl thiocarbamoyl chloride on 2-hydroxylacetophenone derivative (Scheme 2, Route A) can allow direct access to 3-nitro-thioflavanones. Newmann-Kwart rearrangement of compound 4 led to the thiocarbamate 5, which was further hydrolysed to give thioacetophenone 6. However, in our hands, starting from 2-nitro-substituted derivative (R = -NO₂), no expected thiocarbamate was formed, probably due to side reactions occurring with activated methylene. Therefore, Route A was abandoned to focus on Route B (Scheme 2). This latest led to the expected thioflavanone 10 (R = H) but when nitro-substituted benzaldehyde (R = 2'-NO₂ or 2'-NO₂-5'-OMe) was used, chalcone formation (8–9) was very limited and subsequent cyclisation reaction failed. Therefore, direct condensation of 2'-mercaptoacetophenone with corresponding benzaldehyde (Scheme 2, Route C) was tested and selected as the preferred synthetic route.

More precisely, 2'-mercaptoacetophenone 6 was obtained by treatment of thiosalicylic acid with methyllithium. Condensation reaction with appropriate benzaldehyde derivatives mediated by lithium diisopropylamide (LDA) provided thioflavanones 10–13. Contrary to what described in the literature,¹³ in our hands, yields were very moderate for this condensation, thus various reaction conditions were tested to improve reaction yield, as shown in Table 1. An increase in the amount of base (entry 2) and the presence of an electron-withdrawing group on benzaldehyde derivatives, either in *ortho*- (entry 3) or *meta*- (entry 5) position, led to lower reaction yields. More drastically, a nitro-substituted benzaldehyde in *ortho*- (entry 4) or *meta*- (entry 6) position did not lead to the expected compounds. A decrease of *meta*-nitro-benzaldehyde equivalent (entry 7) slightly favoured the formation of thioflavanone but no similar effect was observed with *ortho*-nitro-benzaldehyde (entry 8).

Nitration of thioflavanones was carried out with nitric acid in acetic acid and led to 3-nitro-thioflavanones **14–17** as a mixture of diastereoisomers. Compounds **14–17** were easily identified by ¹H NMR with two characteristic doublets corresponding to H-2 (δ = 5.3–5.6 ppm, J<sub>H2-H3</sub> = 13.1 Hz) and H-3 (δ = 6.1–6.2 ppm, J<sub>H2-H3</sub> = 13.1 Hz). Due to purification complexity, both diastereoisomers were not purified and were used as such for halogenation reaction. Halogenation using *N*-bromosuccinimide (NBS) afforded expected compounds **SFlav1902–1905** and **SFlav2001–2003**. Interestingly, considering that halogenation occurred on both sides of 3-nitro-thioflavanones, both diastereoisomers of 3-bromo-3-nitro-thioflavanones can be formed, since 3-nitro-thioflavanones starting material **14–17** were used as mixtures of diastereoisomers. For all 3-nitro-thioflavanones, except **SFlav2003**, both diastereoisomers were obtained and separated by flash
<figure><img src="image_8.png" /><figcaption>**Scheme 2.** Synthesis pathways of 3-bromo-3-nitro-thioflavanones **SFlav1902–1905** and **SFlav2001–2003**. a) ClC(S)NMe₂ (3 equiv.), Dabco (3 equiv.), DMF, 50 °C, 6 h, quantitative; b) m.w., 41 %; c) aqueous NaOH (1 N, 12 equiv), MeOH, 100 °C, 3 h, 49 %; d) benzaldehyde derivative (1 equiv.), NaOH (1.5 equiv), EtOH/H₂O, 0 °C to r.t., 6 h, 16–90 %; e) xanthate (1 equiv.), Cu(OAc)₂ (10 mol%), DMSO, 100 °C, 6 h, 0–36 %; f) aqueous HNO₃ solution (65 % w/w, 6.5 equiv. to 10 equiv.), AcOH, 10 °C to 20 °C, overnight, 30–40 %; g) NBS or NCS (1.5 equiv.), Na₂CO₃ (1.5 equiv.), THF, 0 °C, 1 h, 33–77 %; h) MeLi (4 equiv.), THF, –15 °C to 0 °C, 1 h, 49 %; i) LDA (2 equiv.), benzaldehyde derivative (1.6 equiv.), THF, –15 °C to rt, 3 h, 11–51 %.</figcaption></figure>

<caption>**Table 1**</caption>

Reaction conditions for thioflavanone synthesis from 2'-mercaptoacetophenone and benzaldehyde derivatives.

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

<table><thead><tr><th>Entry</th><th>R</th><th>Aldehyde (equiv.)</th><th>LDA (equiv.)</th><th>C<sub>final</sub> (M)</th><th>Isolated yield (cpd)</th></tr></thead><tbody><tr><td>1</td><td>H</td><td>1.6</td><td>2.0</td><td>0.23</td><td>51% (10)</td></tr><tr><td>2</td><td>H</td><td>1.6</td><td>3.4</td><td>0.19</td><td>29% (10)</td></tr><tr><td>3</td><td>o-F</td><td>1.6</td><td>2.0</td><td>0.23</td><td>22% (11)</td></tr><tr><td>4</td><td>o-NO<sub>2</sub></td><td>1.6</td><td>3.4</td><td>0.19</td><td>0%</td></tr><tr><td>5</td><td>m-CF<sub>3</sub></td><td>1.6</td><td>2.0</td><td>0.19</td><td>14% (12)</td></tr><tr><td>6</td><td>m-NO<sub>2</sub></td><td>1.6</td><td>2.0</td><td>0.29</td><td>0%</td></tr><tr><td>7</td><td>m-NO<sub>2</sub></td><td>1.0</td><td>2.3</td><td>0.12</td><td>11% (13)</td></tr><tr><td>8</td><td>o-NO<sub>2</sub></td><td>1.0</td><td>2.2</td><td>0.12</td><td>0%</td></tr></tbody></table>

chromatography. To note, syn- and anti-diastereoisomers, referred to compound with bromine and hydrogen atoms, are in anti- and syn-periplanar position, respectively. Since it was previously reported that for 3-bromo-3-nitroflavanones, anti-diastereoisomer was associated with a more shielded H-2 signal in ¹H NMR compared to syn-analogue,¹⁰ we determined syn- or anti-diastereoisomers comparing ¹H NMR spectra. Regarding ¹³C NMR, C-2 signal is less shielded for anti-diastereoisomers compared to syn-analogues, in accordance to ¹³C NMR description of 3-bromo-3-nitroflavanones. Interestingly, compound **SFlav2003** was

prepared with the same synthesis pathway but in this case only the syn-diastereoisomer was obtained, as confirmed by comparison of the C-3 signal (δ = 104.4 ppm) in ¹³C NMR with the other thioflavanones. In fact, the C-3 signal of the final syn- and anti-thioflavanones was observed at δ(ppm) ∈ [103.7, 105.1] and δ(ppm) ∈ [99.2, 99.5], respectively. All final compounds were characterised by NMR and MS and show characteristic signals corresponding to H-2 as singlet at δ (ppm) ∈ [5.1, 6.0] and C-2 at δ(ppm) ∈ [45, 57]. Nitration and bromination yield for these compounds range from 28 to 87 %, strongly affected by the substituents on the phenyl ring and the general stability of final compounds, as showed by meta-CF₃-substituted **SFlav2001–2002** (see details below and Fig. 3, Entry 8).

### 2.3. Stability

Stability of 3-halo-3-nitro-aza/thioflavanones was assessed by HPLC over time in the DNMT methylation buffer (Fig. 3).

Regarding 3-halo-3-nitro-azaflavanones (Fig. 3A and C), substitution of cycle B with a methoxy group in *para*-position led to a 2-fold increase of stability (NFlav2019 vs NFlav2017) as observed for 3-chloro-3-nitroflavanones.¹⁰ Interestingly, replacing the bromine atom (NFlav2017) by a chlorine (NFlav2018) resulted in a significant 3-fold stability increase with $t_{1/2}$ = 2.3 h, better than the previously reported 3-chloro-3-nitroflavanones (Flv69, $t_{1/2}$ = 0.75 h).¹⁰ The impact of buffer pH on stability was also assessed and compound NFlav2017 was shown to be more stable at pH 5.3 than at pH 7.2 (Fig. S1). LC-MS analysis of the degradation mixture of compounds of NFlav2017–2019 enabled the identification of flavone analogues as the main degradation compounds (Fig. S1). Other degradation compounds were detected by HPLC but their identification was not possible.

The stability of 3-halo-3-nitro-thioflavanones (Fig. 3B and C) is globally in the same range as the one previously observed with 3-halo-3-nitroflavanones. Degradation of either syn- or anti-isomers led to the same main compound, identified as the corresponding flavones by LC-MS. Interestingly, syn-derivatives (SFlav1902, SFlav1905 and SFlav2001) are more stable than their anti-analogues (SFlav2009, SFlav1904 and SFlav2002) with the most significant increase (about 15-fold) observed for meta-trifluoromethyl substitution (SFlav2002 vs SFlav2001). Considering that syn-derivatives, where bromine and hydrogen atoms are in anti-periplanar position, are more stable than their anti-analogues it can be hypothesised that the degradation does not
<figure><img src="image_10.png" /><figcaption>**Fig. 3.** Stability of 3-halo-3-nitro-aza- (A) and 3-bromo-3-nitro-thio- (B) flavanones in enzyme buffer, (C) calculated half-life times ($t_{1/2}$).</figcaption></figure>

occur through an E2 elimination mechanism. This hypothesis still need to be confirmed since the constraints of the flavonoid scaffold do not allow exact axial orientation (Fig. 2),¹⁰ which can impact the reactivity of the substituents. To note, alternative or multiple degradation pathways may exist, supporting our inability, despite our efforts, to isolate degradation products. Notably, para-trifluoromethane substitution (SFlav2001) led to a 3-fold increase stability compared to unsubstituted ring B analogue (SFlav1902) for syn-analogues whereas ortho-fluoro substitution did not lead to any increase of stability (SFlav1905).

Thus, although the stability of the 3-halo-3-nitroflavanones was not increased by changing the heteroatom for a nitrogen or sulfur atom, several compounds, e.g. NFlav2018 and SFlav2001, showed a stability in the same order of magnitude as previously described 3-halo-3-nitroflavanones.

## 2.4. Evaluation of hDNMT3A-c inhibition activity

Considering the interest of flavonoides in DNMT inhibition, inhibition activity of this series of 3-halo-3-nitro-aza/thioflavanones was determined on purified hDNMT3A-c, using a fluorescence-based assay (Table 2).

First, the lack of halide at 3-position led to inactive compounds (entry 3 and 4), as previously observed with 3-halo-3-nitroflavanones.¹⁰ 3-Bromo-3-nitro-azaflavanone (entry 7) is more active than its chlorine analogue (entry 6) and the presence of meta-methoxide substitution on cycle B (entry 7) leads to an increase of inhibition activity compared to non-substituted analogues (entry 5). Finally, the inhibition activity of 3-bromo-3-nitro-azaflavanones (entries 5 and 7) is similar to that reported for 3-chloro-3-nitroflavanones (entries 1 and 2).

Regarding 3-bromo-3-nitro-thioflavanones, they globally show similar DNMT3A-c inhibition activity regardless of the nature of the substituent on ring B. Interestingly, the syn-derivatives are slightly to significantly more potent than anti-analogues, which is probably correlated to their higher stability, as discussed above. Finally, comparison of aza- vs thioflavanones (entries 5, 8, 9) show that 3-bromo-3-nitro-azaflavanones are more active than their thio-analogues.

Due to the relatively low stability of some derivatives in the enzyme buffer, as discussed before, and to ensure that potential degradation compounds are not responsible for inhibition activity, inhibition

<caption>**Table 2**</caption>

hDNMT3A-c inhibition activity of 3-halo-3-nitro-aza/thioflavanones compared to 3-chloro-3-nitroflavanones.

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

<table><thead><tr><th rowspan="2">Entry</th><th rowspan="2">Cpd</th><th rowspan="2">Y</th><th rowspan="2">X</th><th rowspan="2">syn/ anti</th><th rowspan="2">R</th><th colspan="2">% inhibition <sup>a</sup></th></tr><tr><th>32 µM</th><th>10 µM</th></tr></thead><tbody><tr><td>1</td><td>Flv69</td><td>O</td><td>Cl</td><td>syn</td><td>2'-NO<sub>2</sub></td><td>84 ± 8</td><td>69±12</td></tr><tr><td>2</td><td>MLo1505</td><td>O</td><td>Br</td><td>syn</td><td>H</td><td>92 ± 3</td><td>69±4</td></tr><tr><td>3</td><td>NFlav2023</td><td>-NH</td><td>H</td><td>.</td><td>H</td><td colspan="2">Inactive <sup>b</sup></td></tr><tr><td>4</td><td>NFlav2022</td><td>-NH</td><td>H</td><td>.</td><td>3'-OMe</td><td colspan="2">Inactive <sup>b</sup></td></tr><tr><td>5</td><td>NFlav2019</td><td>-NH</td><td>Br</td><td>.</td><td>H</td><td>89 ± 1</td><td>55 ± 15</td></tr><tr><td>6</td><td>NFlav2018</td><td>-NH</td><td>Cl</td><td>.</td><td>3'-OMe</td><td>56 ± 3</td><td>42 ± 1</td></tr><tr><td>7</td><td>NFlav2017</td><td>-NH</td><td>Br</td><td>.</td><td>3'-OMe</td><td>100 ± 9</td><td>59 ± 3</td></tr><tr><td>8</td><td>SFlav1902</td><td>S</td><td>Br</td><td>syn</td><td>H</td><td>86 ± 6</td><td>51 ± 13</td></tr><tr><td>9</td><td>SFlav1903</td><td>S</td><td>Br</td><td>anti</td><td>H</td><td>68 ± 21</td><td>42 ± 7</td></tr><tr><td>10</td><td>SFlav1904</td><td>S</td><td>Br</td><td>syn</td><td>2'-F</td><td>67 ± 17</td><td>42 ± 20</td></tr><tr><td>11</td><td>SFlav1905</td><td>S</td><td>Br</td><td>anti</td><td>2'-F</td><td>67 ± 21</td><td>32 ± 15</td></tr><tr><td>12</td><td>SFlav2001</td><td>S</td><td>Br</td><td>syn</td><td>3'-CF<sub>3</sub></td><td>78 ± 8</td><td>34 ± 10</td></tr><tr><td>13</td><td>SFlav2002</td><td>S</td><td>Br</td><td>anti</td><td>3'-CF<sub>3</sub></td><td>63 ± 9</td><td>35 ± 13</td></tr><tr><td>14</td><td>SFlav2003</td><td>S</td><td>Br</td><td>syn</td><td>3'-NO<sub>2</sub></td><td>59 ± 9</td><td>27 ± 12</td></tr></tbody></table>

potency of compounds 10, 13 as well as that of thioflavone and 3-nitro-thioflavone, selected as representatives of potential degradation compounds of SFlav1902–1903 and SFlav2003, was evaluated. These four

<footnote><sup>a</sup> % values were determined on human DNMT3A-c as a mean of with at least two independent experiments. Mean deviation is reported.</footnote>

<footnote><sup>b</sup> les than 20 % and 10 % inhibition at 32 µM and 10 µM, respectively</footnote>
compounds are totally inactive against hDNMT3A-c (Fig. S2), which confirms that potential degradation compounds are not involved in DNMT inhibition activity. To further validate the absence of activity of degradation compounds that can be formed during the DNMT assay, compound NFlav2017 was intentionally degraded, at 37 °C in HEPES buffer for 2 h. Its degradation was confirmed by HPLC analysis (Fig. S3A). The resulting solution was then tested in DNMT inhibition assay at 32 and 10 µM and no significant activity was detected (Fig. S3B).

All together, these assays showed that 3-halo-3-nitro-aza/thioflavanones have moderate to good DNMT3A-c inhibition potency with activities comparable to those of previously described flavanone DNMTi for NFlav2017, NFlav2019 and SFlav1902. Nevertheless, these compounds need further optimisation to be used in in-cell assay since they have low effect in the first in-cell experiments performed (Fig. S4).

## 2.5. Docking studies

### 2.5.1. 3-chloro-3-nitroflavanones could bind in two different pockets

For an understanding of the molecular basis of the aza- and thioflavanone activities against DNMT3A, we performed molecular docking calculations and compared them with those described for 3-chloro-3-nitro-flavanones. Among the existing DNMT3A structures,¹⁶⁻¹⁸ we decided to use the DNMT3A-3L dimer co-crystallised with the cofactor SAM (PDB id: 2QRV).¹⁶ Our method was first validated by docking SAM, Flv62 and Flv69, previously described by Ceccaldi et al. (Table S1).⁹ Initially, only 2S,3R stereoisomers where considered. Surprisingly, besides the pose described by Ceccaldi et al. at the interface of the DNA and SAM pockets (Pkt1), we found also an equivalent number of poses with the compounds Flv62 and Flv69 located in the upper part of the (Pkt2), with ring B of flavanones placed in the binding site of the adenine of SAM. This binding pose allows the nitro group to establish hydrogen bonds (HBs) with Arg887, which does not occur when the compounds are in Pkt1 (Flv62, Table S1). Interestingly, energy scores for Flv62 are similar in both Pkt1 and Pkt2. Therefore, we hypothesise that flavanones and their derivatives could bind in both pockets by establishing transient interactions. This would be still in line with the ligand competition studies described in Ceccaldi et al., in which flavanones showed a mixed inhibition kinetics on DNMT3A, concluding that their affinity for DNMT3A could change depending on SAM binding.⁹

### 2.5.2. 3-bromo-3-nitroflavanones interact in a similar fashion than 3-chloro-3-nitroflavanones

Since current aza/thioflavanones bear a bromine atom at 3-position, as a reference, we docked first MLo1509, the 3-bromo-3-nitroflavanone analogue of Flv62, previously reported by our group to inhibit DNMT3A and reinduce luciferase gene expression in cells.¹⁰ MLo1509 bound like Flv62, in Pkt1 and Pkt2 (Table S1 and Fig. 4). However, the pose with the best energy score for MLo1509 is the one in Pkt2, differently for Flv62, and with significantly lower scores than those obtained in Pkt1 (−7.8 kcal/mol vs −6.9 kcal/mol, Table S1). Thus, we suggest that the large bromine at 3-position hampers the molecule access deeper into Pkt1, without altering its affinity for Pkt2.

### 2.5.3. Replacement of the oxygen at 1-position of flavanones with sulfur or nitrogen implies a difference in orientation in the two binding pockets

MLo1505,¹⁰ SFlav1902, and NFlav2019 were docked to assess the effects of the replacement of the oxygen at 1-position of flavanones with nitrogen and sulfur of the aza- and thioflavanone, respectively. The three of them were found in similar positions in both Pkt1 and Pkt2 (Table S1). In Pkt1, the free amine of NFlav2019 establishes a HB with Gly703 (Fig. 5, top panel). SFlav1902 shares the same position as both other derivatives but with increased binding score (−6.3 vs −7.1 and −7.6 kcal/mol for SFlav1902, NFlav2019 and MLo1505, respectively). Regarding Pkt2, the nitro and carbonyl groups of the three compounds are placed at the same position and are engaged in HB, but MLo1505 is

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

<figure><img src="image_13.png" /><figcaption>**Fig. 4.** Electrostatic surface and ribbon representation of 3-bromo-3-nitroflavanone MLo1509 docking poses in the DNMT3A-3L crystal (PDB: 2QRV), at the interface of DNA and SAM pockets (Pkt1, top panel) and at SAM pocket (Pkt2, bottom panel).</figcaption></figure>

tilted (Fig. 5, bottom panel). In that position oxygen atom of MLo1505 may fit better in the pocket than sulfur atom or -NH group, influencing compound binding scores (−6.2 vs −6.8 and −7.6 kcal/mol for SFlav1902, NFlav2019 and MLo1505, respectively).

### 2.5.4. ortho-Fluoro substitution thioflavanone ring B increase the affinity for the cofactor SAM pocket (Pkt2)

Differences were observed comparing thioflavanones without substitution on ring B (SFlav1902) and ortho-fluoro substituted compound (SFlav1905). In fact, we noticed that SFlav1902 bound at both Pkt1 and Pkt2, differently from SFlav1905 that was positioned only in Pkt2 (Table S1). We thus suggest that fluorine-substitution on ring B increases the affinity to the lipophilic Pkt2, which is surrounded by Phe636, Val661 and Val683. This was confirmed by the docking of SFlav2001 that has a larger fluorinated group (-CF₃) and whose poses always occupy the same position in Pkt2 (Table S1). Interestingly, compound SFlav1902, with no substituent on ring B, has higher potency than the other derivatives in our DNMT3A-c assay. This suggests that poses found in Pkt2 could represent just a transitory binding state, and that those found in Pkt1 would be responsible of the inhibition observed in the experimental data.
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 5.** Electrostatic surface and ribbon representation of aza-, thio- and flavanones docking poses in the DNMT3A-3L crystal (PDB: 2QRV). NFlav2019 in Pkt1, forming a HB with Gly703 (top panel); MLo1505 (yellow backbone), SFlav1902 (orange backbone) and NFlav2019 (green backbone) binding Pkt2 (bottom panel). HBs between nitro and carbonyls groups and Arg887 are shown in yellow.</figcaption></figure>

### 2.5.5. Enantiomers in both syn- and anti-isomers show different contribution depending on substituents of ring B

2R,3S enantiomers were docked to assess whether they had more impact on compound activities in respect to 2S,3R isomers. We observed that they all docked in the same pose in Pkt2, with ring B interacting with the adenine binding site and the flavanone core stretching toward Arg887. 2R,3S-derivative energies are lower than those of 2S,3R enantiomers, suggesting that a stretched pose in the pocket is favoured over a bent one, because it can establish more interactions in the binding pocket, including lipophilic interactions of ring B in SAM adenine pocket.

To tend to rationalise the differences in potency between syn- and anti-diastereomers, we docked SFlav1904, the anti-analogue of SFlav1905 (Fig. 6A). 2R,3R-isomer, which showed a better docking score than 2R,3S-isomer (−7.7 kcal/mol vs −6.2 kcal/mol), was found at the same place and orientation. However, 2R,3R-isomer activity may

<figure><img src="image_16.png" /><figcaption>**Fig. 6.** Docking comparison between pairs of stereoisomers of SFlav2004 and SFlav2005 in the ribbon representation of DNMT3A-3L crystal (PDB: 2QRV). The stretched pose of 2R,3S-isomer (pink) and the bent one of 2S,3R-isomer (light green) (A); anti-2R,3R-isomer (cyan), engaging the nitro group in an interaction with Glu 660, and syn-2R,3S-isomer (pink) (B).</figcaption></figure>

benefit from the interaction of the electron deficient 3-nitro group of the thioflavanone with the lone pair of sulfur and oxygen atoms of Cys662 and Glu660, respectively (Fig. 6B).¹⁹ This could explain its increased activity in the experimental assays.

In summary, we identified that: 1) flavanones and their aza- and thio-analogues can bind both at the interface of the DNA and SAM pocket (Pkt1) or completely at the top of the SAM pocket (Pkt2); 2) the only replacement of the oxygen at 1-position of the flavanone with a sulfur or amine decreases the efficiency of the binding; 3) stretched 2R,3S-enantiomers could theoretically contribute more to compound activity than 2S,3R-ones; 4) syn-compounds presented more efficient binding than their corresponding anti-isomers, i.e. 2R,3R. However, the relevance of compound binding to Pkt1 and Pkt2 would need further experimental validation, but unfortunately, the binding mode of existing DNMT3A inhibitors has never been confirmed so far, for instance by crystal structure.

## 3. Conclusion

In conclusion, this work aimed at exploring molecular diversity of 3-halo-3-nitroflavanones by changing the intracycle heteroatom, evaluating their DNMT inhibition activity and the deciphering their possible mode of interaction by molecular docking. To do so, we described synthesis pathways to access 3-halo-3-nitro-aza/thioflavanones with moderate to good yield depending on the substituents on cycle B. Interestingly, when diastereoisomers were obtained, both compounds could be easily separated. Newly synthesised compounds showed similar stability levels and maintained a good DNMT inhibition activity compared to 3-halo-3-nitroflavanones. In particular, 3-bromo-2-nitro-azaflavanones NFlav2017 and NFlav2019, and 3-bromo-2-nitro-thioflavanones SFlav1902 were the most potent inhibitors against hDNMT3A-c with micromolar potency. Finally, docking studies enabled to identify a new interaction pocket (Pkt2), where nitro group could form hydrogen bonds with Arg887 residue. This is highly relevant for bromo-analogues where bromine atom might limit the entrance to Pkt1
favouring interaction in Pkt2. Altogether, this shows the impact of intracyclic heteroatom of 3-halo-3-nitroflavanones and could lead to the development of new DNMTi with optimised interactions in docking-identified Pkt2.

# 4. Experimental section

## 4.1. Material and methods

All reagents and solvents were purchased from commercial suppliers, Sigma-Aldrich, TCI or Alfa-Aesar. All reactions were monitored by TLC on silica gel 60 F<sub>254</sub> plates (Merck Art. No. 1.05554). Purifications by flash chromatography were carried out on a Purilflash 430 instrument (Interchim) using 30 µm spheric silica or 40–60 µm irregular silica cartridges. Preparative and semi-preparative HPLC were performed on an VWR instrument equipped with a LaPrep P110 pump and LaPrep P314 Dual absorbance detector. A C18 reversed-phase column (Waters, XSelect CSH C18 OBD, 30 × 250 mm, 5 µm) was used for preparative HPLC and C18 reversed-phase column (Macherey-Nagel, Nucleodur C18 HTec, 21 × 250 mm, 7 µm) was used for semi-preparative HPLC. Elution was performed using a binary gradient (solvent A: H<sub>2</sub>O + 0.1 % TFA; solvent B: ACN + 0.1 % TFA). Compound purity and stability were measured by high-performance liquid chromatography (HPLC, Thermo Scientific, UltiMate3000) on C18 reversed-phase column (Thermo Scientific, Hypersil GOLD aQ, 2.1 × 50 mm, 1.9 µm). Compound purity was confirmed as ≥95 % before biological evaluation. Elution was performed using a binary gradient (solvent A: H<sub>2</sub>O + 0.1 % TFA; solvent B: ACN + 0.1 % TFA). Low resolution mass spectra were acquired using electrospray in positive and negative modes. All MS analysis samples were prepared as solutions in ACN. LCMS analyses were performed 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 × 750 mm, 2.6 µm, 100 Å) was used. Elution was performed using a binary gradient (solvent A: H<sub>2</sub>O + 0.1 % formic acid; solvent B: ACN + 0.1 % formic acid). Final compounds were analysed by high-resolution ESI mass spectrometry (HRMS) using a microTOF-Q mass spectrometer fitted with an electrospray ion source. Some compounds did not give MS signal due to the presence of nitro group.<sup>20</sup> NMR spectra were recorded on a Bruker DRX-400 spectrometer at 400 MHz for <sup>1</sup>H and 101 MHz for <sup>13</sup>C. 2D NMR experiments such as <sup>1</sup>H–<sup>1</sup>H COSY, <sup>1</sup>H–<sup>13</sup>C HSQC, <sup>1</sup>H–<sup>13</sup>C HMBC experiments were performed to enable signal attributions. Chemical shifts (δ) are given in ppm, coupling constant (J) are measured in hertz (Hz). Multiplicity is indicated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; dd, doublet of doublet;ddd, doublet of doublet of doublet; m, multiplet. Compound numbering is reported in the *supplementary data*. Flavonoid derivatives are numbered as below using the conventional numbering:

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

## 4.2. Chemical synthesis

(2-Aminophenyl)(1H-benzo[d][1,2,3]triazol-1-yl)methanone (1). Benzotriazole (3.0 g, 25.2 mmol, 0.9 equiv.) and DCC (35.0 mmol, 1.2 equiv.) were added to a solution of the anthranilic acid (4.0 g, 29.2 mmol, 1.0 equiv.) in CH<sub>2</sub>Cl<sub>2</sub> (60 ml) and stirred overnight at rt. Resulting mixture was filtered, solid was washed with CH<sub>2</sub>Cl<sub>2</sub>, and filtrate was evaporated under reduced pressure. Then, residue was purified by silica gel chromatography using 100 % CH<sub>2</sub>Cl<sub>2</sub> as eluent to give title compound **1** (2.90 g, 14.0 mmol, 48 % yield) as a yellow solid. R<sub>F</sub> =

0.55 (CH<sub>2</sub>Cl<sub>2</sub>). ESI-MS *m/z* (Da) = 239 [M + H]<sup>+</sup>, 280 [M + ACN + H]<sup>+</sup>. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) 8.24 (dd, *J* = 8.3, 1.0 Hz, 1H, H-8 or H-5); 8.15 (dd, *J* = 8.3, 1.0 Hz, 1H, H-5 or H-8); 8.03 (dd, *J* = 8.2, 1.5 Hz, 1H, H-6'); 7.66 (ddd, *J* = 8.2, 7.1, 1.1 Hz, 1H, H-7 or H-6); 7.51 (ddd, *J* = 8.2, 6.3, 1.0 Hz, 1H, H-6 or H-7); 7.38 (ddd, *J* = 8.5, 7.1, 1.5 Hz, 1H, H-4); 6.79 (m, 1H, H-3'); 6.75 (m, 1H, H-5'); 5.78 (s, 2H, NH<sub>2</sub>). <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 167.4 (CO); 152.1 (C-2'); 145.8 (C-9); 135.5 (C-4'); 134.4 (C-6'); 132.8 (C-10); 130.0 (C-6 or C-7); 126.0 (C-7 or C-6); 120.2 (C-8 or C-5); 117.1, 116.6 (C-3', C-5'); 114.6 (C-5 or C-8); 111.9 (C-1').

3-Nitro-2-phenyl-2,3-dihydroquinolin-4(1H)-one (2). trans-Nitrostyrene (375 mg, 2.5 mmol, 1.2 equiv.) was added to a solution of 1-(2-aminobenzoyl)-1H-benzotriazole 1 (500 mg, 2.1 mmol, 1.0 equiv.) in a mixture of H<sub>2</sub>O/ACN 1:1 (15 ml). Reaction mixture was heated at 80 °C until completion, monitored by TLC. ACN was evaporated under reduced pressure and the aqueous layer was extracted with EtOAc (×3). Combined organic layers were washed with brine, dried over anhydrous Na<sub>2</sub>SO<sub>4</sub>, and concentrated under reduced pressure. Crude residue was purified by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 80:20) to give title compound **2** (200 mg, 0.8 mmol, 36 % yield). R<sub>F</sub> = 0.51 (cyclohexane/EtOAc 7:3). ESI-MS *m/z* (Da) = 269 [M + H]<sup>+</sup>, 310 [M + ACN + H]<sup>+</sup>; 267 [M–H]<sup>−</sup>. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 7.91 (dd, *J* = 8.0, 1.5 Hz, 1H, H-5); 7.57–7.48 (m, 2H, H<sub>arom.</sub> B); 7.48–7.38 (m, 4H, H-7 and H<sub>arom.</sub> B); 6.89 (dd, *J* = 7.2 Hz, 1H, H-6); 6.75 (d, *J* = 8.3 Hz, 1H, H-8); 5.63 (d, *J* = 12.8 Hz, 1H, H-3); 5.30 (d, *J* = 12.8 Hz, 1H, H-2). <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 182.1 (CO); 150.5 (C-9); 137.0 (C-7); 135.3 (C-1'); 130.1 (C-4'); 129.4 (2C)(C-2', C-6'); 128.6 (C-5); 127.6 (2C)(C-3', C-5'); 119.7 (C-6); 117.0 (C-10); 116.0 (C-8); 93.9 (C-3); 61.7 (C-2).

2-(3-Methoxyphenyl)-3-nitro-2,3-dihydroquinolin-4(1H)-one (3). trans-3-Methoxynitrostyrene (451 mg, 2.5 mmol, 1.2 equiv.) was added to a solution of 1-(2-aminobenzoyl)-1H-benzotriazole 1 (500 mg, 2.1 mmol, 1.0 equiv.) in a mixture of H<sub>2</sub>O/ACN 1:1 (15 ml) and heated at 80 °C until the completion of the reaction monitored by TLC. ACN was evaporated under reduced pressure and aqueous layer was extracted with EtOAc (×3). Combined organic layers were washed with brine, dried over anhydrous Na<sub>2</sub>SO<sub>4</sub>, and concentrated under reduced pressure. Crude residue was purified by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0–80:20). Compound **3** was obtained as a mixture with a by-product with a ratio of 53/47, estimated by NMR, estimated yield 52 %. The mixture of both compounds was used without further purification for the steps but a portion was purified by HPLC preparative to give **3** (77 mg) for the full characterization and biological evaluation. R<sub>F</sub> = 0.40 (cyclohexane/EtOAc 7:3). HRMS (ESI) Calcd for C<sub>16</sub>H<sub>15</sub>N<sub>2</sub>O<sub>4</sub> *m/z* (Da) = 299.1032, found *m/z* (Da) = 299.1027 [M + H]<sup>+</sup>. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 7.79 (br dd, *J* = 8.0, 1.6 Hz, 1H, H-5); 7.46 (ddd, *J* = 8.5, 7.1, 1.6 Hz, 1H, H-7); 7.39–7.33 (m, 1H, H-6'); 7.14–7.07 (m, 2H, H-2' and H-3'); 6.98 (ddd, *J* = 8.3, 2.6, 0.9 Hz, 1H, H-4'); 6.88 (ddd, *J* = 8.4, 1.0, 0.6 Hz, 1H, H-8); 6.84 (ddd, *J* = 8.1, 7.1, 1.0 Hz, 1H, H-6); 5.95 (d, *J* = 12.9 Hz, 1H, H-3); 5.84 (s, 1H, NH); 5.25 (t, *J* = 12.0 Hz, 1H, H-2); 3.80 (s, *J* = 1.9 Hz, 3H, CH<sub>3</sub>). <sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 183.8 (CO); 160.9 (C-5'); 152.5 (C-9); 138.3 (C-1'); 137.9 (C-7); 131.1 (C-6'); 128.5 (C-5); 120.9 (C-3'); 119.6 (C-6); 117.3 (C-10); 117.2 (C-8); 115.7 (C-4'); 114.4 (C-2'); 94.6 (C-3); 62.0 (C-2); 56.0 (CH<sub>3</sub>).

### 4.2.1. General procedure 1 – Synthesis of 3-halo-3-nitro-azaflavanones (NFlav2017-NFlav2019)

NBS or NCS (1.6 equiv.) and Na<sub>2</sub>CO<sub>3</sub> (1.4 equiv.) were added to a solution of appropriate 3-nitro-azaflavanones (1.0 equiv.) in anhydrous THF (*C*<sub>f</sub> = 65 mM) under Ar atmosphere. Reaction mixture was stirred at rt until disappearance of starting material, monitoring by TLC. After addition of CH<sub>2</sub>Cl<sub>2</sub> and H<sub>2</sub>O, aqueous layer was acidified to pH 6 using a 5 % citric acid aqueous solution and was extracted with CH<sub>2</sub>Cl<sub>2</sub> (×3). Combined organic layers were dried over anhydrous Na<sub>2</sub>SO<sub>4</sub>, filtered and concentrated under reduced pressure. Purification by flash
chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 80:20) afforded title compounds **NFlav2017-NFlav2019**.

**3-Bromo-2-(3-methoxyphenyl)-3-nitro-2,3-dihydroquinolin-4(1H)-one** (NFlav2017). Compound NFlav2017 (18 mg, 0.06 mmol, 29 % yield) was obtained from **3** (50 mg, 0.2 mmol) following general procedure 1. $R_F$ = 0.39 (cyclohexane/EtOAc 7:3). **HRMS (ESI)** Calcd for C₁₆H₁₂BrN₂O₄ m/z (Da) = 374.9981, found m/z (Da) = 374.9980 [M-H]⁻. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 8.03 (dd, *J* = 8.1, 1.3 Hz, 1H, H-5); 7.49 (td, *J* = 7.7, 1.5 Hz, 1H, H-7); 7.35 (m, 1H, H-4'); 7.05 (br t *J* = 7.8 Hz, 1H, H-5'); 7.02-6.96 (m, 3H, H-6, H-2', H-4'); 6.80 (d, *J* = 8.2 Hz, 1H, H-8); 5.11 (s, 1H, H-2); 4.93 (s, 1H, NH); 3.82 (s, 3H, CH₃). **¹³C NMR** (101 MHz, CDCl₃) δ (ppm) = 178.7 (CO); 159.8 (C-3'); 150.4 (C-9); 136.9 (C-7); 135.0 (C-1'); 130.0 (C-4'); 130.0 (C-5); 120.9 (C-2'), 120.8 (C-5'), 116.6 (C-10); 116.4 (C-8); 115.9 (C-6); 99.7 (C-3); 69.5 (C-2); 55.6 (s, OCH₃).

**3-Chloro-2-(3-methoxyphenyl)-3-nitro-2,3-dihydroquinolin-4(1H)-one** (NFlav2018). Compound NFlav2018 (30 mg, 0.14 mmol, 45 % yield) was obtained from **3** (100 mg, 0.3 mmol) following general procedure 1. $R_F$ = 0.40 (cyclohexane/EtOAc 7:3). **HRMS (ESI)** Calcd for C₁₆H₁₂ClN₂O₄ m/z (Da) = 331.0486, found m/z (Da) = 331.0490 [M-H]⁻. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 8.03 (ddd, *J* = 8.1, 1.6, 0.4 Hz, 1H, H-5); 7.49 (ddd, *J* = 8.3, 7.2, 1.6 Hz, 1H, H-7); 7.35 (dd, *J* = 8.0 Hz, 1H, H-4'); 7.04 (m, 1H, H-5'); 7.02-6.96 (m, 3H, H-6, H-2', H-6'); 6.81 (ddd, *J* = 8.3, 0.9, 0.5 Hz, 1H, H-8); 5.01 (s, 1H, H-2); 4.93 (s, 1H, NH); 3.83 (s, *J* = 5.1 Hz, 3H, CH₃). **¹³C NMR** (101 MHz, CDCl₃) δ (ppm) = 178.7 (CO); 159.9 (C-3'); 150.5 (C-9); 137.0 (C-7); 134.3 (C-1'); 130.2 (C-4'); 129.9 (C-5); 120.7, 120.7 (C-2', C-5'); 117.1 (C-10); 116.5 (C-8); 116.0 (C-6); 114.4 (C-6'); 102.6 (C-3); 68.7 (C-2); 55.6 (OCH₃).

**3-Bromo-3-nitro-2-phenyl-2,3-dihydroquinolin-4(1H)-one** (NFlav2019). Compound NFlav2019 (9,3 mg, 0.03 mmol, 14 % yield) was obtained from **2** (51 mg, 0.2 mmol) following general procedure 1. **HRMS (ESI)** Calcd for C₁₅H₁₀BrN₂O₃ m/z (Da) = 344.9875, found m/z (Da) = 344.9727 [M-H]⁻. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 8.08 (ddd, *J* = 8.1, 1.6, 0.4 Hz, 1H, H-5); 7.56-7.44 (m, 6H, H-7, H-2', H-3', H-4', H-5', H-6'); 7.00 (ddd, *J* = 8.1, 7.1, 1.0 Hz, 1H, H-6); 6.83 (ddd, *J* = 8.3, 1.0, 0.5 Hz, 1H, H-8); 5.18 (s, 1H, H-2); 4.95 (s, 1H, NH). **¹³C NMR** (101 MHz, CDCl₃) δ (ppm) = 178.5 (CO); 150.2 (C-9); 136.7 (C-7); 133.5 (C-1'); 130.5 (C-4'); 129.8 (C-5); 128.8 (2C) (C-3', C-5'), 128.5 (2C) (C-2', C-6'); 120.6 (C-6); 116.5 (C-10); 116.2 (C-8); 99.5 (C-3); 69.4 (C-2).

**O-(2-Acetylphenyl) dimethylcarbamothioate (4).** N,N-Dimethylthiocarbamoyl chloride (837 mg, 6.6 mmol, 3.0 equiv.) was added in one portion to a solution of 2'-hydroxyacetophenone (300 mg, 2.2 mmol, 1.0 equiv.) in DMF (3 ml) containing DABCO (741 mg, 6.6 mmol, 3.0 equiv.). Reaction mixture was stirred at 50 °C for 6 h. Then, it was quenched with HCl 2 N and extracted with CH₂Cl₂ (×3). Organic layer was washed with brine, dried over anhydrous Na₂SO₄, and evaporated under reduced pressure. Crude product was purified by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 70:30) to give title compound **4** (557 mg, 2.2 mmol, 100 % yield) as a light-pink solid. $R_F$ = 0.47 (cyclohexane/EtOAc 95:5). **ESI-MS m/z** (Da) = 224 [M + H]⁺. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 7.79 (dd, *J* = 7.8, 1.7 Hz, 1H, H-6'); 7.53 (ddd, *J* = 8.1, 7.4, 1.7 Hz, 1H, H-5'); 7.33 (dd, *J* = 7.7, 1.1 Hz, 1H, H-4'); 7.10 (dd, *J* = 8.1, 1.1 Hz, 1H, H-3'); 3.46 (s, 3H, N(CH₃)₂); 3.40 (s, 3H, N(CH₃)₂); 2.55 (s, 3H, COCH₃). **¹³C NMR** (101 MHz, CDCl₃) δ (ppm) = 197.7 (CO), 187.0 (CS), 152.3 (C-1'), 133.0 (C-5'), 131.9 (C-2'), 129.8 (C-6'), 126.0 (C-4'), 124.6 (C-3'), 43.3 (N(CH₃)₂), 38.9 (N(CH₃)₂), 29.5 (COCH₃).

**S-(2-Acetylphenyl) dimethylcarbamothioate (5).** O-(2-Acetylphenyl) dimethylcarbamothioate **4** (436 mg, 2.0 mmol, 1.0 equiv.) was placed in a closed vial and irradiated in a focused MW (power (p) = 250 kW; temperature (T) = 200 °C; time = 1h 30). Reaction conversion was monitored by TLC. Reaction mixture was then cooled and purified by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 50:50) to give title compound **5** (179 mg, 0.9 mmol, 41 % yield). $R_F$ = 0.38 (cyclohexane/EtOAc 95:5). **ESI-MS m/z** (Da) = 224 [M + H]⁺. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 7.61-7.55 (m, 2H, H₄O₃).

7.47-7.41 (m, 2H, H₄O₃), 3.11 (s, 3H, NCH₃), 3.01 (s, 3H, NCH₃), 2.59 (s, 3H, COCH₃).

**1-(2-mercaptophenyl)ethan-1-one (6).** **Method A.** S-(2-Acetylphenyl) dimethylcarbamothioate **5** (168 mg, 0.8 mmol, 1.0 equiv.) was dissolved in MeOH (3 ml). A 1 N aqueous solution of NaOH (9 ml, 9.0 mmol, 12.0 equiv.) was added, and reaction mixture was heated to reflux for 2 h 30. Reaction mixture was cooled down to rt and acidified to pH 2 with 1 N aqueous HCl. Resulting mixture was extracted with EtOAc (×3), and combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Crude mixture was purified by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0-50:50) to give title compound **6** (59 mg, 0.4 mmol, 51 % yield). $R_F$ = 0.72 (CH₂Cl₂). **Method B.** Methyl lithium (1.6 M in Et₂O, 97 ml, 156.0 mmol, 4.0 equiv.) was slowly added to a solution of thiosalicylic acid (6 g, 39.0 mmol, 1.0 equiv.) in anhydrous THF (50 ml) at -15 °C under Ar atmosphere. After 1 h stirring from -15 °C to 0 °C, resulting mixture was quenched with 1 N HCl, and THF was evaporated under reduced pressure. Mixture was poured into 1 N HCl and extracted with CH₂Cl₂ (×3). Combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Then, residue was purified by silica gel chromatography using CH₂Cl₂ to give title compound **6** (2.87 g, 19.1 mmol, 49 % yield) as a light-yellow liquid. $R_F$ = 0.72 (CH₂Cl₂). **ESI-MS m/z** (Da) = 151 [M-H]⁻; 194 [M + ACN + H]⁺. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 7.87 (br d, *J* = 7.8 Hz, 1H, H-6'); 7.32-7.28 (m, 2H, H-3', H-4'); 7.20 (m, 1H, H-5'); 4.45 (s, 1H, SH); 2.62 (s, 3H, CH₃). **¹³C NMR** (101 MHz, CDCl₃) δ (ppm) = 198.9 (CO); 137.6 (C-1'); 132.8 (C-2'); 132.2, 131.8 (C-3', C-4'); 131.7 (C-6'); 124.7 (C-5'); 27.8 (CH₃).

### 4.2.2. General procedure 2 – Synthesis of chalcones (7–9)

2'-Bromoacetophenone (1.0 equiv.) and appropriate benzaldehydes (1.0 equiv.) were successively added dropwise to a solution of NaOH (2.3 M, 1.5 equiv.) in water:ethanol 1:1 ($C_f$ (2'-bromoacetophenone) = 1.56 M) at 0 °C. Reaction mixture was allowed to warm up to rt overnight, then diluted with diethyl ether. Organic layer was washed with a saturated aqueous solution of NH₄Cl, brine and dried over anhydrous Na₂SO₄ and filtered. Solvent was evaporated and crude mixture was purified by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 80:20) to give title compounds **7–9**.

**(E)-1-(2-Bromophenyl)-3-phenylprop-2-en-1-one (7).** Title compound **7** (649 mg, 2.3 mmol, 91 % yield) was obtained as a pale-yellow oil from 2'-bromoacetophenone (339 µl, 2.5 mmol, 1.0 equiv.) and benzaldehyde (256 µl, 2.5 mmol, 1.0 equiv.) using general procedure 2. $R_F$ = 0.71 (cyclohexane/EtOAc 7:3). **ESI-MS m/z** (Da) 287 [M + H]⁺, 328 [M + ACN + H]⁺. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 7.65 (ddd, *J* = 7.9, 1.1, 0.5 Hz, 1H, H-2'); 7.58-7.54 (m, 2H), 7.46-7.39 (m, 6H), 7.37-7.32 (m, 1H) (H-2, H-5, H-6, H-7, H-8, H-9, H-3', H-4', H-5'); 7.10 (d, *J* = 16.1 Hz, 1H, H-3).

**(E)-1-(2-Bromophenyl)-3-(2-nitrophenyl)prop-2-en-1-one (8).** Title compound **8** (201 mg, 0.6 mmol, 24 % yield) was obtained as a dark solid from 2'-bromoacetophenone (339 µl, 2.5 mmol, 1.0 equiv.) and 2-nitrobenzaldehyde (256 µl, 2.5 mmol, 1.0 equiv.) using general procedure 2. $R_F$ = 0.62 (cyclohexane/EtOAc 7:3). **ESI-MS m/z** (Da) = 373 [M + ACN + H]⁺. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 8.05 (dd, *J* = 8.2, 1.0 Hz, 1H, H-6); 7.83 (d, *J* = 16.0 Hz, 1H, H-2); 7.74-7.65 (m, 3H), 7.56 (dd, *J* = 15.2, 1.7 Hz, 1H) (H-2', H-7, H-8, H-9); 7.48 (dd, *J* = 7.6, 1.8 Hz, 1H, H-5'); 7.43 (td, *J* = 7.4, 0.9 Hz, 1H, H-4'); 7.34 (td, *J* = 7.7, 1.8 Hz, 1H, H-3'); 6.97 (d, *J* = 16.0 Hz, 1H, H-3).

**(E)-1-(2-Bromophenyl)-3-(5-methoxy-2-nitrophenyl)prop-2-en-1-one (9).** Title compound **9** (61 mg, 0.4 mmol, 16 % yield) was obtained as a pale-yellow oil from 2'-Bromoacetophenone (136 µl, 1 mmol, 1.0 equiv.) and 2-nitro-5-methoxybenzaldehyde (182 ml, 1 mmol, 1.0 equiv.) using general procedure 2. $R_F$ = 0.44 (cyclohexane/EtOAc 7:3). **ESI-MS m/z** (Da) = 362 [M + H]⁺, 403 [M + ACN + H]⁺. **¹H NMR** (400 MHz, CDCl₃) δ (ppm) = 8.14 (d, *J* = 9.1 Hz, 1H, H-6); 7.90 (d, *J* = 16.0 Hz, 1H, H-2); 7.65 (ddd, *J* = 8.0, 1.2, 0.4 Hz, 1H, H-2'); 7.50 (dd, *J* = 7.6, 1.9, 0.4 Hz, H₄O₃).
1H, H-5'); 7.44 (td, *J* = 7.4, 1.1 Hz, 1H, H-4'); 7.35 (ddd, *J* = 8.0, 7.8, 1.8 Hz, 1H, H-3'); 7.08 (d, *J* = 2.8 Hz, 1H, H-9); 6.99 (dd, *J* = 9.1, 2.8 Hz, 1H, H-7); 6.88 (d, *J* = 15.9 Hz, 1H, H-3); 3.93 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 194.7 (CO); 163.6 (C-8); 143.7 (C-2); 141.0 (C-5); 140.1 (C-1'); 134.0 (C-4); 133.6 (C-2'); 131.8 (C-3'); 130.6 (C-3); 129.5 (C-5'); 127.9 (C-6); 127.5 (C-4'); 119.6 (C-6'); 115.3 (C-7); 114.2 (C-9); 56.2 (CH₃).

### 4.2.3. General procedure 3 – Synthesis of thioflavanones (10–13)

Lithium diisopropylamide (1.5 M in cyclohexane, 2.0 equiv.) was added to a solution of **6** (1.0 equiv.) in anhydrous THF under Ar atmosphere at -15 °C and stirred for 1 h at -15 °C before addition of a solution of appropriate benzaldehyde derivative (1.6 equiv.) solubilised in anhydrous THF. After 2 h stirring at -15 °C to RT, resulting mixture was quenched with 1 N HCl and THF and evaporated under reduced pressure. Residue was poured into 1 N HCl aqueous solution, extracted with CH₂Cl₂ (×3). Combined organic layers were washed with saturated NaHCO₃ solution, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (cyclohexane/EtOAc 95:5) to afford the title thioflavanones **10–13**.

**2-Phenylthiochroman-4-one (10).** *Method A.* (E)-2'-Bromochalcone **8** (144 mg, 0.5 mmol, 1.0 equiv.), ethyl xanthate (160 mg, 1.0 mmol, 2.0 equiv.) and Cu(OAc)₂ (9 mg, 0.05 mmol, 0.1 equiv.) were mixed in a round bottom flask under Ar atmosphere. Then, DMSO (2 ml) was added and reaction mixture was stirred at 100 °C for 6 days, then cool down to rt before addition of water and extraction with ethyl acetate (×3). Combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated. Purification by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 50:50) afforded title compound **10** (31 mg, 0.1 mmol, 27 % yield). *Method B.* Compound **10** was obtained from **6** (500 mg, 3.3 mmol, 1.0 equiv.) and benzaldehyde (531 µl, 5.3 mmol, 1.6 equiv.) following general procedure **3** (404 mg, 1.7 mmol, 51 % yield). *R*<sub>F</sub> = 0.75 (cyclohexane/EtOAc 8:2). *ESI-MS* *m/z* (*Da*) = 239 [M-H]<sup>-</sup>, 241 [M + H]<sup>+</sup>, 282 [M + ACN + H]<sup>+</sup>. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 8.18 (dd, *J* = 8.0, 1.5 Hz, 1H, H-5); 7.49–7.34 (m, 6H, H<sub>arom.</sub>); 7.30 (m, *J* = 7.92 Hz, 1H, H<sub>arom.</sub>); 7.22 (td, *J* = 7.86, 1.13 Hz, 1H, H-6); 4.74 (dd, *J* = 13.0, 3.1 Hz, 1H, H-2); 3.33 (dd, *J* = 13.0, 16.4 Hz, 1H, H-3a); 3.22 (dd, *J* = 16.4, 3.1 Hz, 1H, H-3b). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 194.4 (CO); 142.1 (C-9); 138.5 (C-1'); 133.7 (CH<sub>arom.</sub>); 130.4 (C-10); 129.1 (CH<sub>arom.</sub>); 129.1 (2C)(C-5, CH<sub>arom.</sub>); 128.5 (CH<sub>arom.</sub>); 127.5 (CH<sub>arom.</sub>); 127.3 (CH<sub>arom.</sub>); 125.3 (CH<sub>arom.</sub>); 46.7 (C-3); 45.5 (C-2).

**2-(2-Fluorophenyl)thiochroman-4-one (11).** Title compound **11** was obtained from compound **6** (500 mg, 3.3 mmol, 1.1 equiv.) and 2-fluorobenzaldehyde (553 µl, 5.3 mmol, 1.6 equiv.) following general procedure **3** (189 mg, 0.7 mmol, 22 % yield). *R*<sub>F</sub> = 0.75 (CH₂Cl₂). *ESI-MS* *m/z* (*Da*) = 257 [M-H]<sup>-</sup>. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 8.16 (dd, *J* = 7.2, 1.2 Hz, 1H, H-5); 7.47 (dd, *J* = 7.6, 1.7 Hz, 1H, H-6); 7.42 (ddd, *J* = 8.76, 7.22, 1.59, 1H, H7 or H8); 7.35–7.27 (m, 2H, H-8 or H-7, H-4'); 7.22 (dddd, *J* = 8.32, 7.22, 1.22, 1H, H-6); 7.16 (td, *J* = 7.60, 1.18, 1H, H<sub>5</sub>); 7.10 (dddd, *J* = 10.25, 8.24, 1.19, 1H, H-3'); 5.06 (dd, *J* = 12.4, 3.2 Hz, 1H, H-2); 3.33 (dd, *J* = 16.5, 12.4 Hz, 1H, H-3a); 3.19 (dd, *J* = 16.5, 3.2 Hz, 1H, H-3b). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 194.1 (s, CO); 160.3 (d, *J*<sub>C-F</sub> = 248.3 Hz, C-2'); 141.8 (C-9); 133.8 (C-7 or C-8); 130.4 (C-10); 130.1 (d, *J*<sub>C-F</sub> = 8.7 Hz, C-4'); 129.3 (C-5); 128.5 (d, *J*<sub>C-F</sub> = 3.1 Hz, C-6'); 127.5 (C-7 or C-8); 125.7 (d, *J*<sub>C-F</sub> = 13.7 Hz, C-1'); 125.5 (C-6); 124.7 (d, *J*<sub>C-F</sub> = 3.8 Hz, C-5'); 116.1 (d, *J*<sub>C-F</sub> = 22.0 Hz, C-3'); 45.8 (C-3); 38.1 (d, *J*<sub>C-F</sub> = 3.4 Hz, C-2).

**2-(3-(Trifluoromethyl)phenyl)thiochroman-4-one (12).** Title compound **12** was obtained from compound **6** (1.0 g, 6.6 mmol, 1.0 equiv.) and 3-(trifluoromethyl)benzaldehyde (1.4 ml, 11.0 mmol, 1.6 equiv.) following general procedure **3** (282 mg, 0.9 mmol, 14 % yield). *R*<sub>F</sub> = 0.55 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 8.17 (dd, *J* = 8.0, 1.5 Hz, 1H, H-5); 7.73 (s, 1H, H-2'); 7.64 (t, *J* = 7.3 Hz, 2H, H-4', H-5'); 7.54 (t, *J* = 7.7 Hz, 1H, H-6'); 7.44 (td, *J* = 7.8, 1.6 Hz,

1H, H-7); 7.30 (dd, *J* = 8.0, 0.8 Hz, 1H, H-8); 7.24 (td, *J* = 8.0, 1.1 Hz, 1H, H-6); 4.79 (dd, *J* = 12.8, 3.1 Hz, 1H, H-2); 3.33 (dd, *J* = 16.4, 12.8 Hz, 1H, H-3a); 3.23 (dd, *J* = 16.4, 3.2 Hz, 1H, H-3b). ¹³C NMR (101 MHz, CDCl₃) = δ (ppm) 193.6 (CO); 141.4 (C-9); 139.5 (C-1'); 133.9 (C-7); 131.4 (q, *J* = 32.2 Hz, C-3'); 130.9 (q, *J* = 1.3 Hz, C-5'); 130.3 (C-10); 129.7 (C-6'); 129.3 (C-5); 127.3 (C-8); 125.6 (C-6); 125.4 (q, *J* = 3.8 Hz, C-4'); 124.4 (q, *J* = 3.8 Hz, C-2'); 123.8 (d, *J* = 271.5 Hz, CF₃); 46.3 (C-3); 44.9 (C-2).

**2-(3-Nitrophenyl)thiochroman-4-one (13).** Title compound **13** was obtained from compound **6** (400 mg, 2.6 mmol, 1.0 equiv.) and 3-nitrobenzaldehyde (397 mg, 2.6 mmol, 1.0 equiv.) following general procedure **3** using lithium diisopropylamide (2.3 equiv.) (83 mg, 0.3 mmol, 11 % yield). *R*<sub>F</sub> = 0.40 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 8.33 (t, *J* = 2.0 Hz, 1H, H-2'); 8.21 (ddd, *J* = 8.2, 2.2, 1.0 Hz, 1H, H-4'); 8.15 (dd, *J* = 8.0, 1.3 Hz, 1H, H-5); 7.77 (br d, *J* = 7.7 Hz, 1H, H-6'); 7.58 (t, *J* = 8.0 Hz, 1H, H-5'); 7.45 (ddd, *J* = 8.0, 7.3, 1.6 Hz, 1H, H-7); 7.30 (dd, *J* = 8.0, 0.7 Hz, 1H, H-8); 7.25 (ddd, *J* = 8.3, 5.3, 1.2 Hz, 1H, H-6); 4.82 (dd, *J* = 12.2, 3.3 Hz, 1H, H-2); 3.35 (dd, *J* = 16.3, 12.2 Hz, 1H, H-3a); 3.26 (dd, *J* = 16.3, 3.4 Hz, 1H, H-3b). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 193.3 (CO); 148.6 (C-3'); 140.9 (C-9); 140.6 (C-1'); 134.1 (C-7); 133.6 (C-6'); 130.4 (C-10); 130.2 (C-5'); 129.4 (C-5); 127.4 (C-8); 125.8 (C-6); 123.6 (C-4'); 122.7 (C-2'); 46.2 (C-3); 44.7 (C-2).

### 4.2.4. General procedure 4 – Synthesis of 3-nitro-thioflavanones (14–17)

A 65 % aqueous solution of nitric acid (10.0 equiv.) was slowly added to a solution of appropriate thioflavanone **10–13** (1.0 equiv.) in acetic acid. Reaction mixture was stirred between 10 °C and 20 °C until completion, monitored by TLC. Reaction mixture was poured into water, aqueous layer was extracted with CH₂Cl₂ (×3) and combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Crude residue was purified by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 95:5) to give 3-nitro-thioflavanones **14–17** in mixture with a secondary product. Characteristic NMR signals were observed for H-2 and H-3, i.e. two doublets at 6.1–6.3 ppm and 5.3–5.6 ppm with *J*<sub>H2-H3</sub> ≈ 13 Hz (NMR spectra SI). Compounds **14–17** were used as such for the following step.

**3-Nitro-2-phenyl-2,3-dihydro-1-benzothiopyran-4-one (14).** Compound **14** was obtained from compound **10** (200 mg, 0.8 mmol) following general procedure **4** using nitric acid (10.0 equiv.) (104 mg). *R*<sub>F</sub> = 0.2 (cyclohexane/EtOAc 8:2). *ESI-MS* *m/z* (*Da*) = 284 [M-H]<sup>-</sup>. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 8.19 (d, *J* = 7.9 Hz, 1H); 7.53 (t, *J* = 7.5 Hz, 1H); 7.48 (m, 2H); 7.40 (m, 3H); 7.30 (m, 2H); 7.23 (m, 1H); 6.14 (d, *J* = 13.1 Hz, 1H); 5.28 (d, *J* = 13.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 184.1 (CO); 140.7 (C-9); 135.3 (C<sub>arom.</sub> A); 133.0 (C-1'); 130.7 (C-5); 129.9 (C<sub>arom.</sub>); 129.5 (C<sub>arom.</sub>); 128.9 (C<sub>arom.</sub>); 128.9 (C<sub>arom.</sub>); 128.0 (C<sub>arom.</sub>); 126.9 (C<sub>arom.</sub>); 126.3 (C<sub>arom.</sub>); 126.3 (C<sub>arom.</sub>); 95.2 (C-3); 48.3 (C-2). Title compound **14** could not be isolated and was used in the next step without further purification.

**2-(2-Fluorophenyl)-3-nitro-2,3-dihydro-1-benzothiopyran-4-one (15).** Compound **15** was obtained from **11** (312 mg, 1.2 mmol) following general procedure **4** using nitric acid (10.0 equiv.) (112 mg). *R*<sub>F</sub> = 0.13 (cyclohexane/EtOAc 8:2). *ESI-MS* *m/z* (*Da*) = 302 [M-H]<sup>-</sup>, 630 [M + Na]<sup>+</sup>. Title compound **15** could not be isolated and was used in the next step without further purification.

**3-Nitro-2-[3-(trifluoromethyl)phenyl]-2,3-dihydro-1-benzothiopyran-4-one (16).** Compound **16** was obtained from **12** (280 mg, 0.9 mmol) following general procedure **4** using nitric acid (10.0 equiv.) (134 mg). *R*<sub>F</sub> = 0.13 (cyclohexane/EtOAc 8:2). Title compound **16** could not be isolated and was used in the next step without further purification.

**3-Nitro-2-(3-nitrophenyl)-2,3-dihydro-1-benzothiopyran-4-one (17).** Compound **17** was obtained from **13** (133 mg, 0.5 mmol) following general procedure **4** using nitric acid (10.0 equiv.) (47 mg). *R*<sub>F</sub> = 0.1 (cyclohexane/EtOAc 7:3). Title compound **17** was obtained as a mixture of diastereoisomers that were used in the next step without further purification.
### 4.2.5. General procedure 5 – Synthesis of 3-bromo-3-nitro-thioflavanones (SFlav1902–1905 and SFlav2001–2003)

Na₂CO₃ (1.5 equiv.) was added to a solution of appropriate 3-nitrothioflavanones (1.0 equiv.) in anhydrous THF under Ar atmosphere at 0 °C. Reaction mixture was stirred for 5 min at 0 °C, then NBS (1.5 equiv.) was added and the stirring was extended at 0 °C until disappearance of starting material monitored by TLC, typically 1 h. After addition of CH₂Cl₂ and H₂O, aqueous layer was acidified to pH 6 using a 5 % citric acid aqueous solution and was extracted with CH₂Cl₂ (×3). Combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Purification by flash chromatography using a linear-gradient elution (cyclohexane/EtOAc 100:0 to 97:3) afforded title 3-bromo-3-nitro-thioflavanones SFlav1902–1905 and SFlav2001–2003.

**syn/anti-3-Bromo-3-nitro-2-phenyl-2H-1-benzothiopyran-4-one (SFlav1902–1903).**

Title compounds SFlav1902 (21 mg, 0.06 mmol, 28 % yield) and SFlav1903 (3 mg, 0.008 mmol, 4 % yield) were obtained from compound 14 (60 mg, 0.21 mmol) following general procedure 5 with an overall yield of 32 %.

**syn-3-Bromo-3-nitro-2-phenyl-2H-1-benzothiopyran-4-one SFlav1902.**
R<sub>F</sub> = 0.39 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 8.29 (dd, *J* = 8.0, 1.5 Hz, 1H, H-5); 7.59–7.51 (m, 3H, H<sub>arom.</sub>); 7.45–7.32 (m, 5H, H<sub>arom.</sub>); 5.53 (s, 1H, H-2). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 180.6 (CO); 141.1 (C-9); 135.1 (C-7); 132.3 (C-5); 131.0 (C-1'); 130.6 (C-4'); 129.5 (2C) (C-3', C-5'); 129.3 (2C) (C-2', C-6'); 127.3, 126.6 (C-6, C-8); 126.3 (C-10); 105.1 (C-3); 54.1 (C-2).

**anti-3-Bromo-3-nitro-2-phenyl-2H-1-benzothiopyran-4-one SFlav1903.**
R<sub>F</sub> = 0.53 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 8.26 (br d, *J* = 8.1 Hz, 1H, H-5); 7.55 (br t, *J* = 7.7 Hz, 1H, H<sub>arom.</sub>); 7.49–7.33 (m, 7H, H<sub>arom.</sub>); 5.12 (s, 1H, H-2).

**anti/syn-3-Bromo-2-(2-fluorophenyl)-3-nitro-2H-1-benzothiopyran-4-one (SFlav1904–1905).**
Title compounds SFlav1904 (31 mg, 0.08 mmol, 20 % yield) and SFlav1905 (12 mg, 0.03 mmol, 8 % yield) were obtained from compound 15 (120 mg, 0.4 mmol) following general procedure 5 with an overall yield of 28 %.

**anti-3-Bromo-2-(2-fluorophenyl)-3-nitro-2H-1-benzothiopyran-4-one SFlav1904.**
R<sub>F</sub> = 0.55 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 8.29 (dd, *J* = 8.1, 1.4 Hz, 1H, H-5); 7.55 (td, *J* = 7.5, 1.3 Hz, 1H, H-7); 7.43 (m, 1H, H-4'); 7.40–7.31 (m, 3H, H-6, H-8, H-6'); 7.20 (br t, *J* = 7.7 Hz, 1H, H-5'); 7.16 (br t, *J* = 8.9 Hz, 1H, H<sub>3</sub>); 5.59 (s, 1H, H-2). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 179.9 (CO); 160.1 (d, *J*<sub>C-F</sub>= 249.7 Hz, C-2'); 140.5 (C-9); 134.7 (C-7); 132.3 (C-5); 131.9 (d, *J* = 8.8 Hz, C-4'); 129.5 (d, *J* = 1.5 Hz, C-6'); 128.0 (C-10); 127.6, 127.2 (C-6, C-8); 124.7 (d, *J* = 3.7 Hz, C-5'); 120.9 (d, *J* = 12.9 Hz, C-1'); 116.0 (d, *J* = 22.6 Hz, C-3'); 99.2 (C-3); 48.5 (C-2).

**syn-3-Bromo-2-(2-fluorophenyl)-3-nitro-2H-1-benzothiopyran-4-one SFlav1905.**
R<sub>F</sub> = 0.52 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 8.30 (dd, *J* = 8.0, 1.3 Hz, 1H, H-5); 7.92 (td, *J* = 7.6, 1.5 Hz, 1H, H-6'); 7.56 (td, *J* = 7.8, 1.5 Hz, 1H, H-7); 7.42 (m, 1H, H-4'); 7.39–7.32 (m, 2H, H-6, H-8); 7.21 (br t, *J* = 7.7 Hz, 1H, H-5'); 7.11 (br t, *J* = 9.2 Hz, 1H, H-3'); 5.97 (d, *J* = 1.3 Hz, 1H, H-2). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 180.3 (CO); 160.2 (d, *J* = 251.2 Hz, C-2'); 140.6 (C-9); 135.0 (C-7); 132.3 (C-5); 132.1 (d, *J* = 8.8 Hz, C-4'); 129.6 (d, *J* = 1.5 Hz, C-6'); 127.2, 126.7 (C-6, C-8); 126.5 (C-10); 124.7 (d, *J* = 3.8 Hz, C-5'); 119.1 (d, *J* = 13 Hz, C-1'); 116.4 (d, *J* = 22.4 Hz, C-3'); 103.7 (C-3); 45.5 (d, *J* = 4.0 Hz, 1H, C-2).

**syn-/anti-3-Bromo-3-nitro-2-[3-(trifluoromethyl)phenyl]-2H-1-benzothiopyran-4-one (SFlav2001–2002).**
Title compounds SFlav2002 (41 mg, 0.09 mmol, 42 % yield) and SFlav2001 (44 mg, 0.1 mmol, 45 % yield) were obtained from compound 16 (120 mg, 0.3 mmol) following general procedure 5, with an overall yield of 87 %.

**syn-3-Bromo-3-nitro-2-[3-(trifluoromethyl)phenyl]-2H-1-benzothiopyran-4-one SFlav2001.**
R<sub>F</sub> = 0.45 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 8.29 (dd, *J* = 8.4, 1.6 Hz, 1H, H-5); 7.85 (br s, 1H, H-2'); 7.71 (m, 2H, H-4', H-6'); 7.58 (ddd, *J* = 8.6, 7.4, 1.5 Hz, 1H, H-7); 7.54 (br t, *J* = 7.8 Hz, 1H, H-5'); 7.39 (m, 1H, H-6); 7.37 (m, 1H, H-8); 5.61 (s, 1H, H-2). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 180.4 (CO); 140.0 (C-9); 135.2 (C-7); 132.7 (d, *J* = 1.0 Hz, C-6'); 132.2 (C-5); 131.8 (C-10); 131.5 (d, *J* = 33 Hz, C-3'); 129.7 (C-5'); 127.3 (q, *J* = 3.8 Hz, C-4'); 127.1 (C-8); 126.7 (C-6); 126.2 (q, *J* = 3.9 Hz, C-2'); 126.0 (C-1'); 123.5 (d, *J* = 272.6 Hz, CF<sub>3</sub>); 104.6 (C-3); 53.3 (C-2).

**anti-3-Bromo-3-nitro-2-[3-(trifluoromethyl)phenyl]-2H-1-benzothiopyran-4-one SFlav2002.**
R<sub>F</sub> = 0.40 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 8.28 (dd, *J* = 8.1, 1.0 Hz, 1H, H-5); 7.73 (br t, *J* = 3.4 Hz, 1H, H-4'); 7.64 (br s, 1H, H-2'); 7.59–7.56 (m, 3H, H-7, H-5', H-6'); 7.46–7.34 (m, 2H, H-6, H-8); 5.16 (1H, H-2). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 179.9 (CO); 139.9 (C-9); 134.9 (C-7); 134.0 (C-10); 132.6 (C-6'); 132.4 (C-5); 131.2 (d, *J* = 32.9 Hz, C-3'); 129.4 (C-5'); 128.0 (C-1'); 127.7, 127.4 (C-6, C-8); 127.1 (q, *J* = 3.7 Hz, C-4'); 126.8 (q, *J* = 3.8 Hz, C-2'); 123.7 (d, *J* = 272.6 Hz, CF<sub>3</sub>); 99.5 (C-3); 56.5 (C-2).

**syn-3-Bromo-3-nitro-2-(3-nitrophenyl)-2H-1-benzothiopyran-4-one (SFlav2003).**
Title compound SFlav2003 (19 mg, 0.03 mmol, 33 % yield) was obtained from compound 17 (47 mg, 0.1 mmol) following general procedure 5. R<sub>F</sub> = 0.34 (cyclohexane/EtOAc 8:2). ¹H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm) = 8.51 (s, 1H, H-2'); 8.31 (t, *J* = 7.8 Hz, 1H, H-4'); 8.31 (br d, *J* = 8.1 Hz, 1H, H-5); 7.83 (br d, *J* = 7.8 Hz, 1H, H-8); 7.61 (t, *J* = 8.3 Hz, 1H, H-5'); 7.60 (dd, *J* = 1.6, 7.7 Hz, 1H, H-6'); 7.40 (m, 2H, H-6, H-7); 5.67 (s, 1H, H-2). ¹³C NMR (101 MHz, CDCl<sub>3</sub>) δ (ppm) = 180.3 (CO); 148.4 (C-3'); 139.7 (C-9); 135.5 (C-6'); 135.4 (C-8); 132.9 (C-1'); 132.4 (C-5); 130.4 (C-5'); 127.3, 127.0 (C-6, C-7); 126.0 (C-10); 125.5 (C-4'); 124.6 (C-2'); 104.4 (C-3); 53.0 (C-2).

### 4.3. X-ray data collection and structural refinement

Crystal evaluation and data collection for 2 and 3 were performed on a Rigaku Oxford-Diffraction Gemini-S diffractometer with sealed-tube Mo-Kα radiation using the CrysAlis Pro program (Rigaku Oxford-Diffraction, 2012). The same program was used for the integration of the data using default parameters, for the empirical absorption correction using spherical harmonics employing symmetry-equivalent and redundant data, and the correction for Lorentz and polarization effects. The crystal structure was solved using the ab-initio iterative charge flipping method with parameters described elsewhere²¹ using the Superflip program.²² The structure of 2 was refined using full-matrix least-squares procedures as implemented in CRYSTALS²³ and that of 3 using SHELXL²⁴ interfaced by OLEX2²⁵ on all independent reflections with *I* > -3s(*I*). The structure of appeared to be twinned 3 around a two-fold rotation axis along the reciprocal *c* axis. This structure has been refined with the overlapping and non-overlapping reflections of both domains (i.e. with a hklf5 reflection file) The refined twin fractions were 0.681(2)/0.318. 3 crystallizes in the chiral space group P2₁ and 2 in the chiral space group P2₁2₁2₁, but their absolute structures could not be determined because of the absence of elements with sufficient anomalous scattering power. The H atoms were all located in a difference map, but repositioned geometrically. They were initially refined with soft restraints on the bond lengths and angles to regularize their geometry (C—H in the range 0.93–0.98 Å) and *U*<sub>iso</sub>(H) (in the range 1.2–1.5 times *U*<sub>eq</sub> of the parent atom); after which the positions were refined with riding constraints.²⁶

<table><thead><tr><th></th><th>2</th><th>3</th></tr></thead><tbody><tr><td>formula</td><td>C<sub>15</sub>H<sub>12</sub>N<sub>2</sub>O<sub>3</sub></td><td>C<sub>16</sub>H<sub>14</sub>N<sub>2</sub>O<sub>4</sub></td></tr><tr><td>moiety</td><td>C<sub>15</sub>H<sub>12</sub>N<sub>2</sub>O<sub>3</sub></td><td>C<sub>16</sub>H<sub>14</sub>N<sub>2</sub>O<sub>4</sub></td></tr><tr><td><i>T</i> (K)</td><td>293</td><td>293</td></tr><tr><td>Space group</td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td>P2<sub>1</sub></td></tr><tr><td>crystal system</td><td>orthorhombic</td><td>monoclinic</td></tr><tr><td><i>a</i> (Å)</td><td>5.31056(18)</td><td>5.1705(2)</td></tr><tr><td><i>b</i> (Å)</td><td>13.7898(4)</td><td>13.5160(3)</td></tr><tr><td><i>c</i> (Å)</td><td>18.2348(5)</td><td>10.1389(3)</td></tr><tr><td><i>a</i> (°)</td><td>90</td><td>90</td></tr><tr><td><i>b</i> (°)</td><td>90</td><td>101.014(3)</td></tr><tr><td><i>g</i> (°)</td><td>90</td><td>90</td></tr><tr><td><i>V</i> (Å<sup>3</sup>)</td><td>1335.36(7)</td><td>695.50(4)</td></tr></tbody></table>

(continued on next page)
(continued)

<table><thead><tr><th></th><th>2</th><th>3</th></tr></thead><tbody><tr><td>Z</td><td>4</td><td>2</td></tr><tr><td>r (gcm<sup>-3</sup>)</td><td>1.334</td><td>1.424</td></tr><tr><td>M<sub>r</sub> (gmol<sup>-1</sup>)</td><td>268.27</td><td>298.30</td></tr><tr><td>m (mm<sup>-1</sup>)</td><td>0.095</td><td>0.104</td></tr><tr><td>R<sub>int</sub></td><td>0.052</td><td>0.0521</td></tr><tr><td>Q<sub>max</sub> (°)</td><td>29.085</td><td>29.045</td></tr><tr><td>N<sub>tot</sub> (measured)</td><td>39,952</td><td>5698</td></tr><tr><td>N<sub>ref</sub> (unique)</td><td>2000</td><td>5698</td></tr><tr><td>N<sub>ref</sub> (I > 2 s(I))</td><td>1795</td><td>4146</td></tr><tr><td>N<sub>ref</sub> (least-squares)</td><td>1998</td><td>5698</td></tr><tr><td>N<sub>par</sub></td><td>185</td><td>214</td></tr><tr><td>&lt;s(I)/I&gt;</td><td>0.053</td><td>0.0318</td></tr><tr><td>R<sub>1</sub> (I > 2 s(I))</td><td>0.0795</td><td>0.0507</td></tr><tr><td>wR<sub>2</sub> (I > 2 s(I))</td><td>0.1555</td><td>0.1483</td></tr><tr><td>R<sub>1</sub> (all)</td><td>0.0878</td><td>0.0702</td></tr><tr><td>wR<sub>2</sub> (all)</td><td>0.1600</td><td>0.1808</td></tr><tr><td>GOF</td><td>1.0793</td><td>0.979</td></tr><tr><td>Dr (eÅ<sup>-3</sup>)</td><td>-0.29/0.37</td><td>-0.210/0.212</td></tr><tr><td>crystal size (mm<sup>3</sup>)</td><td>0.18 × 0.23 × 0.35</td><td>0.12 × 0.22 × 0.30</td></tr></tbody></table>

CCDC 2321310 (2) and 2321311 (3) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures.

### 4.4. Stability assay

Tested compounds were first stocked at 10 mM in DMSO and then diluted at 100 µM in enzyme buffer (1 % DMSO final concentration). Enzyme buffer is composed by HEPES pH 7.2 (20 mM), KCl (50 mM), EDTA (1 mM). The proportion of remaining compound was measured over time by HPLC analysis using our standard HPLC method described above.

### 4.5. hDNMT3A inhibition activity assay

Compound inhibitory activities were determined with a fluorescence-based assay previously described.⁹ In brief, a DNA oligonucleotide with a unique CpG site overlaying an endonuclease restriction site for methylation-sensitive enzyme was used. It presents the fluorescent 6-FAM at one end and biotin in the other one, allowing immobilization into a 384-well plate (PerkinElmer) pre-coated with avidin. Compounds, SAM and hDNMT3A-c were added to start the methylation reaction (1 h 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 EnVision 2103 Multilabel Reader (PerkinElmer). Inhibition percentages are defined as 100× (100(X<sub>meth</sub> - X<sub>restri</sub>)/(X<sub>DNA</sub> - X<sub>restri</sub>)), where X<sub>meth</sub>, X<sub>restri</sub> and X<sub>DNA</sub> are respectively the fluorescence signals of the compound methylation, restriction and DNA controls. Data were collected as duplicates of at least two independent experiments (n ≥ 2).

### 4.6. Docking experiments

The ligand was docked in the crystal structure of DNMT3A-3L (PDB: 2QRV).¹⁶ Molecules 3D-conformations of all compounds were generated using Open Babel.²⁷ The ligand was docked in the catalytic-cofactor binding pocket of DNMT3A-3L using a squared grid with side of 24 Å. The method was validated by obtaining the same poses for SAM, Flv62 and Flv69 as described in the published crystal structure or by Ceccaldi et al.⁹ For NFlav2019, considering the nitrogen protonation prediction in physiological conditions by Marvin Sketch (MarvinSketch, version 21.13, ChemAxon, https://www.chemaxon.com), neutral form was docked. All structural files were prepared with AutodockTools. For each run, 9 docking poses were generated with Autodock Vina,²⁸ ranked by score, using an exhaustiveness of 64. All docking figures were generated with UCSF Chimera.²⁹

## CRediT authorship contribution statement

Alexandra Serhouni: Investigation. Francesco Calzaferri: Investigation, Data curation. Arie van der Lee: Formal analysis. Paola B. Arimondo: Conceptualization. Maxime Louet: Validation. Jean-Yves Winum: Conceptualization. Marie Lopez: Writing – review & editing, Writing – original draft, Funding acquisition, Conceptualization.

## Funding

Dr Alexandra Serhouni PhD fellowship was funded by the Ministère de l'enseignement supérieur et de la recherche – France (MESR). Dr Francesco Calzaferri was funded by the CNRS via an Emergence 2021 project granted to Dr Marie Lopez and by the ARC Foundation Post-doctoral Fellowship for the following period.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marie Lopez reports financial support was provided by National Centre for Scientific Research. Francesco Calzaferri reports financial support was provided by ARC Foundation for Cancer Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bmc.2024.117988.

## Data availability

Data will be made available on request.

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