# Quinoline-Based DNA Methyltransferase Inhibitors Featuring Basic Side Chains: Design, Synthesis, and Insight in Biochemical and Anticancer Cell Properties

Clemens Zwergel, Chiara Lambona, Rossella Fioravanti, Alessia Raucci, Francesco Fiorentino, Guilaine Nchugoua Tchieh, Corinne Jallet, Laurent Lacroix, Michela Pierrettori, Francesca Romana Pellegrini, et al.

► To cite this version:

Clemens Zwergel, Chiara Lambona, Rossella Fioravanti, Alessia Raucci, Francesco Fiorentino, et al. Quinoline-Based DNA Methyltransferase Inhibitors Featuring Basic Side Chains: Design, Synthesis, and Insight in Biochemical and Anticancer Cell Properties. Journal of Medicinal Chemistry, 2025, 68 (22), pp.23886-23909. 10.1021/acs.jmedchem.5c01029. pasteur-05389623v2

HAL Id: pasteur-05389623

https://pasteur.hal.science/pasteur-05389623v2

Submitted on 1 Dec 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Quinoline-Based DNA Methyltransferase Inhibitors Featuring Basic Side Chains: Design, Synthesis, and Insight in Biochemical and Anticancer Cell Properties

Clemens Zwergel, Chiara Lambona, Rossella Fioravanti, Alessia Raucci, Francesco Fiorentino, Guilaine Nchugoua Tchieh, Corinne Jallet, Laurent Lacroix, Michela Pierrettori, Francesca Romana Pellegrini, Yan Xiong, Jian Jin, Paola Arimondo, Daniela Trisciuoglio, Antonello Mai,* and Sergio Valente*

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

Cite This: J. Med. Chem. 2025, 68, 23886-23909

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

Read Online

## ACCESS

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

Metrics & More

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

Article Recommendations

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

Supporting Information

**ABSTRACT:** The quinoline DNMT inhibitors 4–21, incorporating basic chains, were designed, synthesized, and evaluated for their ability to inhibit DNMT1 and DNMT3A/3L by directly measuring DNA methylation. Pharmacomodulation yielded nanomolar inhibitors with selectivity for either DNMT1 or DNMT3A/3L. The *meta/metaphenotype* analogs 7–14 exhibited the highest inhibition, with compounds 10 and 14 being the most potent and selective for DNMT3A/3L and DNMT1, respectively. DNA thermal denaturation experiments demonstrated for selected compounds strong DNA interaction. COBRA analysis in HCT-116 colon cancer cells revealed a selective reduction in P16INK4A methylation, a tumor suppressor gene reactivated by DNMT inhibition. Among the tested cancer cell lines, HCT-116 was the most sensitive, and 14 showed the strongest antiproliferative effect. In isogenic HCT-116 P53<sup>-/-</sup> cells, 14 exhibited reduced antiproliferative activity, lower apoptosis, and decreased levels of cleaved Caspase 3, P53, and γH2AX, confirming its P53-dependent mechanism of action linked to DNA damage.

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

## INTRODUCTION

The onset and progression of cancer result from dysregulations linked to various interconnected factors, including lifestyle, environmental influences, and molecular mechanisms driven by genetic and/or epigenetic alterations.<sup>1,2</sup> Epigenetic factors play a crucial role in the development of multiple human diseases, including cancer. These factors encompass aberrant expression or mutations in chromatin remodeling enzymes and alterations in the epigenome due to dysregulated writing, erasing, or reading activities of DNA- and histone-modifying proteins.<sup>3-5</sup> DNA methylation is a key epigenetic mechanism that shapes chromatin structure and regulates gene expression, significantly impacting human genetics, development, and diseases.<sup>4,6,7</sup> In mammals, this process predominantly occurs at the C5 position of cytosines within CpG dinucleotides and is mediated by a specialized group of 5-methylcytosine (5mC) methyltransferases known as DNA methyltransferases (DNMTs).<sup>8</sup> In humans, three primary DNMT family members have been identified, playing essential roles in establishing (DNMT3A/B, de novo methyltransferases) or maintaining (DNMT1, maintenance methyltransferase) DNA methylation patterns in embryonic stem cells. These enzymes influence gene expression,

genome reprogramming, organismal development, and cellular differentiation.<sup>9-11</sup>

Abnormal DNMT activity, including overexpression and/or mutations, has been implicated in a range of human diseases, including neurological,<sup>12,13</sup> autoimmune,<sup>14</sup> and metabolic disorders,<sup>15</sup> as well as cancer.<sup>16</sup> Specifically, tumor suppressor gene promoter hypermethylation leads to their silencing, while hypomethylation of proto-oncogenes results in their overexpression - both of which contribute to various solid and hematological malignancies.<sup>17</sup> For instance, high expression levels of DNMT1 have been reported in some cancer cell lines including HCT-116 colon carcinoma, MDA-MB-231 triple negative breast cancer, and U937 histiocytic lymphoma.

Currently, azacytidine and decitabine are the only two nucleoside DNMT inhibitors (DNMTi) approved for clinical treatment of hematological malignancies. However, these agents

Received: April 11, 2025

Revised: October 6, 2025

Accepted: November 4, 2025

Published: November 11, 2025

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

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>**Figure 1.** Structures of 1–3 and of the compounds 4–21 bearing basic side chain(s).</figcaption></figure>

suffer from chemical instability and severe side effects. Consequently, research efforts are increasingly focused on developing non-nucleoside DNMTi with distinct chemical structures and mechanisms of action.¹⁸

Among non-nucleoside DNMTi, SGI-1027 (1) is widely recognized and extensively used in various research contexts (https://pubmed.ncbi.nlm.nih.gov/?term=SGI-1027&sort date). This molecule consists of four ring fragments (A–D, Figure 1) linked in a para/para orientation. Structural modifications, such as shifting the linkage position from para to meta or ortho, or duplicating the quinoline (fragment A) or pyrimidine (fragment D) moiety, led to a series of new regioisomers and analogs described by our research group in 2014.¹⁹ Among these derivatives, the meta/meta analog MC3343 (2) demonstrated greater potency and selectivity against DNMT1 and DNMT3A than 1. In cancer cell panels, 2 exhibited slightly lower (U937 histiocytic lymphoma and MDA-MB-231 breast cancer) or comparable (RAJI Burkitt's lymphoma and PC-3 prostate cancer) antiproliferative effects compared to 1. Additionally, 2 was 2-fold less cytotoxic in peripheral blood mononuclear cells, used as a model for noncancerous healthy cells.¹⁹ In mouse medulloblastoma stem cells, 2 preferentially inhibited cell growth, while its para/meta regioisomer induced strong cell differentiation.¹⁹ In osteosarco-

ma (OS) and Ewing sarcoma (EWS), 2 effectively inhibited tumor proliferation in vitro (OS, EWS) and in vivo (OS). It also displayed synergistic effects when combined with doxorubicin and cisplatin (OS) or doxorubicin and talazoparib (EWS), a PARP inhibitor targeting DNA repair mechanisms.²⁰,²¹ Further structural modifications of 2 led to derivatives with enhanced flexibility, achieved by introducing methylene units between different fragments or modifying the amide bond between fragments B and C. These compounds exhibited moderate activity in terms of DNMT inhibition and anticancer potential.²²

Lastly, replacing the 2,6-dimethylamino-4-methylpyrimidine moiety (fragment D) in 1 with a benzyl carbamate led to MC3353 (3, Figure 1). Although 3 exhibited weaker DNMT inhibitory activity than 1 and 2, it demonstrates significant dose- and time-dependent antiproliferative activity in multiple cancer cell types, reaching submicromolar concentration.²³ Notably, most of these compounds, including 1–3, not only inhibited DNMT enzymatic activity but also led to DNMT protein downregulation in various cancer cell lines.²¹⁻²³

Since some of these compounds exhibit low water solubility, we aimed to introduce a basic side chain into one of the phenyl rings of their structures. Among the synthesized compounds 4–21 (Figure 1), compounds 4–6 are analogs of 1 maintaining a para/para orientation between rings B and C, with a N,N-
<figure><img src="image_12.png" /><figcaption>Scheme 1. Synthesis of Compounds 4–7<sup>a</sup></figcaption></figure>

<sup>a</sup>(a) dimethylamine, dry THF, rt; (b) SnCl<sub>2</sub>·2H<sub>2</sub>O, 37% HCl, EtOH, rt, 1 h; (c) 37% HCl, EtOH, 75 °C, 1 h; (d) 2N KOH, EtOH, rt, overnight; (e) Et<sub>3</sub>N, PyBOP, dry DMF, N<sub>2</sub>, rt, 2.45 h; (f) 4 M HCl in dioxane, dry THF, 0 °C, 10 min; (g) BH<sub>3</sub> in THF, THF, 70 °C, 6 h.

dimethylaminomethyl (CH<sub>2</sub>NMe<sub>2</sub>) side chain at either ring B (4 and 5) or ring C (6). Compound 5 differs from 4 in that it features an inverse amide function between the B/C rings instead of the carboxamide present in the 1 structure (Figure 1).

Compounds 7–14 are based on the 2 structure (meta/metadivision of rings B and C), and show the insertion of a CH<sub>2</sub>NMe<sub>2</sub> chain (with either the amide (7) or the inverse amide (8) function between B/C rings) at ring B. At ring C, a NMe<sub>2</sub> (9), CH<sub>2</sub>NMe<sub>2</sub> (10, with the amide linkage between the B/C rings, and 11, with the methylamine at the same position), (N-methylpiperazin-1-yl)methyl (12), or (1H-imidazole-1-yl)-methyl (13) basic side chain has been inserted. Compound 14 has two CH<sub>2</sub>NMe<sub>2</sub> side chains, inserted at each of the B and C rings (Figure 1).<sup>22,23</sup>

Compounds 15 and 16 are derived from the 3 structure, with 15 featuring a *para*/*meta* and 16 a *meta*/*meta* orientation between rings B and C, both incorporating a CH<sub>2</sub>NMe<sub>2</sub> side chain at ring C (Figure 1).<sup>22,23</sup>

Additionally, the *bis*-quinoline and *bis*-pyrimidine templates<sup>19,22</sup> were modified to introduce a CH<sub>2</sub>NMe<sub>2</sub> side chain at ring C. Compounds 17–19 belong to the *bis*-quinoline group: in 17 the quinoline is simplified to pyridine linking fragments B and C in a *para*/*para* orientation, while 18 and 19 maintain the

quinoline at ring A with a *para*/*meta* and *meta*/*meta* orientation at B and C rings, respectively. Finally, compounds 20 and 21 are *bis*-pyrimidine analogs incorporating the CH<sub>2</sub>NMe<sub>2</sub> side chain at ring C, with 20 displaying a *para*/*meta* and 21 a *meta*/*meta* orientation (Figure 1).

In a previous study,<sup>24</sup> we reported that six of these compounds (7, 9, 10, 12–14) were able to inhibit DNMT1 at a low micromolar level. Some also exhibited activity against other DNA-interacting enzymes, such as base excision repair glycosylases and DNA/RNA polymerases. Compound 12 was found to induce a DNA damage response via p53 activation in A549 human non-small cell lung carcinoma (NSCLC) cells.

Here, we provide in full detail the synthetic procedures used to obtain all the cited compounds 4–21, introducing 11, 15, and 17 for the first time. Their ability to induce hypomethylation in a cellular context was evaluated using a stable cellular reporter system (CMV-luc) in KG-1 human leukemia cells, in which luciferase expression is controlled by a methylated cytomegalovirus (CMV) promoter.<sup>25</sup> Selected compounds were further assessed for their ability to demethylate p16<sup>INK4A</sup>, a tumor suppressor gene that can be reactivated by DNMT inhibition or deletion,<sup>26</sup> in a solid cancer model, the HCT-116 colon carcinoma cell line, using the Combined Bisulfite Restriction
<figure><img src="image_13.png" /><figcaption>Scheme 2. Synthesis of Compounds 8–13<sup>a</sup></figcaption></figure>

<sup>a</sup>(a) CBr<sub>4</sub>, Ph<sub>3</sub>P, N<sub>2</sub>, rt, 3 h; (b) appropriate amine, dry THF, rt; (c) 4 M HCl in dioxane, dry THF, 0 °C to rt; (d) Et<sub>3</sub>N, PyBOP, dry DMF, N<sub>2</sub>, rt, 2.45 h; (e) 37% HCl, EtOH, 75 °C, 1 h; (f) SnCl<sub>2</sub>·2H<sub>2</sub>O, 37% HCl, EtOH, rt, 1 h; (g) NaBH(OAc)<sub>3</sub>, dry DCE, 60 °C, overnight.

Analysis (COBRA) assay.<sup>27</sup> Thermal denaturation experiments were also conducted to evaluate the DNA-binding capabilities of selected derivatives, based on the detection of changes in DNA absorbance.<sup>28</sup>

Additionally, selected compounds were tested in a panel of cancer cell lines, including A549 and H460 (nonsmall cell lung cancer), HCT-116 (colon cancer), HeLa (cervical cancer), MDA-MB-231 (triple-negative breast cancer), and U937 (histiocytic lymphoma), to determine their antiproliferative

potential. HCT-116 was the most sensitive cell line, prompting further investigation of compounds **9** and **14** regarding their effects on cell cycle progression and apoptosis induction. To explore the role of p53 pathway activation in its anticancer activity, compound **14** - the most potent derivative - was examined in both wild-type p53 and isogenic p53<sup>-/-</sup> HCT-116 cell lines.
<figure><img src="image_14.png" /><figcaption>Scheme 3. Synthesis of Compounds 15 and 16<sup>a</sup></figcaption></figure>

<sup>a</sup>(a) Et<sub>3</sub>N, dry DCM, rt, overnight; (b) Et<sub>3</sub>N, PyBOP, dry DMF, N<sub>2</sub>, rt, 2.45 h; (c) 4 M HCl in dioxane, dry THF, 0 °C, 10 min.

## ■ RESULTS

**Chemistry.** The synthetic procedures used to prepare compounds 4–21 are depicted in **Schemes 1–4**.

For the synthesis of 4 and 7, commercially available methyl 2-(bromomethyl)-4-nitrobenzoate (for 4) and methyl 3-(bromomethyl)-5-nitrobenzoate (for 7) were treated with dimethylamine in THF to yield the nitro intermediates 22a,b. These were subsequently reduced with stannous chloride to obtain the corresponding amines 23a,b. Nucleophilic displacement of 4-chloroquinoline performed by 23a,b in the presence of 37% hydrochloric acid (HCl) in ethanol at 75 °C afforded the methyl esters 24a,b. Hydrolysis with 2N potassium hydroxide (KOH) yielded the corresponding carboxylic acids 25a,b, which were then condensed with either N<sup>4</sup>-(4-aminophenyl)- (26a)<sup>19</sup> or N<sup>4</sup>-(3-aminophenyl)- (26b)<sup>19</sup> 6-methylpyrimidine-2,4-diamine in the presence of benzotriazol-1-yloxytr pyrrolidinophosphonium hexafluorophosphate (PyBOP) and triethylamine in dry DMF. Final treatment with 4 M HCl in dioxane furnished the desired compounds 4 and 7 as hydrochlorides (**Scheme 1A**).

For the synthesis of 5 and 6, the key intermediate 27 was obtained from commercially available 5-amino-N,N-dimethyl-2-nitrobenzamide via reduction with borane (BH<sub>3</sub>) in THF at 70 °C. Compound 27 was reacted with 4-chloroquinoline in the presence of 37% HCl in ethanol at 75 °C, yielding the nitro derivative 28, which was subsequently reduced to the corresponding amine 29 using stannous chloride and 37% HCl in ethanol. Further condensation of 29 with 4-((2-amino-6-methylpyrimidin-4-yl)amino)benzoic acid 30<sup>19</sup> in the presence of PyBOP and triethylamine in dry DMF afforded the reverse amide 5, which was isolated as a hydrochloride following treatment with 4 M HCl. Alternatively, 27 was reacted with commercially available 2-amino-4-chloro-6-methylpyrimidine in the presence of 37% HCl in ethanol at 75 °C to generate the nitro intermediate 31. Reduction with stannous chloride and 37% HCl in ethanol yielded amine 32, which was then condensed with 4-(quinolin-4-ylamino)benzoic acid 33<sup>19</sup> in the presence of PyBOP and triethylamine in dry DMF. Final treatment with 4 M HCl in dioxane produced compound 6 as hydrochloride (**Scheme 1B**).

For the synthesis of compounds 8–14, the key intermediates 36a-c were prepared via an Appel reaction applied on commercially available di-tert-butyl (5-(hydroxymethyl)-1,3-

phenylene) dicarbamate using tetrabromomethane and triphenylphosphine in dry THF. The resulting bromo derivative 34 was then treated with dimethylamine, N-methylpiperazine, or imidazole in dry THF to yield the protected amines 35a-c, which were subsequently deprotected with 4 M HCl in dioxane to afford 36a-c. Subsequent reaction of 36a with the 3-((2-amino-6-methylpyrimidin-4-yl)amino)benzoic acid 37<sup>19</sup> in the presence of triethylamine and PyBOP afforded the intermediate 38, which was then treated with 4-chloroquinoline in an acidic medium to produce compound 8 as a hydrochloride. The reaction of 36a-c with 3-(quinolin-4-ylamino)benzoic acid 39,<sup>19</sup> triethylamine, and PyBOP yielded the intermediates 40a-c, which were subsequently converted into 10, 12, and 13 as hydrochlorides via reaction with 2-amino-4-chloro-6-methylpyrimidine in an acidic medium. The reaction of 36a with intermediate 25b (see **Scheme 1A**) afforded intermediate 41, which, upon treatment with 2-amino-4-chloro-6-methylpyrimidine, yielded the bis-dimethylaminomethyl derivative 14 (**Scheme 2A**).

For the synthesis of compound 9, commercially available N,N-dimethyl-3,5-dinitroaniline was reduced with stannous chloride to yield the corresponding diamine 42, which was reacted with 39<sup>19</sup> to afford intermediate 43. Subsequent treatment of 43 with 2-amino-4-chloro-6-methylpyrimidine furnished compound 9 (**Scheme 2B**).

Compound 11 was synthesized by reacting 3-(quinolin-4-ylamino)benzaldehyde 44<sup>22</sup> with tert-butyl (3-amino-5-((dimethylamino)methyl)phenyl)carbamate 45 - obtained by halting the formation of the free phenylendiamine 36a before completion (see Experimental Section) - in the presence of sodium acetoxyborohydride in dry dichloroethane. The resulting intermediate 46 was immediately deprotected with 4 M HCl to yield 47, which was then treated with 2-amino-4-chloro-6-methylpyrimidine to afford compound 11 (**Scheme 2C**).

Treatment of the key intermediate 36a with benzyl chloroformate and triethylamine in dry DCM furnished the benzyl (3-amino-5-((dimethylamino)methyl)phenyl)carbamate 48, which gave 15 or 16 after reaction with 33<sup>19</sup> or 39,<sup>19</sup> respectively, according to the reported procedure (**Scheme 3**).
<figure><img src="image_15.png" /><figcaption>Scheme 4. Synthesis of Compounds 17–21<sup>a</sup></figcaption></figure>

<sup>a</sup>(a) Et<sub>3</sub>N, PyBOP, dry DMF, N<sub>2</sub>, rt, 2.45 h; (b) 4 M HCl in dioxane, dry THF, 0 °C, 10 min; (c) 37% HCl, EtOH, 75 °C, 1 h.

The synthesis of compound 17 was achieved by treating 4-(pyridin-4-ylamino)benzoic acid 49<sup>29</sup> with 29 in the presence of PyBOP and triethylamine in dry DMF, followed by treatment with 4 M HCl (Scheme 4A). For the synthesis of the bis-quinoline compounds 18 and 19, 5-((dimethylamino)methyl)-benzene-1,3-diamine 36a was treated with 4-(quinolin-4-ylamino)benzoic acid 33<sup>19</sup> to yield intermediate 50, which, upon treatment with 4-chloroquinoline, afforded the para/meta compound 18. The meta/meta analog 19 was obtained through the reaction of 40a and 4-chloroquinoline in 37% HCl and ethanol. Alternatively, the reaction of 36a with ((2-amino-6-methylpyrimidin-4-yl)amino)benzoic acid 30<sup>19</sup> produced intermediate 51, which, upon treatment with 2-amino-4-chloro-6-methylpyrimidine, yielded the para/meta bis-pyrimidine analog 20. Finally, treatment of intermediate 38 with 2-amino-4-chloro-6-methylpyrimidine furnished the meta/meta isomer 21 (Scheme 4B).

Chemical and physical data for the final compounds 4–21 and the intermediates 22–25, 28–32, 34–36, 38, 40–51 are reported in the Experimental Section. Elemental analyses for 4–21 are reported in Supporting Information, Table S1. High-performance liquid chromatography (HPLC) traces for selected compounds 7, 9, 10, 12–14 are reported in Supporting Information, Figures S1–S6.

Human DNMT1 and DNMT3A Inhibition Were Assessed through Direct Measurement of the DNA Methylation on 40-mer DNA Duplexes. Recently, some of us reported the effects of 10 µM compounds 4–10, 12–14, 16, and 18–21 against human DNMTs and other DNA-interacting enzymes, using an assay that measured the conversion of S-adenosyl-L-methionine (SAM) to S-adenosyl-L-homocysteine

(SAH), which is subsequently transformed into ATP and detected via a luciferase reaction.<sup>24</sup> In these assays, an [E]/[DNA] ratio of 1:10 ([DNA] = 5 µM) was used. Among the 15 tested derivatives, six compounds (7, 9, 10, 12–14) completely abrogated DNMT1 activity at 10 µM, exhibiting IC<sub>50</sub> values in the range of 1.9–3.5 µM. At the same concentration (10 µM), these compounds also reduced the activity of three adenine methyltransferases below 50%.<sup>24</sup> Additionally, they inhibited glycosylases and polymerases.<sup>24</sup> Against the full-length DNMT3A2–3L complex, these compounds were initially almost inactive. However, when tested at a different [E]/[DNA] ratio of 1:2 ([DNA] = 1 µM), all displayed >50% of inhibition at 10 µM.<sup>24</sup>

These findings prompted us to re-evaluate the effects of the entire set of compounds 4–21 (including 11, 15, and 17) against human DNMT3A. For comparison, we also included compounds 1 and 2, together with the 3-(quinolin-4-ylamino)-N-(3-(quinolin-4-ylamino)phenyl)benzamide [2b] previously reported by us as a DNMT3A-selective inhibitor.<sup>22</sup> We employed an alternative method developed by some of us,<sup>30</sup> which detects methylation at a single CpG site in a 40-mer DNA duplex using a restriction enzyme inhibited when the DNA is methylated. This method offers the advantage of using an optimal DNA substrate for human catalytic DNMT3A/3L (DNMT3Acat) and directly measuring the methylation event.

We further adapted the assay to evaluate the methylation activity of hDNMT1 on hemimethylated DNA. We optimized the DNA sequence and the restriction enzyme that, for this maintenance DNMT, needs to distinguish between hemimethylated DNA (the initial substrate) and methylated DNA (the product of the reaction). The assay was optimized using the
<table><caption>Table 1. IC₅₀ Values of Compounds 4–21 against DNMT1 and DNMT3A/3L Through the Detection of Direct DNA Methylation</caption><thead><tr><th rowspan="2">Lab code</th><th rowspan="2">cmpd</th><th rowspan="2">structure</th><th colspan="2">IC₅₀, µM</th><th rowspan="2">DNMT1-selectivity</th><th rowspan="2">DNMT3A-selectivity</th></tr><tr><th>DNMT1</th><th>DNMT3A</th></tr></thead><tbody><tr><td>MC3671</td><td>4</td><td></td><td>4.5 ± 0.7</td><td>7.1 ± 3.6</td><td>1.6</td><td>0.6</td></tr><tr><td>MC3682</td><td>5</td><td></td><td>>32</td><td>>10</td><td></td><td></td></tr><tr><td>MC3672</td><td>6</td><td></td><td>>32</td><td>>10</td><td></td><td></td></tr><tr><td>MC3716</td><td>7</td><td></td><td>0.84 ± 0.3</td><td>0.15 ± 0.07</td><td>0.2</td><td>5.6</td></tr><tr><td>MC3653</td><td>8</td><td></td><td>2.1 ± 0.4</td><td>1.2 ± 0.7</td><td>0.6</td><td>1.7</td></tr><tr><td>MC3807</td><td>9</td><td></td><td>0.19 ± 0.08</td><td>0.10 ± 0.05</td><td>0.5</td><td>1.9</td></tr><tr><td>MC3563</td><td>10</td><td></td><td>2.0 ± 0.5</td><td>0.019 ± 0.01</td><td>0.01</td><td>105</td></tr><tr><td>MC3718</td><td>11</td><td></td><td>12.7 ± 0.9</td><td>0.44 ± 0.23</td><td>0.03</td><td>29</td></tr><tr><td>MC3669</td><td>12</td><td></td><td>0.09 ± 0.07</td><td>0.027 ± 0.003</td><td>0.3</td><td>3.3</td></tr><tr><td>MC3821</td><td>13</td><td></td><td>0.051 ± 0.05</td><td>0.058 ± 0.011</td><td>1.1</td><td>0.9</td></tr><tr><td>MC3817</td><td>14</td><td></td><td>0.044 ± 0.03</td><td>>10</td><td>>227</td><td>&lt;0.004</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th rowspan="2">Lab code</th><th rowspan="2">cmpd</th><th rowspan="2">structure</th><th colspan="2">IC<sub>50</sub>, µM</th><th rowspan="2">DNMT1-selectivity</th><th rowspan="2">DNMT3A-selectivity</th></tr><tr><th>DNMT1</th><th>DNMT3A</th></tr></thead><tbody><tr><td>MC3650</td><td>15</td><td>[Image]</td><td>5.5 ± 0.7</td><td>>10</td><td>>1.8</td><td><0.5</td></tr><tr><td>MC3652</td><td>16</td><td>[Image]</td><td>2.7 ± 0.6</td><td>7.6 ± 2.7</td><td>2.8</td><td>0.3</td></tr><tr><td>MC3683</td><td>17</td><td>[Image]</td><td>>32</td><td>>10</td><td></td><td></td></tr><tr><td>MC3646</td><td>18</td><td>[Image]</td><td>2.2 ± 0.8</td><td>4.1 ± 1.5</td><td>1.9</td><td>0.5</td></tr><tr><td>MC3639</td><td>19</td><td>[Image]</td><td>1.9 ± 0.9</td><td>2.7 ± 0.9</td><td>1.4</td><td>0.7</td></tr><tr><td>MC3647</td><td>20</td><td>[Image]</td><td>0.33 ± 0.17</td><td>8.0 ± 2.9</td><td>24</td><td>0.04</td></tr><tr><td>MC3649</td><td>21</td><td>[Image]</td><td>1.3 ± 0.6</td><td>1.2 ± 0.2</td><td>0.9</td><td>1.1</td></tr><tr><td></td><td>1</td><td></td><td>3.5 ± 0.6</td><td>1.1 ± 0.3</td><td>0.3</td><td>3.2</td></tr><tr><td></td><td>2</td><td></td><td>5.7 ± 0.2</td><td>0.7 ± 0.5</td><td>0.1</td><td>8.1</td></tr><tr><td></td><td>GSK-3484862</td><td></td><td>0.11 ± 0.06</td><td>>10</td><td>>90.9</td><td><0.011</td></tr><tr><td></td><td>GSK-3685032</td><td></td><td>0.013 ± 0.005</td><td>>10</td><td>>769.2</td><td><0.001</td></tr><tr><td></td><td>[2b]<sup>22</sup></td><td></td><td>27.0 ± 5.0</td><td>0.8 ± 0.1</td><td>0.03</td><td>33.8</td></tr></tbody></table>

reference inhibitors 1,³¹ GSK3685032³²,³³ and compound #20²⁷ (data not shown), and was subsequently applied to test compounds 4–21. In addition to 4–21, compounds 1, 2, and the DNMT1-selective inhibitors GSK-3484862 and GSK-3685032 were added.³²,³³

The IC₅₀ values reported in Table 1 indicate that introducing a dimethylaminomethyl side chain at ring B of compound 1 resulted in a moderately active DNMT1/3A inhibitor (4), which was 9-fold less potent against DNMT3A and 2-fold more potent against DNMT1 than 1. However, applying the same modification at ring C rendered the inactive compound 6.

Additionally, the inversion of the amide function linking the B/C rings in compound 4 (leading to compound 5) significantly reduced its anti-DNMT activity.

In contrast, the presence of a basic side chain in prototype 2 produced potent DNMT inhibitors (7–14), with some selectivity for DNMT3A (10, 11) or DNMT1 (14). Specifically, introducing the dimethylaminomethyl chain at ring B of 2 yielded compound 7, potent at submicromolar level against both the DNMTs. However, inverting the amide function between the B/C rings (compound 8) slightly decreased its potency. Among 2-analogs with a basic chain at ring C, the most potent
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>**Figure 2.** Effects of 2, 7, 9, 10, 12, 13, and 14 on the methylation status of the tumor suppressor gene P16<sup>INK4A</sup> (A) and of the gene LINE-1 (global DNA) (B) in HCT-116 cells after 10 days of treatment at 0.32 and 1 µM.</figcaption></figure>

DNMT1/3A inhibitors were compound 9 (which contained a dimethylamino group and exhibited low submicromolar potency against both enzymes) and compound 10 (which replaced the dimethylamino group with a dimethylaminomethyl moiety, making it 10-fold more potent against DNMT3A and 10-fold less potent against DNMT1 than 9, thereby achieving >100-fold selectivity for DNMT3A). Reducing the amide function between the B/C rings further diminished potency (compare 11 with 10). Meanwhile, inserting (N-methylpiperazin-1-yl)methyl (compound 12) or (1H-imidazole-1-yl)methyl (compound 13) side chains produced highly potent inhibitors with nanomolar activity against both DNMTs. Finally, introducing two dimethylaminomethyl groups at rings B and C (compound 14) maintained nanomolar potency against DNMT1 while drastically reducing activity against DNMT3A, making 14 a potent DNMT1-selective inhibitor (selectivity index >227 for DNMT1). To confirm its high selectivity toward DNMT1, 14 was also tested against G9a and PRMT1, as representative samples of lysine and arginine methyltransferases, respectively. The IC₅₀ values of 14 were >50 µM for G9a, with selectivity index >1136, and >100 µM for PRMT1, with selectivity index >2272 (Table S2, Supporting Information).

The two analogs of compound 3 featuring a basic chain at ring C (15 and 16) displayed moderate activity against DNMT1

(15) or both DNMTs (16). In the bis-quinoline series, inserting a basic chain at ring C while replacing the quinoline with a pyridine ring reduced DNMT inhibition (17). However, reintroducing the quinoline ring restored activity, yielding compounds 18 and 19, which were effective at low micromolar levels. The meta/meta-oriented 19 exhibited slightly greater potency than its para/meta analog 18.

In the bis-pyrimidine series, adding a basic chain at ring C resulted in compounds 20 and 21. Compound 20 showed submicromolar potency against DNMT1 and micromolar potency against DNMT3A, with a DNMT1 selectivity index of 24. Meanwhile, compound 21 displayed similar potency (~1 µM) against both enzymes.

**Effect of Quinoline-Based DNMT Inhibitors on DNA Methylation in Cells.** To assess the impact of the inhibitors on the DNA methylation levels in cells, we tested compounds 4–21 using a luciferase reporter construct under the control of a methylated cytomegalovirus (CMV) promoter integrated in KG-1 leukemia cells (CMV-luc assay).<sup>22,23</sup> Previous studies by our group<sup>21</sup> and others<sup>32</sup> reported difficulties in detecting cellular hypomethylation with the lead compound 2 during 24–72 h of treatment, and several compounds from the 4–21 series failed to reduce methylation levels in murine embryonic stem cells (48 h of treatment).<sup>24</sup> When tested in KG-1 cells at 3.2 µM
for 24 h, most compounds 4–21 did not reactivate luciferase expression, confirming their inability to induce DNA hypomethylation in cells (data not shown). This could be attributed to their pharmacokinetic properties and/or their ability to inhibit other DNA-interacting enzymes,²⁴ but also to the short duration of the experiment (24 h), during which only active DNA demethylation would be expected.³⁴

Next, we examined selected compounds 7, 9, 10, 12–14, with IC₅₀ values ≤150 nM against at least one DNMT, along with the structurally related prototype 2, for their ability to demethylate an endogenous tumor suppressor gene in a solid cancer model, the colon carcinoma HCT-116 cell line. Compound 10 was chosen for its selectivity toward DNMT3A/3L, while compound 14 was selected for its DNMT1 selectivity. Compounds 12 and 13 were included because they exhibited high potency against both DNMT1 and DNMT3A/3L. We employed the Combined Bisulfite Restriction Analysis (COBRA) assay²⁷ to monitor the methylation status of P16INK4A, a tumor suppressor gene reactivated by DNMT inhibition/deletion,²⁶ and Long Interferenced Nucleotide Element 1 (LINE-1) repeats, a well-established surrogate measure of global DNA methylation.³⁵ HCT-116 cells were treated for 10 days, with media changes and fresh compound additions every 3 days to allow for passive DNA demethylation and dilution of the DNA methylation signal.³⁴ Decitabine (5-azadC) was used as a positive control. As shown in Figure 2, after 10 days of treatment at 0.32 and 1 µM, compound 12, which demonstrated strong in vitro activity against both DNMT1 and DNMT3A/3L (Table 1), was the most effective in this assay specifically decreasing P16 methylation while having no effect on LINE-1 methylation. Compounds 7, 9, and 10 gave only slight effect in the same assay. Prototype 2 exhibited no hypomethylating effect on either P16 or LINE-1. The reference compound 5-azadC reduced both P16 (by approximately 40–50%) and LINE-1 (60%) methylation levels, confirming its broad, nonspecific demethylating activity. Compound 13 failed to reduce methylation levels of either LINE-1 or P16, while 14 was tested only at 0.32 µM due to its high cytotoxicity at higher concentration (Figure 2, and see below).

**DNA Thermal Denaturation Experiments in the Presence of Quinoline Compounds.** The previous studies by our group demonstrated that quinoline-based compounds 7, 9, 10, 12–14 inhibit not only DNMTs but also DNA-interacting enzymes. Additionally, crystallographic experiments showed that compound 10 (9 in ref.) binds in the minor groove of DNA.²⁴ To investigate the potential mechanism underlying this broad inhibition activity, we conducted DNA thermal denaturation experiments, measuring changes in DNA absorbance as the transition occurs from the hairpin duplex state (at low temperature) to unfolded single-stranded DNA (at high temperature).²⁸ Representative compounds 7, 10, 12, and 14 were tested at 10 µM using a 2 µM DNA duplex, while compound 8, a potent DNMT inhibitor (IC₅₀ = 1–2 µM, see Table 1) but a weak inhibitor of other DNA-interacting enzymes,²⁴ was included for comparison. Table 2 reports the differences in the melting temperature (T<sub>m</sub>) of the DNA duplex in the presence and in the absence of the tested compounds (ΔT<sub>m</sub>). The ΔT<sub>m</sub> data indicate that 7, 10, 12, and 14 significantly stabilized the DNA duplex, suggesting strong DNA interaction, whereas 8 did not (Figure 3). Structural analysis of 7 [N-(3-((2-amino-6-methylpyrimidin-4-yl)amino)-phenyl)-3-((dimethylamino)methyl)-5-(quinolin-4-ylamino)-benzamide] revealed that replacing the amide linker connecting

<table><caption>**Table 2.** ΔT<sub>m</sub> Values Determined for 7, 8, 10, 12, and 14 at 10 µM Using the DNA Duplex at 2 µM</caption><thead><tr><th>lab code</th><th>compd</th><th>ΔT<sub>m</sub> (°C)</th></tr></thead><tbody><tr><td>MC3716</td><td>7</td><td>11.3</td></tr><tr><td>MC3653</td><td>8</td><td>0.7</td></tr><tr><td>MC3563</td><td>10</td><td>12.5</td></tr><tr><td>MC3669</td><td>12</td><td>8.2</td></tr><tr><td>MC3817</td><td>14</td><td>15.8</td></tr><tr><td>SGI</td><td>1</td><td>1.0<sup>a</sup></td></tr><tr><td>MC3343</td><td>2</td><td>3.0<sup>a</sup></td></tr></tbody></table>

a Ref 31.

the B/C rings with an inverted amide (compound 8) abolished DNA binding ability and eliminated broad inhibition of DNA-interacting enzymes while maintaining DNMT inhibition. The prototype compounds 1 and 2 were previously tested under the same DNA thermal denaturation conditions and exhibited weak (1) or moderate (2) DNA binding properties.³¹ Both compounds were also weak or inactive against adenine methyltransferases.²⁴

**Anticancer Effects of Selected Quinoline Derivatives.**

To evaluate their antiproliferative potential, selected compounds 7, 9, 10, 12–14 were tested against a panel of cancer cells, including A549 and H460 (nonsmall cell lung cancer), HCT-116 (colon cancer), HeLa (cervical cancer), MDA-MB-231 (triple-negative breast cancer), and U937 (histiocytic lymphoma). Prototype 2 was used for comparison. Cells were treated with increasing concentrations of each compound (0.05–100 µM) for 48 h, after which cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay. The IC₅₀ values for each compound in all cancer cell lines were determined. Figure 4 displays the proliferation inhibition curves, while Table 3 summarizes the IC₅₀ values. Under these conditions, most compounds exhibited similar or higher potency than 2 in inhibiting cancer cell proliferation, mainly in specific cancer cell lines (HeLa, MDA-MB-231) where 2 was less effective. In detail, against the triple-negative breast cancer MDA-MB-231 cells, compounds 9, 10, 12, and 14 demonstrated significantly enhanced potency, with IC₅₀ values in the single-digit micromolar range (1.0–8.8 µM). Compound 9 was 35-fold more potent than 2, while 10, 12, and 14 were 8-, 4-, and 19-fold more potent, respectively. Against HeLa cervical cancer, compound 14 was 4-fold more potent than 2, exhibiting an IC₅₀ of 4.2 µM. In A549 nonsmall cell lung cancer cells, compounds 9 and 14 displayed slightly increased potency than 2. The most sensitive cell line was the HCT-116 colon cancer cell line, with 2 showing an IC₅₀ of 2.3 µM. Compounds 7, 9, and 10 displayed similar potency, whereas 14 was 5-fold more effective, reaching submicromolar concentration (IC₅₀ = 0.4 µM), in agreement with the COBRA analysis results (see above).

**Cytotoxic Effects of Compounds 9 and 14 in Colorectal Cancer Cells.** When tested in HCT-116 colorectal cancer cell line, compounds 9 and 14 displayed potent cytotoxic effects, with IC₅₀ values of 1.5 and 0.4 µM, respectively, after 48 h of treatment (Table 3). The same two compounds 9 and 14, the first equally potent against both the DNMT isoforms and the latter selective for DNMT1 (Table 1), were the most potent also against MDA-MB-231 cell line (Table 3), which expresses comparable levels of DNMT1 and DNMT3A to HCT-116 cells (Human Protein Atlas data, Table S3 in Supporting Information). Compound 17, practically inactive against both
<figure><img src="image_18.png" /><figcaption>**Figure 3.** DNA thermal denaturation experiments performed on 7, 8, 10, 12, and 14 at 10 µM.</figcaption></figure>

DNMTs (Table 1), when tested in HCT-116 cells for 48 h displayed no cytotoxicity up to 2 µM (Figure S7, Supporting Information). To assess selectivity, compound 14 was tested against two noncancerous cell lines, retinal pigment epithelium (RPE) cells and BJ human fibroblasts. It displayed IC₅₀ values of 5.0 and 8.5 µM, respectively, after 48 h (Figure S8, Supporting Information), showing a 12- to 22-fold selectivity window compared to HCT-116 cells.

**Cell Cycle and Apoptosis Analysis.** Compounds 9 and 14 were further analyzed by fluorescence-activated cell sorting (FACS) to evaluate their effects on the HCT-116 cell cycle. Figure 5A–C present representative FACS histograms (Figure 5A) and relative quantification (Figure 5B,C) of HCT-116 cells treated with 9 and 14 compared to control cultures. At 2 µM, compound 9 induced a significant G0/G1 accumulation, shifting the proportion of cells in this phase from 47.3% to 70.6% after 24 h. Conversely, 14 caused a block in the S/G2M phase associated with apoptotic cell death, affecting over 50% of the cells after 48 h, as indicated by the pre-G1 peak (Figure 5C). Compound 17, used as negative control, at 2 µM after 48 h showed a similar trend to the untreated sample (DMSO) (Figure S9, Supporting Information). The induction of apoptosis was further confirmed by Western blot analysis, showing a time-dependent increase in cleaved PARP levels in HCT-116 cells treated with 14 (2 µM) for 24 and 48 h (Figure 5D).

**Live Cell Imaging and Mitotic Progression Analysis.** To further investigate these findings, we performed live-cell imaging of HCT-116 cultures exposed to 9 and 14 (2 µM), monitoring individual cell fates from the moment of drug administration. In untreated (DMSO) cells, mitosis was typically completed within 40 min, with cells entering a subsequent mitotic cycle 24 h later. Of the 79 cells treated with 9, 22 entered mitosis. However, most exhibited a prolonged metaphase arrest. Among these, a small fraction (~2%) underwent cell death, while others ceased cell cycle progression irreversibly after mitosis. In contrast, only 5 of the 66 cells exposed to 14 initiated mitosis, and all showed a considerable delay in completion time. The majority of 14-treated cells underwent widespread apoptosis in interphase,

primarily characterized by extensive membrane blebbing (Figure 6).

**DNA Damage Response and p53 Activation.** To assess whether 14 induces DNA damage, we examined H2AX phosphorylation (γH2AX)—a marker of DNA strand breaks—and P53 activation in HCT-116 cells following 16–48 h of treatment. Western blot analysis revealed a dose-dependent increase in γH2AX and p53 accumulation upon exposure to 14, indicating P53 stabilization and activation in response to DNA damage (Figure 7). These results suggest that 14 triggers DNA damage and activates the p53/γH2AX pathway, leading to cell-cycle arrest and apoptosis in HCT-116 cells.

**P53-dependent Effects of Compound 14 in HCT-116 Cells.** To confirm the role of P53 in the cellular response to 14, isogenic variants of HCT-116 human colon cancer cells, including P53⁻/⁻ (P53-null) cells, were utilized. First, an MTT assay was performed on both P53 WT and P53⁻/⁻ HCT-116 cells to assess the effects of 14 on cell viability (Figure 8A). Notably, a significantly lower level of cell growth and viability was observed in HCT-116 P53⁺/⁺ cells compared with HCT-116 P53⁻/⁻ cells. To confirm whether this effect was attributed to apoptosis induced by 14, Annexin V flow cytometry analysis was performed on both wild-type and P53-null cells. As shown in Figure 8B, upon exposure to 2.5 µM of 14 for 48 h, significant PI and Annexin V fluorescence was detected, indicating early apoptosis (lower right quadrant) and late apoptosis/necrosis (upper right quadrant). These results confirm that 14 induces apoptotic cell death pathways in both cell lines. However, a much higher level of apoptosis was detected in HCT-116 P53 WT cells compared with HCT-116 P53⁻/⁻ cells. Finally, Western blot analysis of cleaved Caspase 3, P53 accumulation, and γH2AX levels (Figure 8C) further confirmed that 14 induces DNA damage, leading to cell-cycle arrest and apoptosis in HCT-116 cells in a P53-dependent manner.

Since some quinoline analogs have been shown to induce DNMT protein degradation in addition to enzymatic inhibition,²⁰⁻²² we investigated the effect of 14 on DNMT1 and
<figure><img src="image_19.png" /><figcaption>**Figure 4.** Dose-dependent effect curves of compounds 7, 9, 10, 12–14, along with the prototype 2, on cell viability in A549, H460, HCT-116, HeLa, MDA-MB-231, and U937 cell lines upon 48 h of treatment. All experiments were performed in sextuplicate ±SD (standard deviation).</figcaption></figure>

DNMT3A protein level in HCT-116 cells, in comparison with MS9024, a recently reported specific DNMT1 degrader.³⁶ When tested at 0.1, 0.5, and 1 µM for 24 h, 14 failed to induce degradation of either DNMT proteins, whereas MS9024 selectively reduced DNMT1 levels, as expected (Figure S10, Supporting Information).

## DISCUSSION AND CONCLUSIONS

Quinoline-based compounds represent a well-established class of small-molecule DNMTi with significant anticancer activity across various cancer models. Some of these compounds have been shown to induce DNMT protein degradation in addition to enzymatic inhibition,²⁰⁻²² while others inhibit DNA-interacting enzymes, including DNA glycosylases and DNA

polymerases, potentially contributing to their anticancer effects.²⁴

Here, we provide a detailed report on the design and synthesis of our latest series of quinoline-based DNMTi (4–21, Figure 1), incorporating basic side chains to improve water solubility and biological activity. These new compounds were developed based on prototypes 1 (compounds 4–6), 2 (compounds 7–14), 3 (compounds 15, 16), as well as their *bis*-quinoline (compounds 17–19) and *bis*-pyrimidine (compounds 20, 21) analogs.

Compounds 4–21 were evaluated against human DNMT1 and human DNMT3Acat, through a direct measure of the methylation event, obtained by using a restriction enzyme-based methylation assay (Table 1).³⁰ Among these, *para/para*-oriented compounds 4–6 exhibited a loss of DNMT inhibition
<table><caption>Table 3. Antiproliferative Activity (48 h Treatment) of Novel Quinoline-Based DNA-Targeting Enzyme Inhibitors against a Panel of Human Cancer Cell Lines</caption><thead><tr><th rowspan="2">compd</th><th colspan="6">antiproliferative activity (IC<sub>50</sub>, µM)<sup>a</sup></th></tr><tr><th>A549 nonsmall cell lung cancer</th><th>H460 nonsmall cell lung cancer</th><th>HCT-116 colon cancer</th><th>HeLa cervical cancer</th><th>MDA-MB-231 triple negative breast cancer</th><th>U937 histiocytic lymphoma</th></tr></thead><tbody><tr><td>7</td><td>41 ± 4</td><td>63 ± 12</td><td>2.2 ± 0.2</td><td>37 ± 4</td><td>20 ± 3</td><td>14 ± 2</td></tr><tr><td>9</td><td>6.0 ± 0.6</td><td>18 ± 5</td><td>1.5 ± 0.2</td><td>24 ± 1</td><td>1.0 ± 0.1</td><td>16 ± 3</td></tr><tr><td>10</td><td>74 ± 12</td><td>29 ± 5</td><td>2.0 ± 0.1</td><td>61 ± 9</td><td>4.5 ± 0.7</td><td>60 ± 8</td></tr><tr><td>12</td><td>8.5 ± 0.7</td><td>ND<sup>b</sup></td><td>5.9 ± 1.2</td><td>21 ± 1</td><td>8.8 ± 0.7</td><td>4.0 ± 0.3</td></tr><tr><td>13</td><td>32 ± 4</td><td>ND</td><td>8.6 ± 1.4</td><td>25 ± 2</td><td>11.5 ± 0.8</td><td>3.2 ± 0.2</td></tr><tr><td>14</td><td>5.5 ± 0.5</td><td>6.8 ± 0.7</td><td>0.4 ± 0.1</td><td>4.2 ± 0.2</td><td>1.9 ± 0.2</td><td>5.5 ± 0.2</td></tr><tr><td>2</td><td>7.9 ± 0.9</td><td>6.9 ± 1.2</td><td>2.3 ± 0.2</td><td>18 ± 2</td><td>35 ± 5</td><td>2.5 ± 0.2</td></tr></tbody></table>

<figure><img src="image_20.png" /><figcaption>**Figure 5.** Effects of compounds **9** and **14** on HCT-116 cell cycle progression and apoptosis induction. (A,B) Representative histograms of DNA content (A) and relative quantification (B,C) using propidium iodide staining in HCT-116 cells exposed to **9** and **14** for the indicated time points. (A) Red bars indicate the pre-G1 peak. (D) Western blot analysis of cleaved PARP in HCT-116 cells exposed to **14** at the indicated time points. GAPDH was used as a loading control. Experiments were performed in duplicate (n = 2). Results are expressed as mean ± SD. Compounds were tested at 2 µM for 24 and 48 h, as indicated.</figcaption></figure>

compared to prototype **1**, while *meta*/*meta* compounds **7–14** demonstrated enhanced potency against one or both DNMT isoforms. Compound **10** was the most potent and selective DNMT3A inhibitor (IC<sub>50</sub> = 0.019 µM, selectivity index = 105). Compound **14**, the only derivative with two basic chains, was the most potent and selective DNMT1 inhibitor (IC<sub>50</sub> = 0.044 µM, selectivity index = >227). Compounds **15** and **16**, basic-chained analogs of **3**, were potent at the low micromolar level against DNMT1 (**15**) or both enzymes (**16**). Bis-quinoline analogs (**17–19**) exhibited variable potency, with **18** and **19** inhibiting DNMTs in the low micromolar range. Bis-pyrimidine analog **20** was highly potent against DNMT1 (submicromolar potency, selectivity index = 24), whereas **21** inhibited, at approximately 1 µM, both the two DNMT isoforms.

Previous studies, reported by us<sup>21</sup> and others,<sup>32</sup> reported unsuccessful detection of hypomethylation in cells treated with prototype **2**. Similarly, several derivatives in series **4–21** failed to reduce methylation in murine embryonic stem cells<sup>24</sup> or

reactivate luciferase expression in KG-1 leukemia cells (data not shown). Thus, compounds with IC<sub>50</sub> ≤ 150 nM against at least one DNMT isoform (**7**, **9**, **10**, **12–14**) were further evaluated in HCT-116 colon cancer cells using the COBRA assay.<sup>27</sup> After 10 days of treatment, compound **12** was the most effective to selectively demethylate P16<sup>INK4A</sup> (about 60%), leaving LINE-1 levels unchanged (Figure 2). Decitabine (5-azadC), used as a positive control, reduced both the P16 and LINE-1 methylation, confirming its nonspecific demethylating activity.

Further DNA thermal denaturation experiments<sup>28</sup> revealed that **7**, **10**, **12**, and **14** strongly bound to DNA and stabilized the DNA duplex, whereas **8**, an amide-inverted analog of **7**, did not (Figure 3, Table 2). Interestingly, **8**, like prototypes **1** and **2**,<sup>31</sup> showed weaker DNA binding, and was less effective against other DNA-interacting enzymes than **7**, **10**, **12**, and **14**.<sup>24</sup> The interference with a wide range of DNA-interacting enzymes might make it difficult to interpret cellular effects, but it could

<footnote><sup>a</sup>The reported results represent the average ±SD (standard deviation) of sextuplicate experiments. <sup>b</sup>ND, not determined.</footnote>
<figure><img src="image_21.png" /><figcaption>**Figure 6.** Live-cell imaging of HCT-116 cultures exposed to 9 and 14 (2 µM). (A) Still images of representative control (DMSO) and 9-treated cells, or 14-treated cells undergoing apoptotic cell death. Time is given in h/min. Cells undergoing mitosis (DMSO or 9) or starting apoptosis (14) are shown at time 00.00. (B) Box-plots with whiskers showing the time spent in mitosis. DMSO: *n* = 93 cells; 9: *n* = 79 cells; 14: *n* = 66 cells.</figcaption></figure>

also be of added value for compounds with anticancer activity, since it covers different pathways related to the disease.

Compounds 7, 9, 10, and 12–14 were tested against a panel of cancer cell lines (A549, H460, HCT-116, HeLa, MDA-MB-231, and U937) in comparison with prototype 2 (Figure 4 and Table 3). Compounds 9, 10, and 14 exhibited stronger potency than 2, particularly in MDA-MB-231 and HCT-116 cells. In HCT-116 cells, 9 and 14 displayed $IC_{50}$ values of 1.5 µM and 0.4 µM, respectively, after 48 h. Compound 14 showed higher selectivity for cancer cells, with $IC_{50}$ values against two noncancerous cell lines of 5.0 µM (RPE) and 8.5 µM (BJ fibroblasts), corresponding to a 12- to 22-fold selectivity window over HCT-116 cells.

Further studies in HCT-116 cells demonstrated that 9 caused a G0/G1 block, while 14 induced an S/G2M arrest and apoptosis (>50% pre-G1 peak, increased cleaved PARP levels, Figure 5). Live-cell imaging revealed that 14 caused extensive interphase apoptosis with membrane blebbing, whereas 9 induced prolonged metaphase arrest with limited cell death (Figure 6).

We recently reported that, in EWS, treatment with 2 gave DNA damage, as revealed by the induction of the γH2AX foci. Such an effect stimulated activation of P53-dependent signaling and apoptosis in P53 WT cells, while in P53-mutated cells persistent micronuclei and increased DNA instability were observed.²¹ Among 4–21, the (N-methylpiperazin-1-yl)methyl compound 12 determined arrest of proliferation in A549 human nonsmall cell lung carcinoma (NSCLC), joined to increased levels of γH2AX, P53, and P21 as a response to DNA damage. Differently, in P53-mutated or -deleted NSCLC cells we observed no changes in P53/P21 levels and little or no cytotoxicity.²⁴

Here, we compare the effects of 14 in the HCT-116 P53 WT cell line and in its isogenic variant HCT-116 P53⁻/⁻ cell line,

determining cytotoxicity, apoptosis induction, and stimulation of DNA damage. With respect to what was observed in HCT-116 P53 WT cells, in HCT-116 P53⁻/⁻ cells 14 showed reduced impact on cell viability, decreased apoptosis and lower levels of cleaved Caspase 3, P53, and γH2AX accumulation, confirming a P53-dependent mechanism of action in this cell line (Figures 7 and 8) linked to DNA damage.

Altogether, these data suggest that quinoline-based DNMT inhibitors represent a promising therapeutic strategy for targeting P53 WT tumors, leveraging P53 activation to induce DNA damage, cell-cycle arrest, and apoptosis.

■ EXPERIMENTAL SECTION

**Chemistry.** Melting points were determined on a Buchi 530 melting point apparatus and are uncorrected. ¹H NMR spectra were recorded at 400 MHz on a Bruker AC 400 spectrometer; chemical shifts are reported in *δ* (ppm) units relative to the internal reference tetramethylsilane (Me₄Si). All compounds were routinely checked by TLC and ¹H NMR. TLC was performed on aluminum-backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F₂₅₄) with spots visualized by UV light. All solvents were reagent grade and, when necessary, were purified and dried by standard methods. Concentration of solutions after reactions and extractions involved the use of a rotary evaporator operating at a reduced pressure of ca. 20 Torr. Organic solutions were dried over anhydrous sodium sulfate. The purity of the final compounds 4–21 was analyzed by elemental analysis and for selected compounds (7–9, 12–14) also by HPLC. The elemental analysis has been performed on Thermo Fisher FlashSmart CNHS/O. The HPLC system consisted of a Dionex UltiMate 3000 UHPLC (Thermo Fisher) system equipped with an automatic injector and column heater and coupled with a Diode Array Detector DAD-3000 (Thermo Fisher). The analytical controls were performed on a Hypersil GOLD C18 Selectivity 5 µm (4.6 × 250 mm) HPLC Column (Thermo Fisher) in gradient elution. Eluents: (A) H₂O + 0.1% TFA; (B) CH₃CN + 0.1% TFA. A 5 min isocratic elution at 10% solvent B was followed by a 15 min linear gradient elution from 10% to 90% solvent B and 5 min at 90%
<figure><img src="image_22.png" /><figcaption>**Figure 7.** Western blot analysis and relative quantification of p53 and H2AX phosphorylation (γH2AX) in HCT-116 cell cultures exposed to 14 for the indicated doses and time points.</figcaption></figure>

B. The flow rate was 1.0 mL/min, and the column was kept at a constant temperature of 30 °C. Samples were dissolved in their respective solvent A at a concentration of 1.0 mg/mL, and the injection volume was 1 µL. By analyzing the HPLC traces at both 254 and 280 nm, a chemical purity >96% was recorded for all molecules. Analytical results are within ±0.40% of the theoretical values. All chemicals were purchased from Sigma-Aldrich Chemistry, Milan (Italy), Fluorochem, Manchester (UK), Enamine, Kyiv (Ukraine), or from AlfaAesar, Karlsruhe (Germany), and were of the highest purity.

**General Procedure for the Synthesis of the Final Compounds 4–7, 15–17, and of the Amide Intermediates 38, 40a–c, 41, 43, 50, and 51.** Triethylamine (3.98 mmol, 8.5 equiv) and PyBOP (0.57 mmol, 1.2 equiv) were added to a solution of the appropriate benzoic acid 25a,b, 30,¹⁹ 33,¹⁹ 37,¹⁹ 39,¹⁹ or 49²⁹ (0.47 mmol, 1 equiv) in dry DMF (3 mL) under a nitrogen atmosphere. The resulting mixture was stirred for 30 min at room temperature (rt); afterward, the appropriate aniline 26a,b,¹⁹ 36a–c, or 42 (0.47 mmol, 1 equiv) was added under a nitrogen atmosphere, and the reaction was stirred overnight. After, the reaction was quenched with water (50 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic extracts were dried over sodium sulfate, and the residue obtained upon solvent evaporation was purified by column chromatography (SiO₂ eluting with chloroform/

methanol/NH₃:10/1/0.2) to provide the pure product 4–7, 1–17, 38, 40a–c, 41, 43, 50 or 51.

N-(4-((2-amino-6-methylpyrimidin-4-yl)amino)phenyl)-2-((dimethylamino)methyl)-4-(quinolin-4-ylamino)benzamide hydrochloride (4, MC3671). Prepared starting from 25a (0.47 mmol, 0.15 g) and 26a¹⁹ (0.47 mmol, 0.10 g). Mp: 198–200 °C (acetonitrile/methanol); yield: 71%. ¹H NMR (400 MHz; DMSO-d₆) δ: 2.28 (s, 3H, -CH₃), 2.83 (s, 6H, -N(CH₃)₂), 4.49 (s, 2H, -CH₂N(CH₃)₂), 6.24 (s, 1H, pyrimidine proton), 7.14 (d, 1H, *J* = 7.2 Hz, quinoline proton), 7.80–7.88 (m, 6H, benzene and quinoline protons), 7.97 (s, 1H, benzene proton), 8.06–8.10 (m, 2H, benzene proton), 8.15–8.18 (m, 1H, benzene proton), 8.68 (d, 1H, *J* = 6.8 Hz, quinoline proton), 9.00 (d, 1H, *J* = 8.8 Hz, quinoline proton), 9.43 (br s, 1H, -NH-pyrimidine) 10.86 (br s, 1H, NH-quinoline), 10.91 (br s, 2H, -CONH-), 11.42 (br s, 1H, hydrochloride), 12.83 (br s, 1H, hydrochloride), 14.99 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 23.8, 44.8 (2C), 61.6, 97.5, 105.5, 117.1, 118.5, 119.8, 120.1 (2C), 123.3, 124.2 (2C), 125.5, 126.3, 127.3, 128.3, 131.0, 133.7, 135.0, 137.5, 144.2, 145.5, 147.3, 148.0, 158.2, 162.4, 166.0, 166.5.

4-((2-Amino-6-methylpyrimidin-4-yl)amino)-N-(2-((dimethylamino)methyl)-4-(quinolin-4-ylamino)phenyl)-
<figure><img src="image_23.png" /><figcaption>**Figure 8.** P53-dependent effects of **14** on cell viability and apoptosis in HCT-116 cells. (A) Effect of **14** on cell viability of P53 WT and P53-null HCT-116 cells. One day after seeding, cells were treated with increasing concentrations of **14** for 48 h, and proliferation was measured by using the MTT assay. Results are expressed as mean ± SE (n = 2) and are presented as a percentage of vehicle-treated controls. (B) Annexin V/PI staining to assess apoptosis in cells treated with **14** (2.5 µM for 48 h), analyzed via flow cytometry. (C) Western blot analysis of cleaved Caspase 3, P53, and γH2AX in HCT-116 cells treated with **14** for 48 h. Representative results from three independent experiments are shown. α-Tubulin and GAPDH were used as loading controls.</figcaption></figure>

**benzamide hydrochloride (5, MC3682).** Prepared starting from 29 (0.47 mmol, 0.14 g) and 30¹⁹ (0.47 mmol, 0.11 g). Mp: 239–241 °C (methanol); yield: 76%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.32 (s, 3H, -CH₂), 2.77 (s, 6H, -N(CH₃)₂), 4.36 (s, 2H, -CH₂N(CH₃)₂), 6.39 (s, 1H, pyrimidine proton), 7.07 (d, 1H, *J* = 6.4 Hz, quinoline proton), 7.66 (s, 2H, benzene proton), 7.82–7.93 (m, 2H, quinoline protons), 8.05–8.21 (m, 6H, benzene and quinoline protons), 8.57 (d, 1H, *J* = 6.8 Hz, quinoline proton), 8.94 (d, 1H, *J* = 8.4 Hz, quinoline proton), 10.62 (br s, 1H, -NH-pyrimidine) 11.22–28 (bm, 3H, NH-quinoline, -CONH-, hydrochloride), 13.15 (br s, 1H, hydrochloride), 14.87 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 23.8, 44.8 (2C), 60.8, 97.5, 105.5, 117.5 (2C), 119.6, 119.9, 120.1, 121.0, 123.3, 123.9, 125.5, 126.3, 126.9, 128.3, 129.9 (2C), 135.8, 136.8, 143.9, 145.6, 147.3, 148.0, 158.2, 162.4, 166.0, 166.5.

**N-(4-((2-Amino-6-methylpyrimidin-4-yl)amino)-2-((dimethylamino)methyl)phenyl)-4-(quinolin-4-ylamino)-benzamide hydrochloride (6, MC3672).** Prepared starting from 32 (0.47 mmol, 0.13 g) and 33¹⁹ (0.47 mmol, 0.12 g). Mp: 218–220 °C (acetonitrile/methanol); yield: 68%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.32 (s, 3H, -CH₃), 2.78 (s, 6H, -N(CH₃)₂), 4.37 (s, 2H, -CH₂N(CH₃)₂), 6.30 (s, 1H, pyrimidine proton), 7.01 (d, 1H, *J* = 7.2 Hz, quinoline proton), 7.47 (d, 1H, *J* = 8.8 Hz, benzene protons), 7.68–7.72 (m, 2H, benzene protons), 7.87 (t, 1H, *J* = 7.6 Hz, quinoline proton), 8.05–8.15 (m, 2H, benzene and quinoline protons), 8.35–8.37 (m, 3H, benzene and quinoline protons), 8.63 (d, 1H, *J* = 6.8 Hz, quinoline proton), 8.93 (d, 1H, *J* = 8.4 Hz, quinoline proton), 10.60 (br s, 1H, -NH-pyrimidine) 10.91 (br s, 1H, NH-quinoline), 11.02 (br s, 1H, -CONH-), 11.25 (br s, 1H, hydrochloride), 12.91 (br s, 1H, hydrochloride), 14.89 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 23.8, 44.8 (2C), 60.8, 97.4, 105.5, 118.0 (2C), 119.3, 119.9, 120.3, 121.3, 123.3, 123.9, 125.5, 126.3, 128.0, 128.3, 130.1 (2C), 135.9, 136.6, 145.1, 145.6, 147.3, 148.0, 158.2, 162.4, 166.0, 166.5.

**N-(3-((2-Amino-6-methylpyrimidin-4-yl)amino)phenyl)-3-((dimethylamino)methyl)-5-(quinolin-4-ylamino)benzamide hy-

**drochloride (7, MC3716).** Prepared starting from 25b (0.47 mmol, 0.15 g) and 26b¹⁹ (0.47 mmol, 0.10 g). Mp: 182–184 °C (acetonitrile/methanol); yield: 67%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.29 (s, 3H, -CH₃), 2.82 (s, 6H, -N(CH₃)₂), 4.48 (s, 2H, -CH₂N(CH₃)₂), 6.24 (s, 1H, pyrimidine proton), 7.19 (d, 1H, *J* = 6.8 Hz, quinoline proton), 7.37 (t, 1H, *J* = 8.0 Hz, benzene proton), 7.53–7.56 (m, 1H, benzene proton), 7.76 (d, 1H, *J* = 8.4 Hz, benzene proton), 7.86 (t, 1H, *J* = 7.6 Hz, quinoline proton), 7.97 (s, 1H, benzene proton), 8.05–8.18 (m, 3H, benzene and quinoline protons), 8.40–8.44 (m, 2H, quinoline protons), 8.58 (d, 1H, *J* = 6.8 Hz, quinoline proton), 8.92 (d, 1H, *J* = 8.8 Hz, quinoline proton), 10.64 (br s, 1H, -NH-pyrimidine), 10.76 (br s, 1H, NH-quinoline), 11.03 (br s, 1H, -CONH-), 11.27 (br s, 1H, hydrochloride), 12.86 (br s, 1H, hydrochloride), 14.80 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 23.8, 45.2 (2C), 63.3, 97.4, 105.5, 109.4, 116.3, 116.8, 117.6, 119.9, 120.4, 123.3, 125.5, 125.5, 126.3, 128.3, 128.8, 133.2, 139.3, 140.0, 141.8, 142.4, 145.7, 147.3, 148.0, 158.1, 162.4, 166.0, 166.0. HPLC: 98.2% pure (retention time 12.08 min) (Supporting Information Figure S1).

**Benzyl (3-((dimethylamino)methyl)-5-(4-(quinolin-4-ylamino)-benzamido)phenyl)carbamate hydrochloride (15, MC3650).** Prepared starting from 33¹⁹ (0.47 mmol, 0.12 g) and 48 (0.47 mmol, 0.14 g). Mp: 154–156 °C (acetonitrile); yield: 70%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.71 (s, 6H, -N(CH₃)₂), 4.23 (s, 2H, -CH₂N(CH₃)₂), 5.16 (s, 2H, -CH₂-), 7.20 (d, 1H, *J* = 5.2 Hz, quinoline proton), 7.35–7.49 (m, 8H, benzene and quinoline protons), 7.60 (d, 1H, *J* = 7.2 Hz, quinoline proton), 7.67–7.76 (m, 3H, benzene and quinoline protons), 7.94 (d, 1H, *J* = 8.0 Hz, benzene proton), 8.00 (d, 1H, *J* = 8.4 Hz, quinoline proton benzene proton), 8.38 (d, 1H, *J* = 8.4 Hz, quinoline proton), 8.58 (d, 1H, *J* = 5.2 Hz, quinoline proton), 9.23 (br s, 1H, -NH-quinoline), 9.72 (br s, 1H, -OCONH-), 10.08 (br s, 1H, -CONH-) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 45.2 (2C), 63.1, 66.7, 105.5, 111.1, 116.1, 116.3, 118.0 (2C), 119.9, 123.3, 124.2, 125.5, 126.3, 128.0 (3C), 128.1, 128.3, 128.4 (2C), 130.1 (2C), 136.3, 138.9, 139.4, 139.6, 145.1, 145.6, 147.3, 148.0, 153.9, 166.2.
Benzyl 3-((Dimethylamino)methyl)-5-(3-(quinolin-4-ylamino)-benzamido)phenyl)carbamate hydrochloride (16, MC3652). Prepared starting from 39¹⁹ (0.47 mmol, 0.12 g) and 48 (0.47 mmol, 0.14 g). Mp: 233–235 °C (methanol); yield: 66%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.64 (s, 6H, -N(CH₃)₂), 4.10 (s, 2H, -CH₂N(CH₃)₂), 5.18 (s, 2H, -CH₂-), 6.98 (d, 1H, J = 5.2 Hz, quinoline proton), 7.32–7.42 (m, 6H, benzene and quinoline protons), 7.60–7.72 (m, 4H, benzene and quinoline protons), 7.92–8.04 (m, 5H, quinoline and benzene protons), 8.54 (s, 1H, quinoline proton), 8.68 (s, 1H, quinoline proton), 10.71 (br s, 1H, -OCONH-), 10.23 (br s, 1H, -NH-quinoline), 10.52 (br s, 1H, -CONH-) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 45.2 (2C), 63.1, 66.7, 105.5, 111.2, 116.1, 116.3, 117.6, 119.9, 122.7, 123.1, 123.3, 125.5, 126.3, 128.0 (3C), 128.1, 128.3, 128.4, 128.5, 133.7, 136.3, 138.8, 139.2, 139.6, 142.0, 145.7, 147.3, 148.0, 153.9, 165.7.

N-(2-((Dimethylamino)methyl)-4-(quinolin-4-ylamino)phenyl)-4-(pyridin-4-ylamino)benzamide hydrochloride (17, MC3683). Prepared starting from 29 (0.47 mmol, 0.14 g) and 49²⁹ (0.47 mmol, 0.11 g). Mp: 225–227 °C (methanol); yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.77 (s, 6H, -N(CH₃)₂), 4.37 (s, 2H, -CH₂N(CH₃)₂), 7.09 (d, 1H, J = 4.4 Hz, quinoline proton), 7.37 (s, 2H, benzene protons), 7.48 (d, 2H, J = 7.2 Hz, pyridine protons) 7.66 (s, 2H, benzene proton), 7.80–7.84 (m, 1H, quinoline proton), 7.94 (s, 1H, quinoline proton), 8.05–8.10 (m, 1H, benzene proton), 8.16–8.18 (m, 1H, quinoline proton), 8.30–8.37 (m, 4H, benzene and pyridine protons), 8.53 (d, 1H, J = 3.6 Hz, quinoline proton), 9.03 (d, 1H, J = 8.0 Hz, quinoline proton), 10.73 (br s, 1H, -NH-pyridine), 11.29 (bm, 1H, -CONH-), 11.43 (bm, 1H, NH-quinoline), 11.44 (bm, 1H, hydrochloride), 14.41 (br s, 1H, hydrochloride), 15.02 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 44.8 (2C), 60.8, 105.5, 110.2 (2C), 116.7 (2C), 119.6, 119.9, 120.1, 121.0, 123.3, 123.8, 125.5, 126.3, 126.9, 128.3, 130.0 (2C), 135.8, 136.8, 145.6, 146.9, 147.3, 147.9, 148.0, 148.6 (2C), 166.5.

N-(3-Amino-5-((dimethylamino)methyl)phenyl)-3-((2-amino-6-methylpyrimidin-4-yl)amino)benzamide (38). Prepared starting from 37¹⁹ (0.47 mmol, 0.12 g) and 36a (0.47 mmol, 0.08 g). Mp: 235–237 °C (methanol); yield: 67%. ¹H NMR (400 MHz, CD₃OD) δ: 2.21 (s, 3H, -CH₃), 2.29 (s, 6H, N(CH₃)₂), 3.41 (s, 2H, -CH₂N(CH₃)₂), 6.00 (s, 1H, pyrimidine proton), 6.11 (br s, 1H, -NH-), 6.52 (s, 1H, benzene proton), 6.90 (s, 1H, benzene proton), 7.17 (s, 1H, benzene proton), 7.42–7.46 (m, 1H, benzene proton), 7.53–7.56 (m, 1H, benzene proton), 7.77 (d, 1H, J = 7.6 Hz, benzene proton), 8.30 (s, 1H, benzene proton) ppm.

N-(3-Amino-5-((dimethylamino)methyl)phenyl)-3-(quinolin-4-ylamino)benzamide (40a). Prepared starting from 36a (0.47 mmol, 0.08 g) and 39¹⁹ (0.47 mmol, 0.12 g). Mp: 200–202 °C (acetonitrile/methanol); yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.18 (s, 6H, N(CH₃)₂), 3.19 (s, 2H, -CH₂N(CH₃)₂), 5.05 (br s, 2H, -NH₂), 6.43 (s, 1H, benzene proton), 6.95 (s, 1H, benzene proton), 7.14 (d 1H, J = 5.2 Hz, quinoline proton), 7.26 (s, 1H, benzene proton), 7.52–7.61 (m, 3H, benzene and quinoline protons), 7.70–7.78 (m, 3H, benzene and quinoline protons), 8.03 (s, 1H, quinoline proton), 8.36 (d, 1H, J = 7.6 Hz, quinoline proton), 8.57 (d, 1H, J = 4.8 Hz, quinoline proton), 9.30 (br s, 1H, -NH-), 10.38 (br s, 1H, -CONH-) ppm.

N-(3-Amino-5-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(quinolin-4-ylamino)benzamide (40b). Prepared starting from 36b (0.47 mmol, 0.10 g) and 39¹⁹ (0.47 mmol, 0.12 g). Mp: 126–129 °C (toluene); yield: 75%. ¹H NMR (400 MHz, DMSO) δ: 2.14 (s, 3H, -CH₃), 2.33 (s, 8H, piperazine protons), 3.26 (s, 2H, -CH₂-), 5.08 (br s, 2H, -NH₂), 6.29 (s, 1H, benzene proton), 6.81 (s, 1H, benzene proton), 7.52–7.60 (m, 3H, benzene and quinoline protons), 7.69–7.76 (m, 2H, benzene and quinoline protons), 7.90–7.92 (m, 2H, benzene and quinoline protons), 8.41 (d, 1H, J = 8.4 Hz, quinoline proton), 8.51 (d, 1H, J = 4.0 Hz, quinoline proton), 9.14 (br s, 1H, -NH-), 9.97 (br s, 1H, -CONH-) ppm.

N-(3-((1H-Imidazole-1-yl)methyl)-5-aminophenyl)-3-(quinolin-4-ylamino)benzamide (40c). Prepared starting from 36c (0.47 mmol, 0.09 g) and 39¹⁹ (0.47 mmol, 0.12 g). Mp: 130–132 °C (toluene); yield: 81%. ¹H NMR (400 MHz, DMSO-d₆) δ: 5.04 (s, 2H, -CH₂-), 5.12 (br s, 2H, -NH₂), 6.15 (s, 1H, benzene proton), 6.77 (s, 1H,

benzene proton), 7.04 (s, 2h, benzene protons), 7.55–7.50 (m, 4H, benzene, imidazole and quinoline protons), 7.69–7.77 (m, 3H, benzene, imidazole and quinoline protons), 7.91–7.96 (m, 3H, benzene, imidazole and quinoline protons), 8.43 (d, 1H, J = 7.6 Hz, quinoline proton), 8.52–8.57 (m, 1H, quinoline proton), 9.28 (br s, 1H, -NH-), 10.03 (br s, 1H, -CONH-) ppm.

N-(3-Amino-5-((dimethylamino)methyl)phenyl)-3-((dimethylamino)methyl)-5-(quinolin-4-ylamino)benzamide (41). Prepared starting from 36a (0.47 mmol, 0.08 g) and 25b (0.47 mmol, 0.15 g). Mp: 220–222 °C (acetonitrile/methanol); yield: 65%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.15 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 3.22 (s, 2H, -CH₂N(CH₃)₂), 3.49 (s, 2H, -CH₂N-(CH₃)₂), 5.06 (br s, 2H, -NH₂), 6.29 (s, 1H, benzene proton), 6.82 (s, 1H, benzene proton), 7.03–7.05 (m, 2H, benzene and quinoline protons), 7.47–7.50 (m, 1H, benzene proton), 7.54–7.57 (m, 1H, quinoline proton), 7.62 (s, 1H, benzene proton), 7.70–7.74 (m, 1H, quinoline proton), 7.83 (s, 1H, benzene proton), 7.90 (d, 1H, J = 8.8 Hz, quinoline proton), 8.42 (d, 1H, J = 7.6 Hz, quinoline proton), 8.51 (d, 1H, J = 4.8 Hz, quinoline proton), 9.12 (br s, 1H, -NH-), 9.93 (br s, 1H, -CONH-) ppm.

N-(3-Amino-5-((dimethylamino)phenyl)-3-(quinolin-4-ylamino)-benzamide (43). Prepared starting from 39¹⁹ (0.47 mmol, 0.12 g) and 42 (0.47 mmol, 0.07 g). Mp: 115–117 °C (toluene); yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.81 (s, 6H, 2x-CH₃), 4.86 (br s, 2H, -NH₂), 5.78 (s, 1H, benzene proton), 6.39 (s, 1H, benzene proton), 6.59 (s, 1H, benzene proton), 7.03 (d, 1H, J = 5.6 Hz, benzene proton), 7.52–7.58 (m, 3H, benzene and quinoline protons), 7.67–7.77 (m, 2H, benzene and quinoline protons), 7.90–7.92 (m, 2H, benzene and quinoline protons), 8.41 (d, 1H, J = 9.2 Hz, quinoline proton), 8.51 (d, 1H, J = 4.8 Hz, quinoline proton), 9.12 (br s, 1H, -NH-), 9.79 (br s, 1H, -CONH-) ppm.

N-(3-Amino-5-((dimethylamino)methyl)phenyl)-4-(quinolin-4-ylamino)benzamide (50). Prepared starting from 33¹⁹ (0.47 mmol, 0.12 g) and 36a (0.47 mmol, 0.08 g). Mp: 181–183 °C (acetonitrile/methanol); yield: 80%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.15 (s, 6H, N(CH₃)₂), 3.18 (s, 2H, -CH₂N(CH₃)₂), 5.04 (br s, 2H, -NH₂), 6.28 (s, 1H, benzene proton), 6.85 (s, 1H, benzene proton), 7.05 (t, 1H, J = 2.0 Hz, benzene proton), 7.20 (d, 1H, J = 5.2 Hz, quinoline proton), 7.46 (d, 2H, J = 8.8 Hz, benzene proton), 7.60 (t, 1H, J = 2.0 Hz, benzene proton), 7.74 (t, 1H, J = 2.0 Hz, benzene proton), 7.93 (d, 1H, J = 8.4 Hz, benzene proton), 7.98 (d, 2H, J = 8.8 Hz, quinoline protons), 8.38 (d, 1H, J = 7.6 Hz, quinoline proton), 8.58 (d, 1H, J = 5.2 Hz, quinoline proton), 9.21 (br s, 1H, -NH-), 9.82 (br s, 1H, -CONH-) ppm.

N-(3-Amino-5-((dimethylamino)methyl)phenyl)-4-((2-amino-6-methylpyrimidin-4-yl)amino)benzamide (51). Prepared starting from 30¹⁹ (0.47 mmol, 0.11 g) and 36a (0.47 mmol, 0.08 g). mp: >250 °C (methanol); yield: 70%. ¹H NMR (400 MHz, CD₃OD) δ: 2.25 (s, 3H, -CH₃), 2.33 (s, 6H, N(CH₃)₂), 3.45 (s, 2H, -CH₂N(CH₃)₂), 6.04 (s, 1H, pyrimidine proton), 6.12 (br s, 1H, -NH-), 6.51 (s, 1H, benzene proton), 6.89 (s, 1H, benzene proton), 7.15 (s, 1H, benzene proton), 7.87–7.88 (m, 4H, benzene protons) ppm.

General Procedure for the Synthesis of the Final Compounds 8–14, 17–21 and of the Amine Intermediate Compounds 24a,b, 28, and 31. A mixture containing either 4-chloroquinoline (0.39 mmol, 1 equiv) or 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 1 equiv), the appropriate amino derivative (0.39 mmol, 1 equiv) and catalytic amount (4 drops) of 37% HCl in ethanol (30 mL) was refluxed for 2 h. Then, the mixture was allowed to cool down to rt, and the precipitated solid was filtered off, washed with water (3 × 5 mL), and purified by column chromatography (SiO₂ eluting with chloroform/methanol/ NH₃:20/1/0.1) to afford the pure product.

3-((2-Amino-6-methylpyrimidin-4-yl)amino)-N-(3-((dimethylamino)methyl)-5-(quinolin-4-ylamino)phenyl)-benzamide hydrochloride (8, MC3653). Prepared starting from 4-chloroquinoline (0.39 mmol, 0.06 g) and 38 (0.39 mmol, 0.15 g). Mp: 219–221 °C (acetonitrile/methanol); yield: 73%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.29 (s, 3H, -CH₃), 2.77 (s, 6H, -N(CH₃)₂), 4.35 (s, 2H, -CH₂N(CH₃)₂), 6.27 (s, 1H, pyrimidine proton), 7.14 (d, 1H, J = 5.6 Hz, quinoline proton), 7.53 (s, 2H, benzene protons), 7.78–7.83 (m, 2H, benzene protons), 7.97 (s, 1H, benzene proton), 8.05–8.20 (m,
5H, benzene and quinoline protons), 8.58 (d, 1H, *J* = 6.4 Hz, quinoline proton), 8.90 (d, 1H, *J* = 8.4 Hz, quinoline proton), 10.82 (br s, 1H, –NH– pyrimidine quinoline), 10.95 (br s, 1H, –NH– quinoline), 11.20 (br s, 1H, –CONH–) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 23.8, 45.2 (2C), 63.2, 97.4, 105.6, 110.8, 115.3, 116.2, 117.5, 119.9, 122.2, 122.6, 123.3, 125.5, 126.3, 128.3, 128.6, 134.1, 138.9, 139.8, 141.3, 143.1, 145.7, 147.3, 148.0, 158.1, 162.4, 165.6, 166.0.

N-(3-((2-Amino-6-methylpyrimidin-4-yl)amino)-5-(dimethylamino)phenyl)-3-(quinolin-4-ylamino)benzamide hydrochloride (9, MC3807). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 43 (0.39 mmol, 0.15 g). Mp: >250 °C (methanol); yield: 59%. ¹H NMR (400 MHz, DMSO-d₆) *δ*: 2.29 (s, 3H, –CH₃), 2.99 (s, 6H, –N(CH₃)₂), 6.21 (s, 1H, pyrimidine proton), 6.94 (d, 1H, *J* = 6.8 Hz, quinoline proton), 7.27–7.36 (br s, 1H, benzene proton), 7.74–7.78 (m, 3H, benzene and quinoline protons), 7.87 (t, 1H, *J* = 8.0 Hz, quinoline proton), 8.05–8.14 (m, 5H, benzene and quinoline protons), 8.56–8.58 (m, 1H, quinoline proton), 8.98 (d, 1H, *J* = 8.4 Hz, quinoline proton), 10.48 (br s, 1H, –NH–pyrimidine) 10.65 (br s, 1H, NH- quinoline), 11.19 (br s, 1H, –CONH–), 12.84 (br s, 1H, hydrochloride), 14.74 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 23.8, 41.4 (2C), 97.4, 101.8, 102.9, 105.5, 107.1, 117.6, 119.9, 122.7, 123.1, 123.3, 125.5, 126.3, 128.3, 128.5, 133.6, 141.5, 142.0, 143.8, 145.7, 147.3, 148.0, 153.3, 157.8, 162.4, 165.7, 166.0. HPLC: 98.2% pure (retention time 12.51 min) (Supporting Information Figure S2).

N-(3-((2-Amino-6-methylpyrimidin-4-yl)amino)-5-((dimethylamino)methyl)phenyl)-3-(quinolin-4-ylamino)benzamide (10, MC3563). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 40a (0.39 mmol, 0.16 g). Mp: 195–197 °C (acetonitrile/methanol); yield: 68%. ¹H NMR (400 MHz, DMSO-d₆) *δ*: 2.09 (s, 3H, –CH₃), 2.18 (s, 6H, –N(CH₃)₂), 3.35 (s, 2H, –CH₂N(CH₃)₂), 5.92 (s, 1H, pyrimidine proton), 6.02 (br s, 2H, –NH₂-pyrimidine), 7.06–7.07 (d, 1H, *J* = 7.6 Hz, quinoline proton), 7.34 (s, 1H, benzene proton), 7.38 (s, 1H, quinoline proton), 7.55–7.58 (m, 3H, benzene and quinoline protons), 7.71–7.74 (m, 2H, benzene protons), 7.91 (d, 1H, *J* = 8.4 Hz, quinoline proton), 7.96 (s, 1H, benzene proton), 8.04 (s, 1H, benzene proton), 8.41 (d, 1H, *J* = 8.0 Hz, quinoline proton), 8.52 (d, 1H, *J* = 5.2 Hz, quinoline proton), 8.99 (br s, 1H, –NH– pyrimidine), 9.15 (br s, 1H, –NH–quinoline), 10.14 (br s, 1H, –CONH–) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 23.8, 45.2 (2C), 63.2, 97.4, 105.5, 110.7, 115.2, 116.2, 117.6, 119.9, 122.7, 123.1, 123.3, 125.5, 126.3, 128.3, 128.5, 133.7, 138.9, 139.6, 142.0, 142.3, 145.7, 147.3, 148.0, 157.9, 162.4, 165.7, 166.0. HPLC: 97.9% pure (retention time 12.12 min) (Supporting Information Figure S3).

N⁴-(3-((dimethylamino)methyl)-5-((3-(quinolin-4-ylamino)-benzyl)amino)phenyl)-6-methylpyrimidine-2,4-diamine (11, MC3718). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 47 (0.39 mmol, 0.15 g). mp: >250 °C (methanol); yield: 60%. ¹H NMR (400 MHz, DMSO-d₆) *δ*: 2.30 (s, 3H, –CH₃), 2.75 (s, 6H, –N(CH₃)₂), 2.82 (s, 6H, –N(CH₃)₂), 4.33 (s, 2H, –CH₂N(CH₃)₂), 4.48 (s, 2H, –CH₂N(CH₃)₂), 6.33 (s, 1H, pyrimidine proton), 7.21 (d, 1H, *J* = 6.8 Hz, quinoline proton), 7.80–7.87 (m, 2H, benzene and quinoline proton), 8.00–8.09 (m, 3H, benzene and quinoline protons), 8.15–8.19 (m, 2H, benzene and quinoline proton), 8.39 (s, 2H, benzene protons), 8.58 (d, 1H, *J* = 6.0 Hz, quinoline proton), 8.98 (d, 1H, *J* = 8.4 Hz, quinoline proton), 10.75 (br s, 1H, –NH–pyrimidine) 10.86 (br s, 1H, NH- quinoline), 10.97 (br s, 1H, hydrochloride), 11.14 (br s, 1H, hydrochloride), 11.37 (br s, 1H, –CONH–), 12.93 (br s, 1H, hydrochloride), 14.90 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 23.8, 45.2 (3C), 45.3, 63.2, 63.3, 97.4, 105.5, 110.7, 115.2, 116.2, 116.8, 119.9, 120.4, 123.3, 125.5, 125.5, 126.3, 128.3, 133.2, 138.8, 139.3, 139.5, 141.8, 142.3, 145.7, 147.3, 148.0, 157.9, 162.4, 165.9, 166.0.

N-(3-((2-Amino-6-methylpyrimidin-4-yl)amino)-5-((4-methylpiperazin-1-yl)methyl) phenyl)-3-(quinolin-4-ylamino)benzamide (12, MC3669). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 40b (0.39 mmol, 0.18 g). Mp: 243–245 °C (methanol); yield: 67%. ¹H NMR (400 MHz, DMSO-d₆) *δ*: 2.09 (s, 3H, –CH₃, pyrimidine), 2.16 (s, 2H, –CH₃ piperazine), 2.34–2.41 (m, 8H, piperazine protons), 3.43 (s, 2H, –CH₂), 5.92 (s, 1H, pyrimidine proton), 6.01 (br s, 2H, –NH₂), 7.06 (d, 1H, *J* = 4.8 Hz,

quinoline proton), 7.35 (s, 2H, benzene protons), 7.55–7.58 (m, 3H, benzene and quinoline protons), 7.71–7.74 (m, 2H, benzene and quinoline protons), 7.90–7.96 (m, 2H, benzene protons), 8.04 (s, 1H, quinoline protons), 8.42 (d, 1H, *J* = 5.4 Hz, quinoline proton), 8.52 (d, 1H, *J* = 5.2 Hz, quinoline proton), 8.99 (br s, 1H, –NH– pyrimidine), 9.14 (br s, 1H, –NH– quinoline), 10.14 (s, 1H, –CONH–) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 23.8, 45.4, 53.0 (2C), 54.7 (2C), 59.4, 97.4, 105.5, 110.9, 115.6, 116.5, 117.6, 119.9, 122.7, 123.1, 123.3, 125.5, 126.3, 128.3, 128.5, 133.7, 138.2, 139.6, 142.0, 142.3, 145.7, 147.3, 148.0, 157.9, 162.4, 165.7, 166.0. HPLC: 96.4% pure (retention time 11.96 min) (Supporting Information Figure S4).

N-(3-((1H-imidazole-1-yl)methyl)-5-((2-amino-6-methylpyrimidin-4-yl)amino)phenyl)-3-(quinolin-4-ylamino)benzamide hydrochloride (13, MC3821). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 40c (0.39 mmol, 0.17 g). Mp: >250 °C (methanol); yield: 61%. ¹H NMR (400 MHz, DMSO-d₆) *δ*: 2.29 (s, 3H, –CH₃), 5.55 (s, 2H, –CH₂-imidazole), 6.29 (s, 1H, pyrimidine proton), 6.95 (d, 1H, *J* = 6.8 Hz, quinoline proton), 7.69–7.86 (m, 8H, benzene imidazole, and quinoline protons), 8.03–8.16 (m, 4H, benzene and quinoline protons), 8.30 (s, 1H, imidazole proton), 8.55 (d, 1H, *J* = 7.6 Hz, quinoline proton), 8.96 (d, 1H, *J* = 8.4 Hz, quinoline proton), 9.49 (s, 1H, imidazole proton), 10.79 (br s, 1H, –NH–pyrimidine) 10.97 (br s, 1H, NH- quinoline), 11.30 (br s, 1H, –CONH–), 13.05 (br s, 1H, hydrochloride), 14.91 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 23.8, 49.0, 97.4, 105.5, 110.9, 116.2, 117.1, 117.6, 119.9, 121.0, 122.7, 123.1, 123.3, 125.5, 126.3, 128.3, 128.5, 129.1, 133.7, 136.6, 137.3, 139.8, 142.0, 142.0, 145.7, 147.3, 148.0, 157.9, 162.4, 165.7, 166.0. HPLC: 99.4% pure (retention time 12.17 min) (Supporting Information Figure S5).

N-(3-((2-Amino-6-methylpyrimidin-4-yl)amino)-5-((dimethylamino)methyl)phenyl)-3-((dimethylamino)methyl)-5-(quinolin-4-ylamino)benzamide hydrochloride (14, MC3817). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 41 (0.39 mmol, 0.18 g). Mp: >250 °C (methanol); yield: 60%. ¹H NMR (400 MHz, DMSO-d₆) *δ*: 2.30 (s, 3H, –CH₃), 2.75 (s, 6H, –N(CH₃)₂), 2.82 (s, 6H, –N(CH₃)₂), 4.33 (s, 2H, –CH₂N-(CH₃)₂), 4.48 (s, 2H, –CH₂N(CH₃)₂), 6.33 (s, 1H, pyrimidine proton), 7.21 (d, 1H, *J* = 6.8 Hz, quinoline proton), 7.80–7.87 (m, 2H, benzene and quinoline proton), 8.00–8.09 (m, 3H, benzene and quinoline protons), 8.15–8.19 (m, 2H, benzene and quinoline proton), 8.39 (s, 2H, benzene protons), 8.58 (d, 1H, *J* = 6.0 Hz, quinoline proton), 8.98 (d, 1H, *J* = 8.4 Hz, quinoline proton), 10.75 (br s, 1H, –NH–pyrimidine) 10.86 (br s, 1H, NH- quinoline), 10.97 (br s, 1H, hydrochloride), 11.14 (br s, 1H, hydrochloride), 11.37 (br s, 1H, –CONH–), 12.93 (br s, 1H, hydrochloride), 14.90 (br s, 1H, hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 23.8, 45.2 (3C), 45.3, 63.2, 63.3, 97.4, 105.5, 110.7, 115.2, 116.2, 116.8, 119.9, 120.4, 123.3, 125.5, 125.5, 126.3, 128.3, 133.2, 138.8, 139.3, 139.5, 141.8, 142.3, 145.7, 147.3, 148.0, 157.9, 162.4, 165.9, 166.0. HPLC: 97.6% pure (retention time 11.30 min) (Supporting Information Figure S6).

N-(3-((Dimethylamino)methyl)-5-(quinolin-4-ylamino)phenyl)-4-(quinolin-4-ylamino) benzamide hydrochloride (18, MC3646). Prepared starting from 4-chloroquinoline (0.39 mmol, 0.06 g) and 50 (0.39 mmol, 0.16 g). Mp: >250 °C (methanol); yield: 71%. ¹H NMR (400 MHz, DMSO-d₆) *δ*: 2.80 (s, 6H, –N(CH₃)₂), 4.37 (s, 2H, –CH₂N(CH₃)₂), 7.03 (d, 1H, *J* = 6.8 Hz, quinoline proton), 7.15 (d, 1H, *J* = 6.8 Hz, quinoline proton), 7.55 (s, 1H, benzene proton), 7.71 (d, 2H, *J* = 8.4 Hz, benzene protons), 7.84–7.88 (m, 2H, benzene protons), 8.03–8.12 (m, 6H, quinoline and benzene protons), 8.21 (d, 2H, *J* = 8.4 Hz, benzene protons), 8.59–8.64 (m, 2H, quinoline protons), 8.83–8.89 (m, 2H, quinoline protons), 10.86 (br s, 2H, –CONH- and –NH– quinoline), 11.12 (br s, 2H, –NH– quinoline and hydrochloride), 14.66 (br s, 2H, 2x hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) *δ*: 45.2 (2C), 63.2, 105.5, 105.6, 110.9, 115.3, 116.2, 118.0 (2C), 119.9 (2C), 123.3, 123.3, 124.2, 125.5, 125.6, 126.3, 126.4, 128.3, 128.4, 130.1 (2C), 138.6, 139.8, 143.1, 145.1, 145.6, 145.7, 147.3, 147.3, 148.0, 148.0, 166.2.

N-(3-((Dimethylamino)methyl)-5-(quinolin-4-ylamino)phenyl)-3-(quinolin-4-ylamino)benzamide hydrochloride (19, MC3639).
Prepared starting from 4-chloroquinoline (0.39 mmol, 0.06 g) and 40a (0.39 mmol, 0.16 g). Mp: >250 °C (methanol); yield: 73%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.20 (s, 6H, -N(CH₃)₂), 3.41 (s, 2H, -CH₂N(CH₃)₂), 7.03-7.06 (m, 3H, benzene and quinoline protons), 7.51-7.59 (m, 5H, benzene and quinoline protons), 7.68-7.75 (m, 3H, benzene and quinoline protons), 7.87-7.92 (m, 3H, benzene and quinoline protons), 7.99 (s, 1H, benzene proton), 8.40-8.43 (m, 2H, quinoline protons), 8.48-8.52 (m, 2H, quinoline protons), 9.02 (br s, 1H, -NH- quinoline), 9.16 (br s, 1H, -NH-quinoline), 10.33 (br s, 1H, -CONH-) ppm. ¹³C NMR (100 MHz, CD₃OD) δ: 45.3 (2C), 63.7, 105.3, 105.4, 110.5, 115.4, 116.4, 117.6, 119.9, 120.0, 122.6, 123.0, 123.0, 123.2, 125.9, 125.9, 126.3, 126.4, 128.3, 128.3, 128.6, 133.9, 139.0, 139.9, 142.1, 143.1, 145.9, 146.2, 147.2, 147.2, 147.4, 147.4, 147.4, 166.3.

4-((2-Amino-6-methylpyrimidin-4-yl)amino)-N-(3-((2-amino-6-methylpyrimidin-4-yl) amino)-5-((dimethylamino)methyl)phenyl)-benzamide hydrochloride (20, MC3647). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 51 (0.39 mmol, 0.15 g). Mp: 149-151 °C (acetonitrile); yield: 65%. ¹H NMR (400 MHz, CD₃OD) δ: 2.20 (s, 6H, 2x-CH₃), 2.36 (s, 6H, -N(CH₃)₂), 3.55 (s, 2H, -CH₂N(CH₃)₂), 6.02 (s, 1H, pyrimidine proton), 6.04 (s, 1H, pyrimidine proton), 7.28 (s, 1H, benzene proton), 7.39 (s, 1H, benzene proton), 7.86-7.94 (m, 4H, benzene protons), 8.12 (br s, 1H, -CONH-), ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 23.8 (2C), 45.2 (2C), 63.2, 97.4, 97.5, 110.7, 115.2, 116.2, 117.5 (2C), 124.2, 129.9, 138.9, 139.5, 142.3, 143.9, 158.0, 158.2, 162.4, 162.4, 166.0 (2C), 166.1, 166.2.

3-((2-Amino-6-methylpyrimidin-4-yl)amino)-N-(3-((2-amino-6-methylpyrimidin-4-yl) amino)-5-((dimethylamino)methyl)phenyl)-benzamide hydrochloride (21, MC3649). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 38 (0.39 mmol, 0.15 g). Mp: >250 °C (methanol); yield: 69%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.30 (s, 6H, 2x-CH₃), 2.76 (s, 6H, -N(CH₃)₂), 4.33 (s, 2H, -CH₂N(CH₃)₂), 6.29 (s, 2H, pyrimidine protons), 7.52-7.56 (m, 2H, benzene protons), 7.72-7.80 (m, 3H, benzene protons), 8.07-8.20 (m, 4H, benzene protons), 10.68 (br s, 1H, -NH-pyrimidine), 10.77 (br s, 1H, -NH-pyrimidine), 10.91 (br s, 1H, -CONH-), 10.99 (bds, 1H, hydrochloride), 12.92 (br s, 2H, 2x hydrochloride) ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ: 23.8 (2C), 45.2 (2C), 63.2, 97.4, 97.4, 110.7, 115.2, 116.2, 117.5, 122.2, 122.6, 128.6, 134.1, 138.9, 139.6, 141.3, 142.3, 158.0, 158.1, 162.4, 162.4 (2C), 165.6, 166.0 (2C), 166.1.

Methyl 2-((dimethylamino)methyl)-4-(quinolin-4-ylamino)-benzoate (24a). Prepared starting from 4-chloroquinoline (0.39 mmol, 0.06 g) and 23a (0.39 mmol, 0.08 g). Mp: >250 °C (methanol); yield: 75%. ¹H NMR (400 MHz, CDCl₃) δ: 2.30 (s, 6H, -N(CH₃)₂), 3.83 (s, 2H, -CH₂N(CH₃)₂), 3.91 (s, 3H, -CH₃), 7.02 (br s, 1H, -NH-), 7.23-7.25 (m, 2H, benzene protons), 7.46 (s, 1H, quinoline proton), 7.52-7.55 (m, 1H, quinoline proton), 7.70-7.74 (m, 1H, benzene proton), 7.93 (d, 1H, *J* = 8.4 Hz, quinoline proton), 7.99 (d, 1H, *J* = 8.4 Hz, quinoline proton), 8.09 (d, 1H, *J* = 4.4 Hz, quinoline proton), 8.67 (d, 1H, *J* = 4.8 Hz, quinoline proton) ppm.

Methyl 3-((dimethylamino)methyl)-5-(quinolin-4-ylamino)-benzoate (24b). Prepared starting from 4-chloroquinoline (0.39 mmol, 0.06 g) and 23b (0.39 mmol, 0.08 g). Mp: 99-101 °C (toluene); yield: 78%. ¹H NMR (400 MHz, CDCl₃) δ: 2.30 (s, 6H, -N(CH₃)₂), 3.51 (s, 3H, -CH₃), 3.95 (s, 2H, -CH₂N(CH₃)₂), 6.71 (br s, 1H, -NH-), 7.04-7.07 (m, 1H, benzene proton), 7.51-53 (m, 1H, benzene proton), 7.54-7.58 (m, 1H, quinoline proton), 7.72-7.79 (m, 2H, benzene and quinoline protons), 7.92-7.95 (m, 2H, quinoline protons), 8.10 (d, 1H, *J* = 8.4 Hz, quinoline proton), 8.65 (d, 1H, *J* = 5.2 Hz, quinoline proton) ppm.

N-(3-((Dimethylamino)methyl)-4-nitrophenyl)quinolin-4-amine (28). Prepared starting from 4-chloroquinoline (0.39 mmol, 0.06 g) and 27 (0.39 mmol, 0.08 g). Mp: >250 °C (methanol); yield: 74%. ¹H NMR (400 MHz, CD₃OD) δ: 2.27 (s, 6H, -N(CH₃)₂), 3.79 (s, 2H, -CH₂N(CH₃)₂), 7.33 (s, 1H, benzene proton), 7.38 (d, 1H, *J* = 8.4 Hz, quinoline proton), 7.51 (s, 1H, benzene proton), 7.54-7.61 (m, 1H, quinoline proton), 7.71-7.78 (m, 1H, quinoline proton), 7.94 (d, 1H, *J* = 8.4 Hz, benzene proton), 8.04 (d, 1H, *J* = 8.4 Hz, quinoline proton),

8.25 (d, 1H, *J* = 7.6 Hz, quinoline proton), 8.56 (s, 1H, quinoline proton) ppm.

N⁴-(3-((Dimethylamino)methyl)-4-nitrophenyl)-6-methylpyrimidine-2,4-diamine (31). Prepared starting from 2-amino-4-chloro-6-methylpyrimidine (0.39 mmol, 0.06 g) and 27 (0.39 mmol, 0.08 g). Mp: >250 °C (methanol); yield: 70%. ¹H NMR (400 MHz, CDCl₃) δ: 1.65 (s, 3H, -CH₃), 2.30 (s, 2H, -CH₂N(CH₃)₂), 3.82 (s, 2H, -CH₂N(CH₃)₂), 4.91 (br s, 2H, NH₂), 6.04 (s, 1H, pyrimidine proton), 7.73 (br s, 1H, -NH-), 7.73-7.65 (m, 1H, benzene proton), 8.04 (d, 1H, *J* = 9.2 Hz, benzene proton) ppm.

**General Procedure for the Synthesis of the Amine Intermediate Compounds 22a,b, 35a-c.** The appropriate alkyl bromide (0.75 mmol, 1 equiv) was dissolved in dry THF and added with the appropriate amine (2.25 mmol, 3 equiv). After 5 h, the reaction was quenched at rt with water (20 mL), and the mixture was extracted with ethyl acetate (3 × 30 mL) and washed with brine (3 × 30 mL). The combined organic extracts were dried over sodium sulfate, and upon solvent evaporation, the amine product was used without further purification.

**Methyl 2-((Dimethylamino)methyl)-4-nitrobenzoate (22a).** Prepared starting from methyl 2-(bromomethyl)-4-nitrobenzoate (0.75 mmol, 0.20 g) and dimethylamine (2.25 mmol, 0.10 g). Mp: 110-112 °C (toluene); yield: 69%. ¹H NMR (400 MHz, CDCl₃) δ: 2.35 (s, 6H, -N(CH₃)₂), 3.84 (s, 3H, -CH₃), 4.00 (s, 2H, -CH₂N(CH₃)₂), 8.22-8.25 (m, 2H, benzene protons), 8.33-8.35 (m, 1H, benzene proton) ppm.

**Methyl 3-((Dimethylamino)methyl)-5-nitrobenzoate (22b).** Prepared starting from methyl 3-(bromomethyl)-5-nitrobenzoate (0.75 mmol, 0.20 g) and dimethylamine (2.25 mmol, 0.10 g). Mp: 134-136 °C (toluene); yield: 63%. ¹H NMR (400 MHz, CDCl₃) δ: 2.30 (s, 6H, -N(CH₃)₂), 3.58 (s, 2H, -CH₂N(CH₃)₂), 4.00 (s, 3H, -CH₃), 8.33-8.35 (m, 1H, benzene proton), 8.41-8.43 (m, 1H, benzene proton), 8.76-8.78 (m, 1H, benzene proton) ppm.

**Ditert-butyl (5-((Dimethylamino)methyl)-1,3-phenylene)-dicarbamate (35a).** Prepared starting from 34 (0.75 mmol, 0.30 g) and dimethylamine (2.25 mmol, 0.10 g). Mp: 197-199 °C (acetonitrile/methanol); yield: 77%. ¹H NMR (400 MHz, CDCl₃) δ: 1.43 (s, 18H, 2x C(CH₃)₂), 2.16 (s, 6H, -N(CH₃)₂), 3.29 (s, 2H, -CH₂N(CH₃)₂), 6.53 (br s, 2H, -OCONH-), 6.93 (s, 2H, benzene protons), 7.36 (s, 1H, benzene proton) ppm.

**Ditert-butyl (5-((4-Methylpiperazin-1-yl)methyl)-1,3-phenylene)-dicarbamate (35b).** Prepared starting from 34 (0.75 mmol, 0.30 g) and N-methylpiperazine (2.25 mmol, 0.22 g). Mp: 220-222 °C (acetonitrile/methanol); yield: 71%. ¹H NMR (400 MHz, DMSO-d₆) δ: 1.47 (s, 18H, C(CH₃)₃), 2.70 (s, 3H, -CH₃), 3.33 (s, 8H, piperazine protons), 3.40 (s, 2H, -CH₂-), 7.05 (s, 2H, benzene protons) 7.48 (s, 1H, benzene proton), 9.27 (br s, 2H, 2x-OCONH-) ppm.

**Ditert-butyl (5-((1H-imidazole-1-yl)methyl)-1,3-phenylene)-dicarbamate (35c).** Prepared starting from 34 (0.75 mmol, 0.30 g) and imidazole (2.25 mmol, 0.15 g). Mp: 226-228 °C (acetonitrile/methanol); yield: 73%. ¹H NMR (400 MHz, CDCl₃) δ: 1.51 (s, 18H, 2x C(CH₃)₃), 5.06 (s, 2H, -CH₂-), 6.64 (br s, 2H, 2x-OCONH-), 6.88 (s, 2H, benzene protons), 6.92 (s, 1H, imidazole proton), 7.10 (s, 1H, imidazole proton), 7.44 (s, 1H, imidazole proton), 7.55 (s, 1H, benzene proton) ppm.

**General Procedure for the Synthesis of the Amine Intermediate Compounds 23a,b, 29, 32, 42.** A solution of 37% HCl (0.2 mL) was slowly added at 0 °C to a cooled solution of the appropriate nitro derivative (0.03 mmol, 1 equiv) and stannous chloride dihydrate (0.15 mmol, 0.03 g, 5 equiv) in ethanol (5 mL). The reaction was then kept at 80 °C for 1 h. Afterward, the reaction was quenched at rt by 2N sodium carbonate solution (20 mL), and the mixture was extracted with ethyl acetate (3 × 30 mL) and washed with brine (3 × 30 mL). The combined organic extracts were dried over sodium sulfate, and upon solvent evaporation, the residue was purified by column chromatography (SiO₂ eluting with ethyl acetate/methanol/NH₃:10/1/0.1) to afford the desired amine product.

**Methyl 4-Amino-2-((dimethylamino)methyl)benzoate (23a).** Prepared starting from 22a (0.03 mmol, 0.007 g). Mp: 150-153 °C
(acetonitrile); yield: 78%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.76 (s, 6H, -N(CH₃)₂), 3.78 (s, 3H, -CH₃), 4.36 (s, 2H, -CH₂N(CH₃)₂), 4.73 (br s, 2H, -NH₂), 6.63-6.68 (m, 2H, benzene proton), 7.79-7.81 (m, 1H, benzene proton) ppm.

**Methyl 3-Amino-5-((dimethylamino)methyl)benzoate (23b).** Prepared starting from 22b (0.03 mmol, 0.007 g). Mp: 148-149 °C (acetonitrile); yield: 77%. ¹H NMR (400 MHz, CDCl₃) δ: 2.26 (s, 6H, -N(CH₃)₂), 3.77 (br s, 2H, -NH₂), 3.49 (s, 2H, -CH₂N(CH₃)₂), 3.90 (s, 3H, -CH₃), 6.88-6.89 (m, 1H, benzene proton), 7.26-7.29 (m, 1H, benzene proton), 7.35-7.37 (m, 1H, benzene proton) ppm.

**3-((Dimethylamino)methyl)-N¹-(quinolin-4-yl)benzene-1,4-diamine (29).** Prepared starting from 28 (0.03 mmol, 0.01 g). Mp: 145-148 °C (acetonitrile); yield: 75%. ¹H NMR (400 MHz, CDCl₃) δ: 2.25 (s, 6H, -N(CH₃)₂), 3.45 (s, 2H, -CH₂N(CH₃)₂), 5.03 (br s, 2H, -NH₂), 6.61 (s, 1H, benzene proton), 6.85 (d, 1H, *J* = 8.4 Hz, quinoline proton), 7.03 (s, 1H, benzene proton), 7.10 (d, 1H, *J* = 7.6 Hz, benzene proton), 7.49-7.54 (m, 1H, quinoline proton), 7.66-7.73 (m, 1H, quinoline proton), 7.86 (d, 1H, *J* = 7.6 Hz, quinoline proton), 8.23-8.31 (m, 2H, quinoline protons) ppm.

**N⁴-(4-Amino-3-((dimethylamino)methyl)phenyl)-6-methylpyrimidine-2,4-diamine (32).** Prepared starting from 31 (0.03 mmol, 0.01 g). Mp: 96-98 °C (toluene); yield: 70%. ¹H NMR (400 MHz, CDCl₃) δ: 2.07 (s, 3H, -CH₃), 2.30 (s, 2H, -CH₂N(CH₃)₂), 3.43 (s, 2H, -CH₂N(CH₃)₂), 5.05 (br s, 4H, 2x-NH₂), 5.70 (s, 1H, pyrimidine proton), 6.67 (br s, 1H, -NH-), 6.65 (d, 1H, *J* = 8.4 Hz, benzene proton), 6.88 (s, 1H, benzene proton), 6.98-7.00 (m, 1H, benzene proton) ppm.

**N¹,N¹-Dimethylbenzene-1,3,5-Triamine (42).** Prepared starting from N,N-dimethyl-3,5-dinitroaniline (0.03 mmol, 0.006 g). mp: >250 °C (methanol); yield: 76%. ¹H NMR (400 MHz, CDCl₃) δ: 2.89 (s, 6H, 2x-CH₃), 3.52 (br s, 4H, 2xNH₂), 5.54 (s, 1H, benzene proton), 5.59 (s, 2H, benzene protons) ppm.

**General Procedure for the Synthesis of the Acid Intermediate Compounds 25a,b.** A solution of the appropriate ester (1.14 mmol, 1 equiv) and 2N KOH (4.56 mmol, 0.26 g, 4 equiv) in ethanol (15 mL) was stirred overnight at rt. Then, the solvent was evaporated, and 2 N HCl was slowly added until the aqueous phase reached a pH value of 5.0. The solid was filtered, washed with water (2 × 10 mL), and recrystallized from methanol to obtain the pure carboxylic acid.

**2-((Dimethylamino)methyl)-4-(quinolin-4-ylamino)benzoic Acid (25a).** Prepared starting from 24a (1.14 mmol, 0.38 g). Mp: >250 °C (methanol); yield: 88%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.66 (s, 6H, N(CH₃)₂), 4.21 (s, 2H, -CH₂N(CH₃)₂), 7.16 (d, 1H, *J* = 5.6 Hz, benzene proton), 7.50-7.55 (m, 2H, benzene and quinoline protons), 7.63-69 (m, 1H, benzene proton), 7.83-7.89 (m, 1H, quinoline proton), 8.02 (d, 2H, *J* = 8.4 Hz, quinoline protons), 8.58 (d, 1H, *J* = 5.6 Hz, quinoline proton), 8.63 (d, 1H, *J* = 7.6 Hz, quinoline proton), 10.16 (br s, 1H, -NH-), 12.03 (br s, 1H, -COOH) ppm.

**3-((Dimethylamino)methyl)-5-(quinolin-4-ylamino)benzoic Acid (25b).** Prepared starting from 24b (1.14 mmol, 0.38 g). Mp: >250 °C (methanol); yield: 83%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.63 (s, 6H, -N(CH₃)₂), 4.25 (s, 2H, -CH₂N(CH₃)₂), 7.02 (br s, 1H, -NH-), 7.21-7.24 (m, 2H, benzene protons), 7.52-7.55 (m, 1H, quinoline protons), 7.68-7.72 (m, 1H, benzene proton), 7.93-7.95 (m, quinoline proton), 8.05 (d, 1H, *J* = 8.4 Hz, quinoline proton), 8.62 (d, 1H, *J* = 7.6 Hz, quinoline proton), 8.66 (d, 1H, *J* = 5.2 Hz, quinoline proton), 10.19 (br s, 1H, -NH-), 12.03 (br s, 1H, -COOH) ppm.

**Synthesis of 3-((dimethylamino)methyl)-4-nitroaniline (27).** 5-Amino-N,N-dimethyl-2-nitrobenzamide (0.45 mmol, 0.09 g, 1 equiv) was dissolved in dry THF (20 mL), and 1 M borane tetrahydrofuran complex solution (2.72 mmol, 2.72 mL, 6 equiv) was added slowly at 0 °C and subsequently the reaction was heated to 70 °C. After 6 h, the reaction was complete, and water (20 mL) was added at rt. The mixture was extracted with ethyl acetate (3 × 30 mL) and washed with brine (3 × 30 mL), and the combined organic extracts were dried over sodium sulfate. Upon evaporation of the solvent, the crude product was purified by column chromatography (SiO₂ eluting with ethyl acetate/methanol 5:1), giving the pure compound 27. Mp: 214-216 °C (acetonitrile/methanol); yield: 78%. ¹H NMR (400 MHz, CD₃OD) δ: 2.29 (s, 6H, -N(CH₃)₂), 3.78 (s, 2H, -CH₂N(CH₃)₂), 6.58 (d, 1H, *J* = 8.8 Hz,

benzene proton), 6.70 (s, 1H, benzene proton), 7.97 (d, 1H, *J* = 8.8 Hz, benzene proton) ppm.

**Synthesis of Ditert-butyl (5-(Bromomethyl)-1,3-phenylene-dicarbamate (34).** Di-tert-butyl (5-(hydroxymethyl)-1,3-phenylene-dicarbamate (2.96 mmol, 1.00 g, 1 equiv) was dissolved in dry THF (10 mL) under a nitrogen atmosphere, and tetrabromomethane (7.5 mmol, 2.49 g, 2.5 equiv) was added slowly. Next, a solution of triphenylphosphine (8.9 mmol, 2.33 g, 3 equiv) in anhydrous THF (5 mL) was added dropwise to the reaction mixture at 0 °C. The reaction was complete after leaving the mixture overnight at rt. The residue obtained upon solvent evaporation was purified by column chromatography (SiO₂ eluting with ethyl acetate/hexane:1/3), providing the pure 34. Mp: 180-182 °C (acetonitrile/methanol); yield: 69%; ¹H NMR (400 MHz, CDCl₃) δ: 1.53 (s, 18H, 2x C(CH₃)₃), 4.61 (s, 2H, -CH₂Br), 6.53 (br s, 2H, -OCONH-), 7.16 (s, 2H, benzene protons), 7.38 (s, 1H, benzene proton) ppm.

**General Procedure for the Synthesis of the Amine Intermediate Compounds 36a-c, 45, 47.** A solution of 4 N HCl in dioxane (0.2 mL, 30 equiv) was slowly added at 0 °C to a cooled solution of the appropriate di-tert-butyl (1,3-phenylene)dicarbamate 35a-c (0.03 mmol, 1 equiv) in dry THF (3 mL). The reaction was allowed to warm to room temperature and stirred for 10 h; then the mixture was quenched with diethyl ether, and the resulting solid was filtered off. The obtained crude product was purified by column chromatography (SiO₂ eluting with ethyl acetate/methanol:1/1) to afford the pure free amine product (36a, 36b, or 36c). For 35a only, the reaction was not stirred upon completion (10 h), but it was interrupted before to obtain the mono-tert-butyl carbamate derivative 45. When the TLC carried out on the mixture showed the formation of the monocarbamate, the reaction was quenched at room temperature by 2N sodium bicarbonate solution (20 mL), and the mixture was extracted with DCM (3 × 30 mL) and washed with brine (3 × 30 mL). The combined organic layers were then dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product, which was purified by column chromatography (SiO₂ eluting with ethyl acetate/methanol 1/1) to afford the pure 45.

**5-((Dimethylamino)methyl)benzene-1,3-diamine (36a).** Prepared starting from 35a (0.03 mmol, 0.01 g). Mp: 230-232 °C (methanol); yield: 82%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.67 (s, 6H, -N(CH₃)₂), 4.13 (s, 2H, -CH₂N(CH₃)₂), 3.57 (br s, 4H, 2x -NH₂), 6.66 (s, 1H, benzene proton), 6.70 (s, 2H, benzene protons), 10.54 (br s, 1H, -N(CH₃)₂ hydrochloride proton) ppm.

**5-((4-Methylpiperazin-1-yl)methyl)benzene-1,3-diamine (36b).** Prepared starting from 35b (0.03 mmol, 0.01 g). Mp: >250 °C (methanol); yield: 85%. ¹H NMR (400 MHz, CDCl₃) δ: 2.21 (s, 3H, -CH₃), 2.38 (s, 8H, piperazine protons), 3.42 (s, 2H, -CH₂-), 3.57 (br s, 4H, 2x -NH₂), 5.86 (s, 1H, benzene proton), 6.04 (s, 2H, benzene protons) ppm.

**5-((1H-Imidazole-1-yl)methyl)benzene-1,3-diamine (36c).** Prepared starting from 35c (0.03 mmol, 0.01 g). Mp: >250 °C (methanol); yield: 80%. ¹H NMR (400 MHz, DMSO-d₆) δ: 4.63 (br s, 4H, 2x -NH₂), 5.38 (s, 2H, -CH₂-), 6.55 (s, 2H, benzene protons), 6.66 (s, 1H, benzene proton), 7.74-7.77 (m, 2H, imidazole protons), 9.34 (s, 1H, imidazole proton) ppm.

**Tert-butyl (3-amino-5-((dimethylamino)methyl)phenyl-dicarbamate (45).** Prepared starting from 35a (0.03 mmol, 0.01 g) before completion of the reaction. Mp: 235-240 °C (methanol); yield: 50%; ¹H NMR (400 MHz, CDCl₃) δ: 1.51 (s, 9H, -C(CH₃)₃), 2.35 (s, 6H, -N(CH₃)₂), 3.44 (s, 2H, -CH₂N(CH₃)₂), 3.99 (br s, 2H, -NH₂), 6.40 (s, 1H, benzene proton), 6.60-6.62 (s and bs, 2H, -OCONH- and benzene proton), 6.88 (s, 1H, benzene proton) ppm.

**5-((Dimethylamino)methyl)-N¹-(3-(quinolin-4-ylamino)benzyl)-benzene-1,3-diamine (47).** Prepared starting from 46 (0.3 mmol, 0.16 g) Mp: >250 °C (methanol); yield: 95%. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.15 (s, 6H, N(CH₃)₂), 3.18 (s, 2H, -CH₂N(CH₃)₂), 4.18 (d, 2H, *J* = 5.2 Hz, CH₂), 5.04 (br s, 2H, -NH₂), 6.24 (s, 1H, benzene proton), 6.39 (s, 1H, benzene proton), 6.44 (s, 1H, benzene proton), 6.73 (s, 1H, benzene proton), 6.92-6.94 (d, 2H, benzene and quinoline proton), 7.14 (d, 1H, *J* = 6.8 Hz, benzene proton), 7.20 (d, 1H, *J* = 8.0 Hz, benzene proton), 7.35-7.38 (m, 2H, benzene proton and NH),
7.52 (t,1H, *J* = 7.6 Hz, quinoline proton), 7.68 (t, 1H, *J* = 8.0 Hz, quinoline proton), 7.85 (d, 1H, *J* = 8.4 Hz, quinoline proton), 8.34–8.38 (m, 2H, quinoline protons), 8.95 (br s, 1H, -NH-) ppm.

**Synthesis of tert-butyl 3-((dimethylamino)methyl)-5-((3-(quinolin-4-ylamino)benzyl)amino)phenyl)carbamate (46).** The 3-(quinolin-4-ylamino)benzaldehyde 44<sup>22</sup> (0.81 mmol, 0.20 g) and the mono-tert-butyl carbamate 45 (0.80 mmol, 0.21 g) were stirred in anhydrous DCE (5 mL) for 5 min. Afterward, sodium triacetoxyborohydride (1.05 mmol, 0.29 g) was added, and the resulting mixture was stirred overnight at 60 °C. The reaction was quenched with 10 mL of water and extracted with DCM (3 × 20 mL). The organic layer was washed with saturated sodium chloride (20 mL) and dried with sodium sulfate. Upon evaporation of the solvent, the crude product was purified by column chromatography (SiO₂ eluting with ethyl acetate/methanol 5:1), giving the pure compound 46. Mp: >250 °C (methanol); yield: 50%. ¹H NMR (400 MHz, CDCl₃) δ: 1.40 (s, 9H, C(CH₃)₃), 2.13 (s, 6H, -N(CH₃)₂), 3.21 (s, 2H, -CH₂N(CH₃)₂), 4.04 (br s, 1H, -CH₂NH-), 4.28 (d, 2H, -CH₂NH-), 6.24 (s, 1H, benzene proton), 6.39 (s, 1H, benzene proton), 6.44 (s, 1H, benzene proton), 6.73 (s, 2H, benzene protons), 7.06 (d, 1H, *J* = 7.6 Hz, quinoline proton), 7.13 (d, 1H, *J* = 8.0 Hz, benzene proton), 7.29 (t, 1H, *J* = 7.6 Hz, benzene proton), 7.41–7.45 (m, 1H, quinoline proton), 7.59–7.63 (m, 1H, quinoline proton), 7.89 (d, 1H, *J* = 8.4 Hz, quinoline proton), 7.97 (d, 1H, *J* = 8.4 Hz, quinoline proton), 8.47 (d, 1H, *J* = 7.2 Hz, quinoline proton) ppm.

**Synthesis of benzyl (3-amino-5-((dimethylamino)methyl)-phenyl)carbamate (48).** Benzyl chloroformate (1.09 mmol, 0.16 mL, 1 equiv) was added to a solution of 5- ((dimethylamino)methyl)-benzene-1,3-diamine (36a) (1.09 mmol, 0.3 g, 1 equiv) and triethylamine (8.19 mmol, 1.14 mL, 7.5 equiv) in dry THF (7 mL). The resulting mixture was stirred overnight at rt; then, the reaction was quenched with NaHCO₃ (20 mL) and extracted with DCM (3 × 30 mL). The combined organic extracts were dried, and the residue obtained upon solvent evaporation was purified by column chromatography (SiO₂ eluting with ethyl acetate/methanol:1.5/2) to provide pure 48. Mp: >250 °C (methanol); yield: 52%. ¹H NMR (400 MHz, CDCl₃) δ: 2.22 (s, 6H, N(CH₃)₂), 3.30 (s, 2H, -CH₂N(CH₃)₂), 3.61 (br s, 2H, -NH₂), 5.11 (s, 2H, -CH₂-), 6.34 (s, 1H, benzene proton), 6.50 (s, 1H, benzene proton), 6.60 (s, 1H, benzene proton), 6.82 (s, 1H, -OCONH-), 7.26–7.32 (m, 5H, benzene protons) ppm.

## BIOCHEMISTRY

**DNMT Inhibition Assay.** Full-length histidine-tagged human DNMT1 (182 kDa) was produced and purified according to<sup>37</sup> and catalytic human DNMT3A/DNMT3L as described in.<sup>38</sup> Compound activities were determined with a fluorescence-based assay.<sup>30</sup> In brief, a double-stranded DNA, comprising a unique CpG site overlaying an endonuclease restriction site for a methylation-sensitive enzyme, was used. This oligonucleotide comprises a 6-carboxyfluorescein (6-FAM) at one end and biotin on the other end, allowing immobilization into a 384-well plate (PerkinElmer) precoated with avidin. Compounds to be evaluated and SAM as methyl donor were added, followed by DNMT3A/3L to start the methylation reaction, which was prolonged 1 h at 37 °C. After several washes, with PBS tween (0.05%) containing NaCl (0.5 M) and PBS tween (0.05%). The restriction step was performed with HpyCH4IV (New England, BioLabs) to hand on only the specific fluorescence signal. 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. These percentages for a concentration range were then used to calculate the half maximal effective concentration (EC<sub>50</sub>) (average of at least 2 experiments) for the compound of interest by using the nonlinear regression with

sigmoidal dose-response (variable slope) tool of GraphPad Prism software.

For DNMT1, the same assay was optimized using a hemimethylated duplex at 1 CpG site (FAM -5' GCA TAT ATA TGA CGA TmCG TGT AGG TCA CTA CCA GAC ATG CAC TG 3'/5' Biot- CAG TGC ATG TCT GGT AGT GAC CTA CAC GAT CGT CAT ATA TAT GC 3'). The methylation reaction is performed by adding human DNMT1 at a final concentration of 200 nM in a total volume of 30 µL in the presence of the chemical compound to be tested at the desired concentration and the cofactor SAM at the final concentration of 20 µM. After incubation (1 h 37 °C) the plate is washed, and the methylation sensitive restriction enzyme Bsh1285I (Thermo Fisher Scientific) is added. After 1.30 h incubation at 60 °C the plate is washed and the fluorescence measured with a PerkinElmer Envision Multilabel Plate Reader. The data are expressed as percentage of inhibition vs log concentration (M). Data are normalized referring to the “restriction control” (wells coated with labeled duplex not treated nor exposed to DNMT1, but only cleaved by the restriction enzyme) as the maximum of inhibition (100%) and to the “DMSO control” (wells coated with labeled duplex but treated just with DMSO 0.1%, exposed to DNMT1 and then to Bsh1285I) as the minimum of inhibition (0%) (total methylation).

**G9a and PRMT1 Inhibition Assays.** The appropriate methyltransferase substrate (2.5 µM histone H3 (aa 1–21) peptide for G9a, and 5 µM histone H4 for PRMT1) was added in freshly prepared reaction buffer (50 mM Tris–HCl (pH 8.5), 5 mM MgCl₂, 50 mM NaCl, 0.01% Brij35, 1 mM DTT, 1% DMSO). Compound 14 was tested in single-dose mode, in duplicate, at 50 and 100 µM. Control compound, SAH (S-(5'-adenosyl)-L-homocysteine), was tested in 10-dose IC<sub>50</sub> mode with 3-fold serial dilution starting at 100 µM. The methyl-transferase enzyme was delivered into the substrate solution, and the mixture was mixed gently. Afterward, the tested compounds dissolved in DMSO were delivered into the enzyme/substrate reaction mixture by using Acoustic Technology (Echo 550, LabCyte Inc., Sunnyvale, CA, USA) in the nanolitre range, and 1 µM ³H-SAM was also added into the reaction mixture to initiate the reaction. The reaction mixture was incubated for 1 h at 30 °C and then it was delivered to filter-paper for detection. The data were analyzed using Excel and GraphPad Prism software for IC<sub>50</sub> curve fits.

**Thermal Denaturation Assay.** DNA thermal denaturation experiments were conducted as described in.<sup>28</sup> Hairpin DNA duplex (5'-GCCTTCGGTGGCTTTTGCCACCGAAGGC-3') was used at 2 µM in the absence or in the presence of the inhibitor at 10 µM in the Tm assay buffer (100 mM NaCl, 10 mM lithium cacodylate pH 7.2). The temperature at which 50% of the duplex is denatured Tm was calculated as previously described.<sup>28</sup>

## BIOLOGY

**Cell Lines and Culture Conditions.** U937 histiocytic lymphoma, H460 and A549 lung adenocarcinoma, MDA-MB-231 breast carcinoma, and HeLa epithelial cervix carcinoma cell lines were maintained in RPMI 1640 medium (Euroclone, Milan, Italy) contained 10% fetal bovine serum (FBS; Euroclone), 2 mM L-glutamine (Euroclone), and antibiotics (100 U/mL penicillin, 100 g/mL streptomycin, Euroclone) in a humidified atmosphere with 5% CO₂ at 37 °C. HCT-116 colon carcinoma cells were propagated in Dulbecco's modified Eagle medium (DMEM) (Euroclone) with 10% FBS, 2 mM L-
glutamine, and antibiotics. BJ and HCT-116 P53<sup>-/-</sup> cells were kindly provided by Dr. Erica Salvati, CNR IBPM. The human hTERT RPE-1 - epithelial cell line immortalized with hTERT-(kind gift of Dr Giulia Guarguaglini, CNR IBPM) was grown in complete DMEM/F12 (Dulbecco's Modified Eagle Medium F-12). All cell lines were routinely tested for mycoplasma contamination. The experiments were performed with early (3-20) cell passages. Cell lines were treated with compounds at the indicated concentrations in the exponential growth phase.

**Combined Bisulfite Restriction Analysis Methylation Assay.** This technique combines bisulfite conversion-based polymerase chain reaction with restriction digestion. DNA extraction and bisulfite treatment DNA was isolated from cultured cells treated or not with the compounds as indicated using the DNeasy Blood and Tissue Kit according to the manufacturer's specifications (Qiagen). DNA bisulfite conversion was performed on 200 ng of DNA using the EZ DNA Methylation-Gold Kit according to the manufacturer's specifications (Zymo Research) and bisulfited DNA was eluted with water.

**CDKN2A Promoter and LINE-1 PCR Amplification of Bisulfite-Treated DNA.** DNA amplification was set up with 100 ng eluted bisulfited DNA in 50 µL PCR reaction containing 1 × PCR buffer, 2.5 mM MgCl₂, 0.3 mM dNTPs, 400 nM each primer and 1.25 units EpiTaq HS (Takara) on C1000 Touch thermal cycler by 98 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s × 40 cycles. Twenty µL of PCR amplicons were digested in 30 µL by 2 units of BsiEI (for CDKN2A promoter) or Hinfl (for LINE-1) at 60 or 37 °C, respectively, for 90 min. DNA fragments were migrated by 2% agarose gel electrophoresis and each band was quantified by Image Lab Software v2.0 (BioRad Laboratories). The percentage of demethylation is calculated as demethylation (%) = 100[NT-T/NT] where NT = DMSO-treated cells and T = treated cells. Primers used were: for CDKN2A promoter: forward, 5'-GGTTTTTTTAGAGGATTTGAGGGATAGG-3' reverse, 5'-CTACCTAATTC-CAATTCCCCTACAAACTTC-3' for LINE-1: forward, 5'-TTGAGTTGTGGTGGGTTTATTAG-3' reverse, 5'-TCATCTCACTAAAAAATACCAACA-3'

**Analysis of Cell Proliferation.** Cell lines were treated with compounds at the indicated concentrations in the exponential growth phase. Briefly, increasing concentrations of different compounds (ranging from 0.05 to 100 µM) were added, and cell proliferation was evaluated by an MTT assay after 48 h. Control (0 µM) consists of 0.5% DMSO-treated cells. After treatments, MTT solution was added for 5 h at 0.5 mg/mL, the obtained crystals were dissolved in DMSO (Sigma-Aldrich, Milan, Italy), and the absorbance was read at a wavelength of 570 nm with a reader. All the experiments were performed in triplicate and repeated at least three times. IC₅₀ values of compounds against cellular viability were determined by using nonlinear regression fitting curves with GraphPad Prism 8.

**Analysis of Cell Cycle and Apoptosis.** For cell cycle analysis, after treatment, both floating and adherent cells were collected and fixed with cold 70% ethanol. Fixed cells were then resuspended in an RNase A (0.3 mg/mL) and propidium iodide (PI) solution (0.025 mg/mL). 10,000 events were then analyzed with a BD FACSCalibur Flow Cytometer: 4-Color (Becton Dickinson & Co., San Jose, CA, USA). The percentage of apoptosis was measured by analyzing sub-G1 peaks. Apoptotic cell death was also evaluated by using annexin V-fluorescein

isothiocyanate (FITC) apoptosis kit (BD Bioscience) and assayed according to the manufacturer's instructions.

**Live Cell Time-Lapse Microscopy.** A total of 3 × 10⁴ cells were seeded per well of a 4-well µ-slide. Following a 24 h incubation period, the cells were exposed to varying concentrations of compounds. Cells were recorded under an inverted microscope. During observations, cells were kept at 37 °C and 5% CO₂. Images were acquired using 40× objective over 5 days at 7 min intervals. The recorded cells were analyzed over time for cellular processes including entry into mitosis and cell death phenotypes. Processing was conducted using NIS-Elements AR 4.0.

**Analysis of DNMT1 and DNMT3A Protein Levels.** For experiments on DNMT1 and DNMT3A protein levels, HCT-116 5 × 10⁵ cells were plated in 60 mm plate and treated with 14 in comparison with MS9024.³⁶ Compounds were used at 0.1, 0.5, and 1 µM for 24 h. After treatment cells were collected and lysed for Western Blot Analysis.

**Western Blot Analysis.** After each treatment, floating and adherent cells were collected and lysed in RIPA lysis buffer (pH 8) containing a protease and phosphatase inhibitor cocktail. Between 30 and 50 µg of total protein were resolved under reducing conditions, extracted, fractionated by SDS-PAGE in a 10-13.5% gel, transferred, blocked, and subjected to an immunoblot assay. The following antibodies were used: P53 (Cell Signaling #48818, 1:1000); Phospho-γH2AX (Cell Signaling Ser139,#2577, 1:1000); Cleaved Caspase-3 Antibody (Cell Signaling #9661, 1:1000); PARP (Sigma-Aldrich AB3565, 1:200); Vinculin (Santa Cruz Biotechnologies, sc-73614, 1:1000)); α-DNMT1 (Novus Biologicals, Littleton, CO, USA 1:1000); α-DNMT3A (Santa Cruz Biotechnologies, CA, USA 1:200); α-Tubulin (Santa Cruz Biotechnologies, sc-5286, 1:1000); GAPDH (Santa Cruz Biotechnologies sc-32233, 1:1000). Blots are representative of at least two independent experiments. A densitometric evaluation was performed using ImageJ software and values normalized to relative controls, depending on the analysis.

## ASSOCIATED CONTENT

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

### Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c01029.

Elemental analysis of compounds **4–21** (Table S1); HPLC traces for compounds **7, 9, 10, 12–14** (Figure S1–6); Effect of **14** on lysine methyltransferase G9a and arginine methyltransferase PRMT1 (Table S2); Expression levels of DNMT1 and DNMT3A in cancer cell lines (Table S3); Cytotoxic effect of **17** in HCT-116 cells (Figure S7); Cell viability curves relative to noncancerous RPE and BJ cells treated with compound **14** (Figure S8); FACS analysis of the effect of **17** in HCT-116 cells (Figure S9); Effects of **14** on DNMT1 and DNMT3A protein levels (Figure S10) (PDF)

Molecular formula strings (CSV)

## AUTHOR INFORMATION

### Corresponding Authors

Antonello Mai – Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome 00185, Italy; ◦ orcid.org/0000-0001-9176-2382; Email: antonello.mai@uniroma1.it
Sergio Valente – Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome 00185, Italy; ● orcid.org/0000-0002-2241-607X; Email: sergio.valente@uniroma1.it

## Authors

Clemens Zwergel – Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome 00185, Italy; ● orcid.org/0000-0002-3097-0003

Chiara Lambona – Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome 00185, Italy; ● orcid.org/0000-0002-3415-4126

Rossella Fioravanti – Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome 00185, Italy

Alessia Raucci – Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome 00185, Italy

Francesco Fiorentino – Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome 00185, Italy; ● orcid.org/0000-0003-3550-1860

Guilaine Nchugoua Tchieh – Université Paris Cité, CNRS UMR no 3523 Chem4Life, Epigenetic Chemical Biology, Department of Structural Biology and Chemistry, Institut Pasteur, F-75015 Paris, France

Corinne Jallet – Université Paris Cité, CNRS UMR no 3523 Chem4Life, Epigenetic Chemical Biology, Department of Structural Biology and Chemistry, Institut Pasteur, F-75015 Paris, France

Laurent Lacroix – IBENS, Département de biologie, École Normale Supérieure, Université PSL, CNRS, INSERM, Paris 75005, France

Michela Pierrettori – IBPM Institute of Molecular Biology and Pathology, CNR, Rome 00185, Italy

Francesca Romana Pellegrini – IBPM Institute of Molecular Biology and Pathology, CNR, Rome 00185, Italy

Yan Xiong – Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Science, Oncological Science and Neuroscience, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States

Jian Jin – Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Science, Oncological Science and Neuroscience, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States; ● orcid.org/0000-0002-2387-3862

Paola Arimondo – Université Paris Cité, CNRS UMR no 3523 Chem4Life, Epigenetic Chemical Biology, Department of Structural Biology and Chemistry, Institut Pasteur, F-75015 Paris, France

Daniela Trisciuoglio – IBPM Institute of Molecular Biology and Pathology, CNR, Rome 00185, Italy

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.5c01029

## Author Contributions

All authors contributed to the present paper and have given approval to the final version of the manuscript.

## Notes

The authors declare no competing financial interest.

## ACKNOWLEDGMENTS

This work was supported by PRIN2020 (2020CW39SJ to S.V.), PRIN2022 (P2022FESRR to A.M.), Associazione Italiana per la

Ricerca sul Cancro (IG26172 to S.V.; IG24942 to D.T., IG31139 to A.M.), Ateneo Sapienza Project 2020 (RG120172B8E53D03 to S.V.), Sapienza Grandi Attrezzature Scientifiche 2021 (GA12117A8711CC9F - INCENTIVE MS) to A.M.. C.Z. is thankful for the generous funding from FSE REACT-EU within the program PON "Research and Innovation" 2014–2020, Action IV.6 "Contratti di ricerca su tematiche Green", as well as the funding from the Italian Ministry of Health Ricerca Finalizzata GR-2021-12374415. We acknowledge MUR (Ministry of University and Research) PON project "IMPARA, Imaging from molecules to the preclinics" for supporting the development of the IBPM imaging platform. The authors thank Chloé Leblond who during her technological internship contributed to the optimization of the hDNMT1 assay. The authors acknowledge Xiaodong Cheng for the fruitful discussion.

## ABBREVIATIONS

5-azadC, decitabine; CBr4, tetrabromomethane; COBRA, Combined Bisulfite Restriction Analysis; DNMT, DNA methyltransferase; DNMT3Acat, human catalytic DNMT3A/3L; DNMTi, DNMT inhibitors; Et<sub>3</sub>N, triethylamine; EWS, Ewing sarcoma; FACS, fluorescence-activated cell sorting; 6-FAM, 6-carboxyfluorescein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; γH2AX, phosphorylated form of the histone H2AX; LINE-1, Long Interspersed Nucleotide Element 1; MTs, methyltranferases; Me<sub>4</sub>Si, tetramethylsilane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OS, osteosarcoma; PARP, poly-ADP ribose polymerase; Ph<sub>3</sub>P, triphenylphosphine; PyBOP, benzotriazol-1-yloxytrypyrrolidinophosphonium hexafluorophosphate; RPE, retinal pigment epithelium; SAH, S-adenosyl-L-homocysteine; SAM, S-adenosyl-L-methionine.

## REFERENCES

(1) You, J. S.; Jones, P. A. Cancer Genetics and Epigenetics: Two Sides of the Same Coin? *Cancer Cell* **2012**, *22*, 9–20.

(2) Takeshima, H.; Ushijima, T. Accumulation of Genetic and Epigenetic Alterations in Normal Cells and Cancer Risk. *npj Precis Oncol* **2019**, *3*, 7.

(3) Baylin, S. B.; Jones, P. A. A Decade of Exploring the Cancer Epigenome - Biological and Translational Implications. *Nat. Rev. Cancer* **2011**, *11*, 726–734.

(4) Esteller, M. Epigenetics in Cancer. *N. Engl. J. Med.* **2008**, *358*, 1148–1159.

(5) Wang, Y.; Xie, Q.; Tan, H.; Liao, M.; Zhu, S.; Zheng, L. L.; Huang, H.; Liu, B. Targeting Cancer Epigenetic Pathways with Small-Molecule Compounds: Therapeutic Efficacy and Combination Therapies. *Pharmacol. Res.* **2021**, *173*, 105702.

(6) Chen, Z.; Zhang, Y. Role of Mammalian DNA Methyltransferases in Development. *Annu. Rev. Biochem.* **2020**, *89*, 135–158.

(7) Hoang, N. M.; Rui, L. DNA Methyltransferases in Hematological Malignancies. *J. Genet Genomics* **2020**, *47*, 361–372.

(8) Breiling, A.; Lyko, F. Epigenetic Regulatory Functions of DNA Modifications: 5-Methylcytosine and Beyond. *Epigenetics Chromatin* **2015**, *8*, 24.

(9) Subramaniam, D.; Thombre, R.; Dhar, A.; Anant, S. DNA Methyltransferases: A Novel Target for Prevention and Therapy. *Front Oncol* **2014**, *4*, 80.

(10) Okano, M.; Bell, D. W.; Haber, D. A.; Li, E. DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development. *Cell* **1999**, *99*, 247–257.

(11) Robert, M. F.; Morin, S.; Beaulieu, N.; Gauthier, F.; Chute, I. C.; Barsalou, A.; MacLeod, A. R. Dnmt1 Is Required to Maintain Cpg
Methylation and Aberrant Gene Silencing in Human Cancer Cells. *Nat. Genet.* **2003**, *33*, 61–65.

(12) Weng, Y. L.; An, R.; Shin, J.; Song, H.; Ming, G. L. DNA Modifications and Neurological Disorders. *Neurotherapeutics* **2013**, *10*, 556–567.

(13) Zhang, Q.; Li, D.; Zhao, H.; Zhang, X. Decitabine Attenuates Ischemic Stroke by Reducing Astrocytes Proliferation in Rats. *PLoS One* **2022**, *17*, No. e0272482.

(14) Teitell, M.; Richardson, B. DNA Methylation in the Immune System. *Clin Immunol* **2003**, *109*, 2–5.

(15) Arguelles, A. O.; Meruvu, S.; Bowman, J. D.; Choudhury, M. Are Epigenetic Drugs for Diabetes and Obesity at Our Door Step? *Drug Discovery Today* **2016**, *21*, 499–509.

(16) Lee, A. V.; Nestler, K. A.; Chiappinelli, K. B. Therapeutic Targeting of DNA Methylation Alterations in Cancer. *Pharmacol. Ther.* **2024**, *258*, 108640.

(17) Van Tongelen, A.; Loriot, A.; De Smet, C. Oncogenic Roles of DNA Hypomethylation through the Activation of Cancer-Germline Genes. *Cancer Lett.* **2017**, *396*, 130–137.

(18) Yu, J.; Xie, T.; Wang, Z.; Wang, X.; Zeng, S.; Kang, Y.; Hou, T. DNA Methyltransferases: Emerging Targets for the Discovery of Inhibitors as Potent Anticancer Drugs. *Drug Discovery Today* **2019**, *24*, 2323–2331.

(19) Valente, S.; Liu, Y.; Schnekenburger, M.; Zwergel, C.; Cosconati, S.; Gros, C.; Tardugno, M.; Labella, D.; Florean, C.; Minden, S.; Hashimoto, H.; Chang, Y.; Zhang, X.; Kirsch, G.; Novellino, E.; Arimondo, P. B.; Miele, E.; Ferretti, E.; Gulino, A.; Diederich, M.; Cheng, X.; Mai, A. Selective Non-Nucleoside Inhibitors of Human DNA Methyltransferases Active in Cancer Including in Cancer Stem Cells. *J. Med. Chem.* **2014**, *57*, 701–713.

(20) Manara, M. C.; Valente, S.; Cristalli, C.; Nicoletti, G.; Landuzzi, L.; Zwergel, C.; Mazzone, R.; Stazi, G.; Arimondo, P. B.; Pasello, M.; Guerzoni, C.; Picci, P.; Nanni, P.; Lollini, P. L.; Mai, A.; Scotlandi, K. A Quinoline-Based DNA Methyltransferase Inhibitor as a Possible Adjuvant in Osteosarcoma Therapy. *Mol. Cancer Ther.* **2018**, *17*, 1881–1892.

(21) Cristalli, C.; Manara, M. C.; Valente, S.; Pellegrini, E.; Bavelloni, A.; De Feo, A.; Blalock, W.; Di Bello, E.; Pineyro, D.; Merkel, A.; Esteller, M.; Tirado, O. M.; Mai, A.; Scotlandi, K. Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response. *Front Endocrinol (Lausanne)* **2022**, *13*, 876602.

(22) Zwergel, C.; Fioravanti, R.; Stazi, G.; Sarno, F.; Battistelli, C.; Romanelli, A.; Nebbioso, A.; Mendes, E.; Paulo, A.; Strippoli, R.; Tripodi, M.; Pechalrieu, D.; Arimondo, P. B.; De Luca, T.; Del Bufalo, D.; Trisciuoglio, D.; Altucci, L.; Valente, S.; Mai, A. Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation. *Cancers (Basel)* **2020**, *12*, 447.

(23) Zwergel, C.; Schnekenburger, M.; Sarno, F.; Battistelli, C.; Manara, M. C.; Stazi, G.; Mazzone, R.; Fioravanti, R.; Gros, C.; Ausseil, F.; Florean, C.; Nebbioso, A.; Strippoli, R.; Ushijima, T.; Scotlandi, K.; Tripodi, M.; Arimondo, P. B.; Altucci, L.; Diederich, M.; Mai, A.; Valente, S. Identification of a Novel Quinoline-Based DNA Demethylating Compound Highly Potent in Cancer Cells. *Clin Epigenetics* **2019**, *11*, 68.

(24) Zhou, J.; Chen, Q.; Ren, R.; Yang, J.; Liu, B.; Horton, J. R.; Chang, C.; Li, C.; Maksoud, L.; Yang, Y.; Rotili, D.; Jain, A. K.; Zhang, X.; Blumenthal, R. M.; Chen, T.; Gao, Y.; Valente, S.; Mai, A.; Cheng, X. Quinoline-Based Compounds Can Inhibit Diverse Enzymes That Act on DNA. *Cell Chem. Biol.* **2024**, *31* (12), 2112–2127.e6.

(25) Rilova, E.; Erdmann, A.; Gros, C.; Masson, V.; Aussagues, Y.; Poughon-Cassabois, V.; Rajavelu, A.; Jeltsch, A.; Menon, Y.; Novosad, N.; Gregoire, J. M.; Vispe, S.; Schambel, P.; Ausseil, F.; Sautel, F.; Arimondo, P. B.; Cantagrel, F. Design, Synthesis and Biological Evaluation of 4-Amino-N-(4-Aminophenyl)Benzamide Analogues of Quinoline-Based Sgi-1027 as Inhibitors of DNA Methylation. *ChemMedChem* **2014**, *9*, 590–601.

(26) Laranjeira, A. B. A.; Hollingshead, M. G.; Nguyen, D.; Kinders, R. J.; Doroshow, J. H.; Yang, S. X. DNA Damage, Demethylation and Anticancer Activity of DNA Methyltransferase (Dnmt) Inhibitors. *Sci. Rep.* **2023**, *13*, 5964.

(27) 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*, 4665–4679.

(28) Mergny, J. L.; Lacroix, L. Analysis of Thermal Melting Curves. *Oligonucleotides* **2003**, *13*, 515–537.

(29) Gamage, S. A.; Brooke, D. G.; Redkar, S.; Datta, J.; Jacob, S. T.; Denny, W. A. Structure-Activity Relationships for 4-Anilinoquinoline Derivatives as Inhibitors of the DNA Methyltransferase Enzyme Dnmt1. *Bioorg. Med. Chem.* **2013**, *21*, 3147–3153.

(30) Ceccaldi, A.; Rajavelu, A.; Champion, C.; Rampon, C.; Jurkowska, R.; Jankevicius, G.; Sénamaud-Beaufort, C.; Ponger, L.; Gagey, N.; Dali Ali, H.; 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*, 1337–1345.

(31) Gros, C.; Fleury, L.; Nahoum, V.; Faux, C.; Valente, S.; Labella, D.; Cantagrel, F.; Rilova, E.; Bouhlel, M. A.; David-Cordonnier, M. H.; Dufau, I.; Ausseil, F.; Mai, A.; Mourey, L.; Lacroix, L.; Arimondo, P. B. New Insights on the Mechanism of Quinoline-Based DNA Methyltransferase Inhibitors. *J. Biol. Chem.* **2015**, *290*, 6293–6302.

(32) Pappalardi, M. B.; Keenan, K.; Cockerill, M.; Kellner, W. A.; Stowell, A.; Sherk, C.; Wong, K.; Pathuri, S.; Briard, 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*, 1002–1017.

(33) Chen, Q.; Liu, B.; Zeng, Y.; Hwang, J. W.; Dai, N.; Correa, I. R., Jr.; Estecio, M. R.; Zhang, X.; Santos, M. A.; Chen, T.; Cheng, X. Gsk-3484862 Targets Dnmt1 for Degradation in Cells. *NAR Cancer* **2023**, *5*, zcad022.

(34) Erdmann, A.; Halby, L.; Fahy, J.; Arimondo, P. B. Targeting DNA Methylation with Small Molecules: What's Next? *J. Med. Chem.* **2015**, *58*, 2569–2583.

(35) Ardeljan, D.; Taylor, M. S.; Ting, D. T.; Burns, K. H. The Human Long Interspersed Element-1 Retrotransposon: An Emerging Biomarker of Neoplasia. *Clin Chem.* **2017**, *63*, 816–822.

(36) Qian, C.; Lee, Y.; Han, Y.; Zhong, Y.; Zhou, J.; Hrit, J.; Xie, L.; Chen, Q.; Kaniskan, H. U.; Chen, X.; Rothbart, S.; Cheng, X.; Xiong, Y.; Jin, J. Structure-Activity Relationship Studies of DNA Methyltransferase 1 Monovalent Degraders. *J. Med. Chem.* **2025**, *68*, 2903–2919.

(37) Lee, B. H.; Yegnasubramanian, S.; Lin, X.; Nelson, W. G. Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1. *J. Biol. Chem.* **2005**, *280*, 40749–40756.

(38) Gros, C.; Chauvigne, L.; Poulet, A.; Menon, Y.; Ausseil, F.; Dufau, I.; Arimondo, P. B. Development of a Universal Radioactive DNA Methyltransferase Inhibition Test for High-Throughput Screening and Mechanistic Studies. *Nucleic Acids Res.* **2013**, *41*, No. e185.