# Design, synthesis, biophysical and biological evaluation of original condensed pyrrolopyrimidine and pyrrolopyridine ligands as anti-SARS-CoV-2 agents targeting G4

Jean Guillon, Solène Savrimoutou, Nicolas da Rocha, Sandra Albenque-Rubio, Olivier Helynck, Cyrielle Durand, Jeanne Chiaravalli, Noël Pinaud, Luisa Ronga, Stéphane Moreau, et al.

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Jean Guillon, Solène Savrimoutou, Nicolas da Rocha, Sandra Albenque-Rubio, Olivier Helynck, et al.. Design, synthesis, biophysical and biological evaluation of original condensed pyrrolopyrimidine and pyrrolopyridine ligands as anti-SARS-CoV-2 agents targeting G4. European Journal of Medicinal Chemistry, 2025, 292, pp.117655. 10.1016/j.ejmech.2025.117655. hal-05064440

HAL Id: hal-05064440

https://univ-pau.hal.science/hal-05064440v1

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# Design, synthesis, biophysical and biological evaluation of original condensed pyrrolopyrimidine and pyrrolopyridine ligands as anti-SARS-CoV-2 agents targeting G4

Jean Guillon<sup>a,*</sup>, Solène Savrimoutou<sup>a</sup>, Nicolas Da Rocha<sup>b</sup>, Sandra Albenque-Rubio<sup>a</sup>, Olivier Helynck<sup>c</sup>, Cyrielle Durand<sup>c</sup>, Jeanne Chiaravalli<sup>c</sup>, Noël Pinaud<sup>d</sup>, Luisa Ronga<sup>e</sup>, Stéphane Moreau<sup>a</sup>, Simon Chirol<sup>a</sup>, Tshering Zangmo<sup>e</sup>, Melika Arab<sup>a</sup>, Lindita Lari<sup>a</sup>, Jean-Louis Mergny<sup>f</sup>, Hélène Munier-Lehmann<sup>g,**</sup>, Marc Lavigne<sup>b,***</sup>

## ARTICLE INFO

### Keywords:

- Guanine quadruplex

- G4 ligands

- SARS-CoV-2

- Nsp3

- COVID-19

- Antiviral molecule

- Pyrrolopyrimidine

- Pyrrolopyridine

- FRET-melting

- HTRF

## ABSTRACT

The design and synthesis of novel bis[(substituted-aminomethyl)phenyl]phenyl pyrrolopyrimidines, pyrrolopyridines, pyrazolopyrimidines, imidazopyrimidines, and tris[(substituted-aminomethyl)phenyl]phenyl pyrrolopyrimidines are reported here. These original G-quadruplex (G4) ligands have been then subjected to a screening on SARS-CoV-2 using a competition HTRF assay by targeting the SUD-NM/TRF2 RNA G4 interaction. The more promising derivatives have been evaluated *in vitro* to determine their potential antiviral effect on two different cell lines infected by two SARS-CoV-2 strains. This study revealed a clear correlation between their antiviral property and their efficacy to prevent the SUD/G4 interaction. This correlation supports the choice of SUD/RNA G4 complexes formed during SARS-CoV-2 infection as new antiviral targets.

## 1. Introduction

Host-virus interactions represent promising targets of antiviral strategies. In the context of the Covid19 pandemic, we have studied the interaction between the SARS-CoV-2 Nps3 protein and cellular non-canonical nucleic acid structures called Guanine quadruplexes (G4). Nsp3 is the largest protein encoded by coronavirus genomes and several domains of this polyprotein are essential for viral replication and transcription [1], especially for the formation of the double membrane

vesicule pore complex [2]. Interestingly, SARS-CoV and SARS-CoV-2 Nsp3 proteins share the presence of a unique domain called SUD (for SARS Unique Domain), interacting with G4 [3,4]. This domain and its interaction with G4 substrates have been shown to be essential for viral replication and constitute potential targets of viral strategies directed against both viruses [5,6]. Although this SUD/G4 interaction has been demonstrated and quantified by several *in vitro* studies [3,4,6], the precise nature and origin of G4s interacting with SUD in the context of viral infection remain to be determined.

<footnote><sup>a</sup> Univ. Bordeaux, CNRS, INSERM, ARNA, UMR 5320, U1212, F-33076, Bordeaux, France</footnote>

<footnote><sup>b</sup> Institut Pasteur, Université Paris Cité, Département de Virologie, F-75015, Paris, France</footnote>

<footnote><sup>c</sup> Institut Pasteur, Université Paris Cité, CNRS UMR3523, Chemogenomic and Biological Screening Core Facility, Center for Technological Resources and Research (C2RT), Department of Structural Biology and Chemistry, F-75015 Paris, France</footnote>

<footnote><sup>d</sup> Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400, Talence, France</footnote>

<footnote><sup>e</sup> Université de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM, F-64012, Pau, France</footnote>

<footnote><sup>f</sup> Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Inserm U1182, CNRS UMR7645, Institut Polytechnique de Paris, F-91120, Palaiseau, France</footnote>

<footnote><sup>g</sup> Université Paris Cité, INSERM, Health & Functional Exposomics - HealthFex, U1124, Institut Pasteur, Structural Biology and Chemistry Department, F-75006, Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>*** Corresponding author.</footnote>

<footnote>E-mail addresses: jean.guillon@u-bordeaux.fr (J. Guillon), helene.munier-lehmann@u-paris.fr (H. Munier-Lehmann), marc.lavigne@pasteur.fr (M. Lavigne).</footnote>
G4s are non-canonical nucleic acid structures formed by DNA or RNA G-rich sequences and triggered by the formation of Guanine-tetrads (G-quartets) [7,8]. G4s regulate major eukaryotic cell processes and are enriched in key regulatory regions [9]. While DNA G4s regulate the transcription of numerous human genes, RNA G4s participate in several mechanisms linked to RNA metabolism [10–12]. G4s are also present in

the genomes of DNA and RNA viruses and regulate their replication [13–17]. As an example, G4s present in the HIV-1 genome regulate its replication and can be targeted by antiviral molecules [18–20]. Recently, we have shown that the human DNA Topoisomerase 1 (TOP1) interacts with G4s present in the HIV-1 viral promoter and represses its transcriptional activity [21]. In the case of SARS-CoV-2, several G4s

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Overview of previous phenanthrolines JG986 and JG1045 and synthesized compounds in this study, including new substituted pyrrolo[2,3-d]pyrimidines **1** (series A), pyrrolo[2,3-b]pyridine **2** (series B), pyrrolo[3,2-c]pyridines **3** (series C), pyrrolo[2,3-d]pyrimidines **4–5** (series D-E), pyrrolo[3,2-d]pyrimidines **6** (series F), pyrazolo[3,4-d]pyrimidines **7** (series G), imidazo[4,5-d]pyrimidines **8** (series H), and pyrrolo[2,3-d]pyrimidines **9** (series I-J).</figcaption></figure>
have been previously identified in the viral genome but they are char-
acterized by low thermal stabilities and the presence of only two tetrads
in the folded structure (reviewed in Refs. [6,15]) which may favor
multimerization [22]. G4s have also been found in RNA sequences
regulating the expression of cellular proteins essential for viral replica-
tion such as TMPRSS2, ACE2, AXL or FURIN enzymes [23,24].

Because of the major role of G4s in human diseases, including can-
cers and viral infections, intensive research has been carried out to
identify G4-ligands (G4L) or G4-mimics interfering with G4 activity and
cellular partner interactions [25–27]. Thousands of different G4Ls have
been described so far and listed in the G4L database [28]. Three of them
have been tested in the clinic against cancer and several others exhibit
promising antiviral activities in cells [16]. Since the Covid-19 pandemic,
several G4 ligands have been identified to inhibit viral replication, such
as Braco19 [29], TMPyP4 [23,30], Pyridostatin and its derivatives [23,
31], Topotecan and Berbamine [24] as well as Chlorpromazin and
Prochlorperazin [32]. However, the mechanisms responsible for the
observed inhibitions and the origin of targeted G4s (cellular or viral) are
very diverse. Within our project, we have chosen to focus on the iden-
tification of G4Ls which prevent the SUD/G4 interaction and inhibit
SARS-CoV-2 through this protein/nucleic acid complex.

In the course of our work devoted to the design and discovery of new aza heterocyclic derivatives for use in medicinal chemotherapy [33–38], we previously prepared a series of original substituted 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives (Fig. 1). These molecules were initially designed as antimalarial candidates that could bind to *Plasmodium falciparum* DNA G-quadruplexes [36]. Due to the potential role of SUD/G4 complex in SARS-CoV-2 replication and with the goal to find new inhibitors against SARS-CoV-2 replication, a certain number of our antiparasitic G4 ligands were selected and subjected to a screen based on a competition HTRF assay targeting the SUD-NM/TRF2 G4 interaction. The initial screen revealed two novel chemical compounds, JG986 and JG1045 (Fig. 1), which inhibit this interaction [4]. This promising result encouraged us to develop related derivatives that could have the same properties.

By taking into account our experience in the field of the synthesis of new nitrogen heterocyclic compounds, we describe herein the design and synthesis of new substituted pyrrolo[2,3-d]pyrimidines **1** (series A), **1H-pyrrolo[2,3-b]pyridine **2** (series B), and pyrrolo[3,2-c]pyridines **3** (series C) that could be considered as new structural bioisoster analogues of our previously described bioactive phenanthroline compounds JG986 and JG1045 (Fig. 1). Then, we considered new pharmacokinetics around these nitrogen heterocyclic systems by the design and synthesis of other homologues pyrrolo[2,3-d]pyrimidines **4–5** (series D-E); we also performed the ring flip of the pyrrole ring to prepare the homologous pyrrolo[3,2-d]pyrimidine **6** (series F), then the biososters pyrazolo[3,4-d]pyrimidines **7** (series G) and imidazo[4,5-d]pyrimidines **8** (series H) were also studied. In addition, we also investigated the design and synthesis of the trisubstituted pyrrolo[2,3-d]pyrimidines **9** (series I-J) (Fig. 1).

Here, we report on the synthesis of these new nitrogen heterocyclic
compounds, their *in vitro* interaction to G4 RNA, their *in vitro* effect on
the formation of a SUD/RNA G4 complex, and their antiviral activity
against SARS-CoV-2 replication evaluated in different cell lines. Our
study reveals new G4-ligands with a micromolar range antiviral activity
targeting at least two SARS-CoV-2 variants.

**2. Results and discussion**

**2.1. Chemistry**

The reported new *bis*[(substituted-aminomethyl)-pyrrolo[2,3-d]py-
rimidines **1** (series A), pyrrolo[2,3-b]pyridine **2** (series B), pyrrolo[3,2-
c]pyridines **3** (series C), were synthesized respectively starting from the
commercially available 2,4-dichloro-7H-pyrrolo[2,3-b]pyridine, 4,6-
dichloro-1H-pyrrolo[2,3-b]pyridine, or 1H-pyrrolo[3,2-c]pyridine

(Scheme 1). The *N*-arylation of these latters under the Chan-Lam conditions using phenylboronic acid, copper (II) acetate and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine led to the preparation of compounds **10–12** [39,40]. The structures of intermediate compounds **11** and **15** were established by X-ray crystallography (Figs. 2 and 3). The *bis*(4-formylphenyl)-phenyl-7H-pyrrolo[2,3-d]pyrimidines, pyrrolo[2,3-b]pyridines or pyrrolo[3,2-c]pyridines **13–15** were prepared by a direct double-Suzuki-Miyaura cross-coupling reaction of derivatives **10–12** with the 4-formylphenylboronic acid performed in the presence of Pd(PPh₃)₄ as a catalyst, and in the presence of sodium carbonate as the base [34–36]. Reaction of primary substituted aminoalkylamines with dialhedydes **13–15** gave the di-imines **16–18**, which were reduced into the 2,4-*bis*[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines **1–3** using sodium borohydride in methanol as previously described [34–36]. These novel heterocyclic derivatives **1–3** were then converted into their ammonium oxalate salts by treatment with oxalic acid in refluxing isopropanol. These oxalate salts were found less grossopic than the hydrochloride ones and also soluble in water. Table 1 summarizes the physical properties of the **1–9** oxalates.

The synthesis of 2,7-bis[(substituted-aminomethyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidines **4a-b** (series D) was accomplished in five steps according to the following pathway (Scheme 2): this synthetic strategy was based on three orthogonal and chemoselective reactions. The first reaction involved the Chan-Lam copper-catalyzed *N*-arylation at position 7 of the 7H-pyrrolo[2,3-d]pyrimidine heterocycle leading to compound **19**. The second reaction consisted in the preparation of compound **20** through the *C*-arylation at position 4 of the pyrrolo[2,3-d] pyrimidine skeleton by a Suzuki coupling. The third one was a second palladium-catalyzed cross-coupling Suzuki reaction on compound **20** leading to the introduction of a benzaldehyde group at position 2 of the heterocyclic system (derivative **21**) [41]. The 3D structural determinations of the substituted 7H-pyrrolo[2,3-d]pyrimidines **19–21** were established by X-ray crystallography (Fig. 4) [7] and confirmed the structure in the solid state as anticipated on the basis of NMR data. Condensation of primary amines with this latter dialdehyde **21** afforded the di-imines **22a-b**, which were immediately reduced into the 2,7-bis [(substituted-aminomethyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidines **4a-b** using sodium borohydride as reductive agent in refluxing methanol as previously described by our team [34–36]. Both compounds **4a** and **4b** were then converted into ammonium oxalate salts by treatment with oxalic acid in refluxing isopropanol.

The same strategy was used for the synthesis of the 4,5-bis [(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine derivatives **5a-b** (series E) via the key intermediate di-imine **25** and starting from the dialdehyde **24**, previously prepared through a double cross-coupling Suzuki reaction from 4-chloro-5-iodo-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine **23** (Scheme 3). Compound **23** was firstly synthesized starting from commercially 4-chloro-5-iodo-7H-pyrrolo[2,3-d] pyrimidine using a Chan-Lam cross-coupling reaction [42]. The 3D structure of **24** was established by a X-ray single crystal analysis and confirm the structure (Fig. 5).

The *N*-arylation of commercially available 5H-pyrrolo[3,2-d]pyrimidine with phenylboronic acid using copper acetate as catalyst led to the preparation of the 2,4-dichloro-5-phenyl-5H-pyrrolo[3,2-d]pyrimidine **26** (Scheme 4) [40], that was then engaged in a double-Suzuki-Miyaura palladium cross-coupling reaction with an excess of 4-formylphenylboronic acid to give the dialdehyde **27**. A X-ray single crystal analysis was also performed on 2,4-*bis*(4-formylphenyl)-5-phenyl-5H-pyrrolo[3,2-d] pyrimidine **27** in order to confirm the structure (Fig. 6). Reaction of 4-dimethylaminobutylamine with the latter **27** gave **28**, reduced into the pyrrolo[3,2-d]pyrimidine **6** (series F) using sodium borohydride in methanol.

The reported 4,6-bis[(substituted-aminomethyl)-1-phenyl-1H-pyr-
azolo[3,4-d]pyrimidines 7a-d (series G) and 2,4-bis[(substituted-ami-
nomethyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidines 8a-c (series H) were
obtained as depicted in Scheme 5 from 5-amino-1-phenylpyrazole-4-
<figure><img src="image_5.png" /></figure>

<table><thead><tr><th colspan="4">Series A X, Y = N</th><th colspan="4">Series B X = N, Y = CH</th><th colspan="4">Series C X = CH, Y = N</th></tr><tr><th>Compounds</th><th>-N<sub>n</sub>R</th><th>n</th><th>m</th><th>Compounds</th><th>-N<sub>n</sub>R</th><th>n</th><th>m</th><th>Compounds</th><th>-N<sub>n</sub>R</th><th>n</th><th>m</th></tr></thead><tbody><tr><td>16a, 1a</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub><br>CH<sub>3</sub></td><td>1</td><td>4</td><td>17, 2</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>2</td><td>4</td><td>18a, 3a</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>2</td><td>4</td></tr><tr><td>16b, 1b</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>2</td><td>4</td><td></td><td></td><td></td><td></td><td>18b, 3b</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>3</td><td>4</td></tr><tr><td>16c, 1c</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>3</td><td>4</td><td></td><td></td><td></td><td></td><td>18c, 3c</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>2</td><td>4</td></tr><tr><td>16d, 1d</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>2</td><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16e, 1e</td><td>H<sub>3</sub>C<br>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub><br>H<sub>3</sub>C</td><td>3</td><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16f, 1f</td><td>-N<sub>n</sub>N-CH<sub>3</sub></td><td>1</td><td>6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16g, 1g</td><td>-N<sub>n</sub>N-CH<sub>3</sub></td><td>2</td><td>6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16h, 1h</td><td>-N<sub>n</sub>CH<sub>3</sub><br>CH<sub>3</sub></td><td>2</td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Scheme 1.** Synthesis of 2,4-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine derivatives 1a-h (series A), 4,6-bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[2,3-b]pyridine 2 (series B) and 4,6-bis{4-[(4-dialkylaminoalkyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine derivatives 3a-c (series C).

carboxamide and 2,6-dichloro-9H-purine, respectively. Derivative 29 was prepared by the urea fusion of 5-amino-1-phenylpyrazole-4-carboxamide, then chlorination of 29 with POCl₃ in the presence of an excess of PCl₅ gave the corresponding 1-phenyl-4,6-dichloropyrazolo[3,4-d]pyrimidine 30 [43,44] The commercially available 2,6-dichloro-9H-purine was reacted with an excess of phenyl boronic acid in the presence of copper(II) acetate, molecular sieves and phenanthroline to give the 2, 6-dichloro-9-phenyl-9H-purine 31 with 51 % yield (method A) [45]. Compound 31 was also prepared by direct arylation between 2, 6-dichloro-9H-purine and diphenyliodonium chloride through CuBr catalyzed C-N cross-coupling reaction (Yield 33 %) (method B) [46]. The palladium-catalyzed Suzuki–Miyaura cross-coupling reaction of dichloro-derivatives **30–31** led to the 4,6-bis(4-formylphenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine **32** and the 2,4-bis

(4-formylphenyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidine **33**, respectively. The bis-imino derivatives **34–35** were then prepared by condensation of various primary amines with the di-aldehydes **32–33**. Finally, these intermediates **34–35** were reduced into the final compounds **7–8** with a NaBH₄ treatment in methanol (Scheme 5).

The synthesis of 2,4,7-tris{4-[(dimethylamino-alkyl)aminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidines **9a-b** (series I) was realized in four steps according to the chemical pathway described in Scheme 6. The previously synthesized compound **19** (see Scheme 2) was then submitted to our classical double Suzuki coupling reaction to afford the tri-aldehyde **36**, which was then condensed with different substituted primary amines leading to the tri-impines **37a-b** reduced in the amines **9a-b** by using sodium borohydride (Scheme 6).

The synthesis of 4,5,7-tris{4-[(dimethylamino-alkyl)aminomethyl]
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** The ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of the pyrrolo[2,3-b]pyridine derivative **11** with atom labeling and thermal ellipsoids at 30 % probability level. Hydrogen atoms are represented as small spheres of arbitrary radii.</figcaption></figure>

phenyl}-7H-pyrrolo[2,3-d]pyrimidines **9c** (series J) was accomplished according to the same chemical sequence as the one described for compounds **9a-b** (Scheme 7). The first reaction involved a Chan-Lam copper-catalyzed N-arylation at position 7 of the 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine leading to derivative **38**. The second reaction consisted in the preparation of the tri-aldehyde **39** through a double Suzuki coupling. A X-ray single crystal analysis was realized on this tri-aldehyde **39** that confirmed the structure (Fig. 7). Condensation of primary amines with this trialdehyde **39** afforded the tri-imine **40**, which was immediately reduced into the 4,5,7-tris[(substituted-aminomethyl)-7H-pyrrolo[2,3-d]pyrimidine **9c** by using sodium borohydride in refluxing methanol. Amine **9c** was then converted into its ammonium oxalate salt by treatment with oxalic acid in refluxing isopropanol.

## 2.2. Biophysical and biological evaluation

### 2.2.1. FRET-melting experiments

With the aim of demonstrating that our new nitrogen heterocyclic compounds **1–9** are G4 ligands, we evaluated their capacity to stabilize a G4 structure, through an established FRET-melting assay (Table 2) [47]. The well-characterized G4 of intermediate stability, F21T [47], was chosen for this assay.

This stabilization assay was performed on the fluorescently labeled human telomeric G4 sequence F21T (Table 3). To probe the G4 selectivity of our derivatives over duplex DNA, a FRET-melting assay was also performed using a hairpin duplex control sequence, FdxT (also described in Table 3). For comparison, the G4 ligand phenanthroline PhenDC3 was used as a reference positive compound, and theaflavin 3,3'-digallate used as a negative control. To compare levels of selectivity, the difference of melting temperature (ΔT<sub>m</sub>) between the T<sub>m</sub> of the G-quadruplex formed by F21T, or FdxT in the presence or absence of each selected molecules was calculated.

As shown in Table 2, for the new aza heterocycles **1–9**, the ΔT<sub>m</sub> values for the F21T sequences ranged from 4.0 to >36 °C at a 2 µM ligand concentration. The best nitrogen ligands which stabilized the human F21T G-quadruplexes sequences were 2,4,7-tris{4-[(dimethylamino-alkyl)aminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidines **9a-b**, bearing substituted alkylaminoalkylaminobenzyl side chains at positions 2, 4 and 7 of the heterocyclic moiety, and mainly compound **9b** that showed the highest ΔT<sub>m</sub> values (ΔT<sub>m</sub> > 36 °C at 2 µM and 23.8 °C at 1 µM, on this F21T sequence).

We could also observe that series A constituted of 2,4-bis [(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine derivatives **1** displayed relatively potent stabilizations excepted its 2,4-bis {4-[(5-methylhexyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d] pyrimidine analog **1h**, in the structure of which there are no nitrogen atoms at the end of the side chains: ΔT<sub>m</sub> = 19.6–30.9 °C on F21T for **1a-g** versus 4.0 °C on F21T for the compound **1h**. From a general point of view concerning the bis substituted derivatives, it is possible to notice

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** The ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of the disubstituted pyrrolo[3,2-c]pyridine derivative **15** with atom labeling and thermal ellipsoids at 30 % probability level. Hydrogen atoms are represented as small spheres of arbitrary radii.</figcaption></figure>
<table><caption>**Table 1**

Physical properties of ammonium oxalate salts 1–9.</caption><thead><tr><th>Compound</th><th></th><th>Salt<sup>a</sup></th><th>mp (°C)<sup>b</sup></th><th>% Yield<sup>a</sup></th></tr></thead><tbody><tr><td>1a</td><td>Beige crystals</td><td>4 (COOH)<sub>2</sub></td><td>178–180</td><td>70</td></tr><tr><td>1b</td><td>White crystals</td><td>4 (COOH)<sub>2</sub></td><td>145–147</td><td>89</td></tr><tr><td>1c</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>200–202</td><td>76</td></tr><tr><td>1d</td><td>White crystals</td><td>4 (COOH)<sub>2</sub></td><td>150–152</td><td>80</td></tr><tr><td>1e</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>165–167</td><td>71</td></tr><tr><td>1f</td><td>White crystals</td><td>6 (COOH)<sub>2</sub></td><td>193–195</td><td>74</td></tr><tr><td>1g</td><td>Beige crystals</td><td>6 (COOH)<sub>2</sub></td><td>191–193</td><td>71</td></tr><tr><td>1h</td><td>Pale-yellow crystals</td><td>2 (COOH)<sub>2</sub></td><td>242–244</td><td>78</td></tr><tr><td>2</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>140–142</td><td>80</td></tr><tr><td>3a</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>164–166</td><td>82</td></tr><tr><td>3b</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>119–121</td><td>75</td></tr><tr><td>3c</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>126–128</td><td>58</td></tr><tr><td>4a</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>150–152</td><td>59</td></tr><tr><td>4b</td><td>White crystals</td><td>4 (COOH)<sub>2</sub></td><td>137–139</td><td>62</td></tr><tr><td>5a</td><td>White crystals</td><td>4 (COOH)<sub>2</sub></td><td>140–142</td><td>92</td></tr><tr><td>5b</td><td>White crystals</td><td>4 (COOH)<sub>2</sub></td><td>129–131</td><td>79</td></tr><tr><td>6</td><td>White crystals</td><td>4 (COOH)<sub>2</sub></td><td>141–143</td><td>74</td></tr><tr><td>7a</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>161–163</td><td>46</td></tr><tr><td>7b</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>128–130</td><td>53</td></tr><tr><td>7c</td><td>Pale-yellow crystals</td><td>4 (COOH)<sub>2</sub></td><td>144–146</td><td>61</td></tr><tr><td>7d</td><td>White crystals</td><td>6 (COOH)<sub>2</sub></td><td>218–220</td><td>76</td></tr><tr><td>8a</td><td>White crystals</td><td>4 (COOH)<sub>2</sub></td><td>159–161</td><td>73</td></tr><tr><td>8b</td><td>Beige crystals</td><td>4 (COOH)<sub>2</sub></td><td>214–216</td><td>76</td></tr><tr><td>8c</td><td>White crystals</td><td>6 (COOH)<sub>2</sub></td><td>173–175</td><td>74</td></tr><tr><td>9a</td><td>White crystals</td><td>6 (COOH)<sub>2</sub></td><td>144–146</td><td>76</td></tr><tr><td>9b</td><td>White crystals</td><td>6 (COOH)<sub>2</sub></td><td>119–121</td><td>86</td></tr><tr><td>9c</td><td>Beige crystals</td><td>6 (COOH)<sub>2</sub></td><td>147–149</td><td>72</td></tr></tbody></table>

<sup>a</sup> The stoichiometry and composition of the salts were determined by elemental analyses and obtained values were within ±0.4 % of the theoretical values.

<sup>b</sup> Crystallization solvent: 2-PrOH-H₂O.

<sup>c</sup> The yields only included the conversions into the ammonium oxalates.

that all of these derivatives **1**, **2**, **4**, **7** and **8** strongly stabilized the F21T G-quadruplex. The ΔTm values ranged from 19.6 to 34.0 °C at 2 µM ligand concentration. In addition, it could be also found that *bis* [(substituted-aminomethyl)-phenylpyrrolo[2,3-d]pyrimidines **5a-b**, some analogues substituted by the two di-alkylaminoalkylaminobenzyl side chains at positions 4 and 5 of the heterocyclic core, displayed lower stabilization on F21T sequence with ΔTm = 13.1–14.8 °C. Moreover, it also appears beneficial that the central heterocyclic system has an imidazo[4,5-d]pyrimidine-type skeleton (ligands **8a-c**) for which some ΔTm of 30.2–34.0 °C were found on the F21T sequence.

Hence, FRET assays revealed a lower stabilization of most of our heterocyclic nitrogen structures (except **1d-e**, **3a**, **8a-c** and **9a**) on duplex DNA sequences; i.e. ΔTm = 0.6–6 °C on the FdxT sequence.

### 2.2.2. Homogenous time resolved fluorescence (HTRF) assays to evaluate the inhibitory potencies of the newly synthesized molecules reported herein

We then investigated whether our new synthesized molecules would perturb the SUD/G4 interaction as previously shown for a few other G4-ligands [4]. For this purpose, we used the previously set up HTRF assay. Briefly, we took advantage of the presence of a His-tag at the N-terminal part of the SUD protein and a Biotin-TEG tag at the 3' end of the TRF2 G4, each tag being recognized by specific antibodies coupled to fluorophores (Revvity®, Bussy-Saint-Martin, France). The assay was performed in the presence of a fixed concentration of both interacting partners (10 and 50 nM of SUD-NM and TRF2 G4, respectively) and increasing concentrations of the tested molecules (in the range of 0–500 nM). Fig. 8 presents HTRF ratios measured at different concentrations of at least one representative of each series. Theaflavin 3,3'-digallate (theaflavin) has been included in these experiments as a positive control. Indeed, it has been identified as a potent disruptor of the SUD/G4 interaction and an inhibitor of SARS-CoV2 replication in Vero E6-TMPRSS2 cells [6]. The curves allowed us to calculate the half-maximal inhibitory concentrations (IC₅₀) values as described in the experimental section. These values obtained for all tested derivatives are

listed in Table 4. It should be noted that a significant % residual HTRF ratio (between 20 and 60 %) is observed for some compounds even at the highest tested concentrations (up to 1500 nM). As references for G4 ligands belonging to the phenanthroline family, the previously reported compounds JG986 and JG1045 exhibited IC₅₀ values around 30 nM in the same conditions [4].

Surprisingly, theaflavin had a low impact on the interaction between SUD-NM and TRF2 G4 (Fig. 8C), with an IC₅₀ greater than 500 nM. On the other hand, the IC₅₀ values decreased down to 46 nM with the newly synthesized molecules tested in this study.

In the sub-series of the pyrrolo[2,3-d]pyrimidine **1** (series A) disubstituted by dialkylaminoalkylaminobenzyl chains in positions 2 and 4 of the heterocycle core, this study revealed a clear inhibition of the SUD/G4 interaction by these G4-ligands **1**, with IC₅₀ values ranging between 48 and 90 nM, excepted surprisingly for compound **1f** bearing two 3-(4-methylpiperazin-1-yl)propyl)iminomethyl]phenyl groups (IC₅₀ > 200 nM). In addition, no inhibition of SUD/G4 interaction was observed with derivative **1h** substituted by two (5-methylhexyl)aminomethyl]phenyls, previously identified for its unstable interaction with F21T G4 (Table 2). This result shows the importance of a dialkylamino group at the end of each lateral side chains (IC₅₀ > 500 nM) for both G4 interaction and inhibition of SUD/G4 interaction. These first data also show that the length of the C3, C4 or C5 dialkylaminoalkylaminobenzyl chain does not seem to play a primordial role in the inhibitory activity. However, in this 2,4 disubstituted pyrrolo[2,3-d]pyrimidine series A, it is possible to observe more pronounced activities with a more bulky amino group at the end of each lateral side chains; i.e. diethylamino or diisopropylamino substituents versus dimethylamino one (IC₅₀ of 64 and 48 nM for **1d** and **1e**, respectively versus 75–90 nM for **1a-c**).

A comparison of aza compounds **1b** and **2**, both substituted by (4-dimethylaminobutyl)aminomethylphenyl side chains and also by a phenyl on the pyrrole nitrogen, but different by the basic heterocyclic system, pyrrolo[2,3-d]pyrimidine versus pyrrolo[2,3-b]pyridine, revealed that the pyrrolo[2,3-d]pyrimidine skeleton presented a more promising inhibitory potency (IC₅₀ = 75 nM for **1b** and 223 nM for **2**). In addition, its analogue pyrrolo[3,2-c]pyridine **3a** showed a more interesting inhibition with an IC₅₀ noticed at 87 nM in comparison with pyrrolo[2,3-b]pyridine **2** (IC₅₀ = 223 nM). However, a advantageous inhibitory activity of the SUD/G4 interaction is not observed for the two other disubstituted compounds exhibiting the same heterocyclic pyrrolo[3,2-c]pyridine unit; i.e. IC₅₀ of **3b** and **3c** were observed superior to 200 nM. These first results seem to indicate that a pyrrolopyrimidine core for the heterocyclic moiety is more detrimental than a pyrrolo-pyridine one.

By comparing compounds **4a** and **4b** both bearing two (dimethylaminoalkyl)aminomethylphenyl side chains and a phenyl ring linked to the pyrrolo[2,3-d]pyrimidine skeleton in positions 2, 7 and 4, respectively, with their homologues **1b** and **1c** substituted by the same groups but in the 2, 4 and 7 positions, the same range in the inhibitory potency was observed: IC₅₀ = 97 and 83 nM for **4a-b** versus 75 and 90 nM for **1b** and **1c**. Modulation of these two (3-dimethylaminoalkyl)aminomethylphenyl side substituents in positions 4 and 5 for the 7-phenyl-pyrrolo[2,3-d]pyrimidine skeleton led to a decrease of the inhibitory potency, i.e. IC₅₀ > 500 nM for **5a** and 140 nM for **5b**.

The ring flip of the pyrrole ring to prepare the pyrrolo[3,2-d]pyrimidine **6** resulted in a compound with a potency comparable to that of the structural homolog pyrrolo[2,3-d]pyrimidine **1b** (IC₅₀ values of 90 nM and 75 nM, respectively).

We also noticed that the replacement of the pyrrole ring in the pyrrolo[2,3-d]pyrimidine by a pyrazole (pyrazolo[3,4-d]pyrimidines **7** - series G) or by an imidazole heterocycle (imidazo[4,5-d]pyrimidines **8** - series H) also exhibited interesting inhibitory potencies: IC₅₀ values of derivatives **7a-d** and **8a-c** ranging from 63 to 137 nM and 79–115 nM, respectively. In this pyrazolo[3,4-d]pyrimidine series G, it could be also noticed that the elongation of the alkyl side chains (propyl versus butyl versus pentyl) led to a slight increase in the inhibitory potency (IC₅₀ =
<figure><img src="image_8.png" /><figcaption>**Scheme 2.** Synthesis of the 2,7-bis[(substituted-aminomethyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine derivatives 4a-b (series D).</figcaption></figure>

137 nM for 7a, 88 nM for 7b, and 63 nM for 7c).

In terms of structure-activity relationships, we could notice that the trisubstitution of the pyrrolo[2,3-d]pyrimidine moiety in positions 2, 4 and 7 or 4, 5 and 7 by (dimethylaminoalkyl)aminomethylphenyl side chains (compounds 9a-c) led to the most promising derivatives with IC₅₀ of 46–50 nM. In addition, the trisubstituted pyrrolo[2,3-d]pyrimidine 9a, which was substituted with three C3 dimethylaminoalkylamino-benzyl chains, exhibited a similar inhibitory potency (IC₅₀ = 50 nM) than its C4 homolog, compound 9b, for which the IC₅₀ value was noticed at 48 nM. The transposition of the C3 dimethylaminoalkylaminobenzyl chain from position 2 to position 5 in the pyrrolo[2,3-d]pyrimidine heterocycle core, if we compare derivatives 9a versus 9c, does not really change the potency: IC₅₀ values of 50 nM and 46 nM for 9a and 9c, respectively.

### 2.2.3. *In vitro* evaluation of the antiviral property of compounds on cell lines infection by SARS-CoV-2 virus

A selection of the synthesised molecules were then tested *in vitro* for their antiviral activity in different cell lines and against two SARS-CoV-2 variants. The assays were performed following an infection protocol established by the PF-CCB of the Institut Pasteur and already used to evaluate the antiviral activity of other molecules [48]. Briefly (the assay is described with more details in the experimental section), cells were

incubated with different concentrations of the molecules 2h before infection. Infection was performed at 0.1 MOI. After 1 h of viral contact with the cells, the viral inoculum was removed and a fresh culture medium containing the same concentrations of molecules was added back on the cells. 72 h postinfection, viruses present in the supernatant of infected cells were quantified by quantitative RT-qPCR of the N-gene. These values were measured in triplicate and at different concentrations of G4-ligand. Fig. 9 presents one set of values obtained with molecules 1b, 1c, 1e, 2, 3a, 4b, 5a, 6, 7c, 8a, 9a, theaflavin and remdesivir. This last molecule was used as a positive control of antiviral activity. In parallel, cell viability was assessed using the CellTiter Glo kit (Promega). RT-qPCR and cell viability values measured at the different concentrations of molecules were used to calculate the concentrations of molecules required to inhibit by 50 % the viral replication (EC₅₀) or to kill 50 % of the cells (CC₅₀). Finally, the selectivity index (SI) was calculated as defined in Table 4 using the CC₅₀ and EC₅₀ values. These values are listed in Table 4.

The antiviral assay was first performed in A549 cell lines stably expressing the Ace2 receptor (gift from O. Schwartz, I. Pasteur, France) infected by the original strain BetaCoV/France/IDF0372/2020 or by its Delta variant (B.1.617.2). A549 cells were chosen because they derive from lung carcinoma epithelial cells and mimic cells physiologically infected by SARS-CoV-2 in human patients.
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** The ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of the pyrrolo[2,3-d]pyrimidine derivatives **19–21** with thermal ellipsoids at 30 % level. Hydrogen atoms are represented as small spheres of arbitrary radii.</figcaption></figure>

For a large majority of molecules, HTRF and antiviral IC₅₀ values revealed a good correlation between their efficacy to perturb SUD/G4 interaction and their anti-SARS-CoV-2 activity. Indeed, molecules **1a**, **1b**, **1c**, **1d**, **1e**, **3a**, **4a**, **4b**, **6**, **7c**, **8a** and **9a** showed an efficient inhibition of both SUD/G4 complex formation (HTRF IC₅₀ values below 100 nM) and SARS-CoV-2 replication (antiviral EC₅₀ in the µM range). Consistent with this correlation, two molecules (**1f** and **theaflavin**) show the exact reverse behavior with poor efficiencies to prevent SUD/G4 interaction (HTRF IC₅₀ > 200 or 500 nM) and low antiviral activities (EC₅₀ > 10 µM). Surprisingly, five molecules appear not to satisfy the observed correlation. One molecule (**9c**) shows a low antiviral activity (EC₅₀ > 10 µM) despite its efficiency to prevent SUD/G4 interaction (IC₅₀ of 46 nM) and its stable interaction with G4 (Table 2). Thus, this result against the Delta variant (B.1.617.2) seems to show that a triple substitution in positions 2, 4 and 7 of the pyrrolo[2,3-d]pyrimidine moiety is much more interesting than a triple substitution in positions 4, 5 and 7 of the same heterocyclic skeleton; the EC₅₀ of compounds **9a-b** were noticed ranging from 0.95/1.1–2.4 µM in comparison with EC₅₀ of 11.16 µM for **9c**. Conversely, four molecules (**2**, **3b**, **3c** and **5a**) have an efficient micromolar range antiviral activity (EC₅₀ < 10 µM), although they do not prevent SUD/G4 interaction at the tested concentrations (IC₅₀ over 200 nM). However, these four molecules are good G4 binders, as shown by the FRET melting value measured on F21T G4.

We also tested to the antiviral activity of six molecules in the Caco2 cell line which is derived from colorectal adenocarcinoma patient. Colorectal epithelial cells also constitute a viral target in infected humans. The six tested molecules (**1a-1e**, and **9a**) revealed a micromolar range antiviral activity against the SARS-CoV-2 Delta variant, which correlates very well with the activities measured in the A549-Ace2 cells infected by the Wuhan or Delta variant SARS-CoV-2 strains.

Therefore, except for a few molecules, the large majority of designed and tested G4 ligands shows a clear correlation between their efficacies to perturb SUD/G4 interaction and to inhibit SARS-CoV-2 replication. It confirms our initial hypothesis that the SUD/G4 complexes are

important for viral replication and can be used as new antiviral target [4].

We also observed a high cellular toxicity of some compounds, with $CC_{50}$ below 10 µM. This toxicity is frequently associated with a low selectivity index (SI), which leads to a narrow window of concentrations between the antiviral activity and the cell toxicity and limits the therapeutic potential of these compounds (see Table 4). However, some other G4-ligands were associated with a higher selectivity index. Indeed, in A549-Ace2 cells, seven compounds (**1d**, **3b**, **3c**, **4b**, **5a**, **6** and **9a**) had a selectivity index larger than 8, and antiviral activities in the micromolar range (0.95–1.5 µM). These values encourage us to further investigate the antiviral properties of these molecules and their derivatives in primary cells and in animal models of infection. Indeed, safety assessment of compound **1b** was evaluated in mice by the PCBIS platform (Illkirch, France). At concentrations up to 5 mg/kg, no behavioural disorders have been observed during 72h after peritoneal injection of **1b**.

### 3. Discussion and conclusion

Several G4 ligands have already been characterized for their antiviral activity targeting SARS-CoV-2 replication. These results were mainly obtained *in vitro*, but also in cellular and animal models of viral infection. If some of these ligands directly target G4 structures folded in the viral RNA genome or transcripts [30–32,49], other molecules act through their interaction to cellular RNA G4s and a consecutive disregulation of proteins essential for viral replication [23,24].

In this study, we present a new class of G4 ligands, with promising activity against SARS-CoV-2 viral replication. These molecules are new nitrogen heterocyclic compounds basically derived from condensed pyrrolopyrimidine and pyrrolopyridine moieties. They were initially screened for two *in vitro* properties: their affinity to a stable G4 structure (derived from a telomeric sequence) and their capacity to prevent the formation of a complex formed between the SARS-CoV-2 SUD domain
<figure><img src="image_10.png" /><figcaption>**Scheme 3.** Synthesis of 4,5-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine derivatives 5a-b (series E).</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 5.** The ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of the pyrrolo[2,3-d]pyrimidine derivative 24 with thermal ellipsoids at 30 % level. Hydrogen atoms are represented as small spheres of arbitrary radii.</figcaption></figure>

and a stable RNA G4 derived from the TRF2 transcript. The SUD/G4 interaction was chosen as a screen target, due to its crucial role in the replication of both SARS-CoV and SARS-CoV-2 viral replication [5,6, 50]. Telomeric and TRF2 G4s were choosen for their biochemical

properties which allow to use them in quantitative G4 binding FRET-Melting and SUD/G4 HTRF interaction assays respectively.

Among the 27 synthesized molecules, 24 of them efficiently stabilize F21T G4 structure (ΔT₁/₂ > 15 °C) (1a-g, 2, 3a-c, 4a-b, 6, 7a-d, 8a-c and
<figure><img src="image_12.png" /><figcaption>**Scheme 4.** Synthesis of the 2,4-bis[(substituted-aminomethyl)-5-phenyl-5H-pyrrolo[3,2-d]pyrimidine 6 (series F).</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 6.** The ORTEP drawing of pyrrolo[3,2-d]pyrimidine 27 with thermal ellipsoids at 30 % level. Hydrogen atoms are represented as small spheres of arbitrary radii.</figcaption></figure>

9a-c), with derivative 9b as the most efficient G4 stabilizer (ΔT₁/₂ > 36 °C). Two derivatives (5a-b) show a weak stabilization of G4 structure (ΔT₁/₂ around 13–15 °C) and derivative 1h nearly shows no effect on G4 stability (ΔT₁/₂ around 4 °C). All molecules were also tested for their effect on SUD/G4 interaction, using an established HTRF assay [4]. This study revealed a good correlation between the two measured parameters, 20 molecules showing both a strong G4 stabilization (ΔT₁/₂ > 15 °C) and an efficient inhibition of SUD/G4 complex formation (HTRF IC₅₀ below 200 nM). As expected, derivative 1h has no effect on SUD/G4 interaction (HTRF IC₅₀ > 500 nM). Similarly, our reference molecule

theaflavin shows a very low inhibitory potency (Fig. 8C). These two molecules do not bind to G4 and do not affect the SUD/G4 interaction. Our result obtained with theaflavin was unexpected as it has been previously described to be a potent inhibitor of the SUD-NM/TRF2 G4 interaction using biolayer interferometry technique [6]. Derivatives 5a and 5b, with a weak G4 stabilization potential, has a weak effect on SUD/G4 interaction (HTRF IC₅₀ > 500 nM and 140 nM respectively and less than 60 % of SUD/G4 maximal complex formation at 500 nM of both G4-ligand). Surprisingly, derivatives 1f, 3b and 3c, show a weak effect on SUD/G4 formation, although they interact well with G4 and stabilize
<figure><img src="image_14.png" /></figure>

<table><caption>**Scheme 5.** Synthesis of the substituted pyrazolo[3,4-d]pyrimidine and imidazo[4,5-d]pyrimidine derivatives **7-8** (series G and H).</caption><thead><tr><th colspan="6">Series G</th><th colspan="6">Series H</th></tr><tr><th>Compounds</th><th>-N<sub>R</sub><sup>R</sup></th><th>X</th><th>Y</th><th>n</th><th>m</th><th>Compounds</th><th>-N<sub>R</sub><sup>R</sup></th><th>X</th><th>Y</th><th>n</th><th>m</th></tr></thead><tbody><tr><td>34a, 7a</td><td>-N<sub>R</sub><sup>R</sup>CH<sub>3</sub></td><td>CH</td><td>N</td><td>1</td><td>4</td><td>35a, 8a</td><td>-N<sub>R</sub><sup>R</sup>CH<sub>3</sub></td><td>N</td><td>CH</td><td>2</td><td>4</td></tr><tr><td>34b, 7b</td><td>-N<sub>R</sub><sup>R</sup>CH<sub>3</sub></td><td>CH</td><td>N</td><td>2</td><td>4</td><td>35b, 8b</td><td>-N<sub>R</sub><sup>R</sup>CH<sub>3</sub></td><td>N</td><td>CH</td><td>3</td><td>4</td></tr><tr><td>34c, 7c</td><td>-N<sub>R</sub><sup>R</sup>CH<sub>3</sub></td><td>CH</td><td>N</td><td>3</td><td>4</td><td>35c, 8c</td><td>-N<sub>R</sub><sup>R</sup>N-CH<sub>3</sub></td><td>N</td><td>CH</td><td>2</td><td>6</td></tr><tr><td>34d, 7d</td><td>-N<sub>R</sub><sup>R</sup>N-CH<sub>3</sub></td><td>CH</td><td>N</td><td>2</td><td>6</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

efficiently this structure (ΔT<sub>1/2</sub> around 20 °C). This result could indicate that some G4 ligands do not behave similarly with F21T and TRF2 G4s or that the G4 surface targeted by these ligands does not perturb SUD interaction. However, for the vast majority of tested G4-ligands, their binding to the RNA TRF2 G4 prevents its recognition by the SARS-CoV-2 SUD domain.

These results led us to investigate the antiviral activities of the most efficient SUD/G4 inhibitors (**1a-e**, **1g**, **2**, **3a**, **4a-b**, **6**, **7c**, **8a** and **9a-c**) but also G4-ligands with minor (**1f**, **3b-c**) or null effects (**5a**, theaflavin) on SUD/G4 interaction. Remdesivir was chosen as a positive control of infection. This analysis was performed using two viral strains (original Wuhan and Delta) and two human cell lines frequently used in SARS-CoV-2 infection assays. First, the different infection assays gave very similar results, showing that the G4-ligands antiviral activities do not depend on the viral strain nor the infected cell lines. Second, the large

majority of tested G4 ligands show a clear correlation between their efficacies to perturb SUD/G4 interaction and their negative effect on SARS-CoV-2 replication (Table 4). The best examples of this correlation are derivatives **1a-e**, **1g**, **3a**, **4a-b**, **6**, **7c**, **8a-c** and **9a-c**, which show a micromolar range antiviral activity (0.5 µM < EC<sub>50</sub> < 3 µM). As expected, larger concentrations of derivative **1f** and theaflavin are required to observe an inhibition of viral replication (EC<sub>50</sub> > 10 µM). Again, for this compound, our data did not reproduce the findings by Qin et al., which reported anti SARS-CoV-2 activity in Vero E6-TMPRSS2 cells [6]. This discrepancy might be due to differences in experimental conditions. The result obtained with compound **1f** probably shows that the alkyl link between the amine function and the piperazine ring must be longer than a C<sub>3</sub> alkyl chain, when compared with its C<sub>4</sub> substituted analogue **1g**, i.e. EC<sub>50</sub> = 10.94 µM for **1f** versus 1.16 µM for **1g** against the Delta variant (B.1.617.2). Surprisingly, derivatives **2**, **3b**, **3c** and **5a**
<figure><img src="image_15.png" /></figure>

<table><caption>**Scheme 6.** Synthesis of the 2,4,7-tris{4-[(dimethylamino-alkyl)aminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidines 9a-b (series I).</caption><thead><tr><th rowspan="2">Compounds</th><th colspan="3">Series I</th></tr><tr><th>-N<sub>2</sub>R</th><th>n</th><th>m</th></tr></thead><tbody><tr><td>37a, 9a</td><td>-N<sub>2</sub>CH<sub>3</sub></td><td>1</td><td>6</td></tr><tr><td>37b, 9b</td><td>-N<sub>2</sub>CH<sub>3</sub></td><td>2</td><td>6</td></tr></tbody></table>

show a micromolar range antiviral activity, although they do not inhibit efficiently the formation of SUD/G4 complexes. Conversely, derivative 9c has a low antiviral activity (EC₅₀ > 10 µM) despite its effect on G4 stability and SUD/G4 interaction. Altogether, these results reveal that the SUD/G4 interaction can be used as a new antiviral target but also that preventing this interaction is not sufficient for G4-ligands to efficiently inhibit SARS-CoV-2 replication.

Further studies are required to characterize the mechanisms associated with the observed antiviral properties of the synthezised compounds and to identify their precise targets. A low stability of a non-G4 structure by some studied compounds herein suggests that they could have other targets than SUD/G4 complexes formed during viral infection. A particular attention will be given to G4s present in the viral genome or its various replication products but also in cellular RNAs interacting with viral proteins or essential for viral replication. Toxicity and pharmacodynamic properties of the synthezised G4 ligands will also be evaluated in small animal models of SARS-CoV-2 infection. Preliminary data on 1b are very promising as no toxicity was observed in mice after peritoneal injection at concentrations up to 5 mg/kg. These studies will be complementary to the ones performed in cellular model of infection as presented in this study.

In Nsp3, the SUD domain is under mutated with regards of the rest of the protein [51], arguing in favor of its role during viral replication and its use as antiviral target. In this study, we observed very good similarities between antiviral activities measured against two viral strains (Wuhan and Delta), which suggests that our compounds could have a larger spectrum of activity against several SARS-CoV-2 variants. More globally, compounds targeting the SUD/G4 interaction in the context of SARS-CoV-2 replication may exhibit antiviral activity against other highly pathogenic coronaviruses. Our strategy targeting SUD/G4 interaction should therefore be considered as part of preventive research strategies against future coronavirus outbreaks.

# 4. Experimental section

## 4.1. Chemistry

Commercially available reagents were used as received without additional purification. Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope (Leitz GMBH, Midland, ON, USA) and are uncorrected. IR spectra were recorded on a NICOLET 380FT-IR spectrophotometer (Thermo Electron Scientific Instruments LLC,
<figure><img src="image_16.png" /><figcaption>**Scheme 7.** Synthesis of the 4,5,7-tris{4-[(dimethylamino-alkyl)aminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidine 9c (series J).</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>**Fig. 7.** The ORTEP drawing of pyrrolo[2,3-d]pyrimidine 39 with thermal ellipsoids at 30 % level. Hydrogen atoms are represented as small spheres of arbitrary radii.</figcaption></figure>
<table><caption>**Table 2**
FRET-melting values for compounds **1–9**, PhenDC3, JG986, JG1045 and Theaflavin 3,3'-digallate.</caption><thead><tr><th rowspan="2">Compound</th><th colspan="2">ΔT<sub>1/2</sub> (°C)<sup>a</sup></th></tr><tr><th>F21T</th><th>FdxT</th></tr></thead><tbody><tr><td>PhenDC3</td><td>21.2 ± 0.5</td><td>0.6 ± 0.1</td></tr><tr><td>JG986</td><td>18.5 ± 1.5</td><td>1.6 ± 0.5</td></tr><tr><td>JG1045</td><td>24.1 ± 0.1</td><td>1.3 ± 0.1</td></tr><tr><td>1a</td><td>22.8 ± 0.4</td><td>1.2 ± 0.3</td></tr><tr><td>1b</td><td>23.7 ± 14</td><td>1.5 ± 0.1</td></tr><tr><td>1c</td><td>30.9 ± 0.8</td><td>5.2 ± 0.3</td></tr><tr><td>1d</td><td>28.7 ± 1.3</td><td>6.0 ± 0.5</td></tr><tr><td>1e</td><td>31.9 ± 0.3</td><td>8.5 ± 0.4</td></tr><tr><td>1f</td><td>19.6 ± 1.3</td><td>0.7 ± 0.4</td></tr><tr><td>1g</td><td>20.4 ± 1.9</td><td>0.6 ± 0.3</td></tr><tr><td>1h</td><td>4.0 ± 0.7</td><td>0.2 ± 0.2</td></tr><tr><td>2</td><td>26.3 ± 1.5</td><td>4.9 ± 0.6</td></tr><tr><td>3a</td><td>21.0 ± 0.8</td><td>2.9 ± 0.3</td></tr><tr><td>3b</td><td>22.6 ± 0.8</td><td>3.1 ± 0.3</td></tr><tr><td>3c</td><td>28.7 ± 0.9</td><td>6.8 ± 0.2</td></tr><tr><td>4a</td><td>26.4 ± 2.8</td><td>4.4 ± 0.7</td></tr><tr><td>4b</td><td>27.2 ± 3.7</td><td>3.5 ± 1.0</td></tr><tr><td>5a</td><td>13.1 ± 0.3</td><td>0.6 ± 0.1</td></tr><tr><td>5b</td><td>14.8 ± 0.9</td><td>1.9 ± 1.5</td></tr><tr><td>6</td><td>18.6 ± 0.4</td><td>2.0 ± 0.2</td></tr><tr><td>7a</td><td>25.9 ± 0.7</td><td>3.8 ± 0.6</td></tr><tr><td>7b</td><td>25.9 ± 3.0</td><td>5.3 ± 1.0</td></tr><tr><td>7c</td><td>27.7 ± 2.0</td><td>5.2 ± 1.3</td></tr><tr><td>7d</td><td>30.4 ± 0.9</td><td>5.5 ± 0.4</td></tr><tr><td>8a</td><td>31.7 ± 1.5</td><td>8.5 ± 1.1</td></tr><tr><td>8b</td><td>34.0 ± 0.5</td><td>9.7 ± 0.4</td></tr><tr><td>8c</td><td>30.2 ± 0.5</td><td>6.8 ± 0.3</td></tr><tr><td>9a</td><td>34.9 ± 0.2</td><td>8.7 ± 0.4</td></tr><tr><td>9b</td><td>&gt;36<sup>a</sup>/23.8<sup>b</sup> ± 1.3</td><td>3.6 ± 1.4</td></tr><tr><td>9c</td><td>20.3 ± 0.9</td><td>2.2 ± 0.2</td></tr><tr><td>Theaflavin 3,3'-digallate</td><td>0.9 ± 0.5</td><td>0.1 ± 0.1</td></tr></tbody></table>

<sup>a</sup> ΔT1/2 of F21T and FdxT (0.2 µM) were reported at 2 µM ligand concentration in 10 mM lithium cacodylate (pH 7.2), 10 mM KCl, and 90 mM LiCl. PhenDC3 was used as control compound at 0.5 µM. Error margins correspond to SD of three replicates.

<sup>b</sup> Experiments were conducted in K<sup>+</sup> conditions at 1 µM.

<table><caption>**Table 3**
Labeled DNA sequences used in the present study.</caption><thead><tr><th>Name</th><th>Sequences<sup>a</sup></th><th>Topology</th></tr></thead><tbody><tr><td>F21T</td><td>FAM-GGGTTAGGGTTAGGGTTAGGG-TAMRA</td><td>Hybrid</td></tr><tr><td>FdxT</td><td>FAM-TATAGCTATA-HEX-TATAGCTATA-TAMRA</td><td>Hairpin duplex</td></tr></tbody></table>

<sup>a</sup> All sequences are provided in the 5' => 3'direction. FAM and TAMRA are the donor and acceptor fluorophores for FRET, respectively. HEX stands for Hexaethylene glycol.

Madison, WI, USA). NMR spectra were recorded with tetramethylsilane as an internal standard using a BRUKER AVANCE 300 spectrometer (Bruker BioSpin, Wissembourg, France). Splitting patterns have been reported as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; ddd = double doublet; qt = quintuplet; m = multiplet. Analytical TLCs were carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) and visualization of compounds after UV light irradiation. Silica gel 60 (70–230 mesh) was used for column chromatography. Mass spectra were recorded on an ESI LTQ Orbitrap Velos mass spectrometer (ThermoFisher, Bremen, Germany). Elemental analyses were found within ±0.4 % of the theoretical values.

### 4.1.1. 4,6-Dichloro-1-phenyl-1H-pyrrolo[2,3-b]pyridine (11)

To a solution of 4,6-dichloro-1H-pyrrolo[2,3-b]pyridine (2.7 mmol) in dichloromethane (25 mL) were added copper (II) acetate (0.54 mmol, 0.2 equiv.) and DBU (2.7 mmol, 1 equiv.). The mixture was stirred at room for 30 min, then phenylboronic acid (3.2 mmol, 1.2 equiv.) was

added. The reaction mixture was continue for another 16 h. After completion of the reaction, dichloromethane (200 mL) was added to the reaction mixture. This reaction mixture was then washed with saturated ammonium chloride solution (2 x 100 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated in vacuo. The residue was then purified by silica gel column chromatography (AcOEt/cyclohexane – 5/95 – v/v) to give the solid product. White crystals (58 %); Mp = 56 °C; ¹H NMR δ (300 MHz, CDCl₃): 7.72 (dd, 2H, *J* = 8.70 and 1.50 Hz, H-2' and H-6'), 7.58-7.53 (m, 2H, H-3' and H-5'), 7.52 (d, 1H, *J* = 3.90 Hz, H-2), 7.39 (m, 1H, H-4'), 7.23 (s, 1H, H-5), 6.75 (d, 1H, *J* = 3.90 Hz, H-3).

### 4.1.2. 4,6-Dichloro-1-phenyl-1H-pyrrolo[3,2-c]pyridine (12)

To a solution of 4,6-dichloro-1H-pyrrolo[3,2-c]pyridine (2.7 mmol) in dichloromethane (6 mL) were added phenylboronic acid (3 mmol, 1.1 equiv.), copper (II) acetate (5.4 mmol, 2.2 equiv.) and Et₃N (27 mmol, 10 equiv.). The mixture was stirred at room for 24 h. Water (25 mL) was added to quench the reaction, and the resulting mixture was extracted with EtOAc (x2). The combined organic phases were washed with saturated brine, dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated in vacuo. The residue was then purified by silica gel column chromatography (CH₂Cl₂) to give the solid product. White crystals (49 %); Mp = 117 °C; ¹H NMR δ (300 MHz, CDCl₃): 7.63-7.59 (m, 2H, H-2' and H-6'), 7.51-7.44 (m, 3H, H-3', H-4' and H-5'), 7.41 (d, 1H, *J* = 3.90 Hz, H-2), 7.37 (s, 1H, H-7), 6.82 (d, 1H, *J* = 3.90 Hz, H-3).

### 4.1.3. 2,4-Bis(4-formylphenyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (13)

To a solution of 4.37 mmol of 2,4-dichloro-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine 10, 1.44 g of 4-formylphenyl boronic acid (9.63 mmol, 2.2 eq.) and 506 mg (0.437 mmol, 0.1 eq.) of tetrakis(triphenylphosphine) palladium in 45 mL of 1,2-dimethoxyethane, 5 mL of 2 M Na₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with water (20 mL x 3 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and EtO₂ and filtered on sintered glassware to give the crude product. The residue was purified by silica gel column chromatography (CH₂Cl₂/CH₃OH 95:5), then cooled and triturated again in EtOH, filtered on sintered glassware, washed with a minimum of EtOH, EtO₂ and petroleum ether and dried under pressure to give the solid product. Pale-yellow crystals (76 %); Mp = 146 °C; IR νmax (KBr)/cm⁻¹ 1690 and 1686 (C=O); ¹H NMR δ (300 MHz, CDCl₃): 10.17 (s, 1H, CHO), 10.09 (s, 1H, CHO), 8.70 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.52 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.25 (d, 1H, *J* = 3.60 Hz, H-6), 8.16 (d, 2H, *J* = 8.10 Hz, H-2''' and H-6'''), 8.05 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.98 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.66 (t, 2H, *J* = 8.10 Hz, H-3''' and H-5''), 7.49 (t, 1H, *J* = 8.10 Hz, H-4'''), 7.25 (d, 1H, *J* = 3.60 Hz, H-5).

### 4.1.4. 4,6-Bis(4-formylphenyl)-1-phenyl-1H-pyrrolo[2,3-b]pyridine (14)

A mixture of 4,6-dichloro-1-phenyl-1H-pyrrolo[2,3-b]pyridine 11 (1.14 mmol), 4-formylphenylboronic acid (3.43 mmol, 3.0 eq.), tetrakis (triphenylphosphine)palladium (0.114 mmol, 0.1 equiv.), Na₂CO₃ (3.43 mmol, 3 equiv.) and 13 mL of a mixture dioxane/H₂O (4/1; v/v) was warmed at 90 °C and stirred for 24 h under nitrogen pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with water (15 mL x 2 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH, filtered on sintered
<figure><img src="image_18.png" /><figcaption>**Fig. 8.** Competition HTRF assays of the interaction between TRF2 G4 (50 nM) and SARS-CoV-2 SUD-NM (10 nM) in the presence of increasing concentrations (from 5 to 500 nM) of compounds **1b**, **1c**, **1e** and **2** in A), **3a**, **4b**, **5a** and **6** in B), **7c**, **8a**, **9a** and theaflavin in C). Each data point represents the mean of three individual measurements. Kaleidagraph software was used to fit the experimental data and to calculate HTRF IC₅₀ values which are reported in Table 4.</figcaption></figure>

glassware, and then washed with petroleum ether and dried under pressure to give the crude product that was then purified by silica gel column chromatography (CH₂Cl₂) to give the solid product. Yellow crystals (25 %); Mp = 205 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.17 (s, 1H, CHO), 10.10 (s, 1H, CHO), 8.35 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.13 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.05 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 8.02 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.93 (dd, 2H, *J* = 8.60 and 1.10 Hz, H-2'' and H-6''), 7.85 (s, 1H, H-5), 7.73 (d, 2H, *J* = 3.60 Hz, H-2), 7.62 (t, 2H, *J* = 8.60 Hz, H-3''' and H-5'''), 7.43 (t, 1H, *J* = 8.60 Hz, H-4'''), 6.85 (d, 1H, *J* = 3.60 Hz, H-3).

### 4.1.5. 4,6-Bis(4-formylphenyl)-1-phenyl-1H-pyrrolo[3,2-c]pyridine (15)

To a solution of 1.1 mmol of 4,6-dichloro-1-phenyl-1H-pyrrolo[3,2-c]pyridine 10, 0.50 g of 4-formylphenyl boronic acid (3.35 mmol, 3.0 eq.) and 130 mg (0.112 mmol, 0.1 eq.) of tetrakis(triphenylphosphine) palladium in 14 mL of a mixture dioxane/H₂O (4/1 – v/v), 0.355 mg (3.35 mmol, 3.0 eq.) of Na₂CO₃ were added at room temperature. Then, the mixture was warmed at 90 °C and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with warm CHCl₃ and the organic phase was filtered on filter paper, then washed with water (20 mL), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and filtered on sintered glassware to give the crude product which was then washed with a minimum of EtOH, EtO₂ and petroleum ether and dried under pressure to give the solid product. Yellow crystals (70 %); Mp > 260 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.17 (s, 1H, CHO), 10.09 (s, 1H, CHO), 8.37 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.34 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.12 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 8.00 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.95 (s, 1H, H-7), 7.69-7.63 (m, 2H, H-2''' and H-6'''), 7.60-7.54 (m, 4H, H-3'''', H-4'''', H-5'''', and H-2), 7.06 (d, 1H, *J* =

3.90 Hz, H-3).

### 4.1.6. General procedure for 2,4-bis[(substituted-iminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines (16a-g)]

To a solution of the appropriate diamines (0.126 mmol, 2.1 eq.) in ethanol (7 mL) was added the 2,4-bis(4-formylphenyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine 13 (0.6 mmol). The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (40 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

#### 4.1.6.1. 2,4-Bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16a). Pale-yellow crystals (97 %) Mp = 141 °C; ¹H NMR δ (300 MHz, CDCl₃): 8.67 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.39 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.30 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6'), 7.93 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.88-7.82 (m, 4H, H-2'''', H-6'''', H-3'''' and H-5'''), 7.57 (t, 2H, *J* = 7.80 Hz, H-3'''', and H-5'''), 7.56 (d, 1H, *J* = 3.60 Hz, H-6), 7.40 (t, 1H, *J* = 7.80 Hz, H-4''''), 6.94 (d, 1H, *J* = 3.60 Hz, H-5), 3.74-3.65 (m, 4H, 2NCH₂), 2.42-2.35 (m, 4H, 2NCH₂), 2.27 (s, 6H, N(CH₃)₂), 2.25 (s, 6H, N(CH₃)₂), 1.98-1.85 (m, 4H, 2CH₂).

#### 4.1.6.2. 2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16b). Yellow oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 8.67 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.39 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.31 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.94 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.88 (dd, 2H, *J* = 8.40 and 1.20 Hz, H-2'''', H-6'''', 7.83 (d, 2H, *J* = 8.40 Hz, H-3'''' and H-5'''), 7.59 (d, 1H, *J* = 3.90 Hz, H-6), 7.58 (t, 2H, *J* = 8.40 Hz, H-3'''', and H-5'''), 7.41 (t, 1H, *J* = 7.80 Hz, H-4'''), 6.96 (d, 1H, *J* = 3.90 Hz, H-5), 3.73-3.65 (m, 4H,
<caption>**Table 4**</caption>

Structure and effects of the synthesized compounds on SUD/G4 interaction, SARS-CoV-2 replication and cellular viability. HTRF assays were performed in the presence of serial dilutions of the compounds. IC₅₀ values report their inhibitory potency on the SUD/G4 interaction as presented in Fig. 8. Antiviral EC₅₀ values quantify their effect on viral replication evaluated on different virus/cell combinations, as presented in Fig. 9 for one of these combinations. Cytotoxicity CC₅₀ values reflect their effect on cell viability measured by CellTiter Glo assays as described in the experimental section. Selectivity index (SI) was defined as the ratio between the CC₅₀ value of cytotoxicity and the EC₅₀ value of viral replication.

<table><thead><tr><th>Compound</th><th>Structure</th><th>HTRF<br>IC₅₀ (nM)</th><th>Viral replication<sup>a</sup><br>EC₅₀ (µM)</th><th>Cytotoxicity<sup>a</sup><br>CC₅₀ (µM)</th><th>Selectivity Index</th></tr></thead><tbody><tr><td>JG986</td><td></td><td>31.2 ± 3.9</td><td>9.17</td><td>&gt;10</td><td>&gt;1.09</td></tr><tr><td>JG1045</td><td></td><td>31.4 ± 2.3</td><td>&gt;10</td><td>&gt;10</td><td>-</td></tr><tr><td>1a</td><td></td><td>89 ± 20</td><td>1.21<br>1.13</td><td>2.96<br>1</td><td>2.45<br>0.88</td></tr><tr><td>1b</td><td></td><td>75 ± 13</td><td>0.528<br>0.681/1.91<br>0.787/2.41</td><td>2.83/3.59<br>2.14/2.24</td><td>-<br>4.16/1.88<br>2.72/0.93</td></tr><tr><td>1c</td><td></td><td>90 ± 17</td><td>1.32<br>1.28/1.08<br>0.946/1.6</td><td>3.01/4.33<br>2.32/3.01</td><td>-<br>2.35/4.01<br>2.45/1.88</td></tr><tr><td>1d</td><td></td><td>64 ± 25</td><td>0.547<br>1.5<br>0.591/3.08</td><td>&gt;16<br>2.49</td><td>-<br>&gt;10.67<br>4.21/0.81</td></tr><tr><td>1e</td><td></td><td>48 ± 7</td><td>0.554<br>0.908/0.704<br>0.867/0.494</td><td>2.78/3.57<br>1.67/1.38</td><td>-<br>3.06/5.07<br>1.93/2.79</td></tr></tbody></table>

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

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

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

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

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

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

<figure><img src="image_25.png" /></figure>
<table><tr><td>1f</td><td></td><td>> 200</td><td>10.94</td><td>>16</td><td>>1.46</td></tr><tr><td>1g</td><td></td><td>74 ± 9</td><td>1.16</td><td>3.18</td><td>2.74</td></tr><tr><td>1h</td><td></td><td>> 500</td><td>nd</td><td>nd</td><td>-</td></tr><tr><td>2</td><td></td><td>223 ± 100</td><td>1.08</td><td>7.97</td><td>7.38</td></tr><tr><td>3a</td><td></td><td>87 ± 7</td><td>1.06</td><td>2.31</td><td>2.18</td></tr><tr><td>3b</td><td></td><td>> 200</td><td>1.08</td><td>8.84</td><td>8.18</td></tr><tr><td>3c</td><td></td><td>446 ± 74</td><td>1.15</td><td>>16</td><td>>13.91</td></tr><tr><td>4a</td><td></td><td>97 ± 23</td><td>0.92<br>0.96</td><td>2.53</td><td>-<br>2.64</td></tr></table>

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

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

<figure><img src="image_28.png" /></figure>

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

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

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

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

<figure><img src="image_33.png" /></figure>
<table><tr><td>4b</td><td></td><td>83 ± 31</td><td>0.41/0.56<br>1.95<br>1.22</td><td>&gt;16<br>2.27</td><td>&gt;8.21<br>1.86</td></tr><tr><td>5a</td><td></td><td>&gt; 500</td><td>1.72</td><td>&gt;16</td><td>&gt;9.30</td></tr><tr><td>5b</td><td></td><td>140 ± 46</td><td>nd</td><td>nd</td><td>-</td></tr><tr><td>6</td><td></td><td>90 ± 12</td><td>1.06</td><td>&gt;16</td><td>&gt;15.10</td></tr><tr><td>7a</td><td></td><td>137 ± 16</td><td>nd</td><td>nd</td><td>-</td></tr><tr><td>7b</td><td></td><td>88 ± 19</td><td>nd</td><td>nd</td><td>-</td></tr><tr><td>7c</td><td></td><td>63 ± 9</td><td>&gt;1<br>1.17</td><td>2.31</td><td>1.97</td></tr><tr><td>7d</td><td></td><td>133 ± 85</td><td>nd</td><td>nd</td><td>-</td></tr></table>

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

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

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>
<table><tbody><tr><td>8a</td><td></td><td>79 ± 10</td><td>0.5&lt;&lt;1</td><td>2.68</td><td>5.36&gt;&gt;2.68</td></tr><tr><td>8b</td><td></td><td>103 ± 10</td><td>nd</td><td>nd</td><td>-</td></tr><tr><td>8c</td><td></td><td>115 ± 16</td><td>nd</td><td>nd</td><td>-</td></tr><tr><td>9a</td><td></td><td>50 ± 9</td><td>0.436<br>0.95/1.1<br>1.54/2.19</td><td>&gt;16/&gt;16<br>7.2/&gt;10</td><td>-<br>&gt;16.84/&gt;14.55<br>4.68/&gt;4.57</td></tr><tr><td>9b</td><td></td><td>48 ± 3</td><td>2.4</td><td>&gt;16</td><td>6.67</td></tr><tr><td>9c</td><td></td><td>46 ± 10</td><td>11.16</td><td>&gt;16</td><td>&gt;1.43</td></tr><tr><td>Remdesivir</td><td></td><td></td><td>0.12</td><td>&gt;10</td><td>&gt;83.33</td></tr><tr><td>Theaflavin</td><td></td><td>&gt;500</td><td>25.34</td><td>&gt;30</td><td>&gt;1.18</td></tr></tbody></table>

<footnote>a in purple: A549-Ace2-Cl33 + Wuhan strain; in red: A549-Ace2-Cl33 + Delta strain (B1.617.2), assay #1; in orange: A549-Ace2-Cl33 + delta strain (B1.617.2), assay #2; in green: Caco2 + Wuhan strain assay #1; in blue: Caco2 + Wuhan strain assay #2.</footnote>

<footnote>nd: not determined</footnote>
<figure><img src="image_42.png" /><figcaption>**Fig. 9.** Antiviral activity of compounds **1b**, **1c**, **1e**, **2** (Figs. **9A**), **3a**, **4b**, **5a**, **6** (Figs. **9B**), and **7c**, **8a**, **9a**, and theaflavin (Fig. **9C**) measured in A549-Ace2 cells infected by the Delta SARS-CoV-2 variant (B.1.617.2). Infection assays were performed as described in the experimental section. Viruses present in the supernatant of infected cells 72 h post infection, at each concentration of compounds, were quantified by RT-qPCR assay targeting viral N RNA coding sequence. Measured Cq data were then normalized against appropriate negative (0 %) and positive (remdesivir) controls (100 %) and expressed as % of relative infection inhibition. Each data point represents the mean of three individual measurements. Plots presented in this figure were performed using GraphPad Prism software. This software was also used to calculate the antiviral EC₅₀ values presented in Table 4, using the variable Hill slope model.</figcaption></figure>

2NCH₂), 2.34 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.32 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.81-1.71 (m, 4H, 2CH₂), 1.65-1.54 (m, 4H, 2CH₂).

4.1.6.3. **2,4-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16c).** Yellow oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 8.63 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.33 (s, 1H, CH=N), 8.29 (s, 1H, CH=N), 8.25 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.88 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.82-7.78 (m, 4H, H-2''', H-6''', H-3''' and H-5''), 7.52 (t, 2H, *J* = 7.80 Hz, H-3''' and H-5'') 7.50 (d, 1H, *J* = 3.60 Hz, H-6), 7.33 (t, 1H, *J* = 7.80 Hz, H-4'''), 6.87 (d, 1H, *J* = 3.60 Hz, H-5), 3.64 (t, 2H, *J* = 7.20 Hz NCH₂), 3.62 (t, 2H, *J* = 7.20 Hz NCH₂), 2.28-2.19 (m, 4H, 2NCH₂), 2.20 (s, 6H, N(CH₃)₂), 2.18 (s, 6H, N(CH₃)₂), 1.80-1.68 (m, 4H, 2CH₂), 1.55-1.47 (m, 4H, 2CH₂), 1.45-1.35 (m, 4H, 2CH₂).

4.1.6.4. **2,4-Bis{4-[(4-diethylaminobutyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16d).** Yellow oil (92 %); ¹H NMR δ (300 MHz, CDCl₃): 8.68 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.41 (s, 1H, CH=N), 8.36 (s, 1H, CH=N), 8.33 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.96 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.89 (dd, 2H, *J* = 8.40 and 1.20 Hz H-2''', H-6'''), 7.85 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.63 (d, 1H, *J* = 3.60 Hz, H-6), 7.61 (t, 2H, *J* = 8.40 Hz, H-3''' and H-5'''), 7.43 (t, 1H, *J* = 7.80 Hz, H-4'''), 7.00 (d, 1H, *J* = 3.60 Hz, H-5), 3.76-3.64 (m, 4H, 2NCH₂), 2.60-2.45 (m, 12H, 6NCH₂), 1.80-1.71 (m, 4H, 2CH₂), 1.63-1.52 (m, 4H, 2CH₂), 2.24 (t, 6H, *J* = 7.20 Hz, 2CH₃), 2.23 (t, 6H, *J* = 7.20 Hz, 2CH₃).

4.1.6.5. **2,4-Bis{4-[(5-diisopropylaminopentyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16e).** Yellow oil (98 %); ¹H NMR δ

(300 MHz, CDCl₃): 8.70 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.41 (s, 1H, CH=N), 8.36 (s, 1H, CH=N), 8.35 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.97 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.90 (d, 2H, *J* = 7.80 Hz, H-2''' and H-6'''), 7.86 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.64 (d, 1H, *J* = 3.60 Hz, H-6), 7.62 (t, 2H, *J* = 7.80 Hz, H-3''' and H-5''), 7.44 (t, 1H, *J* = 7.80 Hz, H-4'''), 7.01 (d, 1H, *J* = 3.60 Hz, H-5), 3.73-3.64 (m, 4H, 2NCH₂), 3.09-3.04 (m, 4H, 4NCH), 2.46-2.40 (m, 4H, 2NCH₂), 1.85-1.72 (m, 4H, 2CH₂), 1.56-1.50 (m, 4H, 2CH₂), 1.45-1.36 (m, 4H, 2CH₂), 1.06-1.02 (m, 24H, 8CH₃).

4.1.6.6. **2,4-Bis{4-[(3-(4-methylpiperazin-1-yl)propyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16f).** Yellow oil (58 %); ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.38 (s, 1H, CH=N), 8.33 (s, 1H, CH=N), 8.30 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.92 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.87 (dd, 2H, *J* = 7.85 and 1.20 Hz, H-2''', H-6'''), 7.82 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.58 (d, 1H, *J* = 3.60 Hz, H-6), 7.57 (t, 2H, *J* = 7.85 Hz, H-3''' and H-5'''), 7.40 (t, 1H, *J* = 7.85 Hz, H-4'''), 6.95 (d, 1H, *J* = 3.60 Hz, H-5), 3.73-3.64 (m, 4H, 2NCH₂), 2.57-2.35 (m, 20H, 10NCH₂), 2.25 (s, 3H, NCH₃), 2.35 (s, 3H, NCH₃), 1.98-1.85 (m, 4H, 2CH₂).

4.1.6.7. **2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16g).** Yellow oil (97 %); ¹H NMR δ (300 MHz, CDCl₃): 8.65 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.36 (s, 1H, CH=N), 8.31 (s, 1H, CH=N), 8.29 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.91 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.85 (d, 2H, *J* = 8.10 Hz, H-2''', H-6'''), 7.80 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.57 (d, 1H, *J* = 3.60 Hz, H-6), 7.55 (t, 2H, *J* = 8.10 Hz, H-3''' and H-5'''), 7.38 (t, 1H, *J* = 8.10 Hz, H-4'''), 6.94 (d, 1H, *J* = 3.60 Hz, H-5), 3.67-3.63 (m, 4H, 2NCH₂), 2.54-2.36 (m, 20H, 10NCH₂), 2.26 (s, 3H, NCH₃), 2.25 (s, 3H,
NCH₃), 1.82-1.66 (m, 4H, 2CH₂), 1.62-1.50 (m, 4H, 2CH₂).

4.1.6.8. **2,4-Bis{4-[(5-methylhexyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16h).** Yellow oil (79 %); ¹H NMR δ (300 MHz, CDCl₃): 8.70 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.41 (s, 1H, CH=N), 8.37 (s, 1H, CH=N), 8.34 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.97 (d, 2H, *J* = 8.10 Hz, H-3' and H-5'), 7.90 (d, 2H, *J* = 8.10 Hz, H-2'', H-6'''), 7.86 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5''), 7.63 (d, 1H, *J* = 3.90 Hz, H-6), 7.61 (t, 2H, *J* = 8.10 Hz, H-3'' and H-5''), 7.44 (t, 1H, *J* = 7.40 Hz, H-4''), 6.99 (d, 1H, *J* = 3.90 Hz, H-5), 3.71 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.67 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.34 (t, 2H, *J* = 7.20 Hz, NCH₂), 1.79-1.62 (m, 4H, 2CH₂), 1.63-1.54 (m, 2H, 2CH), 1.46-1.36 (m, 4H, 2CH₂), 1.32-1.22 (m, 4H, 2CH₂), 0.92 (d, 6H, *J* = 6.60 Hz, 2CH₃), 0.91 (d, 6H, *J* = 6.60 Hz, 2CH₃).

4.1.7. **4,6-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-1-phenyl-1H-pyrrolo[2,3-b]pyridine (17)**

To a solution of the appropriate diamine (0.407 mmol, 2.15 eq.) in ethanol (6 mL) was added the 4,6-bis(4-formylphenyl)-1-phenyl-1H-pyrrolo[2,3-b]pyridine **16** (0.19 mmol). The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (40 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification. Yellow oil (78 %); ¹H NMR δ (300 MHz, CDCl₃): 8.39 (s, 1H, CH=N), 8.34 (s, 1H, CH=N), 8.21 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.96-7.92 (m, 2H, H-2'', H-6''), 7.93 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.86 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.82 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.78 (s, 1H, H-5), 7.64 (d, 2H, *J* = 3.60 Hz, H-2), 7.57 (t, 2H, *J* = 7.80 Hz, H-3'' and H-5''), 7.37 (t, 1H, *J* = 7.80 Hz, H-4''), 6.82 (d, 1H, *J* = 3.60 Hz, H-3), 3.73-3.65 (m, 4H, 2NCH₂), 2.35 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.31 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.83-1.72 (m, 4H, 2CH₂), 1.63-1.54 (m, 4H, 2CH₂).

4.1.8. **General procedure for 4,6-bis{4-[(4-dialkylaminoalkyl)iminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (18a-c)**

To a solution of the appropriate diamines (0.552 mmol, 2.15 eq.) in ethanol (11 mL) was added the 4,6-bis(4-formylphenyl)-1-phenyl-1H-pyrrolo[3,2-c]pyridine **15** (0.26 mmol). The reaction mixture was then heated under reflux for 14 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (25 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

4.1.8.1. **4,6-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (18a).** Yellow oil (87 %); ¹H NMR δ (300 MHz, CDCl₃): 8.40 (s, 1H, CH=N), 8.33 (s, 1H, CH=N), 8.22 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.19 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.93 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.86 (s, 1H, H-7), 7.81 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.63-7.52 (m, 4H, H-2'', H-3'', H-5'' and H-6''), 7.47 (t, 1H, *J* = 7.80 Hz, H-4''), 7.43 (d, 1H, *J* = 3.90 Hz, H-2), 7.00 (d, 1H, *J* = 3.90 Hz, H-3), 3.70 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.66 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.35 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.33 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.81-1.73 (m, 4H, 2CH₂), 1.64-1.54 (m, 4H, 2CH₂).

4.1.8.2. **4,6-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (18b).** Yellow oil (92 %); ¹H NMR δ (300 MHz, CDCl₃): 8.39 (s, 1H, CH=N), 8.32 (s, 1H, CH=N), 8.23 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.19 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.93 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.83 (s, 1H, H-7), 7.81 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.64-7.53 (m, 4H, H-2'', H-3'', H-5'' and H-6''), 7.47 (t, 1H, *J* = 7.85 Hz, H-4''), 7.44 (d, 1H, *J* = 3.90 Hz, H-2), 7.00 (d,

1H, *J* = 3.90 Hz, H-3), 3.68 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.65 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.30 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.28 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.82-1.73 (m, 4H, 2CH₂), 1.59-1.49 (m, 4H, 2CH₂), 1.47-1.36 (m, 4H, 2CH₂).

4.1.8.3. **4,6-Bis{4-[(4-diethylaminobutyl)iminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (18c).** Yellow oil (95 %); ¹H NMR δ (300 MHz, CDCl₃): 8.40 (s, 1H, CH=N), 8.33 (s, 1H, CH=N), 8.22 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.19 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6'), 7.93 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.86 (s, 1H, H-7), 7.81 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.63-7.53 (m, 4H, H-2'', H-3'', H-5'' and H-6''), 7.47 (t, 1H, *J* = 7.80 Hz, H-4''), 7.43 (d, 1H, *J* = 3.30 Hz, H-2), 7.00 (d, 1H, *J* = 3.30 Hz, H-3), 3.70 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.66 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.56 (q, 4H, *J* = 5.70 Hz, NCH₂), 2.52 (q, 4H, *J* = 5.70 Hz, NCH₂), 2.48 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.46 (t, 2H, *J* = 6.90 Hz, NCH₂), 1.79-1.70 (m, 4H, 2CH₂), 1.63-1.52 (m, 4H, 2CH₂), 1.04 (t, 6H, *J* = 5.70 Hz, 2CH₃), 1.00 (t, 6H, *J* = 5.70 Hz, 2CH₃).

4.1.9. **General procedure for 2,4-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1a-g)**

To a solution of 2,4-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine **16** (0.4 mmol) in methanol (10 mL) was added portion-wise at 0 °C sodium borohydride (3.2 mmol, 8 eq.). The reaction mixture was then stirred at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (40 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the 2,4-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines.

4.1.9.1. **2,4-Bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1a).** Pale-yellow oil (96 %); ¹H NMR δ (300 MHz, CDCl₃): 8.59 (d, 2H, *J* = 8.25 Hz, H-2' and H-6'), 8.24 (d, 2H, *J* = 8.25 Hz, H-2'' and H-6''), 7.89 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.59-7.52 (m, 5H, H-6, H-3', H-5', H-3'' and H-5''), 7.43 (t, 2H, *J* = 8.10 Hz, H-3'' and H-5''), 7.38 (t, 1H, *J* = 8.10 Hz, H-4''), 6.96 (d, 1H, *J* = 3.90 Hz, H-5), 3.91 (s, 2H, NCH₂), 3.86 (s, 2H, NCH₂), 2.73 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.69 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.35 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.32 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.77-1.64 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.6 (C-2), 158.9 (C-4), 154.0 (C-7a), 144.1 (C-4''), 143.5 (C-4'), 139.2 (C-1''), 139.0 (C-1'), 138.7 (C-1''), 130.7 (C-2'' and C-6''), 130.5 (C-2' and C-6'), 129.8 (C-3'', C-5'' and C-4''), 129.6 (C-3' and C-5), 129.5 (C-3' and C-5''), 127.9 (C-6), 125.1 (C-2'' and C-6''), 116.2 (C-4a), 103.6 (C-5), 59.5 (NCH₂), 55.2 (NCH₂), 49.3 (NCH₂), 49.2 (NCH₂), 47.0 (N(CH₃)₂), 29.4 (CH₂); ESI MS *m/z* [M+H]⁺ Calculed for C₃₆H₄₆N₇: 576.3814, Found: 576.3778.

4.1.9.2. **2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1b).** Yellow oil (67 %); ¹H NMR δ (300 MHz, CDCl₃): 8.57 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.21 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.84 (d, 2H, *J* = 7.80 Hz, H-2'' and H-6''), 7.56-7.49 (m, 5H, H-3', H-5', H-3'', H-5'' and H-6), 7.40 (d, 2H, *J* = 7.80 Hz, H-3'' and H-5''), 7.32 (t, 1H, *J* = 7.80 Hz, H-4''), 6.91 (d, 1H, *J* = 3.60 Hz, H-5), 3.87 (s, 2H, NCH₂), 3.83 (s, 2H, NCH₂), 2.68-2.61 (m, 4H, 2NCH₂), 2.28 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.26 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.20 (s, 6H, N(CH₃)₂), 2.17 (s, 6H, N(CH₃)₂), 1.54-1.49 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.5 (C-2), 158.8 (C-4), 153.9 (C-7a), 143.9 (C-4'), 143.3 (C-4'), 139.2 (C-1''), 139.0 (C-1'), 138.7 (C-1'), 130.6 (C-2'' and C-6''), 130.4 (C-2' and C-6'), 129.8 (C-3'', C-5'' and C-4''), 129.6 (C-3' and C-5'), 129.5 (C-3'' and C-5''), 127.9 (C-6), 125.0 (C-2'' and C-6''), 116.1 (C-4a), 103.6 (C-5), 61.0 (NCH₂), 55.1 (NCH₂), 50.6 (NCH₂), 50.5 (NCH₂), 46.8 (NCH₃), 29.3 (CH₂), 26.9 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₃₈H₅₀N₇: 604.4128, Found: 604.4089.
4.1.9.3. **2,4-Bis{4-[(5-dimethylaminopentyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1c).** Pale-yellow oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 8.56 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.19 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.83 (d, 2H, *J* = 8.10 Hz, H-2''' and H-6'''), 7.55-7.48 (m, 5H, H-3', H-5', H-3'', H-5'' and H-6), 7.41-7.33 (m, 3H, H-3'', H-4'' and H-5''), 6.90 (d, 1H, *J* = 3.60 Hz, H-5), 3.86 (s, 2H, NCH₂), 3.82 (s, 2H, NCH₂), 2.67-2.58 (m, 4H, 2NCH₂), 2.18 (s, 6H, N(CH₂)₂), 2.17 (s, 6H, N(CH₂)₂), 1.56-1.41 (m, 8H, 4CH₂), 1.36-1.28 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.5 (C-2), 158.8 (C-4), 153.9 (C-7a), 144.0 (C-4'), 143.5 (C-4'), 139.2 (C-1''), 138.9 (C-1'), 138.6 (C-1'), 130.6 (C-2'' and C-6'), 130.4 (C-2' and C-6), 129.8 (C-3'', C-5'' and C-4''), 129.6 (C-3' and C-5'), 129.5 (C-3'' and C-5'), 127.9 (C-6), 125.0 (C-2''' and C-6''), 116.1 (C-4a), 103.6 (C-5), 61.1 (NCH₂), 55.2 (NCH₂), 50.8 (NCH₂), 50.7 (NCH₂), 46.9 (N(CH₃)₂), 31.4 (CH₂), 29.0 (CH₂), 26.6 (CH₂); ESI MS *m/z* [M+H]⁺ Calculed for C₄₀H₅₆N₇: 632.4441, Found: 632.4437.

4.1.9.4. **2,4-Bis{4-[(4-diethylaminobutyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1d).** Yellow oil (78 %); ¹H NMR δ (300 MHz, CDCl₃): 8.61 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.27 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.93 (d, 2H, *J* = 7.80 Hz, H-2''' and H-6'''), 7.64-7.56 (m, 5H, H-3', H-5', H-3'', H-5'' and H-6), 7.46 (d, 2H, *J* = 7.80 Hz, H-3''' and H-5'''), 7.43 (t, 1H, *J* = 7.80 Hz, H-4''), 7.01 (d, 1H, *J* = 3.60 Hz, H-5), 3.95 (s, 2H, NCH₂), 3.90 (s, 2H, NCH₂), 2.76-2.66 (m, 4H, 2NCH₂), 2.57 (q, 4H, *J* = 7.20 Hz, 2NCH₂), 2.55 (q, 4H, *J* = 7.20 Hz, 2NCH₂), 2.50-2.45 (m, 4H, 2NCH₂), 1.60-1.52 (m, 8H, 4CH₂), 1.06 (t, 6H, *J* = 7.20 Hz, 2CH₃), 1.03 (t, 6H, *J* = 7.20 Hz, 2CH₃); ¹³C NMR δ (75 MHz, CDCl₃): 159.6 (C-2), 158.9 (C-4), 154.0 (C-7a), 144.0 (C-4''), 143.4 (C-4'), 139.2 (C-1''), 139.0 (C-1'), 138.7 (C-1'), 130.7 (C-2'' and C-6''), 130.5 (C-2' and C-6'), 129.8 (C-3'', C-5'' and C-4''), 129.6 (C-3' and C-5'), 129.5 (C-3'' and C-5'), 127.9 (C-6), 125.1 (C-2'' and C-6''), 116.2 (C-4a), 103.6 (C-5), 55.2 (NCH₂), 54.2 (NCH₂), 50.7 (NCH₂), 50.6 (NCH₂), 48.2 (NCH₂), 29.6 (CH₂), 26.2 (CH₂), 13.0 (CH₃); ESI MS *m/z* [M+2H]⁺ Calculated for C₄₂H₅₉N₇: 661.4831, Found: 661.4852.

4.1.9.5. **2,4-Bis{4-[(5-diisopropylaminopentyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1e).** Yellow oil (75 %); ¹H NMR δ (300 MHz, CDCl₃): 8.61 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.26 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.92 (d, 2H, *J* = 8.10 Hz, H-2''' and H-6'''), 7.63-7.54 (m, 5H, H-3', H-5', H-3'', H-5'' and H-6), 7.46-7.38 (m, 3H, H-3'', H-4'' and H-5''), 7.00 (d, 1H, *J* = 3.90 Hz, H-5), 3.94 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 3.10-3.00 (m, 4H, 4NCH), 2.73-2.63 (m, 4H, 2NCH₂), 2.45-2.37 (m, 4H, 2NCH₂), 1.61-1.53 (m, 4H, 2CH₂), 1.49-1.39 (m, 4H, 2CH₂), 1.37-1.28 (m, 4H, 2CH₂), 1.04 (d, 12H, *J* = 6.50 Hz, 4CH₃), 1.03 (d, 12H, *J* = 6.50 Hz, 4CH₃); ¹³C NMR δ (75 MHz, CDCl₃): 159.6 (C-2), 158.9 (C-4), 154.0 (C-7a), 144.1 (C-4''), 143.6 (C-4'), 139.2 (C-1''), 139.0 (C-1'), 138.7 (C-1'), 130.7 (C-2'' and C-6''), 130.5 (C-2' and C-6'), 129.8 (C-3'', C-5'' and C-4''), 129.6 (C-3' and C-5'), 129.5 (C-3'' and C-5'), 127.9 (C-6), 125.1 (C-2'' and C-6''), 116.1 (C-4a), 103.6 (C-5), 55.3 (NCH₂), 50.1 (NCH₂), 50.8 (NCH₂), 50.0 (NCH), 46.8 (NCH₂), 32.8 (CH₂), 31.5 (CH₂), 26.6 (CH₂), 22.0 CH₃); ESI MS *m/z* [M+H]⁺ Calculated for C₄₈H₇₀N₇: 744.5693, Found: 744.5674.

4.1.9.6. **2,4-Bis{4-[(3-(4-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1f).** Yellow oil (84 %); ¹H NMR δ (300 MHz, CDCl₃): 8.55 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.19 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.83 (d, 2H, *J* = 8.50 Hz, H-2''' and H-6'''), 7.53-7.46 (m, 5H, H-3', H-5', H-3'', H-5'' and H-6), 7.38 (d, 2H, *J* = 8.40 Hz, H-3''' and H-5''), 7.29 (t, 1H, *J* = 8.50 Hz, H-4''), 6.89 (d, 1H, *J* = 3.90 Hz, H-5), 3.84 (s, 2H, NCH₂), 3.80 (s, 2H, NCH₂), 2.67 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.64 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.41-2.35 (m, 20H, 10NCH₂), 2.23 (s, 3H, NCH₃), 2.21 (s, 3H, NCH₃), 1.71-1.64 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.5 (C-2), 158.8 (C-4), 153.9 (C-7a), 144.0 (C-4''), 143.5 (C-4'), 139.2 (C-1''), 138.9 (C-1'), 138.6 (C-1'), 130.6 (C-2'' and C-6'), 130.4 (C-2' and C-6'), 129.7 (C-3'', C-5'' and C-4''),

129.6 (C-3' and C-5'), 129.4 (C-3'' and C-5'), 127.9 (C-6), 125.0 (C-2''' and C-6''), 116.1 (C-4a), 103.6 (C-5), 58.3 (NCH₂), 56.5 (NCH₂), 55.1 (NCH₂), 54.6 (NCH₂), 49.5 (NCH₂), 49.4 (NCH₂), 47.4 (NCH₃), 28.3 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₂H₅₆N₆: 686.4658, Found: 686.4621.

4.1.9.7. **2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1g).** Yellow oil (76 %); ¹H NMR δ (300 MHz, CDCl₃): 8.59 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.25 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.90 (d, 2H, *J* = 7.80 Hz, H-2''' and H-6'''), 7.62-7.53 (m, 5H, H-3', H-5', H-3'', H-5'' and H-6), 7.43 (t, 2H, *J* = 7.80 Hz, H-3''' and H-5''), 7.40 (t, 1H, *J* = 7.8 Hz, H-4''), 6.99 (d, 1H, *J* = 3.60 Hz, H-5), 3.92 (s, 2H, NCH₂), 3.87 (s, 2H, NCH₂), 2.72-2.66 (m, 4H, 2NCH₂), 2.56-2.30 (m, 20H, 10NCH₂), 2.28 (s, 3H, NCH₃), 2.27 (s, 3H, NCH₃), 1.58-1.52 (m, 8H, 4CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₄H₆₁N₉: 715.5050, Found: 715.5823.

4.1.9.8. **2,4-Bis{4-[(5-methylhexyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1h).** Pale-yellow oil (91 %); ¹H NMR δ (300 MHz, CDCl₃): 1H NMR δ (300 MHz, CDCl₃): 8.62 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.27 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.91 (d, 2H, *J* = 7.90 Hz, H-2''' and H-6''), 7.63-7.55 (m, 5H, H-3', H-5', H-3'', H-5'' and H-6), 7.46 (d, 2H, *J* = 7.90 Hz, H-3''' and H-5''), 7.41 (t, 1H, *J* = 7.90 Hz, H-4''), 6.98 (d, 1H, *J* = 3.60 Hz, H-5), 3.94 (s, 2H, NCH₂), 3.89 (s, 2H, NCH₂), 2.71 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.67 (t, 2H, *J* = 7.20 Hz, NCH₂), 1.60-1.47 (m, 8H, 4CH₂), 1.42-1.30 (m, 2H, 2CH), 1.26-1.17 (m, 4H, 2CH₂), 0.91 (d, 6H, *J* = 6.60 Hz, 2CH₃), 0.88 (d, 6H, *J* = 6.60 Hz, 2CH₃); ¹³C NMR δ (75 MHz, CDCl₃): 159.7 (C-2), 159.0 (C-4), 154.0 (C-7a), 144.2 (C-4'), 143.6 (C-4'), 139.3 (C-1''), 139.0 (C-1'), 138.7 (C-1'), 130.7 (C-2'' and C-6''), 130.5 (C-2' and C-6'), 129.8 (C-3'', C-5'' and C-4''), 129.6 (C-3' and C-5'), 129.5 (C-3'' and C-5''), 128.0 (C-6), 125.1 (C-2'' and C-6''), 116.2 (C-4a), 103.7 (C-5), 55.3 (NCH₂), 51.0 (NCH₂), 50.8 (NCH₂), 40.3 (CH₂), 31.8 (CH₂), 29.4 (CH), 26.6 (CH₂), 24.0 (CH₃); ESI MS *m/z* [M+H]⁺ Calculated for C₄₀H₅₂N₅: 602.4223, Found: 602.4216.

4.1.10. **4,6-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[2,3-b]pyridine (2)**

To a solution of 4,6-bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-1-phenyl-1H-pyrrolo[2,3-b]pyridine **17** (0.15 mmol) in methanol (7 mL) was added portion-wise at 0 °C sodium borohydride (1.2 mmol, 8 eq.). The reaction mixture was then stirred at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (20 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the pyrrolo[2,3-b]pyridine **2**. Yellow oil (95 %); ¹H NMR δ (300 MHz, CDCl₃): 8.12 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.96 (d, 2H, *J* = 7.50 Hz, H-2'', H-6''), 7.78 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6'), 7.73 (s, 1H, H-5), 7.61 (d, 2H, *J* = 3.60 Hz, H-2), 7.57 (t, 2H, *J* = 7.50 Hz, H-3'' and H-5'), 7.52 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.43 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5'), 7.34 (t, 1H, *J* = 7.80 Hz, H-4''), 6.83 (d, 1H, *J* = 3.60 Hz, H-3), 3.92 (s, 2H, NCH₂), 3.87 (s, 2H, NCH₂), 2.77-2.65 (m, 4H, 2NCH₂), 2.30 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.28 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.60-1.53 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 153.1 (C-6), 152.4 (C-4), 152.2 (C-7a), 152.0 (C-1''), 144.7 (C-4'), 142.2 (C-4'), 140.2 (C-1'), 140.0 (C-1''), 130.6 (C-2'' and C-6''), 130.0 (C-2', C-6', C-3'' and C-5''), 129.8 (C-3' and C-5'), 129.4 (C-4''), 128.5 (C-3'' and C-5'), 127.3 (C-2), 125.0 (C-2'' and C-6''), 119.9 (C-3a), 114.5 (C-5), 102.7 (C-3), 61.0 (NCH₂), 55.1 (NCH₂), 50.8 (NCH₂), 50.6 (NCH₂), 46.9 (NCH₃), 29.4 (CH₂), 27.0 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₃₉H₅₁N₆: 603.4175, Found: 603.4158.

4.1.11. **General procedure for 4,6-bis{4-[(4-dialkylaminoalkyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (3a-c)**

To a solution of 4,6-bis[(substituted-iminomethyl)-1-phenyl-1H-
pyrrolo[3,2-c]pyridine **18** (0.22 mmol) in methanol (8 mL) was added portion-wise at 0 °C sodium borohydride (1.76 mmol, 8 eq.). The reaction mixture was then stirred at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (20 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the 4,6-bis[(substituted-aminomethyl)-1-phenyl-1H-pyrrolo[3,2-d]pyridines.

**4.1.11.1. 4,6-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (3a).** Pale-yellow oil (83 %); ¹H NMR δ (300 MHz, CDCl₃): 8.14 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.09 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.80 (s, 1H, H-7), 7.61-7.39 (m, 10H, H-3', H-5', H-3'', H-5'', H-2'', H-3'', H-4'', H-5'', H-6'' and H-2), 7.00 (d, 1H, *J* = 3.95 Hz, H-3), 3.92 (s, 2H, NCH₂), 3.85 (s, 2H, NCH₂), 2.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.66 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.28 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.26 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.57-1.50 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 151.9 (C-6), 150.1 (C-4), 142.0 (C-1''), 140.9 (C-1'), 140.2 (C-7a), 139.5 (C-1'), 139.2 (C-4'), 138.9 (C-4'), 129.9 (C-3'' and C-5''), 129.5 (C-4''), 129.1 (C-2'' and C-6''), 128.4 (C-2' and C-6'), 128.3 (C-3'' and C-5''), 127.4 (C-2), 127.0 (C-3' and C-5'), 124.6 (C-2'' and C-6''), 122.4 (C-7), 103.8 (C-3a), 100.7 (C-3), 59.8 (NCH₂), 53.9 (NCH₂), 53.7 (NCH₂), 49.3 (NCH₂), 49.2 (NCH₂), 45.5 (N(CH₃)₂), 45.4 (N(CH₃)₂) 28.1 (CH₂), 28.0 (CH₂), 25.6 (CH₂); ESI MS *m/z* [M+H]⁺ Calculed for C₃₉H₅₁N₆: 603.42, Found: 603.42.

**4.1.11.2. 4,6-Bis{4-[(5-dimethylaminopentyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (3b).** White crystals (88 %); mp < 40 °C; ¹H NMR δ (300 MHz, CDCl₃): 8.13 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.09 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.79 (s, 1H, H-7), 7.61-7.37 (m, 10H, H-3', H-5', H-3'', H-5'', H-2'', H-3'', H-4'', H-5'', H-6'' and H-2), 6.99 (d, 1H, *J* = 4.00 Hz, H-3), 3.90 (s, 2H, NCH₂), 3.83 (s, 2H, NCH₂), 2.70 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.64 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.29 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.24 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.20 (s, 6H, N(CH₃)₂), 1.61-1.31 (m, 12H, 6CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 151.9 (C-6), 150.1 (C-4), 141.9 (C-1''), 140.9 (C-1'), 140.3 (C-7a), 139.5 (C-1'), 139.2 (C-4'), 138.8 (C-4'), 129.9 (C-3'' and C-5''), 129.5 (C-4''), 129.1 (C-2'' and C-6''), 128.4 (C-2' and C-6'), 128.3 (C-3'' and C-5''), 127.4 (C-2), 127.0 (C-3' and C-5'), 124.6 (C-2'' and C-6''), 122.4 (C-7), 103.8 (C-3a), 100.7 (C-3), 59.8 (NCH₂), 54.0 (NCH₂), 53.9 (NCH₂), 49.3 (NCH₂), 45.5 (N(CH₃)₂), 30.1 (CH₂), 27.7 (CH₂), 25.2 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₁H₅₅N₆: 631.45, Found: 632.45.

**4.1.11.3. 4,6-Bis{4-[(4-diethylaminobutyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (3c).** Yellow oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 8.12 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.09 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.79 (s, 1H, H-7), 7.60-7.37 (m, 10H, H-3', H-5', H-3'', H-5'', H-2'', H-3'', H-4'', H-5'', H-6'' and H-2), 7.00 (d, 1H, *J* = 4.00 Hz, H-3), 3.90 (s, 2H, NCH₂), 3.84 (s, 2H, NCH₂), 2.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.64 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.54 (q, 4H, *J* = 5.65 Hz, NCH₂), 2.52 (q, 4H, *J* = 5.65 Hz, NCH₂), 2.45 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.42 (t, 2H, *J* = 6.90 Hz, NCH₂), 1.56-1.48 (m, 8H, 4CH₂), 1.03 (t, 6H, *J* = 5.65 Hz, 2CH₃), 1.00 (t, 6H, *J* = 5.65 Hz, 2CH₃); ¹³C NMR δ (75 MHz, CDCl₃): 151.9 (C-6), 150.1 (C-4), 142.0 (C-1''), 141.0 (C-1'), 140.2 (C-7a), 139.5 (C-1'), 139.2 (C-4''), 138.9 (C-4'), 129.9 (C-3'' and C-5''), 129.5 (C-4''), 129.1 (C-2'' and C-6''), 128.3 (C-2' and C-6'), 128.2 (C-3'' and C-5''), 127.4 (C-2), 127.0 (C-3' and C-5'), 124.6 (C-2'' and C-6''), 122.4 (C-7), 103.8 (C-3a), 100.7 (C-3), 53.8 (NCH₂), 53.7 (NCH₂), 53.4 (NCH₂), 52.9 (NCH₂), 52.8 (NCH₂), 46.9 (NCH₂), 46.8 (NCH₂), 28.3 (CH₂), 28.2 (CH₂), 24.9 (CH₂), 24.8 (CH₂), 11.7 (CH₃); ESI MS *m/z* [M+H]⁺ Calculated for C₄₃H₅₉N₆: 659.48, Found: 659.48.

**4.1.12. 2,4-Dichloro-7-(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine (19)**

2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (5 mmol), 4-formylphenylboronic acid (6 mmol, 1.2 equiv.) and copper (II) acetate (5 mmol) in methanol (75 mL) was stirred at room temperature for 4 h. Then, the reaction mixture was evaporated to dryness. The residue was extracted with chloroform, filtered and evaporated under reduced pressure. The residue was then purified by silica gel column chromatography (CHCl₃), then cooled and triturated in EtOH, filtered on sintered glassware, washed with a minimum of EtOH, then EtO₂ and petroleum ether and dried under pressure to give the solid product. White crystals (36 %); Mp = 224 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.11 (s, 1H, CHO), 8.11 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.97 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.64 (d, 1H, *J* = 3.90 Hz, H-6), 6.87 (d, 1H, *J* = 3.90 Hz, H-5).

**4.1.13. 2-Chloro-7-(4-formylphenyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (20)**

To a solution of 4.0 mmol of 2,4-dichloro-7-(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine **19**, 4.4 mmol of phenylboronic acid (1.1 eq.) and 0.40 mmol (0.05 eq.) of tetrakis(triphenylphosphine) palladium in 15 mL of 1,2-dimethoxyethane, 2.5 mL of 2 M Na₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The solvent was then evaporated under vacuum. The residue was extracted with CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with water (15 mL x 2 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and EtO₂ and filtered on sintered glassware to give the crude product. The residue was purified by silica gel column chromatography (CH₂Cl₂), then cooled and triturated again in EtOH, filtered on sintered glassware, washed with a minimum of EtOH, EtO₂ and petroleum ether and dried under pressure to give the solid product. White crystals (72 %) Mp = 175 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.10 (s, 1H, CHO), 8.15-8.10 (m, 2H, H-2' and H-6'), 8.08 (d, 2H, *J* = 8.40 Hz, H-2'', H6''), 8.02 (d, 2H, *J* = 8.40 Hz, H-3'' and H5''), 7.64 (d, 1H, *J* = 3.90 Hz, H-6), 7.61-7.55 (m, 3H, H-3', H-4' and H-5'), 7.05 (d, *J* = 3.90 Hz, H-5).

**4.1.14. 2,7-Bis(4-formylphenyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (21)**

To a solution of 8.0 mmol of 2-chloro-4-phenyl-7-(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine **19**, 143 mg of 4-formylphenyl boronic acid (0.96 mmol, 1.2 eq.) and 46 mg (0.04 mmol, 0.05 eq.) of tetrakis (triphenylphosphine) palladium in 10 mL of 1,2-dimethoxyethane, 1.5 mL of 2 M Na₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with water (12 mL x 3 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and EtO₂ and filtered on sintered glassware to give the crude product. The residue was purified by silica gel column chromatography (CH₂Cl₂) to give the solid product. White crystals (63 %); Mp > 260 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.14 (s, 1H, CHO), 10.13 (s, 1H, CHO), 8.83 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.29 (dd, 2H, *J* = 7.80 and 1.65 Hz, H-2'' and H-6''), 8.21 (d, 2H, *J* = 8.85 Hz, H-2'' and H-6''), 8.16 (d, 2H, *J* = 8.85 Hz, H-3'' and H-5''), 8.04 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.74 (d, 1H, *J* = 3.90 Hz, H-6), 7.66-7.61 (m, 3H, H-3', H-4'' and H-5''), 7.13 (d, 1H, *J* = 3.90 Hz, H-5).
### 4.1.15. General procedure for 2,7-bis[(substituted-iminomethyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidines (22a-b)

To a solution of the appropriate diamines (0.126 mmol, 2.1 eq.) in ethanol (7 mL) was added the 2,7-bis(4-formylphenyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine 21 (0.6 mmol). The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (40 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

#### 4.1.15.1. 2,7-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (22a).
Yellow oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.34 (s, 1H, CH=N), 8.33 (s, 1H, CH=N), 8.23 (dd, 2H, *J* = 7.80 and 1.70 Hz, H-2'' and H-6''), 7.94 (d, 2H, *J* = 8.70 Hz, H-3''' and H-5'''), 7.82 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.59-7.50 (m, 4H, H-6, H-3', H-4'' and H-5'), 6.93 (d, 1H, *J* = 3.60 Hz, H-5), 3.67 (t, 4H, *J* = 6.90 Hz, 2NCH₂), 2.36-2.30 (m, 4H, 2NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.79-1.72 (m, 4H, 2CH₂), 1.61-1.55 (m, 4H, 2CH₂).

#### 4.1.15.2. 2,7-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (22b).
Yellow oil (95 %); ¹H NMR δ (300 MHz, CDCl₃): 8.68 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.36 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.26 (dd, 2H, *J* = 7.80 and 1.70 Hz, H-2'' and H-6''), 7.99 (d, 2H, *J* = 8.80 Hz, H-2''' and H-6'''), 7.94 (d, 2H, *J* = 8.80 Hz, H-3''' and H-5'''), 7.84 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.64-7.55 (m, 4H, H-6, H-3', H-4'' and H-5'), 7.00 (d, 1H, *J* = 3.60 Hz, H-5), 3.68 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.66 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.33-2.26 (m, 4H, 2NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.82-1.75 (m, 4H, 2CH₂), 1.60-1.49 (m, 4H, 2CH₂), 1.47-1.38 (m, 4H, 2CH₂).

### 4.1.16. General procedure for 2,7-bis[(substituted-aminomethyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (4a-b)

To a solution of 2,4-bis[(substituted-iminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine 22 (0.4 mmol) in methanol (10 mL) was added portion-wise at 0 °C sodium borohydride (3.2 mmol, 8 eq.). The reaction mixture was then stirred at room temperature for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (40 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the 2,7-bis [(substituted-aminomethyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine 4.

#### 4.1.16.1. 2,7-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (4a).
Pale-yellow oil (82 %); ¹H NMR δ (300 MHz, CDCl₃): 8.61 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.28 (dd, 2H, *J* = 8.10 and 1.60 Hz, H-2'' and H-6''), 7.86 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.63-7.52 (m, 6H, H-6, H-3', H-4', H-5', H-3''' and H-5'''), 7.44 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 6.98 (d, 1H, *J* = 3.60 Hz, H-5), 3.90 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.78-2.66 (m, 4H, 2NCH₂), 2.34-2.24 (m, 4H, 2NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.60-1.48 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.5 (C-2), 159.0 (C-4), 153.9 (C-7a), 143.2 (C-1'''), 140.2 (C-4'), 140.0 (C-4'''), 139.0 (C-1''), 138.0 (C-1'), 131.4 (C-4'''), 130.4 (C-3'', C-5'', C-3''' and C-5'''), 130.1 (C-3' and C-5'), 129.9 (C-6), 129.6 (C-2', C-6', C-2'' and C-6''), 125.0 (C-2''' and C-6'''), 116.2 (C-4a), 103.5 (C-5), 61.0 (NCH₂), 55.1 (NCH₂), 54.9 (NCH₂), 50.8 (NCH₂), 50.5 (NCH₂), 46.8 (NCH₂), 29.3 (CH₂), 26.9 (CH₂); ESI MS *m/z* [M+H]⁺ Calculed for C₃₈H₅₀N₇: 604.4128, Found: 604.4165.

#### 4.1.16.2. 2,7-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (4b).
Pale-yellow oil (91 %); ¹H NMR δ (300 MHz, CDCl₃): 8.61 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.29

(dd, 2H, *J* = 8.10 and 1.60 Hz, H-2'' and H-6''), 7.86 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.64-7.53 (m, 6H, H-6, H-3', H-4'', H-5'', H-3''' and H-5'''), 7.44 (d, 2H, *J* = 8.10 Hz, H-3' and H-5'), 7.00 (d, 1H, *J* = 3.90 Hz, H-5), 3.91 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.76-2.63 (m, 4H, 2NCH₂), 2.34-2.24 (m, 4H, 2NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.24 (s, 6H, N(CH₃)₂), 1.63-1.33 (m, 12H, 6CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.6 (C-2), 159.0 (C-4), 154.0 (C-7a), 143.5 (C-1'''), 140.4 (C-4'), 140.0 (C-4'''), 139.0 (C-1'), 137.9 (C-1'), 131.4 (C-4''), 130.4 (C-3'', C-5'', C-3''' and C-5''), 130.1 (C-3' and C-5'), 129.6 (C-6), 129.5 (C-2', C-6', C-2'' and C-6''), 125.0 (C-2''' and C-6'''), 116.2 (C-4a), 103.5 (C-5), 61.1 (NCH₂), 55.2 (NCH₂), 54.9 (NCH₂), 50.9 (NCH₂), 50.6 (NCH₂), 46.9 (NCH₃), 31.4 (CH₂), 29.0 (CH₂), 26.6 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₀H₅₄N₇: 632.4440, Found: 632.4421.

### 4.1.17. 4,5-Bis(4-formylphenyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (24)

To a solution of 0.6 mmol of 4-chloro-5-iodo-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine 23, 0.27 g of 4-formylphenyl boronic acid (1.81 mmol, 3.0 eq.) and 70 mg (0.06 mmol, 0.1 eq.) of tetrakis(triphenylphosphine) palladium in 13 mL of a mixture dioxane/H₂O (4/1 - v/v), 192 mg (1.81 mmol, 3.0 eq.) of Na₂CO₃ were added at room temperature. Then, the mixture was warmed at 90 °C and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with warm CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with water (20 mL), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and filtered on sintered glassware to give the crude product that was then washed with a minimum of EtOH, EtO₂ and petroleum ether and dried under pressure to give the solid product. Pale-yellow crystals (68 %); Mp = 201 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.01 (s, 1H, CHO), 9.96 (s, 1H, CHO), 9.15 (s, 1H, H-2), 7.84-7.81 (m, 2H, H-2'''' and H-6'''), 7.77 (s, 1H, H-6), 7.73 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.69-7.62 (m, 6H, H-2'', H-6'', H-3', H-5', H-3'' and H-5''), 7.50 (t, 1H, *J* = 7.90 Hz, H-4''').

### 4.1.18. General procedure for 4,5-bis[(substituted-iminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines (25a-b)

To a solution of the appropriate diamines (0.88 mmol, 2.15 eq.) in ethanol (15 mL) was added the 4,5-bis(4-formylphenyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine 24 (0.41 mmol). The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (30 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

#### 4.1.18.1. 4,5-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (25a).
Yellow oil (97 %); ¹H NMR δ (300 MHz, CDCl₃): 9.05 (s, 1H, H-2), 8.19 (s, 1H, CH=N), 8.17 (s, 1H, CH=N), 7.77 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.62 (s, 1H, H-6), 7.56 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.53-7.51 (m, 4H, H-2''', H-3''', H-5'''' and H-6''), 7.47 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.42 (t, 1H, *J* = 7.85 Hz, H-4'''), 7.08 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 3.63-3.57 (m, 4H, 2NCH₂), 2.29 (t, 4H, *J* = 6.90 Hz, 2NCH₂), 2.20 (s, 12H, 2 N(CH₃)₂), 1.73-1.66 (m, 4H, 2CH₂), 1.58-1.50 (m, 4H, 2CH₂).

#### 4.1.18.2. 4,5-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (25b).
Yellow oil (93 %); ¹H NMR δ (300 MHz, CDCl₃): 9.05 (s, 1H, H-2), 8.19 (s, 1H, CH=N), 8.17 (s, 1H, CH=N), 7.77 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.62 (s, 1H, H-6), 7.57 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6'), 7.55-7.50 (m, 4H, H-2'', H-3'', H-5'' and H-6''), 7.46 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.43 (t, 1H, *J* = 7.80 Hz, H-4'''), 7.08 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 3.62-3.55 (m, 4H,
2NCH₂), 2.25 (t, 4H, *J* = 6.90 Hz, 2NCH₂), 2.20 (s, 12H, 2 N(CH₃)₂), 1.69 (qt, 4H, *J* = 6.90 Hz, 2CH₂), 1.51 (qt, 4H, *J* = 6.90 Hz, 2CH₂), 1.42-1.34 (m, 4H, 2CH₂).

### 4.1.19. General procedure for 4,5-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines (5a-b)

To a solution of 4,5-bis[(substituted-iminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine 25 (0.4 mmol) in methanol (12 mL) was added portion-wise at 0 °C sodium borohydride (3.2 mmol, 8 eq.). The reaction mixture was then stirred at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (40 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the 4,5-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines.

#### 4.1.19.1. 4,5-Bis{[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (5a).
Pale-yellow oil (81 %); ¹H NMR δ (300 MHz, CDCl₃): 9.00 (s, 1H, H-2), 7.75 (d, 2H, *J* = 7.80 Hz, H-2'' and H-6''), 7.53 (s, 1H, H-6), 7.51 (t, 2H, *J* = 7.80 Hz, H-3'' and H-5''), 7.36 (t, 1H, *J* = 7.80 Hz, H-4''), 7.34 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.05 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.02 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 6.94 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 3.70 (s, 2H, NCH₂), 3.69 (s, 2H, NCH₂), 2.60-2.51 (m, 4H, 2NCH₂), 2.26-2.21 (m, 4H, 2NCH₂), 2.17 (s, 12H, 2 N(CH₃)₂), 1.52-1.48 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 160.1 (C-4), 151.9 (C-2), 151.8 (C-7a), 141.6 (C-1''), 138.9 (C-4'), 137.3 (C-4'), 136.2 (C-1'), 132.3 (C-1'), 129.8 (C-3'' and C-5'') 129.5 (C-3' and C-5'), 129.2 (C-3'' and C-5''), 127.5 (C-2' and C-6'), 127.2 (C-2'' and C-6'), 127.1 (C-5), 126.8 (C-4''), 124.2 (C-2'' and C-6''), 118.4 (C-6), 114.7 (C-4a), 59.7 (NCH₂), 53.6 (NCH₂), 53.4 (NCH₂), 49.2 (NCH₂), 49.0 (NCH₂), 45.5 (N(CH₃)₂), 28.0 (CH₂), 25.6 (CH₂); ESI MS *m/z* [M+H]⁺ Calculed for C₃₈H₅₀N₇: 604.4128, Found: 604.4115.

#### 4.1.19.2. 4,5-Bis{[(5-dimethylaminopentyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (5b).
Pale-yellow oil (96 %); ¹H NMR δ (300 MHz, CDCl₃): 8.98 (s, 1H, H-2), 7.73 (d, 2H, *J* = 7.85 Hz, H-2'' and H-6''), 7.51 (s, 1H, H-6), 7.49 (t, 2H, *J* = 7.85 Hz, H-3'' and H-5''), 7.36 (t, 1H, *J* = 7.85 Hz, H-4''), 7.32 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.03 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 6.99 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 6.91 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 3.67 (s, 2H, NCH₂), 3.66 (s, 2H, NCH₂), 2.59-2.49 (m, 4H, 2NCH₂), 2.19 (t, 4H, *J* = 6.90 Hz, 2NCH₂), 2.15 (s, 12H, 2 N(CH₃)₂), 1.53-1.41 (m, 8H, 4CH₂), 1.35-1.27 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 160.1 (C-4), 151.9 (C-2), 151.7 (C-7a), 141.6 (C-1''), 138.9 (C-4'), 137.3 (C-4''), 136.2 (C-1''), 132.3 (C-1'), 129.9 (C-3'' and C-5''), 129.5 (C-3' and C-5'), 129.2 (C-3'' and C-5'), 127.5 (C-2' and C-6'), 127.3 (C-2'' and C-6''), 127.2 (C-5), 126.8 (C-4''), 124.2 (C-2'' and C-6''), 118.4 (C-6), 114.6 (C-4a), 59.7 (NCH₂), 53.7 (NCH₂), 49.2 (NCH₂), 49.1 (NCH₂), 45.5 (N(CH₃)₂), 30.1 (CH₂), 27.7 (CH₂), 25.2 (CH₂); ESI MS *m/z* [M+H]⁺ Calculed for C₄₀H₅₄N₇: 632.448, Found: 632.44.

### 4.1.20. 2,4-Bis(4-formylphenyl)-5-phenyl-5H-pyrrolo[3,2-d]pyrimidine (27)

To a solution of 0.35 g (1.34 mmol) of 2,4-dichloro-5-phenyl-5H-pyrrolo[3,2-d]pyrimidine 26, 0.60 g of 4-formylphenyl boronic acid (4.02 mmol, 3 eq.) and 155 mg (0.134 mmol, 0.1 eq.) of tetrakis(triphenylphosphine) palladium in 17 mL of a mixture dioxane/H₂O (4/1; v/v) 1,2-dimethoxyethane, were added at room temperature 0.43 g (4.02 mmol, 3 equiv.) of Na₂CO₃. Then, the reaction mixture was warmed at 90 °C and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with water (20 mL x 2 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum.

The residue was purified by silica gel column chromatography (CH₂Cl₂) to give the solid product. White crystals (52 %); Mp = 220 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.13 (s, 1H, CHO), 10.00 (s, 1H, CHO), 8.80 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.05 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.76 (d, 1H, *J* = 3.30 Hz, H-7), 7.65 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.55 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.22-7.20 (m, 3H, H-3''', H-4'' and H-5''), 7.08 (d, 1H, *J* = 3.30 Hz, H-6), 7.02 (dd, 2H, *J* = 7.50 and 0.90 Hz, H-2'' and H-6''').

### 4.1.21. 2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-5-phenyl-7H-pyrrolo[3,2-d]pyrimidine (28)

To a solution of the appropriate diamine (0.75 mmol, 2.15 eq.) in ethanol (10 mL) was added the 2,4-bis(4-formylphenyl)-5-phenyl-5H-pyrrolo[2,3-d]pyrimidine 27 (0.35 mmol). The reaction mixture was then heated under reflux for 24 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (30 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification. Yellow oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.35 (s, 1H, CH=N), 8.18 (s, 1H, CH=N), 7.84 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.65 (d, 1H, *J* = 3.30 Hz, H-7), 7.45 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.39 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.14-7.10 (m, 3H, H-3''', H-4'' and H-5''), 7.00-6.95 (m, 3H, H-2''', H-6'' and H-6), 3.69-3.59 (m, 4H, 2NCH₂), 2.33 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.29 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.75-1.69 (m, 4H, 2CH₂), 1.59-1.50 (m, 4H, 2CH₂).

### 4.1.22. 2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-5-phenyl-5H-pyrrolo[2,3-d]pyrimidine (6)

To a solution of 2,4-bis[(substituted-iminomethyl)-5-phenyl-5H-pyrrolo[2,3-d]pyrimidine 28 (0.35 mmol) in methanol (12 mL) was added portion-wise at 0 °C sodium borohydride (2.80 mmol, 8 eq.). The reaction mixture was then stirred at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (35 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the product. Pale-yellow oil (78 %); ¹H NMR δ (300 MHz, CDCl₃): 8.53 (d, 2H, *J* = 8.15 Hz, H-2' and H-6'), 7.60 (d, 1H, *J* = 3.30 Hz, H-7), 7.41 (d, 2H, *J* = 8.15 Hz, H-2'' and H-6''), 7.27 (d, 2H, *J* = 8.15 Hz, H-3' and H-5'), 7.10-7.07 (m, 3H, H-3''', H-4'' and H-5''), 7.01 (d, 2H, *J* = 8.15 Hz, H-3'' and H-5''), 6.95-6.91 (m, 3H, H-2''', H-6'' and H-6), 3.84 (s, 2H, NCH₂), 3.68 (s, 2H, NCH₂), 2.65 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.53 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.25-2.22 (m, 4H, 2NCH₂), 2.20 (s, 6H, N(CH₃)₂), 2.17 (s, 6H, N(CH₃)₂), 1.52-1.47 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 158.8 (C-2), 155.3 (C-4), 151.7 (C-4a), 142.9 (C-4''), 142.6 (C-4'), 140.6 (C-1''), 139.1 (C-1'), 138.2 (C-5), 136.7 (C-1''), 130.7 (C-2'' and C-6''), 130.0 (C-2' and C-6'), 129.5 (C-3''', C-5'' and C-4''), 128.5 (C-3' and C-5'), 128.3 (C-3'' and C-5'), 126.7 (C-2'' and C-6''), 125.1 (C-7a), 105.5 (C-6), 61.1 (NCH₂), 55.1 (NCH₂), 54.8 (NCH₂), 50.6 (NCH₂), 50.2 (NCH₂), 46.9 (NCH₃), 29.4 (CH₂), 26.9 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₃₈H₅₀N₇: 604.4128, Found: 604.4105.

### 4.1.23. 4,6-Bis(4-formylphenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (32)

To a solution of 2.26 mmol of 4,6-dichloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine 30, 0.75 g of 4-formylphenyl boronic acid (5.0 mmol, 2.2 eq.) and 260 mg (0.226 mmol, 0.1 eq.) of tetrakis(triphenylphosphine)palladium in 25 mL of 1,2-dimethoxyethane, 3.5 mL of 2 M Na₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with
water (12 mL x 3 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and Et₂O and filtered on sintered glassware, washed with a minimum of EtOH, EtO₂ and petroleum ether and dried under pressure to give the solid product. Pale-yellow crystals (48 %); Mp = 264 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.22 (s, 1H, CHO), 10.17 (s, 1H, CHO), 8.91 (d, 2H, *J* = 8.25 Hz, H-2' and H-6'), 8.59 (s, 1H, H-3), 8.53 (d, 2H, *J* = 8.25 Hz, H-2'' and H-6''), 8.39 (d, 2H, *J* = 7.50 Hz, H-2''' and H-6'''), 8.20 (d, 2H, *J* = 8.25 Hz, H-3' and H-5'), 8.09 (d, 2H, *J* = 8.25 Hz, H-3'' and H-5''), 7.66 (t, 2H, *J* = 7.50 Hz, H-3''' and H-5'''), 7.47 (t, 1H, *J* = 7.50 Hz, H-4''').

### 4.1.24. General procedure for 4,6-bis[(substituted-iminomethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidines (34a-d)

To a solution of the appropriate diamines (0.126 mmol, 2.1 eq.) in ethanol (7 mL) was added the 4,6-bis(4-formylphenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine 32 (0.6 mmol). The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (40 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

#### 4.1.24.1. 4,6-Bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (34a)
Pale-yellow crystals (98 %); Mp = 145 °C; ¹H NMR δ (300 MHz, CDCl₃): 8.77 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.52 (s, 1H, H-3), 8.44 (s, 1H, CH=N), 8.41-8.36 (m, 5H, CH=N, H-2', H-6', H-2'' and H-6''), 8.00 (d, 2H, *J* = 8.10 Hz, H-3' and H-5'), 7.91 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5''), 7.62 (t, 2H, *J* = 7.65 Hz, H-3''' and H-5'''), 7.41 (t, 1H, *J* = 7.65 Hz, H-4'''), 3.75 (t, 2H, *J* = 7.20 Hz, NCH₂), 3.73 (t, 2H, *J* = 7.20 Hz, NCH₂), 2.45 (t, 4H, *J* = 7.20 Hz, 2NCH₂), 2.31 (s, 12H, 2 N(CH₃)₂), 2.00-1.93 (m, 4H, 2CH₂).

#### 4.1.24.2. 4,6-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (34b)
Yellow oil (70 %); ¹H NMR δ (300 MHz, CDCl₃): 8.76 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.53 (s, 1H, H-3), 8.43-8.37 (m, 6H, 2CH=N, H-2'', H-6'', H-2''' and H-6'''), 8.00 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.90 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.63 (t, 2H, *J* = 7.80 Hz, H-3''' and H-5'''), 7.42 (t, 1H, *J* = 7.80 Hz, H-4'''), 3.73 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.71 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.37 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.35 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.28 (s, 6H, N(CH₃)₂), 2.27 (s, 6H, N(CH₃)₂), 1.83-1.74 (m, 4H, 2CH₂), 1.66-1.56 (m, 4H, 2CH₂).

#### 4.1.24.3. 4,6-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (34c)
Yellow crystals (93 %); Mp = 120 °C; ¹H NMR δ (300 MHz, CDCl₃): 8.74 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.51 (s, 1H, H-3), 8.41-8.35 (m, 6H, 2CH=N, H-2'', H-6'', H-2''' and H-6'''), 7.99 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.89 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.61 (t, 2H, *J* = 7.50 Hz, H-3''' and H-5'''), 7.40 (t, 1H, *J* = 7.50 Hz, H-4'''), 3.71 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.68 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.32 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.29 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.83-1.75 (m, 4H, 2CH₂), 1.59-1.40 (m, 8H, 4CH₂).

### 4.1.25. 4,6-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (34d)

Pale-yellow crystals (80 %); Mp = 132 °C; ¹H NMR δ (300 MHz, CDCl₃): 8.75 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.52 (s, 1H, H-3), 8.41 (s, 1H, CH=N), 8.40-8.36 (m, 5H, CH=N, H-2'', H-6'', H-2''' and H-6''), 7.99 (d, 2H, *J* = 8.10 Hz, H-3' and H-5'), 7.88 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5''), 7.62 (t, 2H, *J* = 7.50 Hz, H-3''' and H-5'''), 7.41 (t, 1H, *J* = 7.50 Hz, H-4''').

3.71 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.50-2.32 (m, 20H, 10NCH₂), 2.29 (s, 3H, NCH₃), 2.28 (s, 3H, NCH₃), 1.81-1.72 (m, 4H, 2CH₂), 1.67-1.45 (m, 4H, 2CH₂).

### 4.1.26. General procedure for 4,6-bis[(substituted-aminomethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidines (7a-d)

To a solution of 4,6-bis[(substituted-iminomethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine 34 (0.4 mmol) in methanol (10 mL) was added portion-wise at 0 °C sodium borohydride (3.2 mmol, 8 eq.). The reaction mixture was then stirred at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (40 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the 4,6-bis[(substituted-aminomethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidines.

#### 4.1.26.1. 4,6-Bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (7a)
Yellow oil (95 %); ¹H NMR δ (300 MHz, CDCl₃): 8.69 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.52 (s, 1H, H-3), 8.42 (dd, 2H, *J* = 8.60 and 1.20 Hz, H-2'' and H-6''), 8.32 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.65-7.59 (m, 4H, H-3', H-5', H-3''' and H-5'''), 7.53 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5'), 7.41 (t, 1H, *J* = 8.60 Hz, H-4'''), 3.97 (s, 2H, NCH₂), 3.95 (s, 2H, NCH₂), 2.79 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.78 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.41 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.40 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.28 (s, 6H, N(CH₃)₂), 2.27 (s, 6H, N(CH₃)₂), 1.78 (qt, 4H, *J* = 6.90 Hz, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 163.1 (C-4), 161.8 (C-6), 145.2 (C-7a), 143.6 (C-4'' and C-4'), 140.4 (C-1''), 138.2 (C-1'), 137.4 (C-1''), 135.8 (C-3), 130.6 (C-2'', C-6'', C-2', C-6', C-3''' and C-5''), 130.2 (C-3' and C-5'), 129.8 (C-3'' and C-5'), 127.9 (C-4''), 122.7 (C-2'' and C-6''), 112.6 (C-3a), 59.3 (NCH₂), 54.9 (NCH₂), 54.8 (NCH₂), 49.3 (NCH₂), 46.9 (N(CH₃)₂), 29.0 (CH₂), 28.5 (CH₂); ESI MS m/z [M+H]⁺ Calculated for C₃₅H₄₅N₈: 577.3767, Found: 577.3762.

#### 4.1.26.2. 4,6-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (7b)
Pale-yellow oil (75 %); ¹H NMR δ (300 MHz, CDCl₃): 8.69 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.53 (s, 1H, H-3), 8.43 (dd, 2H, *J* = 8.40 and 1.20 Hz, H-2''' and H-6'''), 8.33 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.66-7.59 (m, 4H, H-3', H-5', H-3''' and H-5'''), 7.51 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5'), 7.41 (t, 1H, *J* = 8.40 Hz, H-4'''), 3.97 (s, 2H, NCH₂), 3.93 (s, 2H, NCH₂), 2.74-2.70 (m, 4H, 2NCH₂), 2.35-2.27 (m, 4H, 2NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.60-1.52 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 163.1 (C-4), 161.7 (C-6), 145.7 (C-7a), 144.8 (C-4'' and C-4'), 140.4 (C-1''), 137.9 (C-1'), 137.2 (C-1''), 135.7 (C-3), 130.5 (C-2'', C-6'', C-2', C-6', C-3''' and C-5''), 130.1 (C-3' and C-5'), 129.6 (C-3'' and C-5'), 127.8 (C-4''), 122.6 (C-2'' and C-6''), 112.5 (C-3a), 61.1 (NCH₂), 55.1 (NCH₂), 50.7 (NCH₂), 46.9 (N(CH₃)₂), 29.4 (CH₂), 26.9 (CH₂); ESI MS m/z [M+H]⁺ Calculated for C₃₇H₄₉N₈: 605.4080, Found: 605.4069.

#### 4.1.26.3. 4,6-Bis{4-[(5-dimethylaminopentyl)aminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (7c)
Yellow oil (92 %); ¹H NMR δ (300 MHz, CDCl₃): 8.68 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.51 (s, 1H, H-3), 8.42 (dd, 2H, *J* = 8.40 and 1.20 Hz, H-2'' and H-6'''), 8.31 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6'), 7.64-7.58 (m, 4H, H-3', H-5', H-3''' and H-5''), 7.50 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5'), 7.40 (t, 1H, *J* = 8.40 Hz, H-4'''), 3.94 (s, 2H, NCH₂), 3.91 (s, 2H, NCH₂), 2.71 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.31-2.27 (m, 4H, 2NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.62-1.34 (m, 12H, 6CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 163.1 (C-4), 161.8 (C-6), 145.7 (C-7a), 144.8 (C-4'' and C-4'), 140.4 (C-1''), 137.9 (C-1'), 137.3 (C-1''), 135.7 (C-3), 130.5 (C-2'', C-6'', C-2' and C-6'), 130.4 (C-3''' and C-5''), 130.1 (C-3' and C-5'), 129.6 (C-3'' and C-5''), 127.8 (C-4'''), 122.6 (C-2'' and C-6''), 112.5 (C-3a), 61.1 (NCH₂), 55.1 (NCH₂), 50.8 (NCH₂), 46.9 (N(CH₃)₂), 31.2 (CH₂), 31.2 (CH₂), 29.0 (CH₂), 26.9 (CH₂), 26.3 (CH₂); ESI MS m/z [M+H]⁺ Calculated for C₃₇H₅₃N₈: 633.4393, Found: 633.4382.
### 4.1.26.4. 2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (7d).
Yellow oil (70 %);
¹H NMR δ (300 MHz, CDCl₃): 8.70 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.52 (s, 1H, H-3), 8.42 (dd, 2H, *J* = 7.80 and 1.20 Hz, H-2'' and H-6''), 8.32 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.68-7.56 (m, 6H, H-3', H-5', H-3'', H-5'', H-3'' and H-5'), 7.41 (t, 1H, *J* = 7.80 Hz, H-4''), 3.98 (s, 2H, NCH₂), 3.96 (s, 2H, NCH₂), 2.75 (t, 4H, *J* = 6.60 Hz, 2NCH₂), 2.59-2.39 (m, 20H, 10NCH₂), 2.29 (s, 3H, NCH₃), 2.27 (s, 3H, NCH₃), 1.69-1.59 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 163.1 (C-4), 161.8 (C-6), 145.6 (C-7a), 144.6 (C-4'' and C-4'), 140.4 (C-1'''), 138.0 (C-1'), 137.3 (C-1'), 135.7 (C-3), 130.5 (C-2'', C-6', C-2', C-6', C-3'' and C-5''), 130.1 (C-3' and C-5'), 129.6 (C-3'' and C-5'), 127.8 (C-4''), 122.7 (C-2'' and C-6''), 112.7 (C-3a), 59.9 (NCH₂), 56.5 (NCH₂), 55.1 (NCH₂), 54.6 (NCH₂), 50.8 (NCH₂), 50.6 (NCH₂), 47.5 (N(CH₃)₂), 29.5 (CH₂), 26.1 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₃H₅₉N₁₀: 715.4924, Found: 715.4910.

### 4.1.27. 2,4-Bis(4-formylphenyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidine (33)

To a solution of 2.26 mmol of 2,4-dichloro-7-phenyl-7H-imidazo[4,5-d]pyrimidine **31**, 0.75 g of 4-formylphenyl boronic acid (5.0 mmol, 2.2 eq.) and 260 mg (0.226 mmol, 0.1 eq.) of tetrakis(triphenylphosphine)palladium in 25 mL of 1,2-dimethoxyethane, 3.5 mL of 2 M Na₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the precipitate was filtered, and washed successively with 1,2-dimethoxyethane, H₂O, EtOH, and Et₂O, and then dried under pressure to give the solid product. White crystals (54 %); Mp = 256 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.20 (s, 1H, CHO), 10.15 (s, 1H, CHO), 9.19 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.84 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 8.52 (s, 1H, H-6), 8.16 (d, 2H, *J* = 8.10 Hz, H-3' and H-5'), 8.06 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5''), 7.89 (d, 2H, *J* = 7.50 Hz, H-2'' and H-6''), 7.71 (t, 2H, *J* = 7.50 Hz, H-3'' and H-5''), 7.59 (t, 1H, *J* = 7.50 Hz, H-4'').

### 4.1.28. General procedure for 2,4-bis[(substituted-iminomethyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidines (35a-c)

To a solution of the appropriate diamines (0.126 mmol, 2.1 eq.) in ethanol (7 mL) was added the 2,4-bis(4-formylphenyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidine **33** (0.6 mmol). The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (40 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

### 4.1.28.1. 2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-phenyl-7H-imidazo[4,5-d]pyrimidine (35a).
Pale-yellow crystals (80 %);
Mp = 140 °C; ¹H NMR δ (300 MHz, CDCl₃): 9.04 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.68 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.42 (s, 1H, H-6), 8.41 (s, 1H, CH=N), 8.37 (s, 1H, CH=N), 7.96 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.90-7.84 (m, 4H, H-2'', H-6'', H-3'' and H-5'), 7.66 (t, 2H, *J* = 7.60 Hz, H-3''' and H-5'''), 7.52 (t, 1H, *J* = 7.60 Hz, H-4''), 3.72 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.36 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.32 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.24 (s, 6H, N(CH₃)₂), 1.83-1.72 (m, 4H, 2CH₂), 1.66-1.56 (m, 4H, 2CH₂).

### 4.1.28.2. 2,4-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-7-phenyl-7H-imidazo[4,5-d]pyrimidine (35b).
Pale-yellow crystals (90 %);
Mp = 115 °C; ¹H NMR δ (300 MHz, CDCl₃): 9.06 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.70 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.46 (s, 1H, H-6), 8.42 (s, 1H, CH=N), 8.39 (s, 1H, CH=N), 7.98 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.92-7.86 (m, 4H, H-2'', H-6'', H-3'' and H-5'), 7.68 (t, 2H, *J* = 7.50 Hz, H-3''' and H-5'''), 7.55 (t, 1H, *J* = 7.50 Hz, H-4''), 3.71 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.50 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.47 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.39 (s, 6H, N(CH₃)₂), 2.38 (s, 6H, N(CH₃)₂), 1.85-1.76 (m, 4H, 2CH₂), 1.70-1.63 (m, 4H, 2CH₂), 1.50-1.43 (m, 4H, 2CH₂).

### 4.1.28.3. 2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-7-phenyl-7H-imidazo[4,5-d]pyrimidine (35c).
White crystals (99 %);
Mp = 135 °C; ¹H NMR δ (300 MHz, CDCl₃): 9.04 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.66 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.40 (s, 1H, H-6), 8.38 (s, 1H, CH=N), 8.34 (s, 1H, CH=N), 7.94 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.88-7.81 (m, 4H, H-2'', H-6'', H-3'' and H-5''), 7.64 (t, 2H, *J* = 7.80 Hz, H-3''' and H-5''), 7.51 (t, 1H, *J* = 7.80 Hz, H-4''), 3.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.67 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.64-2.35 (m, 20H, 10NCH₂), 2.28 (s, 6H, 2NCH₃), 1.80-1.72 (m, 4H, 2CH₂), 1.65-1.51 (m, 4H, 2CH₂).

### 4.1.29. General procedure for 2,4-bis[(substituted-aminomethyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidines (8a-c)

To a solution of 2,4-bis[(substituted-iminomethyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidines **32** (0.4 mmol) in methanol (10 mL) was added portion-wise at 0 °C sodium borohydride (3.2 mmol, 8 eq.). The reaction mixture was then stirred at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (40 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the 2,4-bis[(substituted-aminomethyl)-7-phenyl-7H-imidazo[4,5-d]pyrimidines.

### 4.1.29.1. 2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-phenyl-7H-imidazo[4,5-d]pyrimidine (8a).
Yellow oil (78 %); ¹H NMR δ (300 MHz, CDCl₃) 8.94 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.62 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.40 (s, 1H, H-6), 7.91 (d, 2H, *J* = 7.80 Hz, H-2'' and H-6'''), 7.66 (t, 2H, *J* = 7.80 Hz, H-3'' and H-5''), 7.58 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.52 (t, 1H, *J* = 7.80 Hz, H-4''), 7.49 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5'), 3.94 (s, 2H, NCH₂), 3.91 (s, 2H, NCH₂), 2.72 (t, 2H, *J* = 6.60 Hz, NCH₂), 2.70 (t, 2H, *J* = 6.60 Hz, NCH₂), 2.36-2.28 (m, 4H, 2NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.76-1.52 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 160.5 (C-2), 156.1 (C-4), 154.4 (C-7a), 144.8 (C-4' and C-4''), 143.6 (C-1'''), 138.5 (C-4a), 136.2 (C-1' and C-1''), 131.4 (C-3''' and C-5'''), 131.2 (C-3' and C-5'), 129.9 (C-3'' and C-5''), 129.8 (C-2' and C-6'), 129.7 (C-2'' and C-6''), 129.5 (C-6), 128.2 (C-4'''), 124.8 (C-2'' and C-6''), 61.0 (NCH₂), 55.1 (NCH₂), 50.6 (NCH₂), 46.8 (N(CH₃)₂), 29.4 (CH₂), 27.0 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₃₇H₄₉N₈: 605.4080, Found: 605.4065.

### 4.1.29.2. 2,4-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-7-phenyl-7H-imidazo[4,5-d]pyrimidine (8b).
Yellow oil (93 %); ¹H NMR δ (300 MHz, CDCl₃) 8.93 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.61 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.40 (s, 1H, H-6), 7.90 (d, 2H, *J* = 7.80 Hz, H-2'' and H-6'''), 7.66 (t, 2H, *J* = 7.80 Hz, H-3'' and H-5''), 7.57 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.52 (t, 1H, *J* = 7.80 Hz, H-4''), 7.47 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5'), 3.93 (s, 2H, NCH₂), 3.89 (s, 2H, NCH₂), 2.70 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.67 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.32-2.26 (m, 4H, 2NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.62-1.46 (m, 8H, 4CH₂), 1.41-1.33 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 160.3 (C-2), 156.0 (C-4), 154.3 (C-7a), 145.0 (C-4' and C-4''), 144.0 (C-1'''), 138.3 (C-4a), 136.2 (C-1' and C-1''), 131.3 (C-3''' and C-5'''), 131.1 (C-3' and C-5'), 129.8 (C-3'' and C-5''), 129.7 (C-2' and C-6'), 129.6 (C-2'' and C-6''), 129.4 (C-6), 128.2 (C-4''), 124.6 (C-2'' and C-6''), 61.1 (NCH₂), 55.2 (NCH₂), 50.7 (NCH₂), 46.9 (N(CH₃)₂), 31.4 (CH₂), 29.0 (CH₂), 26.6 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₃₉H₅₃N₈: 633.4393, Found: 633.4379.

### 4.1.29.3. 2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-7-phenyl-7H-imidazo[4,5-d]pyrimidine (8c).
Pale-yellow oil (68 %);
¹H NMR δ (300 MHz, CDCl₃): 8.92 (d, 2H, *J* = 8.10 Hz, H-2' and H-6''), 8.70 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.46 (s, 1H, H-6), 8.42 (s, 1H, CH=N), 8.39 (s, 1H, CH=N), 7.98 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.92-7.86 (m, 4H, H-2'', H-6'', H-3'' and H-5'), 7.68 (t, 2H, *J* = 7.50 Hz, H-3''' and H-5'''), 7.55 (t, 1H, *J* = 7.50 Hz, H-4''), 3.71 (t, 2H, *J* = 6.90 Hz, NCH₂), 3.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.50 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.47 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.39 (s, 6H, N(CH₃)₂), 2.38 (s, 6H, N(CH₃)₂), 1.85-1.76 (m, 4H, 2CH₂), 1.70-1.63 (m, 4H, 2CH₂), 1.50-1.43 (m, 4H, 2CH₂).
6'), 8.59 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6'), 8.38 (s, 1H, H-6), 7.87 (d, 2H, *J* = 7.80 Hz, H-2'' and H-6'), 7.63 (t, 2H, *J* = 7.80 Hz, H-3'' and H-5''), 7.55 (d, 2H, *J* = 8.10 Hz, H-3' and H-5'), 7.49 (t, 1H, *J* = 7.80 Hz, H-4''), 7.45 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5'), 3.91 (s, 2H, NCH₂), 3.87 (s, 2H, NCH₂), 2.69 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.67 (t, 2H, *J* = 6.90 Hz, NCH₂), 2.62-2.34 (m, 20H, 10NCH₂), 2.27 (s, 3H, NCH₃), 2.26 (s, 3H, NCH₃), 1.58-1.48 (m, 8H, 4CH₂), 1.65-1.51 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 160.5 (C-2), 156.1 (C-4), 154.4 (C-7a), 144.8 (C-4' and C-4'), 143.7 (C-1''), 138.5 (C-4a), 136.2 (C-1' and C-1''), 131.3 (C-3'' and C-5''), 131.2 (C-3' and C-5'), 129.9 (C-3'' and C-5''), 129.7 (C-2' and C-6'), 129.6 (C-2'' and C-6'), 129.4 (C-6), 128.3 (C-4''), 124.7 (C-2'' and C-6''), 59.9 (NCH₂), 56.5 (NCH₂), 55.1 (NCH₂), 54.6 (NCH₂), 50.5 (NCH₂), 47.4 (NCH₃), 29.4 (CH₂), 26.1 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₃H₅₉N₁₀: 715.4924, Found: 715.4911.

### 4.1.30. 2,4,7-Tri(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine (36)

To a solution of 2 mmol of 2,4-dichloro-7-(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine 20, 4.4 mmol of 4-formylphenyl boronic acid (2.2 eq.) and 240 mg (0.2 mmol, 0.1 eq.) of tetrakis(triphenylphosphine) palladium in 25 mL of 1,2-dimethoxyethane, 3.5 mL of 2 M Na₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CHCl₃ and the organic phase was filtered on filter paper, then washed with water (20 mL x 3 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was purified by silica gel column chromatography (CHCl₃), then cooled and triturated again in EtOH, filtered on sintered glassware, washed with a minimum of EtOH and EtO₂ and dried under pressure to give the solid product. Pale-yellow crystals (22 %) Mp > 240 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.17 (s, 1H, CHO), 10.11 (s, 1H, CHO), 10.10 (s, 1H, CHO), 8.75 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.51 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.35 (d, 1H, *J* = 3.90 Hz, H-6), 8.33 (d, 2H, *J* = 8.40 Hz, H-2'', H-6''), 8.18 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 8.15 (d, 2H, *J* = 8.40 Hz, H-3'', H-5''), 8.05 (d, 2H, *J* = 8.40 Hz, H-3'', H-5''), 7.29 (d, 1H, *J* = 3.90 Hz, H-5).

### 4.1.31. General procedure for 2,4,7-tris(4-[(dimethylamino-alkyl)iminomethyl]phenyl)-7H-pyrrolo[2,3-d]pyrimidines (37a-b)

To a solution of the dimethylamino-alkylamine (0.726 mmol, 3.3 eq.) in ethanol (7 mL) was added a 2,4,7-tris(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine 36 (0.22 mmol). The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (30 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

#### 4.1.31.1. 2,4,7-Tris(4-[(3-dimethylaminopropyl)iminomethyl]phenyl)-7H-pyrrolo[2,3-d]pyrimidine (37a).
Orange oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.39 (s, 1H, CH=N), 8.37 (s, 1H, CH=N), 8.36 (s, 1H, CH=N), 8.29 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.99-7.92 (m, 6H, H-2'', H-6'', H-3', H-5', H-3'' and H-5''), 7.83 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.62 (d, 1H, *J* = 3.60 Hz, H-6), 6.98 (d, 1H, *J* = 3.60 Hz, H-5), 3.73-3.66 (m, 6H, 3NCH₂), 2.41-2.34 (m, 6H, 3NCH₂), 2.26 (s, 6H, N(CH₃)₂), 2.24 (s, 12H, 2 N(CH₃)₂), 1.94-1.87 (m, 6H, 3CH₂).

#### 4.1.31.2. 2,4,7-Tris(4-[(4-dimethylaminobutyl)iminomethyl]phenyl)-7H-pyrrolo[2,3-d]pyrimidine (37b).
Yellow oil (88 %); ¹H NMR δ (300 MHz, CDCl₃): 8.67 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.39 (s, 1H, CH=N), 8.36 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.30 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 8.00-7.92 (m, 6H, H-2'', H-6'', H-3', H-5', H-3'' and H-5'), 7.84 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.62 (d, 1H, *J* = 3.60 Hz, H-6), 6.98 (d, 1H, *J* = 3.60 Hz, H-5), 3.73-3.66 (m, 6H, 3NCH₂), 2.24 (s, 12H, 2 N(CH₃)₂), 1.94-1.87 (m, 6H, 3CH₂).

(d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.62 (d, 1H, *J* = 3.60 Hz, H-6), 6.98 (d, 1H, *J* = 3.60 Hz, H-5), 3.73-3.66 (m, 6H, 3NCH₂), 2.37-2.24 (m, 6H, 3NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.21 (s, 12H, 2 N(CH₃)₂), 1.85-1.72 (m, 6H, 3CH₂), 1.64-1.54 (m, 6H, 3CH₂).

### 4.1.32. General procedure for 2,4,7-tris(4-[(dimethylamino-alkyl)aminomethyl]phenyl)-7H-pyrrolo[2,3-d]pyrimidines (9a-b)

To a solution of 2,4,7-tris(4-[(dimethylamino-alkyl)iminomethyl]phenyl)-7H-pyrrolo[2,3-d]pyrimidine 37 (0.22 mmol) in methanol (7 mL) was added portion-wise at 0 °C sodium borohydride (1.32 mmol, 6 eq.). The reaction mixture was then stirred at room temperature for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (15 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the product that was purified by HPLC.

#### 4.1.32.1. 2,4,7-Tris(4-[(3-dimethylaminopropyl)iminomethyl]phenyl)-7H-pyrrolo[2,3-d]pyrimidine (9a).
Yellow oil (86 %); ¹H NMR δ (300 MHz, CDCl₃): 8.60 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 8.25 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.87 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.60-7.52 (m, 5H, H-6, H-3', H-5', H-3'' and H-5''), 7.44 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 6.99 (d, 1H, *J* = 3.60 Hz, H-5), 3.93 (s, 2H, NCH₂), 3.91 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.79-2.68 (m, 6H, 3NCH₂), 2.40-2.28 (m, 6H, 3NCH₂), 2.26 (s, 6H, N(CH₃)₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.79-1.66 (m, 6H, 3CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.6 (C-2), 159.0 (C-4), 154.0 (C-7a), 144.0 (C-1''), 143.5 (C-4'), 140.4 (C-4'), 139.0 (C-4''), 138.7 (C-1''), 138.0 (C-1'), 131.2 (C-6), 130.5 (C-3'' and C-5''), 130.4 (C-3' and C-5'), 129.8 (C-3'' and C-5'), 129.6 (C-2' and C-6'), 129.5 (C-2'' and C-6''), 125.1 (C-2'' and C-6''), 116.1 (C-4a), 103.5 (C-5), 59.5 (NCH₂), 55.2 (NCH₂), 55.0 (NCH₂), 54.8 (NCH₂), 49.3 (NCH₂), 49.2 (NCH₂), 47.0 (N(CH₃)₂), 29.5 (CH₂); ESI MS *m/z* [M+2H]⁺ Calculated for C₄₂H₆₁N₉: 691.5050, Found: 691.5035.

#### 4.1.32.2. 2,4,7-Tris(4-[(4-dimethylaminobutyl)iminomethyl]phenyl)-7H-pyrrolo[2,3-d]pyrimidine (9b).
Pale-yellow oil (82 %); ¹H NMR δ (300 MHz, CDCl₃): 8.57 (d, 2H, *J* = 8.10 Hz, H-2' and H-6'), 8.22 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.83 (d, 2H, *J* = 8.10 Hz, H-2'' and H-6''), 7.55-7.48 (m, 5H, H-6, H-3', H-5', H-3'' and H-5''), 7.41 (d, 2H, *J* = 8.10 Hz, H-3'' and H-5''), 6.94 (d, 1H, *J* = 3.60 Hz, H-5), 3.89 (s, 2H, NCH₂), 3.87 (s, 2H, NCH₂), 3.85 (s, 2H, NCH₂), 2.72-2.62 (m, 6H, 3NCH₂), 2.35-2.24 (m, 6H, 3NCH₂), 2.21 (s, 6H, N(CH₃)₂), 2.20 (s, 6H, N(CH₃)₂), 2.19 (s, 6H, N(CH₃)₂), 1.57-1.48 (m, 12H, 6CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.5 (C-2), 158.9 (C-4), 153.9 (C-7a), 143.9 (C-1''), 143.3 (C-4'), 140.3 (C-4'), 139.0 (C-4''), 138.7 (C-1'), 137.9 (C-1'), 133.9 (C-6), 130.4 (C-3'' and C-5''), 130.3 (C-3' and C-5'), 129.8 (C-3'' and C-5''), 129.6 (C-2' and C-6'), 129.5 (C-2'' and C-6'), 125.0 (C-2'' and C-6''), 116.1 (C-4a), 103.5 (C-5), 61.0 (NCH₂), 55.1 (NCH₂), 54.9 (NCH₂), 50.7 (NCH₂), 50.5 (NCH₂), 46.8 (N(CH₃)₂), 29.3 (CH₂), 26.9 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₅H₆₆N₉: 732.5441, Found: 732.5419.

### 4.1.33. 4-Chloro-7-(4-formylphenyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (38)

4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (1.8 mmol), 4-formylphenylboronic acid (2.16 mmol, 1.2 equiv.) and copper (II) acetate (1.8 mmol) in methanol (35 mL) was stirred at 35 °C for 5 h. Then, the reaction mixture was evaporated to dryness. The residue was extracted with chloroform, filtered and evaporated under reduced pressure. The residue was then cooled and triturated in EtOH, filtered on sintered glassware, washed with a minimum of EtOH, then EtO₂ and dried under pressure to give the solid product. White crystals (24 %); Mp = 217 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.11 (s, 1H, CHO), 8.74 (s, 1H, H-2), 8.11 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.96 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.80 (s, 1H, H-6).
### 4.1.34. 4,5,7-Tri(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine (39)

To a solution of 0.65 mmol of 4-chloro-7-(4-formylphenyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 38, 2.0 mmol of 4-formylphenyl boronic acid (3 eq.) and 76 mg (0.065 mmol, 0.1 eq.) of tetrakis(triphenylphosphine) palladium in 14 mL of a mixture dioxane/H₂O (4/1; v/v), 210 mg (2 mmol) of Na₂CO₃ were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The reaction mixture was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CH₂Cl₂ and the organic phase was then washed with water (x2), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was then triturated in EtOH, filtered on sintered glassware, washed with a minimum of EtOH and EtO₂ and dried under pressure to give the solid product. Pale-yellow crystals (68 %) Mp = 218 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.13 (s, 1H, CHO), 10.00 (s, 1H, CHO), 9.96 (s, 1H, CHO), 9.17 (s, 1H, H-2), 8.15-8.13 (m, 4H, H-2', H-6', H-2'' and H-6'), 7.84 (s, 1H, H-6), 7.72 (d, 2H, *J* = 8.40 Hz, H-2''' and H-6'''), 7.67 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.62 (d, 2H, *J* = 8.40 Hz, H-3'', H-5''), 7.24 (d, 2H, *J* = 8.40 Hz, H-3''' and H-5''').

### 4.1.35. 4,5,7-Tris{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidine (40)

To a solution of the dimethylamino-alkylamine (1.32 mmol, 3.3 eq.) in ethanol (13 mL) was added the 4,5,7-tris(4-formylphenyl)-7H-pyrrolo[2,3-d]pyrimidine 39 (0.40 mmol). The reaction mixture was then heated under reflux for 24 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (20 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification. Yellow oil (98 %); ¹H NMR δ (300 MHz, CDCl₃): 9.07 (s, 1H, H-2), 8.35 (s, 1H, CH=N), 8.21 (s, 1H, CH=N), 8.19 (s, 1H, CH=N), 7.93 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.87 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.66 (s, 1H, H-6), 7.55-7.51 (m, 4H, H-2'', H-6'', H-3'' and H-5''), 7.47 (d, 2H, *J* = 8.40 Hz, H-2''' and H-6'''), 7.09 (d, 2H, *J* = 8.40 Hz, H-3''' and H-5'''), 3.71-3.58 (m, 6H, 3NCH₂), 2.34-2.31 (m, 6H, 3NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 12H, 2 N (CH₃)₂), 1.94-1.81 (m, 6H, 3CH₂).

### 4.1.36. 4,5,7-Tris{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidine (9c)

To a solution of 4,5,7-tris{4-[(dimethylamino-alkyl)iminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidine 40 (0.40 mmol) in methanol (15 mL) was added portion-wise at 0 °C sodium borohydride (4.0 mmol, 10 eq.). The reaction mixture was then stirred at room temperature for 30 min then heated at 60 °C for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (20 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the product that was purified by HPLC. Yellow oil (90 %); ¹H NMR δ (300 MHz, CDCl₃): 9.05 (s, 1H, H-2), 7.76 (d, 2H, *J* = 8.40 Hz, H-2' and H-6'), 7.58 (s, 1H, H-6), 7.55 (d, 2H, *J* = 8.40 Hz, H-3' and H-5'), 7.41 (d, 2H, *J* = 8.40 Hz, H-2'' and H-6''), 7.12 (d, 2H, *J* = 8.40 Hz, H-3'' and H-5''), 7.08 (d, 2H, *J* = 8.40 Hz, H-2''' and H-6'''), 7.01 (d, 2H, *J* = 8.40 Hz, H-3''' and H-5'''), 3.90 (s, 2H, NCH₂), 3.77 (s, 2H, NCH₂), 3.76 (s, 2H, NCH₂), 2.78-2.64 (m, 6H, 3NCH₂), 2.40-2.31 (m, 6H, 3NCH₂), 2.29 (s, 6H, N(CH₃)₂), 2.26 (s, 12H, N(CH₃)₂), 1.79-1.68 (m, 6H, 3CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.8 (C-4), 152.0 (C-2), 151.8 (C-7a), 140.5 (C-1''), 138.8 (C-4'), 137.2 (C-4''), 136.1 (C-1''), 132.5 (C-1'), 129.9 (C-3'' and C-5''), 129.3 (C-3' and C-5'), 129.2 (C-3'' and C-5''), 127.6 (C-2' and C-6'), 127.4 (C-2'' and C-6''), 127.0 (C-5), 124.3 (C-4''), 124.2 (C-2'' and C-6''), 118.3 (C-6), 114.6 (C-4a), 58.1 (NCH₂), 53.5 (NCH₂), 47.9 (NCH₂), 45.5 (N(CH₃)₂), 27.9 (CH₂); ESI MS *m/z* [M+H]⁺ Calculated for C₄₂H₆₀N₉: 690.49, Found: 690.50.

### 4.1.37. General procedure for 2,4-bis{4-[(5-dialkylaminoalkyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine (1), 4,6-bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[2,3-b]pyrimidine (2), 4,6-bis{4-[(4-dialkylaminoalkyl)aminomethyl]phenyl}-1-phenyl-1H-pyrrolo[3,2-c]pyridine (3), 2,7-bis[(substituted-aminomethyl)-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (4), 4,5-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines (5), 2,4-bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-5-phenyl-5H-pyrrolo[2,3-d]pyrimidine (6), 4,6-bis[(substituted-aminomethyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidines (7), 2,4-bis{4-[(4-dialkylaminoalkyl)aminomethyl]phenyl}-7-phenyl-7H-imidazo[4,5-d]pyrimidine (8) and tris{4-[(3-dimethylaminoalkyl)aminomethyl]phenyl}-7H-pyrrolo[2,3-d]pyrimidine (9) oxalate salts

To a solution of compounds 1–9 (0.3 mmol) in isopropanol (11 mL) was added oxalic acid (2.4 mmol, 8 eq.). The reaction mixture was heated under reflux for 30 min. The precipitate was filtered, washed with isopropanol then with diethyl ether and dried under reduced pressure to give the ammonium oxalate salts, which were then crystallized using 2-PrOH-H₂O (80/20, v/v) as solvent.

#### 4.1.37.1. 2,4-Bis{4-[(4-dimethylaminopropyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1a•4(COOH)₂). Beige crystals (70 %); ¹H NMR δ (300 MHz, DMSO-d₆): 8.57 (d, 2H, *J* = 7.80 Hz, H-2' and H-6'), 8.40 (d, 2H, *J* = 7.80 Hz, H-2'' and H-6'), 8.20 (d, 1H, *J* = 3.75 Hz, H-6), 8.01 (d, 2H, *J* = 7.80 Hz, H-2''' and H-6''), 7.79 (d, 2H, *J* = 7.80 Hz, H-3' and H-5'), 7.71-7.64 (m, 4H, H-3'', H-5'', H-3''' and H-5'''), 7.48 (t, 1H, *J* = 7.80 Hz, H-4''), 7.23 (d, 1H, *J* = 3.75 Hz, H-5), 4.28 (s, 2H, NCH₂), 4.22 (s, 2H, NCH₂), 3.09-2.97 (m, 8H, 4NCH₂), 2.69 (s, 6H, N(CH₃)₂), 2.68 (s, 6H, N(CH₃)₂), 2.09-1.98 (m, 4H, 2CH₂).

#### 4.1.37.2. 2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1b•4(COOH)₂). White crystals (89 %); ¹H NMR δ (300 MHz, DMSO-d₆): 8.57 (d, 2H, *J* = 7.80 Hz, H-2' and H-6'), 8.40 (d, 2H, *J* = 7.80 Hz, H-2'' and H-6'), 8.21 (d, 1H, *J* = 3.75 Hz, H-6), 8.01 (d, 2H, *J* = 7.80 Hz, H-2''' and H-6''), 7.79 (d, 2H, *J* = 7.80 Hz, H-3' and H-5'), 7.71-7.62 (m, 4H, H-3'', H-5'', H-3''' and H-5'''), 7.49 (t, 1H, *J* = 7.80 Hz, H-4''), 7.24 (d, 1H, *J* = 3.75 Hz, H-5), 4.29 (s, 2H, NCH₂), 4.23 (s, 2H, NCH₂), 3.05-2.94 (m, 8H, 4NCH₂), 2.71 (s, 6H, N(CH₃)₂), 2.69 (s, 6H, N(CH₃)₂), 1.73-1.65 (m, 8H, 4CH₂).

## 4.2. FRET-melting experiments

The FRET melting method is based on the fluorescence resonance energy transfer (FRET) phenomenon between two dyes: a donor and an acceptor (here: FAM and TAMRA). When the nucleotides are folded at low temperature the fluorescence emission of the donor will be minimal due to the acceptor proximity. Fluorescence emission recovery will occur when temperature is increased during the thermally induced melting of the structure. Compounds 1–9 were tested for the subsequent FRET melting experiments. These were performed with the human telomeric sequence F21T (FAM-⁵'GGGTTA)₃-GGG³'-TAMRA), and the human duplex sequence FdxT (FAM-⁵'TATAGCTATA-hexaethyleneglycol-TATAGCTATA³'-TAMRA) (purchased from Integrated DNA Technologies, Leuven, Belgium). The oligonucleotides were pre-folded in 10 mM lithium cacodylate buffer (pH 7.2), with 10 mM KCl and 90 mM LiCl (K⁺ condition). The FAM emissions were recorded at 516 nm using a 492 nm excitation wavelength in the absence and presence of a single compound as a function of temperature (25–95 °C) in 96-well microplates by using a Stratagene MX3000P (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) real-time PCR device. Each experiment was performed in duplicate with 0.2 µM of labeled oligonucleotide and 2 µM of compound 1–9 under K⁺ condition (0.5 µM for PhenDC3) (λ<sub>exc</sub> = 492 nm, λ<sub>em</sub> = 516 nm, T interval = 25–95 °C, ramp: 25 °C for 5 min, then 1 °C/min, measurements every 1 °C, 8x magnification of the fluorescence signal). For each compound, three
independent experiments were carried out. The stabilization (ΔT₁/₂) induced by each compound was calculated as the difference between the mid-transition temperature of the nucleic acid (NA) alone and in the presence of 2 µM compound (0.5 µM for PhenDC3). The data were plotted using OriginPro 9.1 software (OriginLab Corporation, Northampton, MA, USA).

### 4.3. Homogenous time resolved fluorescence (HTRF)

HTRF assays were performed using purified His-Tagged SUD-NM domain (expression and purification as described previously [4], 3'-Biotin-TEG labeled TRF2 RNA oligonucleotide (purchased from Eurogentec) folded into G4 (see sequence ang G4 folding procedure in Ref. [4]) and specific antibodies coupled to fluorophores (Revvity), namely Streptavidin-d2 (acceptor conjugate) and MAb Anti-6HisEu cryptate (donor conjugate). The composition of the assay buffer T is the following: 25 mM Tris-HCl pH 8, 100 mM KCl, 1 mM DTT, 0.05 % Tween, 0.1 % BSA. Each assay was performed in triplicate in a 96-well low-binding surface 1/2 area AlphaPlate (Revvity). 2 µL of the tested compound in water, 4 µL of 5 × SUD-NM domain solution in buffer T and 4 µL of 5 × biotinylated TRF2 G4 solution in buffer T were added to each well. Positive (in the absence of compound) and negative (where both partners were replaced by the assay buffer T) control wells were included in each plate. Plates were incubated 1 h at room temperature. After addition in each well of 10 µL of donor-acceptor conjugate mixture (1:1) diluted in PBS pH 7.4 and BSA 0.1 %, plates were further incubated 1 h at room temperature. Then the fluorescence emission was measured at 620 and 665 nm using an excitation wavelength of 340 nm on a microplate reader (Infinite M1000 Pro, TECAN). The HTRF ratio was calculated as follows: (fluorescence intensity at 660 nm/fluorescence intensity at 620 nm) × 10⁴. The % residual HTRF ratio of each compound was calculated via the following equation: % residual HTRF ratio = (HTRF ratio of the compound - HTRF ratio of the negative controls)/(HTRF ratio of the positive controls - HTRF ratio of the negative controls) × 100. IC₅₀ values were calculated from the experimental data (% residual HTRF ratio) using the KaleidaGraph software (Synergy Software).

### 4.4. In vitro evaluation of the antiviral effect of compounds on cell lines infected by SARS-CoV-2

The anti-SARS-CoV-2 activity of the tested molecules was quantified using the following infection protocol developed by the PF-CCB of the Institut Pasteur. A549 lung cancer cells stably expressing the human ACE2 receptor (gift from O. Schwartz, Institut Pasteur) or Caco2 cells are plated in 384 cells at 5000 cells per well. The following day, in triplicate conditions, cells are incubated with 8 concentrations of G4-ligand (1:3 serial dilutions starting at 16 µM) diluted in cell medium or in cell media alone. 1:2 serial dilutions of remdesivir starting at 10 µM and DMSO are used as positive and negative control compounds for the viral replication assay. Two hours post addition of the molecules, the medium is replaced by the viruses, at 0.1 MOI. Two SARS-CoV-2 strains are tested, the original strain BetaCoV/France/IDF0372/2020 or the Delta variant (B.1.617.2). After 1 h of viral contact with the cells, the inoculum is removed and fresh medium with the tested molecules are added back on the cells. 72 h post-infection, cell supernatant is recovered, and viruses present in the supernatant of infected cells are quantified by quantitative RT-PCR of the N-gene. More precisely, the RT-qPCR is performed in an Applied Biosystems QuantStudio 6 Flex thermocycler, using a Luna Universal One-Step RT-qPCR Kit (NEB, USA) and two oligonucleotides specific of the SARS-CoV-2 N gene (Forward N: TAATCAGAACAGGAACTGATTA and reverse N: CGAAGGTGTGACTTCCATG). The quantity of viral genomes is expressed as Cq and normalized against the Cq value of the positive control and negative controls. More precisely, raw Cq data are normalized against appropriate negative (DMSO, i.e. 0 %) and positive controls (remdesivir, i.e. 100 %) and are expressed in

% of viral replication inhibition. The curve fits are performed using the variable Hill slope model and the GraphPad Prism software. In parallel, cell toxicity is assessed using the CellTiter Glo kit which reports cell viability (Promega). The EC₅₀ and CC₅₀ values calculated for the inhibition of viral replication and the cell toxicity of the molecules, correspond to the concentrations of molecule measured at 50 % of viral replication and cytotoxic response, respectively.

### CRediT authorship contribution statement

**Jean Guillon:** Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. **Solène Savrimoutou:** Methodology, Investigation, Formal analysis. **Nicolas Da Rocha:** Validation, Methodology, Formal analysis. **Sandra Albenque-Rubio:** Methodology, Investigation, Formal analysis. **Olivier Helynck:** Methodology, Investigation, Formal analysis. **Cyrielle Durand:** Methodology, Investigation, Formal analysis. **Jeanne Chiaravalli:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation. **Noël Pinaud:** Methodology, Investigation, Formal analysis. **Luisa Ronga:** Methodology, Investigation, Formal analysis. **Stéphane Moreau:** Investigation, Formal analysis. **Simon Chirolod:** Methodology. **Tshering Zangmo:** Methodology, Investigation, Formal analysis. **Melika Arab:** Methodology. **Lindita Lari:** Methodology, Investigation, Formal analysis. **Jean-Louis Mergny:** Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Funding acquisition. **Hélène Munier-Lehmann:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Marc Lavigne:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Data availability statement

Crystal structure data have been deposited in the Cambridge Crystallographic Data Centre (CCDC, University Chemical Lab, Lensfield Road, Cambridge, CB2 1 EW, UK) with accession code CCDC-2294691, CCDC-2294692, CCDC-2294693, CCDC-2294694, CCDC-2294695, CCDC-2294698, CCDC-2324394 and CCDC-2345480 for 11, 19, 20, 21, 24, 27, 39 and 15, respectively. Available online: https://www.ccdc.cam.ac.uk/.

### Funding

Covid Task force PFR7 Therapeutics program of the Institut Pasteur (Paris, France); ANR RA-COVID-19; internal resources of the involved laboratories originating from the Institut Pasteur, and the French national research institutions (CNRS, Inserm); Université de Bordeaux; Ecole Polytechnique. This research was also partly supported by grants from ANRS-MIE (BIOVAR and PRI projects, EMERGEN research program) awarded to the CCB core facility (JC & CD), directed by F. Agou. J. L.M. would like to express his gratitude to the Fondation de l'Ecole polytechnique and the Agence de l'Innovation de Défense (AID) via the Centre Interdisciplinaire d'Etudes pour la Défense et la Sécurité (CIEDS) [project 2023 - Pathogens] for their financial support and contribution to this research.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2025.117655.

## Data availability

Data will be made available on request.

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