# N,N'-bridged heterocyclic bis-iminium salts as potent antimalarial agents against multi-resistant Plasmodium falciparum

M. C. Casanova, Y. Zhao, Sébastien Hutter, M. Fil, M.H. Do, A. Benech, T.-B. Muller, C. Curti, M. Médebielle, P. Vanelle, et al.

► To cite this version:

M. C. Casanova, Y. Zhao, Sébastien Hutter, M. Fil, M.H. Do, et al., N,N'-bridged heterocyclic bis-iminium salts as potent antimalarial agents against multi-resistant Plasmodium falciparum. European Journal of Medicinal Chemistry, 2025, 292, pp.117633. 10.1016/j.ejmech.2025.117633. hal-05059854

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Research paper

N,N'-bridged heterocyclic bis-iminium salts as potent antimalarial agents
against multi-resistant *Plasmodium falciparum*

M.C. Casanova<sup>a,b</sup>, Y. Zhao<sup>a,c</sup>, S. Hutter<sup>b</sup>, M. Fil<sup>a</sup>, M.H. Do<sup>a,b</sup>, A. Benech<sup>a</sup>, T.-B. Muller<sup>a</sup>, C. Curti<sup>a,d</sup>, M. Médebielle<sup>e</sup>, P. Vanelle<sup>a,d,***</sup>, N. Azas<sup>b,**</sup>, J. Broggi<sup>a,*</sup>

ARTICLE INFO

Keywords:

N-heterocyclic iminium salts

Bis-aminopyridinium salts

K1 multi-resistant *Plasmodium falciparum* strain
(*Pf*K1)

*In vitro* antimalarial activity

Bivalent interactions

A B S T R A C T

The emergence of *Plasmodium* resistance to past and newly introduced antimalarial drugs reinforces the need for new antimalarial agents with innovative mechanisms of action. In this regard, the search for new redox-cycling compounds might offer an interesting approach to treat malaria since *P. falciparum* is sensitive to oxidative stress. Based on our experience in the design of powerful organic reducers, we undertook here the synthesis and evaluation of different families of *N*-heterocyclic iminium salts that could provide more selective redox-cycling candidates against the K1 multi-resistant *Plasmodium falciparum* strain (*Pf*K1). Bis-aminopyridinium salts emerged as the most promising candidates, exhibiting nanomolar antiplasmodial activity comparable to that of methylene blue. The biological study also underlined the positive impact on the *in vitro* activity of electron-donating groups and bis-salt forms, likely due to simultaneous bivalent interactions with the target. Our investigations revealed differences in the mechanism of action according to the heterocycle nature, highlighting an original mechanism for bis-aminopyridinium derivatives, different from that of chloroquine.

# 1. Introduction

The *Plasmodium falciparum* parasite is considered one of the most significant threats in malaria, a disease that caused 249 million cases worldwide, including 608, 000 deaths, in 2022 [1]. Despite major progress in vaccines, the eradication of this parasitosis is still out of reach, and the search for innovative treatments remains a priority [2]. The emergence of resistance to past and newly introduced antimalarial drugs [3] reinforces the need for new antimalarial agents with innovative mechanisms of action. Redox-active antiparasitic drugs [4,5] are, for instance, an interesting approach since *P. falciparum* is particularly sensitive to oxidative stress [6,7].

In the search for new pro-oxidant drugs [8,9], little attention has
been given to the antimalarial redox cycling properties of *N*-heterocyclic
cations. Yet, methylene blue (*MB*) has regained significant interest as a

potent inhibitor of *P. falciparum* and *P. vivax* and to combat
drug-resistant malaria [10,11]. In particular, *MB* has been shown to be a
selective inhibitor of *P. falciparum* glutathione reductase (GR), a vital
enzyme in the parasite, protecting it against oxidative stress [12,13]. *MB*
activity was correlated with its reversible one-electron reduction reac-
tion, promoting the toxic release of reactive oxygen species (ROS) into
the parasite's environment. Several clinical trials have reported no
serious adverse effects for *MB*, apart from mild urogenital and gastro-
intestinal symptoms, as well as blue coloration of urine [14,15]. Meth-
ylene blue treatment is also associated with a slight, but clinically
non-significant, reduction in haemoglobin concentrations.

Other *N*-heterocyclic iminium salts have demonstrated powerful *in vitro* and/or *in vivo* antimalarial activities, but their redox-active properties were not investigated. Though, initial reports suggest original mechanisms of inhibition through membrane processing or signalling,

<footnote><sup>a</sup> Aix Marseille Univ, CNRS, Institut de Chimie Radiculaire ICR - UMR 7273, Faculté de Pharmacie, 13005, Marseille, France</footnote>

<footnote><sup>b</sup> Aix Marseille Univ, IHU Méditerranée Infection, UMR VITROME, 13005, Marseille, France</footnote>

<footnote><sup>c</sup> Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, 250117, Jinan, China</footnote>

<footnote><sup>d</sup> Assistance Publique – Hôpitaux de Marseille (AP-HM), Service central de la qualité et de l'information pharmaceutiques (SCQIP), Pôle pharmacie, Hôpital de la Conception, 13005, Marseille, France</footnote>

<footnote><sup>e</sup> Univ Lyon 1, CNRS, Institut de Chimie et de Biochimie Moléculaires et Supramoléculaires ICBMS-UMR 5246, 69622, Villeurbanne, France</footnote>

<footnote>* Corresponding author.</footnote>

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

<footnote>*** Corresponding author. Aix Marseille Univ, CNRS, Institut de Chimie Radiculaire ICR - UMR 7273, Faculté de Pharmacie, 13005 Marseille, France.</footnote>

<footnote>E-mail addresses: patrice.vanelle@univ-amu.fr (P. Vanelle), nadine.azas@univ-amu.fr (N. Azas), julie.broggi@univ-amu.fr (J. Broggi).</footnote>
whereas conventional antimalarial drugs mainly inhibit classic intra-
cellular processes (like heme detoxification [16] or folate de novo syn-
thesis through inhibition of the dihydrofolate reductase [17]). For
instance, several triazolium (e.g. 1) and imidazolium salts (e.g. 2 and 3)
[18–20] were identified as potent inhibitors of Plasmodium invasion,
that would probably interact with a sulphated or phosphorylated region
of Merozoite Surface Protein 1 (MSP1) (Fig. 1) [21]. Thiazolium salts (e.
g 4 and 5) [22–24], were even evaluated in clinical trials, but stopped at
phase 2 due to lack of in vivo efficacy (SAR97276A) [25]. These choline
mimetics [26,27] could interfere with the de novo biosynthesis of
phosphatidylcholine during the membrane biogenesis of
parasite-infected red blood cells [28]. Quinolinium salts (e.g. 7) were
also compared to their chloroquine counterpart but were less active and
clearly used the same mechanism of action to inhibit hemozoin forma-
tion [29]. Surprisingly, less attention has been paid to pyridinium salts,
although Sasaki reported promising nanomolar activities (e.g. 6, IC₅₀ =
5 nM) [30–32]. Their preliminary investigations foreshadowed a
different mechanism of action for pyridinium salts, with a more pro-
nounced stage-dependent effect between schizont and ring forms.

From the perspective of a structure-redox activity relationship study,
we undertook here a closer evaluation of forty-six *N*-heterocyclic imi-
nium salts that could provide good and more selective redox-cycling
candidates. The antiplasmodial activity of *N*,*N*'-bridged bis-cationic
iminium heterocycles was also compared to the activity of their mono-
cationic counterparts to evaluate the impact of bivalent interactions.

**2. Results and discussion**

**A Chemical syntheses:** The series of aromatic iminium salts was
defined based on our experience in the design of powerful organic re-
ducers and their precursors [33–37]. Indeed, these iminium salts can be
seen as the oxidized forms of dihydrocyclic amines, and could thus *in
vivo* generate potent and selective redox-cyclers [38]. The salts were
prepared following simple one- or two-step procedures (Scheme 1).
(Benz)imidazolium and pyridinium salts were obtained with moderate

to excellent yields (21–99 %) by simple *N*-alkylation of commercially
available heterocycles and filtration treatments. For instance, pyr-
idinium salts **25–34** were efficiently prepared from the nucleophile
substitution of the appropriate halo or dihaloalkanes with 4-aminopyr-
idines. Quinolinium and quinazolinium series were synthesized
through *N*-alkylation and amination steps (S<sub>NAr</sub>) on the corresponding
chloro-heterocycles. Depending on the heterocycle, these two steps
could be reversed. For instance, the first strategy (1/2) was more suit-
able for the preparation of unbridged amino-quinazolinium salts **43–48**
and N,N'-bridged bis-quin(az)olinium salts **49–51**. These were obtained
by first performing the amination (step 1) of 4-chloro-quin(az)olines *via*
nucleophilic aromatic substitution (S<sub>NAr</sub>) followed by *N*-alkylation (step
2) of the resulting amino-quin(az)olines with various (di)haloalkanes.
Alternatively, the 4-chloroquinolines could first be *N*-alkylated (step 2)
to facilitate the amination step (step 1) on the resulting
electron-deficient 4-chloroquinolinium intermediates. This strategy
(2/1) significantly increased the overall yield of unbridged
amino-quinolinium salts **39–42** but was not applicable to quinazolinium
or N,N'-bridged quin(az)olinium salts. In a previous report, we discussed
in detail the implications of each of these strategies [39]. Full chemical
characterizations confirmed the structure and purity of the series of
imidazolium, triazolium, benzimidazolium, pyridinium, quinolinium
and quinazolinium salts.

**B Antiplasmodial activity and cytotoxicity assessment:** The *in vitro* antiplasmodial activity of these *N*-heterocyclic iminium salts against the K1 multi-resistant *Plasmodium falciparum* strain (PfK1) was then evaluated, along with their cytotoxicity on the HepG2 human cell line. Triazolium (8-11) and benzimidazolium (12-13) derivatives with small alkyl substituents did not display significant antiplasmodial activities (IC₅₀ > 50 µM) or cytotoxicity effects (Table 1, entries 1-4). Nevertheless, an increase in aromaticity and lipophilicity in benzyl-substituted triazolium 11, or particularly between the mono cationic-benzimidazolium 12 and *N*,*N*'-tethered bis-benzimidazolium 13, was observed to be detrimental to the cytotoxicity. In the imidazolium series (14-23), similar conclusions were drawn (Table 1, entries 5-8). Despite

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Chemical structures of some antimalarial *N*-heterocyclic iminium salts and their respective IC₅₀ and CC₅₀ values (Strain indicated in parentheses).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Scheme 1.** General procedures for the synthesis of *N*-heterocyclic iminium salts.</figcaption></figure>

an electron-withdrawing or -donating group on the backbone, *N*-methyl imidazolium salts **14–16** and propylene-bridged bis-imidazolium **17** were not active, albeit non-cytotoxic (CC₅₀ > 500 µM and IC₅₀ > 50 µM). In contrast, the more lipophilic and bulkier *N*-aromatic- and *N*-cycloalkyl-(*C*ₙ) substituted imidazolium **18–23** and imidazolinium **24** showed potent *in vitro* antiplasmodial activities, with IC₅₀ up to 800 nM for cyclododecyl-containing imidazolium **23** (Table 1, entries 7–9). However, low selectivity indexes were noted (0.8 < SI < 2.30) related to high cytotoxicities. Hence, the hydrophobicity of the bulky aromatic and cycloalkyl rings seemed to play a key role in the antiplasmodial activity of the azolium cores. Szarek and co-workers also observed that the replacement of a benzene ring by a more hydrophobic ring, such as naphthalene, significantly increased both the antimalarial potency and the cytotoxicity of imidazolium salts [18].

On the other hand, bis-aminopyridinium salts presented very interesting antiplasmodial activities without significant *in vitro* cytotoxicity (Table 2). Compared to the mono-cationic *N*-methyl-aminopyridinium salt **25** showing no significant *in vitro* activity (IC₅₀ = 56.8 µM) (Entry 1), *N*,*N'*-tethering of two aminopyridinium cycles through a polymethylene bridge greatly enhanced the antiplasmodial activity (Entry 2). Interestingly, a minimum of three carbons was required to observe activity against the PfK1 strain, as evidenced by the lack of activity observed with the ethyl-bridged salt **26** (IC₅₀ > 50 µM). Lengthening the alkyl bridge from three to twelve carbons (**27–33**) progressively decreased the IC₅₀ values from 12.5 to 0.0087 µM, reaching the nanomolar scale and improving the antimalarial efficacy by approximately 1500-fold. The longer polymethylene-bridged aminopyridinium compound **33** (n = 12, IC₅₀ = 8.7 nM) exhibited high nanomolar potency against PfK1, comparable to methylene blue (*MB*, IC₅₀ = 8.8 nM) or artesunate (IC₅₀ = 3.7 nM). These polymethylene-bridged aminopyridinium (**27–33**) series was not cytotoxic to Chinese hamster ovary (CHO) cells (CC₅₀ > 250 µM, entry 2) and thus exhibited high selectivity index (SI > 20 to 28 735). Salts **28–33** presented a higher selectivity index against CHO cells than the reference drugs chloroquine or doxycycline. Compound **33** even had a slightly superior selectivity index (SI > 28 735) than artesunate (>27 027).

This lack of cytotoxicity was confirmed on HepG2 cells with salts containing a moderate-length alkyl bridge (**26–29**, n = 2–5). They again exhibited a higher selectivity index (SI up to > 303) against HepG2 cells than the reference drugs chloroquine or doxycycline. Unexpectedly, from six-carbon bridges, an increasingly strong cytotoxicity was measured on HepG2 cells (**30–33**, n = 6–12, CC₅₀ = 5.3–0.4 µM). Two separate studies also observed that methylene blue, while known for its clinically non-significant adverse effects, was more toxic to tumour cell lines, such as HepG2, than to normal peripheral blood mononuclear cells (PBMCs) [41,42]. Although human hepatocytes HepG2 were considered a gold standard for toxicity and drug metabolism studies, they are increasingly seen as less reliable cell lines [43,44]. We also confirmed that this toxicity was not due to the presence of bromide counterions.

Identical values were obtained with a hexafluorophosphate salt form (PF₆) of **30** or **33**. Apart from a difference of solubility [45], exchange of the halogen for a hexafluorophosphate counterion did not usually affect the activity nor the toxicity (e.g. X = I vs. PF₆ for **28** or **29**, entry 3). Removal of the four methyl substituents from the *p*-amino groups in compound **34** also increased cytotoxicity on HepG2 (CC₅₀ = 11.9 µM) and reduced the selectivity index by at least a factor of 18.5 compared to compound **27**.

Hence, the SAR study highlighted that a minimum of three carbons was required to observe activity against the PfK1 strain. Sasaki et al. also underlined the impact of alkyl bridge length on the antiplasmodial activity of pyridinium salt **6** (n = 10) (Fig. 1) [30–32]. These results strongly suggested that our bivalent iminium salts, containing two electron-deficient pyridinium rings, could interact simultaneously with two domains of the same target while maintaining a specific distance between the two heterocycles. Bivalent compounds generally led to higher selectivity of interaction, and higher efficiency. Identical conclusions were drawn in the literature by Szarek and co-workers [19]. Free rotation of the aminopyridinium cycles around the polymethylene bridge also seemed crucial for antiplasmodial activity and supported the existence of bivalent interactions (Table 2, entries 4–5). Indeed, impeding flexibility by introducing a carbon-carbon bond between the two pyridinium rings led to a complete loss of the activity of the corresponding bis-annulated counterpart **35**. Additionally, hindering rotation through a methyl substituent on the butyl bridge slightly decreased the activity (36: 1.4 µM vs **28-PF₆**: 1.1 µM). This rotatability parameter could also explain the lack of activity of ethyl-bridged pyridinium **26**. Interestingly, the partial or total transformation of the iminiums into lactam functions in **37** and **38** led to a complete loss of the activity (Table 2, entries 6–7). This confirmed the significant role of the redox-active iminium function in the mechanism of action.

Based on these findings, we finally evaluated the impact of lipophilicity and *N*,*N'*-bridging on the antiplasmodial activity of aminoquinolinium and aminoquinazolinium series (Table 3). The introduction of a larger *π*-conjugated system could be beneficial for *π*-stacking interactions with the aminoquinolinium oxidant form but also for the stability of the dihydroaminoquinoline reducer form. Compared to the previous 4-aminopyridinium salt **25**, adding a supplementary benzene ring fused with the pyridine ring of 4-aminoquinolinium salt **39** appeared beneficial to the activity but less favourable for the cytotoxicity (Entry 1). Both monomeric quinolinium forms **39** and **40** had modest selectivity indexes, albeit comparable to those of doxycycline and chloroquine, respectively. Interestingly, the insertion of two methoxy groups on the benzene ring to increase the electron-donating properties totally reversed the trend, leading to the active and non-toxic quinolinium salts **41** and **42** (IC₅₀ = 1.9 and 0.7 µM and CC₅₀ = >100 and > 75 µM, respectively).

In the aminoquinazolinium series, similar trends were observed, although there was a notable disparity in toxicity (Table 3, entry 2).
<table><caption>**Table 1**

*In vitro* evaluation of azolium salts.</caption><thead><tr><th>Entry</th><th>Azolium salts</th><th>CC<sub>50</sub> (µM)<br>(HepG2 cells)<sup>[a]</sup></th><th>IC<sub>50</sub> (µM)<br>(Pf K1)<sup>[c]</sup></th><th>SI<sup>[d]</sup></th></tr></thead><tbody><tr><td rowspan="3">1</td><td rowspan="3">R'-N-N-N-I-R</td><td>8, R = H, R' = Me</td><td>&gt;500<sup>[b]</sup></td><td>&gt;50</td></tr><tr><td>9, R = Me, R' = Me</td><td>&gt;500</td><td>&gt;50</td></tr><tr><td>10, R = H, R' = NH<sub>2</sub></td><td>&gt;500</td><td>&gt;50</td></tr><tr><td>2</td><td>Phenyl-N-N-N-PF<sub>6</sub></td><td>11</td><td>167.1</td><td>&gt;50</td></tr><tr><td>3</td><td>Phenyl-N-N-I-R</td><td>12</td><td>273.5</td><td>&gt;50</td></tr><tr><td>4</td><td>Phenyl-N-N-N-I-R</td><td>13</td><td>57.5</td><td>&gt;50</td></tr><tr><td rowspan="2">5</td><td rowspan="2">R'-N-N-N-I-R</td><td>14, R = H</td><td>&gt;500</td><td>&gt;50</td></tr><tr><td>15, R = Me</td><td>&gt;500</td><td>&gt;50</td></tr><tr><td rowspan="2">6</td><td rowspan="2">Phenyl-N-N-I-R</td><td>16, R = NO<sub>2</sub></td><td>&gt;500</td><td>&gt;50</td></tr><tr><td>17</td><td>&gt;500</td><td>&gt;50</td></tr><tr><td>7</td><td>Phenyl-N-N-Cl-R</td><td>18</td><td>4.6 ± 1.5</td><td>2 ± 0.3</td></tr><tr><td rowspan="4">8</td><td rowspan="4">Phenyl-N-N-Cy<sub>n</sub></td><td>19, Cy<sub>5</sub></td><td>24.8 ± 17.9</td><td>25.1 ± 5.7</td><td>0.99</td></tr><tr><td>20, Cy<sub>6</sub></td><td>30.1 ± 16.7</td><td>26.4 ± 3.9</td><td>1.1</td></tr><tr><td>21, Cy<sub>7</sub></td><td>15.3 ± 5.6</td><td>13.0 ± 2.0</td><td>1.2</td></tr><tr><td>22, Cy<sub>8</sub></td><td>6.3 ± 1.9</td><td>8.3 ± 2.9</td><td>0.8</td></tr><tr><td rowspan="2">9</td><td rowspan="2">Phenyl-N-N-BF<sub>4</sub></td><td>23, Cy<sub>12</sub></td><td>0.8 ± 0.4</td><td>0.8 ± 0.1</td><td>1.0</td></tr><tr><td>24</td><td>9.8 ± 6.4</td><td>5.5 ± 0.9</td><td>1.8</td></tr><tr><td colspan="5">Doxorubicine</td></tr><tr><td colspan="5">Chloroquine</td></tr><tr><td colspan="5">Artesunate</td></tr><tr><td colspan="5">Doxycycline</td></tr></tbody></table>

Data represent the mean ± SD of at least three independent experiments carried out in duplicate [a] Cytotoxicity on HepG2 cells. Doxorubicin was used as a cytotoxic reference drug [b] Higher concentrations were not tested due to lack of solubility [c] Activity on K1 P. falciparum strain. Chloroquine, artesunate, and doxycycline were used as antimalarial reference drugs [d] The selectivity index (SI) was calculated according to the formula: $SI = CC_{50}/IC_{50}$. Cy<sub>n</sub>= Cycloalkyl ring of n carbon.

Indeed, the simple introduction of supplementary nitrogen to form a pyrimidine ring led to active and non-toxic derivatives, whatever the substituents on the benzene ring. Better activities against PfK1 were again observed for quinazolinium salts bearing electron-donating methoxy groups (45–48, IC<sub>50</sub> = 1.4–3.9 µM), with a 4-fold gain in some cases (e.g., 44 vs 47). The alkyl N-substituents of the 4-position amino group (NR<sub>2</sub><sup>2</sup>) also influenced *in vitro* activity. Whether in quinolinium and quinazolinium series, a slight improvement in activity was always observed upon increasing bulkiness by using ethyl instead of the methyl group (e.g., 41 vs 42, or 45 vs 47). Hence, combining ethyl amino-substituents and methoxy groups in the quinazolinium core 47 promoted high antiplasmodial activity (IC<sub>50</sub> = 2.9 µM) without causing any cytotoxicity effects (CC<sub>50</sub> > 125 µM). The impact of the alkyl N<sup>+</sup>

-substituent (R<sup>3</sup>) of the iminium seemed less pronounced (45-Me IC<sub>50</sub> = 3.9 µM vs 46-Et IC<sub>50</sub> = 2.9 µM). However, note that the introduction of a N<sup>+</sup>-benzyl substituent in 48 increased the activity, albeit also affecting toxicity. The benzyl moiety probably favoured hydrophobic and/or π-stacking interactions with the biological target. As with the pyridinium series, N,N'-tethering of two quinazolinium cycles with a propyl-bridge in 49 enhanced antiplasmodial activity (IC<sub>50</sub> = 0.8 µM) compared to its unbridged counterpart 47 (IC<sub>50</sub> = 2.9 µM) (Table 3, entry 3). Unexpectedly, this trend was not observed with N,N'-propyl-bridged quinolinium salt 51, which was twice less active (IC<sub>50</sub> = 1.8 µM) than unbridged quinolinium salt 42 (IC<sub>50</sub> = 0.7 µM). Once more, the exchange of the iodide for a hexafluorophosphate counterion did not affect the activity. 51 and 51-PF<sub>6</sub> exhibited similar antiplasmodial
<table><caption>**Table 2**

*In vitro* evaluation of pyridinium salts.</caption><thead><tr><th>Entry</th><th>Pyridinium salts</th><th>CC<sub>50</sub> (µM)<br>(HepG2)<sup>[a]</sup></th><th>CC<sub>50</sub> (µM)<br>(CHO)<sup>[c]</sup></th><th>IC<sub>50</sub> (µM)<br>(Pf K1)<sup>[d]</sup></th><th>SI<sup>[e]</sup><br>(HepG2)</th><th>SI<sup>[e]</sup><br>(CHO)</th><th>% inhibition<br>(heme)<sup>[f]</sup></th><th>IC<sub>50</sub> (µM)<br>(Pf. DHFR)<sup>[g]</sup></th></tr></thead><tbody><tr><td>1</td><td><img src="image_0_0.png" alt="Chemical structure of 25"></td><td>&gt;230<sup>[b]</sup></td><td></td><td>56.8</td><td>&gt;4.0</td><td></td><td></td><td></td></tr><tr><td>2</td><td><img src="image_0_1.png" alt="Chemical structure of 26, n=2, X=Br"></td><td>&gt;100<sup>[b]</sup></td><td></td><td>&gt;50<sup>[b]</sup></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>X=Br</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>27, n=3, X=I</td><td>&gt;230<sup>[b]</sup></td><td>&gt;250</td><td>12.5 ± 1.9</td><td>&gt;18.4</td><td>&gt;20.0</td><td>&lt;5</td><td>&gt;100</td></tr><tr><td></td><td>28, n=4, X=I</td><td>&gt;100<sup>[b]</sup></td><td>&gt;250</td><td>0.5 ± 0.3</td><td>&gt;196.1</td><td>&gt;490.0</td><td>&lt;5</td><td>&gt;100</td></tr><tr><td></td><td>X=PF<sub>6</sub></td><td>&gt;100<sup>[b]</sup></td><td>&gt;250</td><td>1.1 ± 0.5</td><td>&gt;85.9</td><td>&gt;214.7</td><td></td><td></td></tr><tr><td></td><td>29, n=5, X=I</td><td>&gt;100<sup>[b]</sup></td><td>&gt;250</td><td>0.4 ± 0.2</td><td>&gt;221.4</td><td>&gt;553.5</td><td></td><td></td></tr><tr><td></td><td>X=PF<sub>6</sub></td><td>&gt;100<sup>[b]</sup></td><td>&gt;250</td><td>0.3 ± 0.1</td><td>&gt;303.0</td><td>&gt;757.6</td><td>&lt;5</td><td>&gt;100</td></tr><tr><td></td><td>30, n=6, X=Br</td><td>5.3 ± 0.1</td><td>&gt;250</td><td>0.07 ± 0.02</td><td>76.0</td><td>&gt;3571.4</td><td></td><td></td></tr><tr><td></td><td>31, n=7, X=Br</td><td>2.6 ± 1.0</td><td>&gt;250</td><td>0.07 ± 0.00</td><td>36.4</td><td>&gt;3472.2</td><td></td><td></td></tr><tr><td></td><td>32, n=10, X=Br</td><td>0.7 ± 0.2</td><td>&gt;250</td><td>0.05 ± 0.00</td><td>13.7</td><td>&gt;5102.0</td><td></td><td></td></tr><tr><td></td><td>33, n=12, X=Br</td><td>0.4 ± 0.1</td><td>&gt;250</td><td>0.0087 ± 0.0235</td><td>46.9</td><td>&gt;28735.6</td><td></td><td></td></tr><tr><td>3</td><td><img src="image_0_2.png" alt="Chemical structure of 34"></td><td>11.9 ± 0.1</td><td>&gt;250</td><td>12.3 ± 2.6</td><td>1.0</td><td>&gt;20.3</td><td></td><td></td></tr><tr><td>4</td><td><img src="image_0_3.png" alt="Chemical structure of 35"></td><td>&gt;500<sup>[b]</sup></td><td></td><td>&gt;50<sup>[b]</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>5</td><td><img src="image_0_4.png" alt="Chemical structure of 36"></td><td>&gt;100<sup>[b]</sup></td><td></td><td>1.4 ± 0.1</td><td>&gt;74.6</td><td></td><td></td><td></td></tr><tr><td>6</td><td><img src="image_0_5.png" alt="Chemical structure of 37"></td><td>&gt;50<sup>[b]</sup></td><td></td><td>&gt;50<sup>[b]</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td><img src="image_0_6.png" alt="Chemical structure of 38"></td><td>&gt;50<sup>[b]</sup></td><td></td><td>&gt;50<sup>[b]</sup></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>Doxorubicine</td><td>0.2 ± 0.1</td><td>7.1 ± 0.7</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>Chloroquine</td><td>30.0</td><td>162.1 ± 13.8</td><td>0.9 ± 0.2</td><td>33.3</td><td>180.1</td><td>100</td><td></td></tr><tr><td></td><td>Artesunate</td><td>14.7 ± 1.4</td><td>&gt;100<sup>[h]</sup></td><td>0.0037 ± 0.0011</td><td>3973.0</td><td>&gt;27027.0</td><td></td><td></td></tr><tr><td></td><td>Doxycycline</td><td>20.0</td><td>34.2 ± 12.8</td><td>8.5 ± 0.0</td><td>2.4</td><td>4.0</td><td></td><td></td></tr><tr><td></td><td>Cycloguanil</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>Pyrimethamine</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Data represent the mean ± SD of at least three independent experiments carried out in duplicate [a] Cytotoxicity on HepG2 cells. Doxorubicin was used as a cytotoxic reference drug [b] Higher concentrations were not tested due to lack of solubility [c] Cytotoxicity on CHO cells. Doxorubicin was used as a cytotoxic reference drug [d] Activity on K1 P. falciparum strain. Chloroquine, artesunate and doxycycline were used as antimalarial reference drugs [e] The selectivity index (SI) was calculated according to the formula: SI = CC<sub>50</sub>/IC<sub>50</sub> [f] Activity on heme polymerization inhibition. Compounds were tested at 0.5 mM. Chloroquine was used as a reference drug (100 % of inhibition at 0.5 mM). [g] Activity on Saccharomyces cerevisiae-transformed yeasts (with P. falciparum DHFR gene). Cycloguanil and pyrimethamine were used as antifolate reference drugs. [h] Obtained from reference [40].

activities (IC<sub>50</sub> = 1.8 and 1.4 µM, CC<sub>50</sub> > 100 µM, respectively). Surprisingly again, increasing the length of the alkyl bridge to a C4-chain in bis-quinazolinium 50 led to a decrease in antiplasmodial activity (IC<sub>50</sub> = 3.9 µM). One hypothesis could be that, while the space between two

small pentyl-bridged pyridinium rings was optimal for simultaneous bivalent interaction, the larger methoxy-substituted bis-quinazolinium salt might not tolerate a space longer than a propyl bridge to maintain good bivalency with the biological target. Another hypothesis is that
<table><caption>**Table 3**

*In vitro* evaluation of quinazolinium and quinolinium salts.</caption><thead><tr><th>Entry</th><th>Quinolinium and Quinazolinium salts</th><th>CC<sub>50</sub> (µM)<br>(HepG2)<sup>[a]</sup></th><th>IC<sub>50</sub> (µM)<br>(Pf K1)<sup>[c]</sup></th><th>SI<sup>[d]</sup></th><th>% inhibition<br>(heme)<sup>[e]</sup></th><th>IC<sub>50</sub> (µM)<br>(Pf.<br>DHFR)<sup>[f]</sup></th></tr></thead><tbody><tr><td rowspan="4">1</td><td rowspan="4">[Image of 1,3-diphenyl-2,4,5,6-tetrahydro-3H-1,4-benzoxazin-2-one]</td><td>39, R<sup>1</sup>= H, R<sup>2</sup>= Me, R<sup>3</sup>= Me, X= PF<sub>6</sub></td><td>34.3 ± 2.8</td><td>9.1 ± 1.3</td><td>3.1</td><td></td></tr><tr><td>40, R<sup>1</sup>= H, R<sup>2</sup>= Et, R<sup>3</sup>= Me, X= PF<sub>6</sub></td><td>86.6 ± 6.2</td><td>2.5 ± 0.5</td><td>28.2</td><td></td></tr><tr><td>41, R<sup>1</sup>= OMe, R<sup>2</sup>= Me, R<sup>3</sup>= Me, X= PF<sub>6</sub></td><td>>100</td><td>1.9 ± 0.8</td><td>>51.3</td><td></td></tr><tr><td>42, R<sup>1</sup>= OMe, R<sup>2</sup>= Et, R<sup>3</sup>= Me, X= I</td><td>>75</td><td>0.7 ± 0.2</td><td>>107.1</td><td>13.3</td><td>>100</td></tr><tr><td rowspan="5">2</td><td rowspan="5">[Image of 1,3-diphenyl-2,4,5,6-tetrahydro-3H-1,4-benzoxazin-2-one]</td><td>43, R<sup>1</sup>= H, R<sup>2</sup>= Me, R<sup>3</sup>= Me, X= I</td><td>>125</td><td>14.2 ± 0.7</td><td>>8.8</td><td></td></tr><tr><td>44, R<sup>1</sup>= H, R<sup>2</sup>= Et, R<sup>3</sup>= Me, X= PF<sub>6</sub></td><td>>100</td><td>12.2 ± 1.4</td><td>>8.2</td><td></td></tr><tr><td>45, R<sup>1</sup>= OMe, R<sup>2</sup>= Me, R<sup>3</sup>= Me, X= I</td><td>>125</td><td>3.9 ± 0.9</td><td>>31.4</td><td></td></tr><tr><td>46, R<sup>1</sup>= OMe, R<sup>2</sup>= Me, R<sup>3</sup>= Et, X= I</td><td>>100</td><td>2.9 ± 1.0</td><td>>34.5</td><td></td></tr><tr><td>47, R<sup>1</sup>= OMe, R<sup>2</sup>= Et, R<sup>3</sup>= Me, X= I</td><td>>125</td><td>2.9 ± 0.3</td><td>>42.4</td><td>&lt;5</td><td>>100</td></tr><tr><td rowspan="4">3</td><td rowspan="4">[Image of 1,3-diphenyl-2,4,5,6-tetrahydro-3H-1,4-benzoxazin-2-one]</td><td>48, R<sup>1</sup>= OMe, R<sup>2</sup>= Et, R<sup>3</sup>= Bn, X= Br</td><td>>70</td><td>1.4 ± 0.7</td><td>51.8</td><td>&lt;5</td><td></td></tr><tr><td>49, Y= N, n= 3, X= I</td><td>>75</td><td>0.8 ± 0.5</td><td>>125</td><td>15.9</td><td>>100</td></tr><tr><td>50, Y= N, n= 4, X= Br</td><td>>50</td><td>3.9 ± 1.4</td><td>>12.8</td><td></td></tr><tr><td>51, Y= CH, n= 3, X= I</td><td>>100</td><td>1.8 ± 0.5</td><td>>54.9</td><td>74.8</td><td>>100</td></tr><tr><td colspan="2">Doxorubicine</td><td colspan="5">0.2 ± 0.1</td></tr><tr><td colspan="2">Chloroquine</td><td colspan="5">30.0</td></tr><tr><td colspan="2">Artesunate</td><td colspan="5">14.7 ± 1.4</td></tr><tr><td colspan="2">Doxycycline</td><td colspan="5">20.0</td></tr><tr><td colspan="2">Cycloguanil</td><td colspan="5"></td></tr><tr><td colspan="2">Pyrimethamine</td><td colspan="5"></td></tr></tbody></table>

Data represent the mean ± SD of at least three independent experiments carried out in duplicate [a] Cytotoxicity on HepG2 cells. Doxorubicin was used as a cytotoxic reference drug [b] Higher concentrations were not tested due to lack of solubility [c] Activity on K1 P. falciparum strain. Chloroquine, artesunate and doxycycline were used as antimalarial reference drugs [d] The selectivity index (SI) was calculated according to the formula: $SI = CC_{50}/IC_{50}$ [e] Activity on heme polymerization inhibition. Compounds were tested at 0.5 mM. Chloroquine was used as reference drug (100 % of inhibition at 0.5 mM). [f] Activity on Saccharomyces cerevisiae-transformed yeasts (with P. falciparum DHFR gene). Cycloguanil and pyrimethamine were used as antifolate reference drugs. Bn= Benzyl

these different N-heterocyclic iminium salts do not share the same mechanism of action against Plasmodium falciparum.

**C Mechanism of Action Studies** To validate either of these hypotheses, we conducted various physicochemical and biological studies. The physicochemical properties of the pyridinium, quinolinium, and quinazolinium salt series were predicted using the ADMET predictor® 12 software (see section 4, in supporting information). The fraction absorbed (Fa), *ie* crossing the enterocyte membrane, as well as the fraction bioavailable (F), *ie* reaching the systemic blood circulation, were predicted in fasted humans using the advanced compartmental and transit model displayed in GastroPlus® software (version 9.8.3). In terms of solubility, the additional benzene rings of quinolinium and quinazolinium salts made them less soluble than their pyridinium counterparts (e.g. 12.5–13.5 mg/ml for 39 and 43 vs 164 mg/ml for 25). Alkyl bridges also made dicationic structures less soluble than their monocationic counterparts (141.5 mg/ml for 26). Inevitably, solubility decreases with the length of the alkyl bridge. Pyridinium salts with the shortest alkyl bridges (n = 2–6) exhibited the best solubilities (13–141.5 mg/ml), while the most active salt, compound 33, showed the lowest solubility (0.5 mg/ml). Nevertheless, according to the predicted values of Fa, all compounds demonstrated a good ability to cross the gastrointestinal barrier (Fa >82 % at 100 mg), even at high doses for our most active molecules (Fa >80 % at 1 g for compounds 28–33). The Fa values were similar regardless of the length of the alkyl bridge in the pyridinium series. Absolute bioavailability remained good for these same compounds (F = 48–75 % at 100 mg and 44–58 % at 1 g for compounds 28–33), despite a high hepatic first-pass metabolism. The difference between bioavailability (F) and the absorbed fraction (Fa) is more pronounced for pyridinium salts with the longest alkyl bridges (a gap of 37 % for compound 32 (n = 10) and 46 % for compound 33 (n = 12)), which would indicate a higher risk of metabolism during hepatic first-pass for 32 and 33. The lowest bioavailabilities were observed for the

largest compounds, the bis-quinolinium salts 51 and bis-quinazolinium salts 49–50 with dicationic bridges (36–39 %).

To assess cross-resistance, the IC<sub>50</sub> values of our most promising bis-iminium salts against the chloroquine-resistant PfK1 strain were compared to their activity against the drug-sensitive 3D7 P. falciparum strain (Table 4). All salts were equally effective against both strains, suggesting that their mechanism of action differs from that of conventional drugs (chloroquine, pyrimethamine, etc.), thereby minimizing the risk of cross-resistance. Compared to chloroquine (RI = 23), most salts

<table><caption>**Table 4**

Compared activity on 3D7 and K1 Pf strains.</caption><thead><tr><th>Compounds</th><th>3D7 IC<sub>50</sub> (µM)</th><th>K1 IC<sub>50</sub> (µM)</th><th>RI</th></tr></thead><tbody><tr><td>27</td><td>6.4</td><td>12.5</td><td>1.9</td></tr><tr><td>28</td><td>0.78</td><td>1.1</td><td>1.4</td></tr><tr><td>29-I</td><td>1.34</td><td>0.4</td><td>0.3</td></tr><tr><td>29-PF<sub>6</sub></td><td>0.75</td><td>0.3</td><td>0.4</td></tr><tr><td>30</td><td>0.103</td><td>0.07</td><td>0.7</td></tr><tr><td>31</td><td>0.053</td><td>0.07</td><td>1.3</td></tr><tr><td>32</td><td>0.01</td><td>0.05</td><td>5</td></tr><tr><td>33</td><td>0.016</td><td>0.0087</td><td>0.5</td></tr><tr><td>34</td><td>>25</td><td>12.3</td><td>0.5</td></tr><tr><td>42</td><td>1.11</td><td>0.7</td><td>0.6</td></tr><tr><td>47</td><td>2.63</td><td>2.9</td><td>1.1</td></tr><tr><td>48</td><td>0.87</td><td>1.4</td><td>1.6</td></tr><tr><td>49</td><td>0.48</td><td>0.8</td><td>1.7</td></tr><tr><td>51</td><td>2.76</td><td>1.8</td><td>0.6</td></tr><tr><td>Chloroquine</td><td>0.039</td><td>0.9</td><td>23</td></tr><tr><td>Doxycycline</td><td>6.35</td><td>8.5</td><td>1.3</td></tr><tr><td>Artesunate</td><td>0.0022</td><td>0.0037</td><td>1.7</td></tr></tbody></table>

Data represent the mean ± SD of at least three independent experiments carried out in duplicate. Chloroquine, doxycycline and artesunate were used as antimalarial reference drugs. The resistance index (RI) was calculated according to the formula: $RI=IC_{50} (K1)/IC_{50} (3D7)$.
exhibited a resistance index (RI) below 2. Several salts were even showing greater potency against the resistant strain than the sensitive strain (RI < 1).

Further biological mechanistic studies were conducted on some pyridinium, quinazolinium and quinolinium candidates to confirm this distinctive mode of action. N,N'-tethered bis-pyridinium (27, 28, 29-PF₆), bis-quinazolinium (49) and bis-quinolinium salts (51), were selected for their similar polymethylene-bridge length and progressively increasing selectivity indexes (SI ranging from 18.4 to 303 on HepG2 cells). For comparison, we also evaluated their unbridged counterparts (47, 48, and 42). First, an *in vitro* heme polymerization inhibition assay was performed to evaluate how our antiplasmodial N-heterocyclic iminium salts performed on this common mechanism. The inhibition of heme polymerization into hemozoin (β-hematin) is one of the principal mechanisms of action for 4-aminoquinoline-based antimalarial drugs such as chloroquine and amodiaquine. Not surprisingly, the dicationic quinolinium salt 51 strongly inhibited β-hematin formation (75 %), just like the chloroquine reference, which exhibited powerful inhibition at 0.5 mM (Fig. 2, Table 3). Monocationic quinolinium salt 42 and dicationic quinazolinium salt 49 also displayed close inhibitory effects on heme polymerization at 0.5 mM with 13 and 16 % of inhibition, respectively, albeit significantly lower than chloroquine. The inhibiting capacity trend of these quin(az)olinium salts was also in line with their *in vitro* activities on the PfK1 strain. The most active N,N'-bridged quin (az)olinium salts, 49 and 51, also induced the greatest inhibitions of heme polymerization. Interestingly, no inhibition of heme polymerization was noted with benzyl-substituted quinazolinium salt 48 (<5 %), even though it was active on PfK1. Although the presence of a quinazolinium pharmacophore is essential to inhibit heme polymerization, these results suggest that the introduction of a bulky and aromatic substituent on the iminium can bypass this mechanism of action. Finally, bis-pyridinium salts 27, 28 and 29-PF₆, sharing similar activity on PfK1, did not produce any inhibition effect on heme polymerization, even at high concentrations (<5 %) (Fig. 2, Table 2). Hence, bis-pyridinium compounds could present a unique mechanism of action, different from that of chloroquine.

To confirm the distinctive mode of action taken by our pyridinium series, a second *in vitro* inhibition assay was conducted to assess the folate pathway sometimes claimed for the antiplasmodial activity of pyridine derivatives [46]. This assay evaluates the inhibition of *P. falciparum* dihydrofolate reductase (*Pf*). DHFR, an enzyme involved in the folate pathway (de novo synthesis of purines and thymidylate for DNA). In our case, none of the selected *N*-heterocyclic iminium salts, including the pyridinium ones, showed any inhibition effect on the dihydrofolate reductase (Fig. 3, Tables 2 and 3). These results strongly suggested an original mechanism of action for our polymethylene-bridged bis-pyridinium salts.

The shared structural characteristics between our bis

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** *In vitro* heme polymerization inhibition assay. Histograms represent the maximal inhibition of active compounds compared to chloroquine (CQ, 100 %) at 0.5 mM.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** *In vitro* inhibition assay of Plasmodial dihydrofolate reductase (*Pf* DHFR). Curves represent the percentage of *Pf* DHFR-transfected *Saccharomyces cerevisiae* yeasts growth in the presence of active compounds, compared to pyrimethamine (*PYR*, IC₅₀ = 2.6 µM) and cycloguanil (*CG*, IC₅₀ = 14.8 µM) used as antifolate reference drugs.</figcaption></figure>

(dimethylamino)pyridinium salts and the bis(dimethylamino) phenothiazin-5-ium of the methylene blue (MB) or the bis-pyridinium of the paraquat (PQ), led us to consider a redox-cycling mechanism. MB and PQ act as redox cyclers that disrupt the redox balance in *P. falciparum* by facilitating electron transfers with NADPH or other cellular redox systems (Scheme 2) [10,12,13]. The 4,4'-bipyridinium of the paraquat salt PQ is *in vivo* reduced to a dihydropyridine radical cation PQ⁺, capable of reducing the oxygen into reactive oxygen species, such as toxic superoxide (O₂⁻) [47]. These redox disorders represent interesting anti-malarial pathways, especially when parasite enzymes involved in redox homeostasis and antioxidant defense, such as glutathione reductase or thioredoxin reductase, are selectively targeted [48–51]. However, while MB is not toxic, the easy and non-selective redox cycling of paraquat, catalysed within cells by NADPH cytochrome P450 reductase, makes it highly toxic to human cells [52,53]. New redox-cycling drugs with potent and more selective reducing properties for *P. falciparum* parasite enzymes, would thus be of great importance.

The redox-cycling behaviour of our best candidates was thus measured by cyclic voltammetry (CV) to evaluate their potential as precursors of potent dihydropyridine and dihydropyrimidine reducers (Table 5). It should be noted that the trimethylene-bridged amino-pyridinium salt 27 is a known precursor of a super electron donor with a highly negative reduction potential for reactions in organic synthesis [33,34,54], and redox-cycling properties for organic batteries [55]. Electrochemical characterization of all iminium salts showed an irreversible reduction wave at strong negative potentials (Ep<sub>c</sub>, Fig. 4) [56]. The CV profiles then followed a cyclic reduction/oxidation process (Scheme 3), similar to the one reported for other N-heterocyclic iminium salts [57–59]. The first electron-transfer reduction of the iminium salt leads to a dihydro-pyridine or -quin(az)olinium radical cation intermediate with higher reducing power (Ep<sub>c</sub> = -1.72 to - 2.31 V vs Ag/Ag⁺ 0.01 M in CH<sub>3</sub>CN/NBu<sub>4</sub>PF<sub>6</sub>) than PQ⁺ or MB⁺ (Ep<sub>c</sub> ≈ - 0.75 V). A second electron transfer could lead to an even more potent biradical species. The most *in vitro* active bis-pyridinium salts 28–33 (IC<sub>50</sub> = 0.0087–0.5 µM) also exhibited the highest reduction potentials (Ep<sub>c</sub> = - 2.28 to - 2.31 V), showing a significant gap of >0.5 V compared to their quin(az)olinium counterparts (IC<sub>50</sub> = 0.7–2.9 µM, Ep<sub>c</sub> = - 1.72 to - 1.78

<figure><img src="image_7.png" /><figcaption>**Scheme 2.** Reported redox-cycling mechanism of paraquat (PQ).</figcaption></figure>
<table><caption>**Table 5**
Reduction potentials of selected iminium salts.</caption><thead><tr><th>Compounds</th><th>Ep<sub>pc1</sub> (V)<sup>[a]</sup></th><th>Pf K1 IC<sub>50</sub> (µM)</th></tr></thead><tbody><tr><td>26</td><td>-2.16</td><td>&gt;50</td></tr><tr><td>27</td><td>-2.23</td><td>12.5</td></tr><tr><td>28</td><td>-2.28</td><td>0.5</td></tr><tr><td>29</td><td>-2.31</td><td>0.4</td></tr><tr><td>30</td><td>-2.28</td><td>0.07</td></tr><tr><td>31</td><td>-2.28</td><td>0.07</td></tr><tr><td>32</td><td>-2.28</td><td>0.05</td></tr><tr><td>33</td><td>-2.28</td><td>0.0087</td></tr><tr><td>42</td><td>-1.78</td><td>0.7</td></tr><tr><td>47</td><td>-1.77</td><td>2.9</td></tr><tr><td>49</td><td>-1.72</td><td>0.8</td></tr><tr><td>PQ</td><td>-0.76<sup>[b]</sup></td><td>42 (Pf 3D7)</td></tr><tr><td>MB</td><td>-0.73<sup>[c]</sup></td><td>0.0088</td></tr></tbody></table>

<sup>a</sup> Ep<sub>c</sub> of the PF<sub>6</sub>-salt forms = cathodic peak potential vs Ag/Ag<sup>+</sup> 0.01 M in CH<sub>3</sub>CN/NBu<sub>4</sub>PF<sub>6</sub> [56].

<sup>b</sup> Ep<sub>c</sub> = -0.76, -1.18 V vs Ag/Ag<sup>+</sup> 0.01 M in CH<sub>3</sub>CN/NBu<sub>4</sub>PF<sub>6</sub>.

<sup>c</sup> Ep<sub>c</sub> = -0.73, -1.54 V vs Ag/Ag<sup>+</sup> in CH<sub>3</sub>CN/0.1 M TEAP [56,60].

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Cyclic voltammetry of 29-PF<sub>6</sub> as an example.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Scheme 3.** Redox-cycling processes of our N-heterocyclic iminium salts based on CV analysis.</figcaption></figure>

V). The equivalent reduction potential of the non-active ethane-bridged bis-pyridinium **26** (Ep<sub>c</sub> = -2.16 V), compared to the active polymethylene-bridged bis-pyridiniums (Ep<sub>c</sub> = -2.28 V) suggests that a negative redox potential is not the sole parameter required to ensure strong antiplasmodial activity. This underscores the necessity of a minimum three-carbon bridge to facilitate efficient bivalent interaction with the biological target. Propane-bridged bis-quinazolinium **49** and its monocyclic counterpart **47** also had equivalent reduction potentials (Ep<sub>c</sub> = -1.72 and -1.77 V respectively).

*In vivo* generation of such strong dihydro-pyridine reducers could lead to high rates of oxidative stress through the efficient release of toxic ROS.

### 3. Conclusion

Our structure-activity relationship study on a large range of N-heterocyclic iminium salts underlined the positive impact of the pyridinium ring on the *in vitro* antiplasmodial activity against PfK1, while maintaining remarkably low cytotoxicities. The introduction of bulky and hydrophobic substituents and/or the presence of electron-donating substituents were also essential for the activity. The dimeric heterocyclic iminium salts with alkyl-bridge spacer (n = 3–12) were the most potent candidates, likely through simultaneous bivalent interactions with two biological sites. The longer polymethylene-bridged aminopyridinium salt (n = 12) exhibited high nanomolar activity (IC<sub>50</sub> = 8.7 nM) against PfK1, comparable to methylene blue or artesunate. Moreover, the mechanical studies on aminopyridinium salts attested to their potent cyclic reductive potentials and revealed an original mechanism of action, distinct from that of chloroquine. Further biological studies are now underway to confirm their selective redox-cycling mechanism of action on parasite enzymes and strong pro-oxidant properties, which could subject the parasite to high oxidative stress.

### 4. Material and methods

#### 4.1. Synthetic procedures

The synthetic procedures of the active compounds are provided here. All other synthetic procedures and characterization data are documented in the supplementary information file.

General chemistry considerations: Reagents were provided by Sigma Aldrich, Fisher Scientific, Doug Discovery or TCI and used as received. ¹H NMR (200, 250, 300 or 400 MHz), ¹³C NMR (50, 62.9 or 100 MHz), ³¹P (162 MHz) and ¹⁹F (376.5 MHz) spectra were recorded at ambient temperature in solutions of CDCl₃ (δ<sub>H</sub> = 7.26 ppm and δ<sub>C</sub> = 77.16 ppm), CD₃OD (δ<sub>H</sub> = 3.31 ppm and δ<sub>C</sub> = 49.00 ppm) or DMSO-d₆ (δ<sub>H</sub> = 2.50 ppm and δ<sub>C</sub> = 39.52 ppm) on Bruker spectrometers with automatic sampler. HRMS analyses were carried out at the Spectropole, Faculté des Sciences et Techniques de Saint-Jérôme, Aix Marseille université. Compounds **18** (1,3-bis(2,6-diisopropylphenyl)-1H-imidazole-3-ium chloride (IPr.HCl), CAS: 250285-32-6) and **24** (1,3-dimesityl-4,5-dihydro-1H-imidazole-3-ium tetrafluoroborate (SIMes.HBF₄), CAS: 245679-18-9) were commercially available. N-Cycloalkyl-N'-mesityl-substituted imidazolium chlorides **19** to **23** (IMesCy<sub>n</sub>.HCl) were generously provided by Dr M. Mauduit and co-workers from the Ecole Nationale Supérieure de Chimie de Rennes and synthesized according to the procedures reported in the literature [61]. Purity of tested compounds was confirmed by elemental analysis or by liquid chromatography (>95 %).

1,3-Bis(N,N'-dimethyl-4-aminopyridinium)propane diiode **27**: In a sealed flask, a solution of 4-dimethylaminopyridine (10 g, 81.85 mmol, 1 equiv.) and 1,3-diiodopropane (4.2 mL, 36.83 mmol, 0.45 equiv.) was stirred 72 h in anhydrous acetonitrile (20 mL) at 100 °C under N₂ atmosphere. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3x 10 mL) and dried under vacuum to give **27** as white solid (19 g, 96 %). ¹H NMR: (400 MHz, DMSO-d₆) δ 2.36 (2H, q), *J* = 7.2 Hz, CH₂), 3.20 (12H, s, 4 NCH₃), 4.25 (4H, t, *J* = 7.2 Hz, N + CH₂), 7.05 (4H, d, *J* = 7.8 Hz, 2H³ & H⁵), 8.29 (4H, d, *J* = 7.8 Hz, 2H² & H⁶). ¹³C NMR (100 MHz, DMSO-d₆) δ 31.0 (CH₂), 39.8 (NCH₃), 53.6 (N + CH₂), 107.8 (CH³ & CH⁵), 141.8 (CH² & CH⁶), 155.8 (C⁴). HPLC: Purity >96 %

1,4-Bis(N,N'-dimethyl-4-aminopyridinium)butane diiode, **28**:

In a sealed microwave vial, a solution of 4-dimethylaminopyridine (2 g, 16.3 mmol, 1 equiv.) and 1,4-diiodobutane (0.97 mL, 7.34 mmol, 0.45 equiv.) was stirred 72 h in anhydrous acetonitrile (12 mL) at 90°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with
diethyl ether (3x 10 mL) and dried under vacuum to give **28** as a white solid (3.93 g, 96 %). ¹H NMR: (400 MHz, DMSO-d6) δ 1.73 (4H, broad s, 2 CH2), 3.19 (12H, s, 4 NCH3), 4.20 (4H, broad s, 2 N+CH2), 7.05 (4H, broad d, J = 6.6 Hz, 2 H³ & H⁵), 8.30 (4H, m, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 27.2 (CH2), 40.1 (NCH3), 56.1 (N+CH2), 107.9 (CH³ & CH⁵), 142.1 (CH² & CH⁶), 156.01 (C⁴). HRMS (ESI): m/z 150.1152 [M]²⁺, calcd for C₁₈H₂₈N₄²⁺ 150.1151. HPLC: Purity > 99%

**1,4-Bis(N,N'-dimethyl-4-aminopyridinium)butane dihexafluorophosphate, 28-PF6:** A solution of 1,4-bis(N,N'-dimethyl-4-aminopyridinium)butane diiodide **28** (0.12 g, 0.217 mmol, 1 equiv.) was dissolved in a MeOH/H₂O solution (10 mL, 1:1) with potassium hexafluorophosphate (0.39 g, 2.17 mmol, 10 equiv.) and stirred 2 h at 60°C under reflux. Water (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with water (3 x 10 mL) to give **28-PF6** as a white solid (0.10 g, 78 %). ¹H NMR: (400 MHz, DMSO-d6) δ 1.72 (4H, broad s, 2 CH2), 3.18 (12H, s, 4 NCH3), 4.17 (4H, broad s, 2 N+CH2), 7.03 (4H, d, J = 7.7 Hz, 2 H³ & H⁵), 8.25 (4H, d, J = 7.7 Hz, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 27.0 (CH2), 39.7 (NCH3), 56.0 (N+CH2), 107.7 (CH³ & CH⁵), 141.9 (CH² & CH⁶), 155.8 (C⁴). ¹⁹F NMR (DMSO-d6) δ: -69, d, J = 773 Hz. ³¹P NMR (DMSO-d6) δ: -145, sept, J = 773 Hz. HRMS (ESI): m/z 150.1156 [M]²⁺, calcd for C₁₈H₂₈N₄²⁺ 150.1151. Elemental analysis: N : 9.32, C : 36.35, H : 4.65, Theo calcd for C₁₈H₂₈F₁₂N₄P₂: N : 9.49, C : 36.62, H : 4.78

**1,5-Bis(N,N'-dimethyl-4-aminopyridinium)pentane diiodide, 29:** In a sealed microwave vial, a solution of 4-dimethylaminopyridine (1 g, 8.18 mmol, 1 equiv.) and 1,5-diiodopentane (1.17 g, 3.68 mmol, 0.45 equiv.) was stirred 72 h in anhydrous acetonitrile (10 mL) at 90°C. Diethyl ether (25 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give **29** as a white solid (2.09 g, >99 %). ¹H NMR: (400 MHz, DMSO-d6) δ 1.19 (2H, broad qi, J = 7.5 Hz, CH2), 1.80 (4H, qi, J = 7.3 Hz, 2 CH2), 3.19 (12H, s, 4 NCH3), 4.16 (4H, t, J = 7.3 Hz, 2 N+CH2), 7.04 (4H, d, J = 7.7 Hz, 2 H³ & H⁵), 8.30 (4H, d, J = 7.7 Hz, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 21.7 (CH2), 29.6 (CH2), 39.8 (NCH3), 56.1 (N+CH2), 107.7 (CH³ & CH⁵), 141.9 (CH² & CH⁶), 155.7 (C⁴). HRMS (ESI): m/z 157.1235 [M]²⁺, calcd for C₁₉H₃₀N₄²⁺ 157.1230. Elemental analysis: N: 9.66, C: 39.11, H: 5.36; Theo calcd for: C₁₉H₃₀I₂N₄. H₂O: N: 9.56, C: 38.92, H: 5.50.

**1,5-Bis(N,N'-dimethyl-4-aminopyridinium)pentane dihexafluorophosphate 29-PF6:** A solution of 1,5-bis(N,N'-dimethyl-4-aminopyridinium) pentane diiodide **29** (1 g, 1.75 mmol, 1 equiv.) was dissolved in a MeOH/H₂O solution (15 mL, 1:1) with potassium hexafluorophosphate (3.23g, 17.5 mmol, 10 equiv.) and stirred 2 h at 60°C under reflux. Water (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and triturated in water (3 x 10 mL) to give **29-PF6** as a white solid (0.83g, 78 %). ¹H NMR: (400 MHz, DMSO-d6) δ 1.19 (2H, broad qi, J = 7.5 Hz, CH2), 1.79 (4H, qi, J = 7.3 Hz, 2 CH2), 3.18 (12H, s, 4 NCH3), 4.14 (4H, t, J = 7.3 Hz, 2 N+CH2), 7.03 (4H, d, J = 7.7 Hz, 2 H³ & H⁵), 8.26 (4H, d, J = 7.7 Hz, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 21.9 (CH2), 29.6 (CH2), 39.7 (NCH3), 56.3 (NCH2), 107.6 (CHAr), 141.9 (N+CHAr), 155.8 (CAr). ¹⁹F NMR (DMSO-d6) δ: -69, d, J = 656 Hz. ³¹P NMR (DMSO-d6) δ: -144, sept, J = 656 Hz. HRMS (ESI): m/z 157.1230 [M]²⁺, calcd for C₁₉H₃₀N₄²⁺ 157.1230. EA: N: 9.52, C: 37.62, H: 5.12; Theo calcd for C₁₉H₃₀F₆N₄P: N: 9.27, C: 37.76, H: 5.00.

**1,6-Bis(N,N'-dimethyl-4-aminopyridinium)hexane dibromide, 30:** In a sealed microwave vial, a solution of 4-dimethylaminopyridine (0.5 g, 4.09 mmol, 1 equiv.) and 1,6-dibromohexane (0.28 mL, 1.84 mmol, 0.45 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 80°C. Diethyl ether (25 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give **30** as a white solid (0.90 g, >99 %). ¹H NMR: (400 MHz, DMSO-d6) δ 1.25 (4H, broad m, 2 CH2), 1.74 (4H, broad m, 2 CH2), 3.18 (12H, s, 4 NCH3), 4.20 (4H, t, J = 7.3 Hz, 2 N+CH2), 7.05 (4H, d, J = 7.2 Hz, 2 H³ & H⁵), 8.39 (4H, d, J

= 7.2 Hz, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 24.8 (CH2), 30.0 (CH2), 39.7 (NCH3), 56.3 (N+CH2), 107.6 (CH³ & CH⁵), 142.0 (CH² & CH⁶), 155.7 (C⁴). HRMS (ESI): m/z 164.1309[M]²⁺, calcd for C₂₀H₃₂N₄²⁺ 164.1308. Elemental analysis: N: 11.06, C: 47.39, H: 6.74; Theo calcd for: C₂₀H₃₂Br₂N₄. H₂O: N: 11.07, C: 47.44, H: 6.77.

**1,7-Bis(N,N'-dimethyl-4-aminopyridinium)heptane dibromide, 31:** In a sealed microwave vial, a solution of 4-dimethylaminopyridine (1 g, 8.18 mmol, 1 equiv.) and 1,7-dibromoheptane (0.63 mL, 3.68 mmol, 0.45 equiv.) was stirred 24 h in anhydrous acetonitrile (15 mL) at 80°C. Diethyl ether (25 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give **31** as a white solid (1.81 g, >99 %). ¹H NMR: (400 MHz, DMSO-d6) δ 1.20 (4H, m, 2 CH2), 1.29 (2H, m, CH2), 1.75 (4H, m, 2 CH2), 3.19 (12H, s, 4 NCH3), 4.17 (4H, t, J = 7.2 Hz, 2 N+CH2), 7.04 (4H, d, J = 7.7 Hz, 2 H³ & H⁵), 8.33 (4H, d, J = 7.7 Hz, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 25.2 (CH2), 27.8 (CH2), 30.2 (NCH3), 39.7 (CH2), 56.5 (N+CH2), 107.6 (CH³ & CH⁵), 142.0 (CH² & CH⁶), 155.8 (C⁴). HRMS (ESI): m/z 171.1387 [M]²⁺, calcd for C₂₁H₃₄N₄²⁺ 171.1386. Elemental analysis: N: 11.21, C: 50.06, H: 6.88; Theo calcd for: C₂₁H₃₄Br₂N₄: N: 11.15, C: 50.21, H: 6.82.

**1,10-Bis(N,N'-dimethyl-4-aminopyridinium)decane dibromide, 32:** In a sealed microwave vial, a solution of 4-dimethylaminopyridine (0.5 g, 4.09 mmol, 1 equiv.) and 1,10-dibromodecane (0.41 mL, 1.84 mmol, 0.45 equiv.) was stirred 24h in anhydrous acetonitrile (10 mL) at 80°C. Diethyl ether (25 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give **32** as a white solid (0.95 g, 95 %). ¹H NMR: (400 MHz, DMSO-d6) δ 1.22 (12H, broad m, 6 CH2), 1.73 (4H, qi, J = 7.3 Hz, 2 CH2), 3.18 (12H, s, 4 NCH3), 4.16 (4H, t, J = 7.3 Hz, 2 N+CH2), 7.04 (4H, d, J = 7.8 Hz, 2 H³ & H⁵), 8.34 (4H, d, J = 7.8 Hz, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 25.4 (CH2), 28.4 (CH2), 28.8 (CH2), 30.3 (CH2), 39.7 (NCH3), 56.6 (N+CH2), 107.7 (CH³ & CH⁵), 142.0 (CH² & CH⁶), 155.8 (C⁴). HRMS (ESI): m/z 192.1623[M]²⁺, calcd for C₂₄H₄₀N₄²⁺ 192.1621. Elemental analysis: N: 10.19, C: 52.63, H: 7.40; Theo calcd for: C₂₄H₄₀Br₂N₄: N: 10.29, C: 52.95, H: 7.41.

**1,12-Bis(N,N'-dimethyl-4-aminopyridinium)dodecane dibromide, 33:** In a sealed microwave vial, a solution of 4-dimethylaminopyridine (0.5 g, 4.09 mmol, 1 equiv.) and 1,12-dibromododecane (0.6 g, 1.84 mmol, 0.45 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 80°C. Diethyl ether (25 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give **33** as a white solid (1.03 g, 98 %). ¹H NMR: (400 MHz, CD₃OD) δ 1.33-1.40 (16H, m, 8 CH2), 1.89 (4H, qi, J = 7.4 Hz, 2 CH2), 3.29 (12H, s, 4 NCH3), 4.22 (4H, t, J = 7.4 Hz, 2 N+CH2), 7.04 (4H, d, J = 7.8 Hz, 2 H³ & H⁵), 8.23 (4H, d, J = 7.8 Hz, 2 H² & H⁶). ¹³C APT NMR (100 MHz, DMSO-d6) δ 27.2 (CH2), 30.2 (CH2), 30.5 (CH2), 30.6 (CH2), 32.0 (CH2), 40.3 (NCH3), 58.9 (N+CH2), 109.0 (CH³ & CH⁵), 143.1 (CH² & CH⁶), 157.9 (C⁴). HRMS (ESI): m/z 206.1782[M]²⁺, calcd for C₂₆H₄₄N₄²⁺ 206.1778. Elemental analysis: N: 9.67, C: 54.18, H: 7.85; Theo calcd for: C₂₆H₄₄Br₂N₄: N: 9.79, C: 54.55, H: 7.75.

**1,3-Bis(4-aminopyridinium)propane diiodide, 34:** In a sealed microwave vial, a solution of 4-aminopyridine (1 g, 10 mmol, 1 equiv.) and 1,3-diiodopropane (0.52 mL, 4.3 mmol, 0.45 equiv.) was stirred 24 h in anhydrous acetonitrile (15 mL) at 80°C under N₂ atmosphere. Diethyl ether (25 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3x 10 mL) and dried under vacuum to give **34** as a white solid (1.96 g, 94 %). ¹H NMR (400 MHz, DMSO-d6) δ 2.27 (2H, qi, J = 7.2 Hz, CH2), 4.17 (4H, t, J = 7.3 Hz, 2 N+CH2), 6.80 (4H, d, J = 7.4 Hz, 2 H³ & H⁵), 8.09 (4H, s, 2 NH2), 8.15 (4H, d, J = 7.4 Hz, 2 H² & H⁶). ¹³C NMR (100 MHz, DMSO-d6) δ 31.5 (CH2), 54.5 (N+CH2), 110.0 (CH⁴ & CH⁵), 143.3 (CH² & CH⁶), 159.1 (C⁴). HRMS (ESI) m/z 115.0760 [M]²⁺, calcd for C₁₃H₁₈N₄²⁺ 115.0760. Elemental analysis: N: 11.71, C: 31.48,
H : 3.77. Theo calcd for C₁₃H₁₈I₂N₄ : N : 11.57, C : 32.25, H : 3.75.

**1,1'-(Pentane-1,4-diyl)bis(4-(dimethylamino)pyridin-1-ium) dihexa-fluorophosphate, 36:** In a sealed microwave vial, a solution of 4-dimethylaminopyridine (1 g, 8.11 mmol, 1 equiv.) and 1,4-diiodopentane (0.54 mL, 3.65 mmol, 0.45 equiv.) was stirred 72 h in anhydrous acetonitrile (10 mL) at 90°C. Diethyl ether (50 mL) was then added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give a mixture of 1,1'-(pentane-1,4-diyl)bis(4-(dimethylamino)pyridin-1-ium) diiodide **S1** (NMR ratio: 69 %) and 1,4-diiodopentane (NMR ratio: 31 %) as a pale-yellow solid (1.37 g). Then, a solution of this impurified **S1** (0.5 g, 0.88 mmol, 1 equiv.) and potassium hexa-fluorophosphate (1.63 g, 8.88 mmol, 10 equiv.) was stirred 2 h in a MeOH/H₂O solution (20 mL, 1:1) at 60°C under reflux. Water (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with water (3 x 10 mL) to give **36** as a white solid (0.39 g, 74 %). ¹H NMR: (400 MHz, DMSO-d₆) δ 1.45 (3H, d, J = 6.6 Hz, CH₃), 1.47-1.54 (1H, m, CH₂), 1.70-1.80 (3H, m, CH₂), 3.18 (6H, s, 2 NCH₃), 3.19 (6H, s, 2 NCH₃), 4.13-4.15 (2H, m, N⁺CH₂), 4.38-4.43 (1H, m, N⁺CH) 7.02 (4H, d, J = 7.2 Hz, H³, H⁵, H⁸ & H¹⁰), 8.23 (2H, d, J = 7.2 Hz, H² & H⁶), 8.32 (2H, d, J = 7.2 Hz, H⁷ & H¹¹). ¹³C APT NMR (100 MHz, DMSO-d₆) δ 20.3 (CH₃), 26.8 (CH₂), 32.0 (CH₂) 39.7 (NCH₃), 56.0 (N⁺CH₂), 63.0 (N⁺CH), 107.7 (2 CHAr), 107.9 (2 CHAr), 140.2 (2 N⁺CHAr), 141.9 (2 N⁺CHAr), 155.8 (CAr), 156.1 (CAr). ¹⁹F NMR (DMSO-d₆) δ: -69, d, J = 766 Hz. ³¹P NMR (DMSO-d₆) δ: -144, sept, J = 766 Hz. HRMS (ESI): m/z 157.1229 [M]⁺, calcd for C₁₃H₃₀N₄⁺ 157.1230. Elemental analysis: N : 9.12, C : 37.37, H : 4.75; Theo calcd for C₁₃H₃₀F₁₂N₄P₂: N : 9.27, C : 37.76, H : 5.00.

**4-(Dimethylamino)-1-methylquinolin-1-ium hexafluorophosphate, 39:** In a sealed flask, a mixture of 4-(chloro)-1-methylquinolin-1-ium (**S2**) and 4-(iodo)-1-methylquinolin-1-ium (**S3**) chloride or iodide[⁵⁶] (1.0 g, 3.28 mmol, 1.0 equiv.) and a 2M solution of dimethylamine in tetrahydrofuran (16.4 mL, 10 equiv.) was stirred 24 h at 60°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give a pale-yellow solid mixture (1.2 g) of 4-(dimethylamino)-1-methylquinolin-1-ium iodide or chloride **S4**. A solution of **S4** (0.5 g, 1.59 mmol, 1 equiv.) and potassium hexa-fluorophosphate (0.732 g, 3.98 mmol, 2.5 equiv.) was then stirred 2 h in a MeOH/H₂O solution (20 mL, 1:1) at 60°C under reflux. Water (20 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with water (3 x 10 mL) and dried under vacuum to give **39** as a yellow solid (0.25 g, 46 %). ¹H NMR (400 MHz, DMSO-d₆) δ 3.46 (6H, s, 2 NCH₃), 4.13 (3H, s, N⁺CH₃), 7.01 (1H, d, J = 7.5 Hz, H³), 7.71 (1H, ddd, J = 1.5, 6.6, and 8.4 Hz, H⁶), 8.01-8.08 (2H, m, H⁵ & H⁷), 8.41 (1H, dd, J = 0.9 and 8.7 Hz, H⁸), 8.56 (1H, d, J = 7.5 Hz, H²). ¹³C APT NMR (100 MHz, DMSO-d₆) δ 41.8 (N⁺CH₃), 44.3 (NCH₃), 102.7 (CH³), 118.1 (CH⁸), 118.3 (C⁴a), 125.2 (CH⁵), 127.9 (CH⁶), 133.5 (CH⁷), 139.8 (C⁸a), 145.3 (CH²), 159.2 (C⁴). ¹⁹F NMR (DMSO-d₆) δ -69, d, J = 650Hz. ³¹P NMR (DMSO-d₆) δ -144, sept, J = 650 Hz. HRMS (ESI): m/z 187.1230 [M]⁺, calcd for C₁₂H₁₅N₂⁻ 187.1230. Elemental analysis: N : 8.24, C : 42.89, H : 4.79; Theo calcd for C₁₂H₁₅F₆N₂P: N : 8.43, C : 43.38, H : 4.55.

**4-(Diethylamino)-1-methylquinolin-1-ium hexafluorophosphate, 40:** In a sealed flask, a solution of 4-(chloro)-1-methylquinolin-1-ium (**S2**) and 4-(iodo)-1-methylquinolin-1-ium (**S3**) chloride or iodide[⁵⁶] (1.0 g, 3.28 mmol, 1.0 equiv.) and diethylamine (7.4 mL, 71.6 mmol, 20 equiv.) was stirred 24 h in anhydrous dimethylformamide (10 mL) at 115°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give a pale-yellow solid mixture (0.825 g) of 4-(diethylamino)-1-methylquinolin-1-ium iodide and chloride **S5**. A solution of **S5** (0.5 g, 1.46 mmol, 1 equiv.) and potassium hexafluorophosphate (0.67 g, 3.65 mmol, 2.5 equiv.) was stirred 2 h in a MeOH/H₂O solution (20 mL, 1:1) at 60 °C under reflux. Water (20 mL) was added to the cooled suspension to favour the precipitation

of the solid. The suspension was filtered and washed with water (3 x 10 mL) and dried under vacuum to give **40** as an orange solid (0.32 g, 61 %). ¹H NMR: (400 MHz, DMSO-d₆) δ 1.38 (6H, t, J = 7 Hz, 2 CH₃), 3.78 (4H, q, J = 7 Hz, 2 NCH₂), 4.13 (3H, s, N⁺CH₃), 7.06 (1H, d, J = 7.5 Hz, H³), 7.74 (1H, broad t, J = 8.2 Hz, H⁶), 8.01-8.10 (2H, m, H⁵ & H⁷), 8.17 (1H, broad d, J = 8.5 Hz, H⁸), 8.54 (1H, d, J = 7.5 Hz, H²). ¹³C APT NMR: (100 MHz, DMSO-d₆) δ 12.2 (CH₃), 41.8 (N⁺CH₃), 47.3 (NCH₂), 103.1 (CH³), 118.4 (CH⁸), 118.6 (C⁴a), 125.7 (CH⁵), 126.7 (CH⁶), 133.6 (CH⁷), 139.8 (C⁸a), 145.4 (CH²), 158.2 (C⁴). ¹⁹F NMR (DMSO-d₆) δ -70, d, J = 725 Hz. ³¹P NMR (DMSO-d₆) δ -143, sept, J = 725 Hz. HRMS (ESI): m/z 215.1547 [M]⁺, calcd for C₁₄H₁₉N₂⁻ 215.1543. Elemental analysis: N : 7.82, C : 46.02, H : 5.39; Theo calcd for C₁₄H₁₉F₆N₂P: N : 7.78, C : 46.67, H : 5.32.

**4-(Dimethylamino)-6,7-dimethoxy-1-methylquinolin-1-ium hexa-fluorophosphate, 41:** In a sealed flask, 4-(chloro)-6,7-dimethoxy-1-methylquinolin-1-ium (**S6**) and 4-(iodo)-6,7-dimethoxy-1-methylquinolin-1-ium (**S7**) chloride or iodide[⁵⁶] (1.0 g, 2.74 mmol, 1.0 equiv.) and a 2M solution of dimethylamine in tetrahydrofuran (13.7 mL, 10 mL equiv.) was stirred 24 h at 60°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give a white solid mixture (1 g, >99 %) of 4-(dimethylamino)-6,7-dimethoxy-1-methylquinolin-1-ium iodide or chloride **S8**. A solution of **S8** (0.5 g, 1.33 mmol, 1 equiv.) and potassium hexa-fluorophosphate (0.61 g, 3.32 mmol, 2.5 equiv.) was stirred 2 h in a MeOH/H₂O solution (20 mL, 1:1) at 60°C under reflux. Water (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with water (20 mL) and dried under vacuum to give **41** as a yellow solid (0.28 g, 54 %). ¹H NMR (400 MHz DMSO-d₆) δ 3.39 (6H, s, 2 NCH₃), 3.96 (3H, s, OCH₃), 4.06 (3H, s, OCH₃), 4.14 (3H, s, N⁺CH₃), 6.93 (1H, d, J = 7.2 Hz, H³), 7.31 (1H, s, H⁸), 7.54 (1H, s, H⁵), 8.45 (1H, d, J = 7.2 Hz, H²). ¹³C APT NMR (100 MHz DMSO-d₆) δ 42.2 (N⁺CH₃), 43.7 (NCH₃), 56.0 (OCH₃), 56.5 (OCH₃), 98.9 (CH³), 102.9 (CH⁵), 106.7 (CH⁸), 113.1 (C⁴a), 136.7 (C⁸a), 143.4 (CH²), 147.3 (C⁶), 154.2 (C⁷), 158.4 (C⁴). ¹⁹F NMR (DMSO-d₆) δ -69, d, J = 714Hz. ³¹P NMR (DMSO-d₆) δ -144, sept, J = 714Hz. HRMS (ESI): m/z 247.1444 [M]⁺, calcd for C₁₄H₁₉N₂O₂⁻ 247.1441. Elemental analysis: N : 6.81, C : 42.31, H : 5.13; Theo calcd for C₁₄H₁₉F₆N₂O₂P: N : 7.14, C : 42.87, H : 4.88.

**4-(Diethylamino)-6,7-dimethoxy-1-methylquinolin-1-ium iodide, 42:** In a sealed microwave vial, a solution of N,N'-diethyl-6,7-dimethoxyquinolin-4-amine **S9** (1 g, 3.84 mmol, 1 equiv.) and iodomethane (0.7 mL, 11.52 mmol, 3 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 110°C. Diethyl ether (25 mL) was then added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum to give **42** as a pale-yellow solid (1.41 g, 91 %). ¹H NMR: (400 MHz, DMSO-d₆) δ 1.37 (6H, t, J = 6.9 Hz, 2 CH₃), 3.72 (4H, q, J = 6.9 Hz, 2 NCH₂), 3.97 (3H, s, OCH₃), 4.07 (3H, s, OCH₃), 4.17 (3H, s, N⁺CH₃), 7.02 (1H, d, J = 7.4 Hz, H³), 7.33 (1H, s, H⁸), 7.34 (1H, s, H⁵), 8.46 (1H, d, J = 7.4 Hz, H²). ¹³C APT NMR: (100 MHz, DMSO-d₆) δ 12.3 (CH₃), 42.2 (N⁺CH₃), 46.7 (NCH₂), 55.8 (OCH₃), 56.6 (OCH₃), 99.1 (CH³), 103.5 (CH⁵), 105.6 (CH⁸), 113.5 (C⁴a), 136.8 (C⁸a), 143.4 (CH²), 147.5 (C⁶), 154.1 (C⁷), 157.4 (C⁴). HRMS (ESI): m/z 275.1756 [M]⁺, calcd for C₁₆H₂₃N₂O₂⁻ 275.1754. HPLC: Purity > 99%.

**4-(Dimethylamino)-1-methylquinazolin-1-ium iodide, 43:** In a sealed microwave vial, a solution of N,N-dimethylquinazolin-4-amine **S10** (1 g, 5.77 mmol, 1 equiv.) and iodomethane (1.1 mL, 17.31 mmol, 3 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 110°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give **43** as a pale-yellow solid (1.8 g, 99 %). ¹H NMR: (400 MHz, DMSO-d₆) δ 3.52 (3H, broad s, NCH₃), 3.69 (3H, broad s, NCH₃), 3.98 (3H, s, N⁺CH₃), 7.79 (1H, ddd, J = 1.1, 7.2 and 8.5 Hz, H⁶), 7.97 (1H, dd, J = 1.3 and 8.5 Hz, H²), 8.13 (1H, ddd, J = 1.3, 7.2 and 8.6 Hz, H⁷), 8.44 (1H, dd, J = 1.1
and 8.6 Hz, H<sup>8</sup>), 8.87 (1H, s, H<sup>2</sup>). <sup>13</sup>C NMR: (100 MHz, DMSO-d<sub>6</sub>) δ 38.1 (N<sup>+</sup>CH<sub>3</sub>), 41.3 (NCH<sub>3</sub>), 43.9 (NCH<sub>3</sub>), 112.5 (C<sup>4a</sup>), 117.0 (CH<sup>8</sup>), 127.0 (CH<sup>5</sup>), 128.6 (CH<sup>6</sup>), 135.8 (CH<sup>7</sup>), 140.1 (C<sup>8a</sup>), 151.1 (CH<sup>2</sup>), 161.1 (C<sup>4</sup>). HRMS (ESI): m/z 503.1400 [M<sup>+</sup>Γ]M<sup>+</sup>, calcd for C<sub>22</sub>H<sub>28</sub>N<sub>6</sub>O<sub>6</sub> 503.1415. HPLC: Purity > 97%.

**4-(Diethylamino)-1-methylquinazolin-1-ium hexafluorophosphate, 44:** In a sealed microwave vial, a solution of N,N-diethylquinazolin-4-amine S11 (0.4 g, 1.99 mmol, 1 equiv.) and iodomethane (0.37 mL, 5.97 mmol, 3 equiv.) was stirred 36 h in anhydrous acetonitrile (6 mL) at 110°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give 4-(diethylamino)-1-methylquinazolin-1-ium iodide S12 as a pale-yellow solid (0.52 g, 76 %). A solution of S12 (0.4 g, 1.16 mmol, 1 equiv.) and potassium hexafluorophosphate (1.07 g, 5.8 mmol, 5 equiv.) was stirred 2 h in a MeOH/H<sub>2</sub>O solution (20mL, 1:1) at 60°C under reflux. Water (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with water (3 x 10 mL) and dried under vacuum to give 44 as a pale-yellow solid (0.33 g, 79 %). <sup>1</sup>H NMR (400 MHz, DMSO-d<sub>6</sub>) δ 1.42 (6H, broad s, 2 CH<sub>3</sub>), 3.93 (4H, q, J = 7 Hz, 2 NCH<sub>2</sub>), 3.96 (3H, s, N<sup>+</sup>CH<sub>3</sub>), 7.82 (1H, broad t, J = 8.4 Hz, H<sup>6</sup>), 7.98 (1H, d, J = 8.4 Hz, H<sup>5</sup>), 8.14 (1H, broad t, J = 8.4 Hz, H<sup>7</sup>), 8.18 (1H, d, J = 8.4 Hz, H<sup>8</sup>), 8.84 (1H, s, H<sup>2</sup>). <sup>13</sup>C NMR (100 MHz, DMSO-d<sub>6</sub>) δ 12.4 (CH<sub>3</sub>), 38.1 (N<sup>+</sup>CH<sub>3</sub>), 46.3 (NCH<sub>2</sub>), 112.3 (C<sup>4a</sup>), 117.3 (CH<sup>8</sup>), 127.3 (CH<sup>5</sup>), 127.6 (CH<sup>6</sup>), 135.8 (CH<sup>7</sup>), 140.1 (C<sup>8a</sup>), 151.1 (CH<sup>2</sup>), 160.4 (C<sup>4</sup>). <sup>19</sup>F NMR (DMSO-d<sub>6</sub>) δ: -69, d, J = 741Hz.<sup>31</sup>C NMR (DMSO-d<sub>6</sub>) δ: -144, sept, J = 741 Hz. HRMS (ESI): m/z 216.1495 [M]<sup>+</sup>, calcd for C<sub>13</sub>H<sub>18</sub>N<sub>3</sub>O<sub>5</sub> 216.1495. Elemental analysis: N: 11.66, C: 42.52, H: 4.99, Theo calcd for C<sub>13</sub>H<sub>18</sub>F<sub>6</sub>N<sub>3</sub>P: N: 11.63, C: 42.22, H: 5.02.

**4-(Dimethylamino)-6,7-dimethoxy-1-methylquinazolin-1-ium iodide, 45:** In a sealed microwave vial, a solution of 6,7-dimethoxy-N,N'-dimethylquinazolin-4-amine S13 (1 g, 4.28 mmol, 1 equiv.) and iodomethane (0.8 mL, 12.84 mmol, 3.0 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 110°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give 45 as a pale-yellow solid (1.53 g, 95 %). <sup>1</sup>H NMR: (250 MHz, DMSO-d<sub>6</sub>) δ 3.56 (6H, s, 2 NCH<sub>3</sub>), 3.96 (3H, s, OCH<sub>3</sub>), 3.99 (3H, s, N<sup>+</sup>CH<sub>3</sub>), 4.07 (3H, s, OCH<sub>3</sub>), 7.29 (1H, s, H<sup>8</sup>), 7.64 (1H, s, H<sup>5</sup>), 8.77 (1H, s, H<sup>2</sup>). <sup>13</sup>C NMR: (100 MHz, DMSO-d<sub>6</sub>) δ 38.5 (N<sup>+</sup>CH<sub>3</sub>), 42.2 (NCH<sub>3</sub>), 56.2 (OCH<sub>3</sub>), 56.9 (OCH<sub>3</sub>), 98.5 (CH<sup>5</sup>), 106.1 (C<sup>4a</sup>), 107.8 (CH<sup>8</sup>), 137.1 (C<sup>8a</sup>), 147.8 (C<sup>6</sup>), 149.5 (CH<sup>2</sup>), 155.6 (C<sup>7</sup>), 160.2 (C<sup>4</sup>). HRMS (ESI): m/z 248.1393 [M]<sup>+</sup>, calcd for C<sub>13</sub>H<sub>18</sub>N<sub>3</sub>O<sub>2</sub> 248.1394. Elemental analysis: C: 41.61, H: 4.84, N: 11.20; Theo calcd for C<sub>13</sub>H<sub>18</sub>N<sub>3</sub>O<sub>2</sub>: C: 41.26, H: 4.69, N: 11.22.

**4-(Dimethylamino)-6,7-dimethoxy-1-ethylquinazolin-1-ium iodide, 46:** In a sealed microwave vial, a solution of 6,7-dimethoxy-N,N'-dimethylquinazolin-4-amine S13 (1 g, 4.28 mmol, 1 equiv.) and iodoethane (1 mL, 12.84 mmol, 3.0 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 110°C. Diethyl ether (50 mL) was added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give 46 as a yellow solid (1.48 g, 89 %). <sup>1</sup>H NMR: (400 MHz, DMSO-d<sub>6</sub>) δ 1.43 (3H, t, J = 7.1 Hz, CH<sub>3</sub>), 3.55 (6H, s, 2 NCH<sub>3</sub>), 3.96 (3H, s, OCH<sub>3</sub>), 4.07 (3H, s, OCH<sub>3</sub>), 4.54 (2H, q, J = 7.1 Hz, N<sup>+</sup>CH<sub>2</sub>), 7.31 (1H, s, H<sup>8</sup>), 7.63 (1H, s, H<sup>5</sup>), 8.82 (1H, s, H<sup>2</sup>). <sup>13</sup>C NMR: (100 MHz, DMSO-d<sub>6</sub>) δ 14.3 (CH<sub>3</sub>), 42.2 (NCH<sub>3</sub>), 46.0 (N<sup>+</sup>CH<sub>2</sub>), 56.2 (OCH<sub>3</sub>), 57.0 (OCH<sub>3</sub>), 98.2 (CH<sup>5</sup>), 106.4 (C<sup>4a</sup>), 108.0 (CH<sup>8</sup>), 135.9 (C<sup>8a</sup>), 147.8 (C<sup>6</sup>), 149.1 (CH<sup>2</sup>), 155.7 (C<sup>7</sup>), 160.3 (C<sup>4</sup>). HRMS (ESI): m/z 262.1551 [M]<sup>+</sup>, calcd for C<sub>14</sub>H<sub>20</sub>N<sub>3</sub>O<sub>2</sub> 262.1550. Elemental analysis: N: 10.80, C: 43.13, H: 5.06; Theo calcd for C<sub>14</sub>H<sub>20</sub>N<sub>3</sub>O<sub>2</sub>: N: 10.80, C: 43.20, H: 5.18.

**4-(Diethylamino)-6,7-dimethoxy-1-methylquinazolin-1-ium iodide, 47:** In a sealed microwave vial, a solution of N,N'-diethyl-6,7-dimethoxyquinazolin-4-amine S14 (1 g, 3.83 mmol, 1 equiv.) and iodomethane (0.7 mL, 11.5 mmol, 3 equiv.) was stirred 24 h in anhydrous acetonitrile

(10 mL) at 110°C. Diethyl ether (20 mL) was then added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered and washed with diethyl ether (3 x 10 mL) to give 47 as a pale-yellow solid (1.48 g, 96 %). <sup>1</sup>H NMR: (400 MHz, DMSO-d<sub>6</sub>) δ 1.43 (6H, t, J = 6.8 Hz, 2 CH<sub>3</sub>), 3.90 (4H, q, J = 6.8 Hz, 2 NCH<sub>2</sub>), 3.98 (3H, s, OCH<sub>3</sub>), 4.00 (3H, s, N<sup>+</sup>CH<sub>3</sub>), 4.08 (3H, s, OCH<sub>3</sub>), 7.30 (1H, s, H<sup>8</sup>), 7.39 (1H, s, H<sup>5</sup>), 8.78 (1H, s, H<sup>2</sup>). <sup>13</sup>CAPT NMR: (100 MHz, DMSO-d<sub>6</sub>) δ 12.6 (CH<sub>3</sub>), 38.5 (N<sup>+</sup>CH<sub>3</sub>), 45.9 (NCH<sub>2</sub>), 55.8 (OCH<sub>3</sub>), 56.9 (OCH<sub>3</sub>), 98.8 (CH<sup>5</sup>), 105.9 (C<sup>4a</sup>), 106.5 (CH<sup>8</sup>), 137.2 (C<sup>8a</sup>), 148.1 (C<sup>6</sup>), 149.5 (CH<sup>2</sup>), 155.6 (C<sup>7</sup>), 159.5 (C<sup>4</sup>). HRMS (ESI): m/z 276.1707 [M]<sup>+</sup>, calcd for C<sub>15</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub> 276.1707. Elemental analysis: N: 10.42, C: 44.68, H: 5.50; Theo calcd for C<sub>15</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub>: N: 10.61, C: 44.74, H: 5.47.

**4-(Diethylamino)-6,7-dimethoxy-1-benzylquinazolin-1-ium bromide, 48:** In a sealed flask, a solution of N,N'-diethyl-6,7-dimethoxyquinazolin-4-amine S14 (1.0 g, 3.83 mmol, 1.0 equiv.) and benzyl bromide (0.69 g, 4.02 mmol, 1.05 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at room temperature. Diethyl ether (50 mL) was added to the suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give 48 as a white solid (1.46 g, 88 %). <sup>1</sup>H NMR: (400 MHz, DMSO-d<sub>6</sub>) δ 1.45 (6H, broad s, 2 CH<sub>3</sub>), 3.86 (3H, s, OCH<sub>3</sub>), 3.94 (4H, q, J = 7 Hz, 2 NCH<sub>2</sub>), 3.94 (3H, s, overlapping the q, OCH<sub>3</sub>), 5.81 (2H, s, N<sup>+</sup>CH<sub>2</sub>), 7.24 (1H, s, H<sup>8</sup>), 7.32-7.46 (6H, m, H<sup>5</sup> & 5 H<sup>Ph</sup>), 9.11 (1H, s, H<sup>2</sup>). <sup>13</sup>CAPT NMR: (100 MHz, DMSO-d<sub>6</sub>) δ 12.6 (CH<sub>3</sub>), 46.2 (NCH<sub>2</sub>), 53.5 (N<sup>+</sup>CH<sub>2</sub>), 55.8 (OCH<sub>3</sub>), 56.7 (OCH<sub>3</sub>), 99.4 (CH<sup>5</sup>), 106.5 (C<sup>4a</sup>), 106.9 (CH<sup>8</sup>), 127.3 (CH<sup>Ph</sup>), 128.4 (CH<sup>Ph</sup>), 129.0 (CH<sup>Ph</sup>), 134.6 (C<sup>Ph</sup>), 135.9 (C<sup>8a</sup>), 148.0 (C<sup>6</sup>), 149.7 (CH<sup>2</sup>), 155.2 (C<sup>7</sup>), 159.4 (C<sup>4</sup>). HRMS (ESI): m/z 352.2021 [M]<sup>+</sup>, calcd for C<sub>21</sub>H<sub>26</sub>N<sub>3</sub>O<sub>2</sub> 352.2020. Elemental analysis: N: 9.63, C: 57.93, H: 6.03; Theo calcd for C<sub>21</sub>H<sub>26</sub>BrN<sub>3</sub>O<sub>2</sub>: N: 9.72, C: 58.34, H: 6.06.

**1,3-Bis(4-(diethylamino)-6,7-dimethoxy-quinazolinium)propane diiodide 49:** In a sealed microwave vial, a solution of N,N'-diethyl-6,7-dimethoxyquinazolin-4-amine S14 (1 g, 3.83 mmol, 1 equiv.) and 1,3-diiodopropane (0.2 mL, 1.84 mmol, 0.48 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 110°C. Diethyl ether (20 mL) was then added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL), and dried under vacuum to give 49 as a pale-yellow solid (1.41 g, 94 %). <sup>1</sup>H NMR: (400 MHz, DMSO-d<sub>6</sub>) δ 1.42 (12H, broad t, J = 7 Hz, 4 CH<sub>3</sub>), 2.43-2.50 (2H, m, CH<sub>2</sub>), 3.88 (8H, q, J = 7.0 Hz, 4 NCH<sub>2</sub>), 3.98 (6H, s, 2 OCH<sub>3</sub>), 4.03 (6H, s, 2 OCH<sub>3</sub>), 4.70 (4H, t, J = 7.2 Hz, 2 N<sup>+</sup>CH<sub>2</sub>), 7.28 (2H, s, 2 H<sup>8</sup>), 7.38 (2H, s, 2 H<sup>5</sup>), 8.83 (2H, s, 2 H<sup>2</sup>). <sup>13</sup>CAPT NMR: (100 MHz, DMSO-d<sub>6</sub>) δ 12.6 (CH<sub>3</sub>), 27.1 (CH<sub>2</sub>), 46.1 (NCH<sub>2</sub>), 47.8 (N<sup>+</sup>CH<sub>2</sub>), 55.9 (OCH<sub>3</sub>), 57.1 (OCH<sub>3</sub>), 98.6 (CH<sup>5</sup> & CH<sup>5</sup>'), 106.3 (C<sup>4a</sup> & C<sup>4a</sup>'), 106.7 (CH<sup>8</sup> & CH<sup>8</sup>'), 136.0 (C<sup>8a</sup> & C<sup>8a</sup>'), 148.0 (C<sup>6</sup> & C<sup>6</sup>'), 149.8 (CH<sup>2</sup> & CH<sup>2</sup>'), 155.7 (C<sup>7</sup> & C<sup>7</sup>'), 159.6 (C<sup>4</sup> & C<sup>4</sup>). HRMS (ESI): m/z 282.1705 [M]<sup>2+</sup>, calcd for C<sub>31</sub>H<sub>44</sub>N<sub>6</sub>O<sub>2</sub> 282.1707. Elemental analysis: N: 10.04, C: 44.16, H: 5.31; Theo calcd for C<sub>31</sub>H<sub>44</sub>I<sub>2</sub>N<sub>6</sub>O<sub>4</sub>.H<sub>2</sub>O: N: 10.05, C: 44.51, H: 5.54.

**1,4-Bis(4-(diethylamino)-6,7-dimethoxy-quinazolinium)butane dibromide, 50:** In a sealed microwave vial, a solution of N,N'-diethyl-6,7-dimethoxyquinazolin-4-amine S14 (1.0 g, 3.83 mmol, 1.0 equiv.) and 1,4-dibromobutane (0.30 mL, 1.84 mmol, 0.48 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 110°C. Diethyl ether (50 mL) was then added to the cooled suspension to favour the precipitation of the solid. The suspension was filtered and washed with diethyl ether (3 x 10 mL) and dried under vacuum to give a white solid mixture (2.54 g). The mixture was then recrystallized from MeOH to give 50 as a white solid (1.1 g, 80 %). <sup>1</sup>H NMR: (400 MHz, DMSO-d<sub>6</sub>) δ 1.42 (12H, broad t, J = 6.9 Hz, 4 CH<sub>3</sub>), 1.92 (4H, broad s, 2 CH<sub>2</sub>), 3.90 (8H, q, J = 6.9 Hz, 4 NCH<sub>2</sub>), 3.98 (6H, s, 2 OCH<sub>3</sub>), 4.05 (6H, s, 2 OCH<sub>3</sub>), 4.60 (4H, broad s, 2 N<sup>+</sup>CH<sub>2</sub>), 7.30 (2H, s, 2 H<sup>8</sup>), 7.37 (2H, s, 2 H<sup>5</sup>), 8.85 (2H, s, 2 H<sup>2</sup>). <sup>13</sup>CAPT NMR: (100 MHz, DMSO-d<sub>6</sub>) δ 12.6 (CH<sub>3</sub>), 24.7 (CH<sub>2</sub>), 46.0 (NCH<sub>2</sub>), 49.9 (N<sup>+</sup>CH<sub>2</sub>), 55.9 (OCH<sub>3</sub>), 57.0 (OCH<sub>3</sub>), 98.7 (CH<sup>5</sup> & CH<sup>5</sup>'), 106.3 (C<sup>4a</sup> & C<sup>4a</sup>'), 106.6 (CH<sup>8</sup> & CH<sup>8</sup>'), 136.0 (C<sup>8a</sup> & C<sup>8a</sup>'), 148.1 (C<sup>6</sup> & C<sup>6</sup>'), 149.3 (CH<sup>2</sup> & CH<sup>2</sup>'), 155.7 (C<sup>7</sup> & C<sup>7</sup>'), 159.5 (C<sup>4</sup> & C<sup>4</sup>). HRMS (ESI): m/z
289.1784 [M]$^{2+}$, calcd for C$_{32}$H$_{46}$N$_{6}$O$_{4}$$^{2+}$ 289.1785. Elemental analysis: N: 10.96, C: 50.54, H: 6.12; Theo calcd for C$_{32}$H$_{46}$Br$_{2}$N$_{6}$O$_{4}$.H$_{2}$O: N: 11.11, C: 50.80, H: 6.40.

**1,3-Bis(4-(diethylamino)-6,7-dimethoxy-quinolinium)propane diiodide 51:** In a sealed microwave vial, a solution of N,N'-diethyl-6,7-dimethoxyquinolin-4-amine **S9** (0.745 g, 2.86 mmol, 1 equiv) and 1,3-diiodopropane (0.13 mL, 1.14 mmol, 0.4 equiv.) was stirred 24 h in anhydrous acetonitrile (10 mL) at 80°C. Diethyl ether (25 mL) was then added to the cooled suspension to favour the precipitation of the solid. The suspension was then filtered, washed with diethyl ether (3 x 10 mL) and dried under vacuum. The solid was recrystallized from ethanol to **51** as a pale-yellow solid (0.78 g, 84 %).¹H NMR: (400 MHz, DMSO-d$_{6}$) δ 1.38 (12H, t, J = 6.9 Hz, 4 CH$_{3}$), 2.44-2.46 (2H, m, CH$_{2}$), 3.72 (8H, q, J = 6.9 Hz, 4 NCH$_{2}$), 3.96 (6H, s, 2 OCH$_{3}$), 3.98 (6H, s, 2 OCH$_{3}$), 4.79 (4H, broad t, J = 7.2 Hz, 2 N$^{+}$CH$_{2}$), 7.02 (2H, d, J = 7.5 Hz, 2 H$^{3}$), 7.27 (2H, s, 2 H$^{8}$), 7.31 (2H, s, 2 H$^{5}$), 8.45 (2H, d, J = 7.5 Hz, 2 H$^{2}$). ¹³C APT NMR: (100 MHz, DMSO-d$_{6}$) δ 12.4 (CH$_{3}$), 27.9 (CH$_{2}$), 46.8 (NCH$_{2}$), 51.2 (N$^{+}$CH$_{2}$), 55.9 (OCH$_{3}$), 56.7 (OCH$_{3}$), 98.7 (CH$^{3}$), 103.7 (CH$^{5}$), 106.0 (CH$^{8}$), 113.7 (C$^{4a}$), 135.8 (C$^{8a}$), 142.8 (CH$^{2}$), 147.4 (C$^{6}$), 154.3 (C$^{7}$), 157.4 (C$^{4}$). HRMS (ESI): m/z 281.1752 [M]$^{2+}$ calcd for C$_{33}$H$_{46}$N$_{4}$O$_{4}$$^{2+}$ 281.1754. Elemental analysis : N : 6.71, C : 47.11, H : 5.57; Theo calcd for C$_{33}$H$_{46}$I$_{2}$N$_{4}$O$_{4}$.H$_{2}$O: N : 6.71, C : 47.49, H : 5.80.

## 4.2. *In vitro* antimalarial assay

In this study, the K1 culture-adapted P. falciparum strain, resistant to chloroquine, pyrimethamine, and proguanil, and the 3D7 culture-adapted P. falciparum strain, sensitive to chloroquine and pyrimethamine, were used in an *in vitro* culture (obtained through BEI Resources, NIAID, NIH: Plasmodium falciparum, Strain K1, MRA-159, contributed by Dennis E. Kyle and Plasmodium falciparum, Strain 3D7, MRA-102, contributed by Daniel J. Carucci). They were maintained in continuous culture, as described previously by Trager and Jensen [62]. Cultures were maintained in fresh A+ human erythrocytes at 2.5 % hematocrit in complete medium (RPMI 1640 with 25 mM HEPES, 25 mM NaHCO$_{3}$, 10 % of A+ human serum) at 37 °C under reduced O$_{2}$ atmosphere (gas mixture 10 % O$_{2}$, 5 % CO$_{2}$, and 85 % N$_{2}$). Parasitemia was maintained daily between 1 and 3 %. The P. falciparum drug susceptibility assay was carried out by comparing DNA quantities in treated and control co-cultures of the parasite in human erythrocytes according to a SYBR Green fluorescence-based method [63]. The N-heterocyclic iminium salts, previously dissolved in DMSO (final concentration less than 0.5 % v/v) were incubated in a total assay volume of 200 µL (complete medium, 2 % hematocrit and 0.4 % parasitemia) at different concentrations for 72 h in a humidified atmosphere (5 % CO$_{2}$, 10 % O$_{2}$ and 85 % N$_{2}$) at 37 °C, in 96-well flat bottom plates. Triplicate assays were performed for each sample. After incubation, the plates were frozen at -20 °C for at least 24 h to promote erythrocyte lysis. Then, after 1h at 37 °C, for each well, 15 µL of supernatant were transferred to new 96-well flat bottom non-sterile black plates (Greiner Bio-one) already containing 15 µL of the SYBR Green lysis buffer (2X SYBR Green, 20 mM Tris pH 7.5, 20 mM EDTA, 0.008 % w/v saponin, 0.08 % w/v Triton X100). Plates were shaken vigorously at 900 rpm for 30 s and placed at 37 °C for 15min. Negative control, treated by solvents (DMSO) and positive controls (chloroquine diphosphate, artesunate and doxycycline) were added to each set of experiments. Fluorescence intensities of each plate were finally read on a TECAN Infinite F-200 spectrophotometer with excitation and emission wavelengths at 485 and 535 nm, respectively. IC$_{50}$ values were calculated from the dose-response curve by using the TableCurve software 2D v.5.0 for three distinct experiments. All IC$_{50}$ were compared to the chloroquine (IC$_{50}$ PFK1 = 0.9 µM, IC$_{50}$ Pf3D7 = 0.035 µM), artesunate (IC$_{50}$ PFK1 = 3.7 nM, IC$_{50}$ Pf3D7 = 2.1 nM) and doxycycline (IC$_{50}$ PFK1 = 8.5 µM, IC$_{50}$ Pf3D7 = 5.3 µM) chosen as antimalarial reference drugs.

## 4.3. *In vitro* cytotoxicity assay

In vitro cell viability evaluation of the tested molecules was performed on the HepG2 (hepatocarcinoma cell line, ref HB-8065) and CHO-K1 cell lines (ref ATCC CCL-61) purchased from ATCC according to the Mosmann method with slight modifications [64]. The HepG2 cell line was maintained at 37 °C, 5 % CO$_{2}$, at 90 % humidity in MEM supplemented with 10 % fetal bovine serum, 1 % l-glutamine (200 mM), and penicillin (100 U per mL)/streptomycin (100 mg/mL) (complete RPMI medium). The CHO-K1 cell line was maintained at 37 °C, 5 % CO$_{2}$, at 90 % humidity in DMEM-F12 medium supplemented with 10 % fetal bovine serum, 1 % l-glutamine (200 mM), and penicillin (100 U per mL)/streptomycin (100 mg mL-1) (complete RPMI medium). 5000 cells suspended in 100 µL of complete medium were added into each well of 96-well plates and incubated at 37 °C under 5 % CO$_{2}$. After 24h, 100 µL of complete medium with various N-heterocyclic iminium salt concentrations dissolved in DMSO (final concentration less than 0.5 % v/v) or Methanol (final concentration less than 0.5 % v/v) were added to the cells and the plates were incubated for 72 h at 37 °C, 5 % CO$_{2}$. Triplicate assays were performed for each sample. Before removing the supernatant, each plate well was microscope-examined to detect possible precipitate formation. Then, 100 µL of MTT ((3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (0.5 mg/mL in MEM or DMEM F12 medium without FBS) were added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and 100 µL of DMSO was added to dissolve the resulting formazan crystals. Plates were shaken vigorously at 700 rpm for 10 min. The absorbance was finally measured at 570 nm with 630 nm as a reference wavelength using a TECAN Infinite F200 Microplate Reader. DMSO was used as blank and doxorubicin as positive control. The 50 % cytotoxic concentration (CC$_{50}$) was calculated from the dose-response curve by using the TableCurve software 2D v.5.0. CC$_{50}$ values represent the mean value calculated from three independent experiments. To estimate the potential of the N-heterocyclic iminium salts to inhibit the growth of parasites without toxicity, the selectivity indexes (SI) were also calculated following the equation: $SI_{HepG2} = CC_{50}$ (HepG2 cells)/IC$_{50}$ (Plasmodium falciparum K1) and $SI_{CHO} = CC_{50}$ (CHO-K1 cells)/IC$_{50}$ (Plasmodium falciparum K1). All CC$_{50}$ were compared to Doxorubicin, chosen as a cytotoxic reference agent (CC$_{50}$ HepG2 = 0.2 µM and CC$_{50}$ CHO = 7.1 µM)

## 4.4. Dihydrofolate reductase inhibition assay

In vitro cell viability evaluation of the tested molecules was performed on transformed Saccharomyces cerevisiae yeast strains expressing the DHFR plasmodium falciparum gene (Pf. DHFR). The yeasts were maintained at 30 °C in YEPD complete liquid medium (yeast extract, peptone, dextrose). The culture was frequently maintained at a DO (630 nm) of 0.6. 100 µL of a stock solution of $10^{-5}$ yeasts per mL suspended in the complete medium was added into each well of 96-well plates ($10^{-4}$ yeasts/wells). Then, 100 µL of complete medium with various product concentrations, dissolved in DMSO (final concentration less than 0.5 % v/v), were added to the yeast suspension and the plates were incubated at 30 °C for 48 h. Triplicate assays were performed for each sample. The absorbance was finally measured at 630 nm using a TECAN Infinite F200 Microplate Reader. DMSO was used as blank and pyrimethamine (IC$_{50}$ = 2.64 µM) and cycloquanil (IC$_{50}$ = 14.8 µM) as positive controls. IC$_{50}$ values were calculated from the dose-response curve by using the TableCurve software 2D v.5.0 for three distinct experiments.

## 4.5. Heme polymerization inhibition assay

A stock solution of hemin chloride (16.3 mg in 1 mL DMSO) was prepared and passed through a 0.2 µm pore membrane filter to remove insoluble particles. The solution was diluted just before the assay to 1
mM with DMSO and then to 50 µM with 1 M acetate buffer, pH 4.8. Several conditions were tested to determine optimal conditions for the assay, including heme concentration and percentage of inducer of polymerization (Tween 20). In all assays, 50 µM of heme and 0.025 g/L of Tween 20 were used to evaluate the effect of drugs on β-hematin (BH) formation (see supporting information). The assay includes heme control, heme converted to BH control (heme + Tween 20) and samples (heme + Tween 20 + drug). After 4h of incubation at 37 °C, the absorbance was finally measured at 405 and 630 nm wavelengths using a TECAN Infinite F200 Microplate Reader. The inhibition effects were compared to those of the chloroquine reference.

## CRediT authorship contribution statement

**M.C. Casanova:** Writing – original draft, Investigation. **Y. Zhao:** Investigation. **S. Hutter:** Investigation. **M. Fil:** Investigation. **M.H. Do:** Investigation. **A. Benech:** Investigation. **T.-B. Muller:** Formal analysis. **C. Curti:** Supervision. **M. Médebielle:** Investigation. **P. Vanelle:** Writing – review & editing. **N. Azas:** Writing – review & editing, Supervision. **J. Broggi:** Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julie Broggi reports financial support was provided by French National Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The Agence Nationale de la Recherche is gratefully acknowledged for the JCJC ANR grant "iPOD" (17-CE07-000101). Aix-Marseille Université (AMU) and the Centre National de la Recherche Scientifique (CNRS) are gratefully acknowledged for financial support. M.C. thanks the College Doctoral of Aix-Marseille Université (AMU) for her inter-doctoral PhD grant. We also thank Dr M. Mauduit and co-workers from the Ecole Nationale Supérieure de Chimie de Rennes for generously providing the N-cycloalkyl-substituted imidazolium salts 19–23 [61]. Dr K. Mabrouk and S. Aboudou from the Institut de Chimie Radicalaire (AMU) are acknowledged for their valuable help in the separation and purification of compounds 37–38 by HPLC. Dr F. Gattacceca is acknowledged for valuable discussions on pharmacokinetic data.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2025.117633.

### Supplementary data

- Experimental procedures

- NMR and CV spectra

### Data availability

Data will be made available on request.

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