Chemistry of polyhalogenated nitrobutadienes, 17: Efficient synthesis of persubstituted chloroquinolinyl-1H-pyrazoles and evaluation of their antimalarial, anti-SARS-CoV-2, antibacterial, and cytotoxic activities

A series of 26 novel 1-(7-chloroquinolin-4-yl)-4-nitro-1H-pyrazoles bearing a dichloromethyl and an amino or thio moiety at C3 and C5 has been prepared in yields up to 72% from the reaction of 1,1-bisazolyl-, 1-azolyl-1-amino-, and 1-thioperchloro-2-nitrobuta-1,3-dienes with 7-chloro-4-hydrazinylquinoline. A new way for the formation of a pyrazole cycle from 3-methyl-2-(2,3,3-trichloro-1-nitroallylidene)oxazolidine (6) is also described. In addition, the antimalarial activity of the synthesized compounds has been evaluated in vitro against the protozoan malaria parasite Plasmodium falciparum. Notably, the 7-chloro-4-(5-(dichloromethyl)-4-nitro-3-(1H-1,2,4-triazol-1-yl)-1H-pyrazol-1-yl)quinoline (3b) and 7-chloro-4-(3-((4-chlorophenyl)thio)-5-(dichloromethyl)-4-nitro-1H-pyrazol-1-yl)quinoline (9e) inhibited the growth of the chloroquine-sensitive Plasmodium falciparum strain 3D7 with EC50 values of 0.2 ± 0.1 µM (85 ng/mL, 200 nM) and 0.2 ± 0.04 µM (100 ng/mL, 200 nM), respectively. Two compounds (3b and 10d) have also been tested for anti-SARS-CoV-2, antibacterial, and cytotoxic activity.


Introduction
Tropical malaria remains one of the most devastating human diseases with over half of the world's population being at risk of infection. In 2019, there were 229 million cases of malaria worldwide resulting in an estimated 409,000 fatalities [1]. Chloroquine has been utilized extensively for decades because of its efficacy, safety, and low cost. However, the widespread resistance of Plasmodium falciparum to chloroquine has hampered efforts to combat malaria [2] resulting in artemisininbased combination therapies as currently recommended standard. Notably, the use of chloroquine, hydroxychloroquine, and amodiaquine ( Figure 1) against a SARS-CoV-2 infection is currently under discussion [3][4][5][6][7]. As the 4-amino-substituted 7-chloroquinoline is the essential fragment of chloroquine and its derivatives, we have designed and synthesized a series of pyrazoles containing this fragment in position 1 of the pyrazole ring with the aim to obtain new compounds with antimalarial and/or anti-SARS-CoV activity. Polyhalo-1,3-butadienes, carrying at least one nitro group, are valuable starting materials for the directed synthesis of highly functionalized heterocycles. During the past years, we have reported on the syntheses of a wide range of diverse substance classes applying this useful starting material. In the course of our studies concerning polyhalogenated nitrodienes, in many cases 2-nitroperchlorobuta-1,3-diene (1) [8][9][10] or nitrotrichloroethylene [11][12][13] have proved as appropriate precursors for a diverse variety of synthetically and/or physiologically interesting chemical compounds. Recently, we have developed an efficient method for the synthesis of persubstituted nitropyrazoles from diene 1 [14].
We suggest that dienes 4a,l-o, obtained from 2c and primary amines are stabilized by formation of intramolecular hydrogen bonds between the NH and the NO 2 group forming a sixmembered ring system that upon reaction with 7-chloro-4hydrazinylquinoline formed 5-aminopyrazoles 5a,l-o. Here, a nucleophilic attack of the NH 2 group of the arylhydrazine on the C3 position of the butadiene chain is observed due to the sterically hindered rigidized six-membered ring system in the dienes 4a,l-o (Scheme 4). The obtained 5-aminopyrazoles 5a,l-o show the following 13  . On the other hand, the dienes 4b-k, obtained from reaction of 2c and secondary amines, are obviously stabilized by the effective conjugation between the amino and nitro groups and react with 7-chloro-4-hydrazinylquinoline to give 3-aminopyrazoles 5b-k. In this case, the nucleophilic attack of the NH 2 group of ArNHNH 2 on the C1 position of the butadiene chain is more likely due to the possibility of a rotation around the C1-C2 bond in 4b-k. The 3-aminopyrazoles 5b-k show considerable differences of the 13 C shifts of C-NO 2 -, C-CHCl 2 -and CHCl 2 -groups in comparison with the corresponding 13 C shifts of 5-aminopyrazoles 5a,l-o. The 13 C NMR shifts of the pyrazole ring and the dichloromethyl group of 3-aminopyrazoles 5b-k are 151.1-154.0 ppm (C-NRR 1 ), 120.6-122.2 ppm (C-NO 2 ), 136.7-136.9 ppm (C-CHCl 2 ), and 57.5-57.7 ppm (CHCl 2 ).
The obtained 13 C NMR data of pyrazoles 5 match the calculated NMR shifts (Table S1 in Supporting Information File 1). The predicted and found data for compounds 5a and 5b correspond well and thus support our assumptions regarding the orientation of the nucleophilic attack of 7-chloro-4-hydrazinylquinoline to nitrodienes 4.
Further, we developed a new way for the formation of a pyrazole cycle from oxazolidine 6 and a hetarylhydrazine. The oxazolidine 6 was synthetized under mild reaction conditions either from nitrodiene 1 (yield 58%) or from the benzotriazolyl derivative 2c (yield 76%). The increase in yield in case of 2c supports the suggestion that the benzotriazolyl subunit is a better leaving group compared to chlorine [28]. The reaction of oxazolidine 6 with 2.3 equivalents of 7-chloro-4hydrazinylquinoline in toluene at 90-95 °C for 30 h led to the formation of pyrazole 7 (yield 23%) together with considerable amounts of a resin (Scheme 5). We suggest that the lower yield of alcohol 7 is due to the poor leaving group quality of the formal alkoxide.
A conceivable mechanistic pathway for the reaction cascade to pyrazole 7 is shown in Scheme 6. Initially, a nucleophilic attack of the NH 2 group of 7-chloro-4-hydrazinylquinoline on the C2 position of the oxazolidine ring of 6 leads to the formation of imidoacetal D. Due to free rotation of the (O,N,N)C-C(NO 2 ) axis the suitable conformer E can be formed. Thus, the less hindered NH group of intermediate E interacts with the C3 position giving an intramolecular, five-membered addition product F. Upon elimination of HCl from F by means of the second equivalent of 7-chloro-4-hydrazinylquinoline as base the triazaoxaspiro system G is formed. Finally, pyrazole 7 is obtained via intramolecular rearrangement of G under thermodynamic control.
tion of sulfane 9d with m-chloroperbenzoic acid in chloroform at room temperature the sulfoxide 10d was obtained in 43% yield (Scheme 7).

Evaluation of biological properties Antibacterial and cytotoxic properties
The general cytotoxic activities of compounds 3b and 10d were studied, as they influence the applicability of the compounds in other assays comprising mammalian cells. We incubated the murine fibroblast L929 cell line with different concentrations of the compounds for 72 h and quantified the residual viability of the cells. Non-linear regression of the dose-response data led to IC 50 values of 1.4 µM for 3b and of 0.6 µM for 10d. Thus, for both compounds similar cytotoxic properties in the low µM range were determined.
The compounds were also tested in a viral infection model comprising SARS-CoV-2 and VeroE6 cells. However, no specific antiviral properties could be discovered, as the results were dominated by the cytotoxicity of the compounds. For details see Supporting Information File 1.
Furthermore, we evaluated the antibacterial properties of 3b and 10d. Growth of the Gram-positive bacterial strain Staphylococcus aureus was clearly inhibited with IC 50 values of 15 µM (3b) and 30 µM (10d), whereas the growth of Escherichia coli, which was used as representative of Gram-negative strain, was hardly affected.

Antimalarial properties
The activity of the 26 pyrazoles was tested against the asexual blood stages of the malaria parasite P. falciparum (3D7 strain) using the SYBR Green I-based fluorescence assay [35]. Table 1 summarizes the activity of the compounds against the red blood cell stages of P. falciparum with EC 50 values ranging from high nanomolar (200 nM) to low micromolar concentrations (4 µM).
In our experiments we used the established antimalarial drug amodiaquine as control. As shown in Table 1, the EC 50 of amodiaquine was with 6 nM about two orders of magnitude more active than the newly synthesized and tested compounds.
All persubstituted pyrazoles tested in this study showed pronounced antimalarial activity against the asexual blood stages of P. falciparum with EC 50 values ranging from 200 nM to 4 µM (Table 1). Thus, the antiparasitic activity of persubstituted pyrazoles is about two orders of magnitude lower compared to the antimalarial amodiaquine (EC 50 = 6 nM). Also many other antimalarials such as chloroquine, quinine, mefloquine, and artesunate, tested in the SYBR Green or comparable test systems, show EC 50 values in the lower nM range [36][37][38][39]. Thus, the newly reported compounds are a valuable enrichment from the chemical perspective and have promising potential for further optimization or combination with other compounds.