Synthesis and Evaluation of Self-Assembling Properties of 3-(3,5-Diﬂuoro-3,5-bis((alkoxy)carbonyl)-2,6-dioxoheptan-4-yl)-1-methylpyridin-1-iumIodides

: A synthesis of 3-(3,5-difluoro-3,5-bis((alkoxy)carbonyl)-2,6-dioxoheptan-4-yl)-1-methylpyridin-1-ium iodides with ethyl or nonyl ester groups at positions 3 and 5 was performed. Treatment of the corresponding 2’,6’-dimethyl-1’,4’-dihydro-[3,4’-bipyridine]-3’,5’-dicarboxylates with Selectfluor ® followed by quaternization of pyridine moiety in the obtained dialkyl 2,4-diacetyl-2,4-difluoro-3-(pyridin-3-yl)pentanedioates with methyl iodide gave the desired 3-(3,5-difluoro-3,5-bis((alkoxy)carbonyl)-2,6-dioxoheptan-4-yl)-1-methylpyridin-1-ium iodides. This type of compound would be useful as synthetic lipids for further development of the delivery systems. The obtained target compounds were fully characterized by 1 H NMR, 19 F NMR, 13 C NMR, HRMS, IR and UV data. The estimation of self-assembling properties and characterization of the nanoparticles obtained by ethanol solution injection in an aqueous media were performed by dynamic light scattering (DLS) measurements. DLS measurement data showed that 3-(3,5-diﬂuoro-3,5-bis((nonyloxy)carbonyl)-2,6-dioxoheptan-4-yl)-1-methylpyridin-1-ium iodide created liposomes with the average diameter of 300–400 nm and polydispersity index (PDI) value around 0.30–0.40, while 3-(3,5-diﬂuoro-3,5-bis((ethyloxy)carbonyl)-2,6-dioxoheptan-4-yl)-1-methylpyridin-1-ium iodide formed a heterogeneous sample with PDI value 1, which was not prospective for delivery system development.


Introduction
N-Heterocyclic compounds represent a large class of organic molecules, where many representatives possess various biological activities and are widely applied in medicine. Pyridine and structurally related molecules-dihydropyridine and pyridinium derivatives-are suggested as prevalent structural units for pharmaceutical targets [1]. According to the US Food and Drug Administration (FDA) database, a pyridine and dihydropyridine system containing drugs reaches almost 18% of the N-heterocyclic drugs approved by the agency in the major therapeutic areas-infectious diseases, inflammation, the nervous system and oncology [2]. Additionally, structurally diverse pyridinium salts are quite common structures in various pharmaceuticals and many natural compounds. Pyridinium salts are usable in a wide range of research topics. Pyridinium ionic liquids and pyridinium ylides are used in synthetic chemistry and in material science and biological issues related to gene delivery, antimicrobial, anticancer and antimalarial activities [3]. Different SAINT pyridinium salts with alkyl chain variations at the quaternized pyridine N-atom or as substituents at the pyridinium cycle were proposed as active gene delivery agents by several research groups [4][5][6]. Additionally, gemini dioleylbispyridinium-based amphiphiles were elaborated for nucleic acid transfection [7].
The interest in fluorinated surfactants has increased due to their chemical and biological inertness and their hydrophobic and, at the same time, lipophobic character. Fluorouscontaining amphiphiles are important for the formation of uniform nanoparticles, avoiding protein denaturation, efficient endocytosis and maintaining low cytotoxicity [17,18]. Several new fluorinated surfactants on the base of pyridinium salts were recently synthesized and studied as drug carriers and gene delivery systems with very promising results [19][20][21][22].

Results and Discussion
Taking into account the fact that the introduction of fluorine atoms in the synthetic cationic lipid structure may lead to the formation of the original delivery systems with more pronounced properties, the synthesis of 3-(3,5-difluoro-3,5-bis((alkoxy)carbonyl)-2,6dioxoheptan-4-yl)-1-methylpyridin-1-ium iodides 4 was performed.
The structures of compounds 3a,b and 4a,b were established and confirmed on the basis of one-dimensional 1 H, 19 F, 13 C NMR spectral data (Supplementary Materials).
In the 19 F NMR spectra of compounds 3a,b and 4a,b, a signal of the fluorine atom appeared as a doublet in the range of −166.1 ppm to −166.5 ppm with the constants around 3 J F-H = 29 Hz for compounds 3a,b and in the range of −163.2 ppm to −163.3 ppm with the constants around 3 J F-H = 25 Hz for compounds 4a,b. The corresponding constants were also observed in the 1 H NMR spectra for the proton attached to the carbon between the two CF groups, which appeared as a triplet in the range of 4.92−4.93 ppm for compounds 3a,b and at 5.10−5.11 ppm for 4a,b.
CF carbon atoms appeared characteristically as doublet multiplets in the 13 C NMR spectra at 99.2−101.4 ppm for compounds 3a,b and at 97.7−99.9 ppm for 4a,b with the constants around 1 J C-F = 213 Hz and 1 J C-F = 210 Hz, respectively.

Materials and Methods
All reagents were purchased from Acros Organics (Geel, Belgium), Sigma-Aldrich/Merck KGaA (Darmstadt, Germany) or Alfa Aesar (Lancashire, UK) and used without further purification. TLC was performed on silica gel 60 F 254 aluminum sheets 20 cm × 20 cm (Merck KGaA, Darmstadt, Germany). The melting points were recorded on an OptiMelt digital melting point apparatus (Stanford Research Systems, Sunnyvale, CA, USA) and were uncorrected. 1 H, 19 F and 13 C NMR spectra were recorded on a Bruker Avance Neo 400 MHz (Bruker Biospin Gmbh, Rheinstetten, Germany). Chemical shifts of the hydrogen, carbon and fluorine atoms are presented in parts per million (ppm) and referred to the residual signals of the undeuterated CDCl 3 (δ: 7.26) solvent for the 1 H NMR spectra and CDCl 3 (δ: 77.16) solvent for the 13 C NMR, respectively. For the 19 F-NMR experiments, indirect referencing (Bruker standard referencing) was used. The coupling constants J were reported in hertz (Hz). High-resolution mass spectra (HRMS) were determined on an Acquity UPLC H-Class system (Waters, Milford, MA, USA) connected to a Waters Synapt GII Q-ToF operating in the ESI positive or negative ion mode on a Waters Acquity UPLC ® BEH C18 column (1.7 µm, 2.1 mm × 50 mm, using gradient elution with acetonitrile (0.1% formic acid) in water (0.1% formic acid)). Infrared spectra were recorded with a Prestige-21 FTIR spectrometer (Shimadzu, Kyoto, Japan). UV spectra were recorded on a UV-Vis Spectrophotometer (501 UV-Vis CamSpec Spectrophotometer; Spectronic CamSpec Ltd., Leeds, UK). The DLS measurements of the nanoparticles in aqueous solution were carried out on a Zetasizer Nano ZSP (Malvern Panalytical Ltd., Malvern, UK) instrument with Malvern Instruments Ltd. Software 8.01.4906.
The DLS measurements of the nanoparticles in aqueous solution were carried out on a Zetasizer Nano ZSP (Malvern Panalytical Ltd., Malvern, UK) instrument with Malvern Instruments Ltd. Software 8.01.4906, using the following specifications: medium-water; refractive index-1.33; viscosity-0.8872 cP; temperature-25 • C; dielectric constant-78.5; nanoparticles-liposomes; refractive index of materials-1.60; detection angle-173 • ; wavelength-633 nm. Data were analyzed using the multimodal number distribution software that was included with the instrument. The measurements were performed in triplicate in order to check their reproducibility. The measurements were performed in triplicate in order to check their reproducibility. Example of DLS data for freshly prepared nanoparticles of 4b see in the Supplementary Materials.

Statistical Analysis
Results are expressed as mean standard deviation (SD). All experiments were performed in triplicate.
Supplementary Materials: The following are available online. File S1. 1 H NMR, 19 F NMR, 13 C NMR and HRMS spectra of compounds 3a,b and 4a,b, example of DLS data for freshly prepared nanoparticles of 4b.
Author Contributions: Conceptualization was conducted by N.P. and A.P.; methodology and experimental works were conducted by N.P., M.R., M.P., K.P., D.L., A.S. and A.P.; data analysis, writing and editing of the paper were conducted by A.S., M.R., N.P. and A.P.; project administration and supervision were conducted by N.P., A.S. and A.P. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in this article.