Pyridinium chlorides equipped with long chains as amphiphiles for potential nonviral gene delivery

Pyridinium chlorides with long alkyl substituents were prepared, sometimes in quantitative yields, using a macroporous, strongly basic, anion exchange resin of the styrene-divinylbenzene type. This procedure opens the way to convert readily available and highly pure pyrylium salts with tailored amphiphilicity inducing substituents, into pyridinium perchlorates, and then by this optimized anion exchange procedure into the corresponding chlorides. Previous transfection studies using long chain pyridinium perchlorates have proven acute cell toxicity whereas the corresponding chlorides were benign and had good to high transfection efficiencies, superior to commercial nonviral vectors. All new compounds were fully characterized and can now be made available in large quantities.


Introduction
Recent in depth studies of cationic amphiphiles have demonstrated practical utility for the latter for delivery of DNA and RNA into cells.The groups of Jan Engberts and Dick Hoekestra describe the synthesis of pyridinium chlorides as amphiphiles by the quaternization of 4methylpyridine, in order to identify lead structures and to optimize the efficiency of these quaternary salts in transfection experiments.Some characteristics of amphiphile/DNA complex formation were also presented. 1,2Although their synthetic methods are non demanding and highyielding, the pyridinium polar head group in their constructs was minimally substituted.Sandy Balaban, to whom this work is dedicated, has used the 2,4,6-trimethylpyridinium scaffold for attaching various lipophilic chains via the well known conversion of pyrylium salts into pyridinium salts by reaction with primary amines. 3Constructs using glycerolamines, by esterification with fatty acids of the two glycerol hydroxyl groups, have provided 2,4,6trimethylpyridinium-substituted compounds, which were tested in vivo, 4,5 and have allowed structure-activity correlations. 6Marc Ilieş and Sandy Balaban have also reviewed their work as well as recent developments in non-viral gene delivery. 7A very recent comprehensive study by Jean-Pierre Vigneron, Jean-Marie Lehn and Pierre Lehn and their coworkers summarizes the present status of cationic lipids in the field of non-viral gene therapies. 8ur group has been involved since 1991 in synthesizing amphiphilic pyridinium salts with the long chains attached directly to the pyridinium nucleus.This design could combine in a unique manner (i) ionic pairing with the anionic nucleic acid strands, (ii) π−π stacking interactions of the pyridinium cation, and (iii) hydrophobic packing between the long chains which in an aqueous environment leads to micellar aggregates.More than fifty new compounds were assessed and previous papers describe their preparation from either 2, 4, 6-trisubstituted or 2, 3, 4, 6-tetra-substituted pyrylium salts having either one, or two linear α/γ-alkyl side chains. 9- 15All these pyrylium salts were synthesized as perchlorates, which although hazardous compounds that could lead potentially to powerful explosions, when handled with care, allow an unprecedented and easy purification from the complex reaction mixtures.A comprehensive review on the available methods for the synthesis of pyrylium salts, including detailed experimental procedures involving different anions, has been recently published in a father-son co-authorship. 16mmonium perchlorate, which has been widely used as a solid rocket propellant and missile fuel, is known to contaminate drinking water, groundwater or soil.The perchlorate anion is a powerful thyroid toxin that can affect the thyroid's ability to take up the essential nutrient iodide used to make thyroid hormones.Edwards reported earlier on substrate inhibition of active iodide secretion in saliva by nitrate and also by thiocyanate and perchlorate anions. 17The perchlorate anion has been primarily studied in human patients with Graves' disease.Graves' disease is an autoimmune disorder in which patients have antibodies to the TSH receptors in the thyroid.9][20] All our cell culture assays involving pyridinium perchlorates proved an enhanced cytotoxicity and thus these compounds were abandoned for transfection studies.Thus, it was highly desirable to develop a facile, highyielding anion exchange method, chloride being among the most used anion.Unfortunately, the synthesis of pyrylium chlorides is non-practical leading to impure and usually hygroscopic oils.Pyridinium salts with long alkyl substituents are expected to have surfactant properties 21,22 and are microbiologically active compounds. 23On the other hand, pyridinium chlorides have antibacterial activity 24 and cetylpyridinum chloride was useful for isolation of Mycobacterium tuberculosis from sputa. 25For Ν−octyl pyridinum chloride, practical uses as surfactant, 26 disinfectant 27 and industrial applications as cleaning agent, 28 for modification of properties of ion exchange membranes 29 or water-dispersible agents were also described. 30on exchange processes can be very useful in organic chemistry and can be simply carried out on various solid supports in high yields.Some of the known industrial processes utilizing ion exchange materials are 31 : (i) conversion of one salt to another, (ii) removal of ionic impurities from organic compounds, (iii) separation of both inorganic and organic ions by chromatography, (iv) catalysis.One example of converting a pyridinium perchlorate, which is not a pharmaceutically acceptable ingredient, to the corresponding chloride salt was reported. 32The present paper describes the synthesis of 2,4,6-trisubstituted and 2,3,4,6-tetrasubstituted pyridinium chlorides having two various linear and long side chains in the 1,2-or 2,6-positions.An experimental procedure, spectral data and elemental analyses for all new compounds are presented.These novel chlorides are expected to be useful as efficient transfection vectors in nonviral gene therapy.

Conversion of pyrylium salts 1 into N-alkyl-pyridinium perchlorates (3−10) followed by anion exchange to N-alkyl-pyridinium chlorides (3Cl−10Cl)
0][11][12][13][14][15] Table 2 presents elemental analyses and melting points and the synthetic procedure for the anion exchange.Yields were 60-80%.These compounds usually crystallize as colorless crystals (N-methylpyridinium perchlorates 3−4, 9,11 , 7 and 9, 14 8 and 10, 15 ) or are waxy oils which crystallized in the refrigerator (N-dodecylpyridinium perchlorates 5-6).All these compounds are soluble in fairly polar solvents such as lower alcohols, acetone, chloroform, methylene chloride, etc. N-Dodecylpyridinium perchlorates 5, 6 having two long alkyl side chains in positions 1-and 2-are highly soluble also in weakly polar solvents such as diethyl ether, a fact quite uncommon for organic salts.][11][12][13][14][15] The conversion of pyridinum perchlorates 3−10 was easily performed by passing the perchlorate salts through the strongly basic ion exchange resin Vionit AT14 which was brought in the chloride form in order to afford the chloride salts 3Cl-10Cl in almost quantitative yields.All chlorides compounds are waxy oils but again the N-methyl pyridinium chlorides proved to have a higher crystallinity because although they were obtained as waxy oils, after prolonged standing they crystallized in the refrigerator exhibiting low melting points (35-48 o C) as detailed in Table 2.All the chlorides were fairly soluble in diethyl ether.
Two mixtures of solvents were useful for the anion exchange to pyridinium chlorides namely methanol: water and acetone: water.The influence of the solvent and water concentration on the yield in pyridinium chloride was studied in some detail for 4Cl and 5Cl and is presented in Table 3.In all cases the use of a methanol : water mixture gave less satisfactory results.It should be noted, however, that slow exchange, presumably via decyclization to the 1-amino-1,3-dien-5one 12, 3 occurred when compounds were eluted with solvent mixtures containing methanol (Scheme 2).A mixture of Cl -/ ClO 4 -pyridinium salts and ring-opened products 12-13 was obtained in these cases, showing that this system is inefficient, due not only to the incomplete anion exchange, but also due to an undesired ring opening.
Scheme 2. Side reactions leading to a mixture of ring opened products.
High concentrations of water and high elution rates increased the anion exchange yields to pyridinium chlorides.The amount of water in the elution solvent mixtures is an important factor in determining the relative selectivity of the resin for ClO 4 /Cl ions.The use a mixture of acetone: water elution solvent in the second case gave the best results.The decrease in conversion into chlorides 4Cl and 5Cl with increasing water concentration is due to the differences between polarity of solvent and pyridinium solubility and also probably to the base catalyzed ring opening to the ketodiamine.We have not yet tried to suppress this side reaction by controlling the pH with various buffers.
The purification of the products sometimes met with difficulty so that after solvent evaporation, the reaction mixture had to be separated by thin layer chromatography (silica gel Merck type 60G, solvent diethyl ether: acetone = 9: 1), when the pyridinium perchlorates remain adsorbed.For these cases, a mixture of solvents acetone : water 65:5 (for 4Cl) or 50:4 (for 5Cl) affords excellent separation results and the pyridinium chlorides thus obtained were analytically pure compounds.The purity of pyridinium chlorides was confirmed by 1 H-NMR, IR spectra and elemental analyses.It is interesting to note that the long chains on our compounds allow easy purification of organic salts by chromatography, which ensures highly pure compounds of GMP quality.It is not so common practice in preparative organic chemistry to perform chromatographic separations, which may include also reverse phase HPLC, on ionic compounds.

NMR and IR spectra
0][11][12][13][14][15] The atom numbering for NMR assignments follows the one shown in Chart 1. Unambiguous assignments for 3Cl−10Cl were accomplished using two-dimensional correlation spectroscopy (COSY).Table 4 presents the proton chemical shifts and should allow an easy comparison of the data for the various compounds.Most of the signals for 3Cl−10Cl were similar to those observed for the analogous perchlorates 3-10.A small systematic difference in chemical shifts values for the β−protons (3H/5H) of the heterocyclic ring and the first alkyl side chain protons was observed (see Table 4).Comparison of the spectral data within 3Cl−10Cl and 3−10 indicates that the different anion pairing was responsible for differentiated 1 H-NMR spectra and this could be used to quantify the mixtures with partially exchanged anions.The chloride anion leads to slightly more deshielded signals than the perchlorate.
The 13 C-NMR spectra of pyridinium chlorides 3Cl−10Cl are summarized in table 5 where only those signals which could be safely assigned are listed.For all these compounds, similar spectra to those observed for the analogous perchlorates were obtained.
The only difference observed in the IR spectra was the absence of the sharp band at 625 cm -1 (when measured in KBr pellets) and of the broad and intense band at 1100 cm -1 , both which correspond to the ClO 4 anion.In Table 6 IR spectra for some typical compounds are presented.

Experimental Section
General Procedures.The NMR spectra have been recorded on a Bruker Avance DRX 400 instrument, equipped with a 5 mm inverse detection multinuclear probe head and field gradients on the z axis, operating at 400 MHz for 1 H and at 100 MHz for 13 C nuclei.All spectra have been recorded in deuterated chloroform, and the chemical shifts have been reported as δ values referenced to TMS as internal standard.Infrared spectra were run on a Zeiss UR 20 instrument.Melting points were measured in open capillary tubes (for the low melting compounds) or on a hot-stage melting point apparatus which was equipped with a polarizer in order to check for nematic properties.All other reagents were obtained from commercial sources and purified by standard methods as necessary.

Materials and Methods
0][11][12][13][14][15] Briefly, these reactions were performed by refluxing of reactants in ethanol / dimethylformamide, or according to the method optimized by Katritzky, at room temperature in methylene chloride. 33Pyridinium chlorides were conveniently prepared by passing the perchlorate salts through a strongly basic ion exchange resin of the styrenedivinylbenzene type and afforded the chloride salt 3Cl−10Cl in yields higher than 99%.Scheme 1 presents the synthesis of the new compounds which are described in this paper, while the atom numbering is shown in Chart 1.
All new compounds were fully characterized by 1 H-and 13 C-NMR (at 400 MHz for protons and at 100 MHz for carbons), by IR spectra and by elemental analysis.

Chart 1 .
The compounds discussed in this work and their atom numbering scheme.