Synthesis, Spectroscopic and Theoretical Studies of New Dimeric Quaternary Alkylammonium Conjugates of Sterols

New dimeric quaternary alkylammonium conjugates of sterols were obtained by two step reactions of ergosterol, cholesterol and cholestanol with bromoacetic acid bromide, followed by bimolecular nucleophilic substitution with N, N, N', N'-tetramethyl-1,3-propanediamine, N, N, N', N'', N''-pentamethyldiethylenetriamine and 3,3'-iminobis-(N, N-dimethylpropylamine). The product structures were conﬁrmed by spectral (1 H-NMR, 13 C-NMR, FT-IR) analysis, mass spectrometry (ESI-MS) and PM5 semiempirical methods. Additionally in silico studies have been conducted for the synthesized compounds on the basis of Prediction of Activity Spectra for Substances (PASS).


Introduction
Steroids are very important natural products which play a crucial role in living organisms. This class of compounds comprises hormones, bile acids, steroidal saponins, cardioactive glycosides, steroid alkaloids and sterols [1][2][3]. One of the most important group among steroids are sterols, which can be divided into three groups: (1) zoosterols-the animal sterols (e.g., cholesterol, coprosterol); (2) phytosterols-the plant or algae sterols (e.g., sitosterol, stigmasterol); and (3) mycosterols-the sterols found in fungi (e.g., ergosterol, fungisterol) [4][5][6]. These compounds are crystalline secondary alcohols. Depending on the orientation of the condensed A and B rings of the steroid skeleton we can OPEN ACCESS can distinguished between normal and allo sterols. In the first of them the A/B rings have a cis orientation, while the second have in trans geometry. Furthermore, all representative sterols have a 3β-hydroxyl group in ring A, endocyclic double bonds, usually located in the C(5)=C(6) position and side chains which various degrees of unsaturation and branching [7,8].
Among sterols, three of them are ubiquitous, i.e., ergosterol (1), cholesterol (2) and cholestanol (3), which is a metabolite of cholesterol (Scheme 2). Ergosterol is a vital sterol for fungal survival. It serves two purposes: a bulk membrane function and a sparking function [1,2,7,10]. Since ergosterol is part of the fungal cell membrane, fulfilling the same function as cholesterol (2) in animal cells, it is a target for antifungal drugs. Furthermore ergosterol is also a biological precursor to vitamin D 2 [8,9,11,12].
An extremely important and well-known sterol is cholesterol. This sterol is an important component of mammalian cell membranes as in the ester form it stabilizes and stiffens protein-lipid membranes. Cholesterol is also present in significant concentrations in the brain and nervous tissue [13,14]. This animal sterol is the biosynthetic precursor of the steroid hormones, bile acids, vitamin D and lipoproteins [15][16][17][18]. On the other hand, gemini alkylammonium salts represent a new class of dimeric surfactants made up of two identical or different amphiphilic moieties having a monomeric quaternary alkylammonium salt connected by a linker (spacer) group structure [19,20]. The linker may be hydrophobic (aliphatic or aromatic) or hydrophilic (polyether, hydroxyalkyl). It can be rigid (stilbene) or flexible (a polymethylene chain). The gemini molecules have a neutral charge which is associated with the positive charge neutralization by negative ions such as e.g., halide anions [19][20][21][22][23][24]. The gemini alkylammonium salts show unique micelle-forming and surface-adsorbing properties in aqueous solution. The critical micelle concentration (CMC) for gemini surfactants is usually two orders lower than for corresponding monomeric surfactants [19,20,[24][25][26][27][28][29]. The gemini alkylammonium compounds show also a very good antimicrobial activity against bacteria, viruses, molds and yeasts [30,31]. In some cases, the minimal inhibitory concentrations (MIC) of the discussed compounds are even three orders of magnitude lower in comparison to non-gemini connections [25,32,33]. The mechanism of biocidal activity of quaternary alkylammonium salts is based on adsorption of alkylammonium cations on the bacterial cell surface, diffusion through the cell wall and then binding and disruption of cytoplasmatic membrane. Membrane damage results in a release of potassium ions and other cytoplasmatic constituents, finally leading to cell death [25,34,35]. Frequently use of microbiocides, especially at sublethal concentrations, can lead to increasing microorganism resistance. One of the ways to overcome this serious negative side effect is the periodic application of new microbiocides with modified structures. Scheme 2. Synthesis of dimeric quaternary alkylammonium conjugates 4-12 of sterols 1-3.
HO HO (2) (1) (3) Potential pharmacological activities of the synthesized compounds have been found on the basis of computer-aided drug discovery approach with in silico of the successful use of the PASS approach leading to new pharmacological agents [36][37][38][39][40]. Through the use of PASS the biological activity of the obtained compounds may be analyzed. This approach can be used at the earliest stages of investigation, because only the structural formula of the compound is necessary to obtain a PASS prediction.
The structures of all synthesized compounds were determined by 1 H-and 13 C-NMR, FT-IR and ESI-MS spectra. Moreover, PM5 calculations [43][44][45] were performed for all compounds. Additionally the pharmacotherapeutic potential of the synthesized compounds has been estimated on the basis of Prediction of Activity Spectra for Substances (PASS). The 1 H-and 13 C-NMR data spectra were assigned on the basis of our previous study [41,42]. The 1 H-NMR spectra of 4-12 show a signal in the range 4.65-4.48 ppm for the protons of the COCH 2 N + group. The signals for six methyl protons of the N + (CH 3 ) 2 and two methylene protons of the N + CH 2 occurred as singlets and triplets in the 3.58-3.46 and 4.44-3.89 ppm range, respectively ( Figure 1).
The 1 H-NMR spectra of compounds 4-12 show characteristic multiplets in the 4.92-4.61 ppm range assigned to the C3α-H protons of the sterol skeleton. Characteristic hydrogen singlets ranging from 0.69-0.65 ppm for 7-9 and 10-12 are assigned to CH 3 -18. Moreover, very characteristic triplets appear in the 0.84 ppm region for CH 3  The 13 C-NMR spectra of compounds 4-12 are summarized in Table 1. The spectra show characteristic signals at 15.79-15.63 ppm (compounds 4-6) and 12.12-11.65 ppm (compounds 7-12), which are assigned to CH3-18. In a series of 10-12 derivatives the signals from the CH3-18 group carbon atoms from both steroid skeletons are observed ( Table 2) The solid-state IR spectra of representative conjugates 6, 9 and 12 are shown in Figure 2. The strong band in the 3,350-3,330 cm −1 region has been assigned to the NH group with no hydrogen bond to bromide anion. The intense bands in the 1,740-1,739 cm −1 region are due to the carbonyl group ν(C=O) stretching vibrations ( Figure 3). The presence of two strong band at 1,739 and 1,721 cm −1 for 12 indicates the non-equivalence of the carbonyl groups. The split of the carbonyl bands suggest that both carbonyl groups in 12 are involved in different interactions in the supramolecular structure. Further coupling has little or no effect on the carbonyl group vibration. Some more strong characteristic bands in the 1,248-1,227 cm −1 region are present, which are assigned to the ν(C-O). The ν(C=C) stretching vibration bands of compounds 6 and 9 occur at 1,665 cm −1 and 1,670 cm −1 respectively; for compound 12 this band is absent. The δ(N-H) deformation vibration bands are observed at 1,629 cm −1 , 1,635 cm −1 , 1,634 cm −1 for 6, 9 as well as 12, respectively (Figure 3).     The ESI-MS spectra were recorded in methanol. The ESI-MS spectra of compounds 6, 9 and 12 are presented in Figure 4. The ESI mass spectrometry fragmentation pathways of these compounds is poor. It is rather more diagnostic than analytical. The molecular ion [M] + is not observed. Furthermore, in the spectra of these compounds, the [M] 2+ ion peaks are observed at m/z 531 (69%), 521 (28%) as well as 523 (23%) for 6, 9 and 12, respectively. In the discussed compounds, the presence of [M 2+ +2Br − +amine+2H + +AcOEt] 2+ ions (100%) was observed at m/z 748, 732 and 741 for 6, 9 as well as 12, respectively. For all other compounds (4, 5, 7, 8, 10 Table 2. Representative compounds 7, 8 and 9 are shown in Figure 5. In ergosterol derivatives 4-6 and cholesterol ones 7-9 where double bonds are present increasing HOF values are observed. The lowest HOF value is observed for cholestanol and its derivatives 10-12 where there are no double bonds to stabilize the molecules and hinder their reactivity. Two low energy syn and anti conformers are possible for the dimeric-type quaternary alkylammonium conjugates of sterols as a consequence of the free rotation of bonds in the linker fragment. Semiempirical calculations on compounds 4-12 showed that the syn conformer was more stable than the anti conformer by 0.05-19.07 kcal/mol. The distances between the quaternary nitrogen and the anion bromide are 3.70-4.66 Å for syn and 3.46-3.88 Å for anti conformers, respectively. The distances between the quaternary nitrogen atoms and the oxygen atoms are 2.89-3.35 Å for syn and 2.93-3.88 Å for anti conformers, respectively. Compensation charges occurs only through intramolecular electrostatic interaction. In optimized structures, the anion is not engaged in hydrogen bond but in electrostatic interactions what is also confirmed by FT-IR studies in the solid phase. The variations of dihedral angles reflects the flexibility of the N + CH 2 COOR moiety. The PM5 calculations indicate that the conformations of all molecules are controlled by the electrostatic interactions of the positively charged nitrogen atom with the oxygen atoms of the COOR moiety and bromide anions. This is a very good confirmation of the conclusion that interactions reduce HOF. Additionally, analyses of the biological prediction activity spectra for the new esters prepared herein are good examples of in silico studies of chemical compounds. We also selected the types of activity that were predicted for a potential compound with the highest probability (focal activities) ( Table 3). Table 3. PA (Probability "to be Active") values for predicted biological activity of compounds 4-12. The most important parameter is the predicted activity (PA). PA > 0.7 indicate a high probability of occurrence of a particular activity, which give a high chance to confirm it in real clinical tests. For most cases, PA > 0.7 have a structure similar to the structure of the known drug substance. In turn, 50 < PA < 70, are compounds for which the probability of confirmation of biological activity in biological research is smaller. On the other hand PA < 0.5 pertain to compounds for which the confirmation of biological activity in biological research is small, but this group connection may include new compounds structurally. We focused on the first compartment.
According to these data the most frequently predicted types of biological activity are: glyceryl-ether monooxygenase inhibitor, cholesterol antagonist as well as antihypercholesterolemic, respectively. The gemini quaternary alkylammonium conjugates 4-6 of ergosterol addition showed inhibition of oxidoreductase and alcohol O-acetyltransferase. On the other hand conjugates of 10-12 showed inhibition of alkylacetylglycerophosphatase and alkenylglycerophosphocholine hydrolase.

General Information
The NMR spectra were measured with a Spectrometer NMR Varian Mercury 300 MHz (Oxford, UK), operating at 300.07 and 75.4614 for 1 H and 13 C, respectively. Typical conditions for the proton spectra were: pulse width 32°, acquisition time 5 s, FT size 32 K and digital resolution 0.3 Hz per point, and for the carbon spectra pulse width 60°, FT size 60 K and digital resolution 0.6 Hz per point, the number of scans varied from 1200 to 10,000 per spectrum. The 13 C and 1 H chemical shifts were measured in CDCl 3 with methanol relative to an internal standard of TMS. Infrared spectra were recorded in the KBr pellets using a FT-IR Bruker IFS 66 spectrometer (Karlsruhe, Germany). The ESI (electron spray ionization) mass spectra were recorded on a Waters/Micromass (Manchester, UK) ZQ mass spectrometer equipped with a Harvard Apparatus (Saint Laurent, QC, Canada), syringe pump. The sample solutions were prepared in methanol at the concentration of approximately 10 −5 M. The standard ESI-MS mass spectra were recorded at the cone voltage 30 V. The sterols and amines are the commercial products from Sigma-Aldrich (Saint Louis, MO, USA).