Synthesis, Spectroscopic and Semiempirical Studies of New Quaternary Alkylammonium Conjugates of Sterols

New quaternary alkylammonium conjugates of steroids were obtained by two step reaction of sterols (ergosterol, cholesterol, dihydrocholesterol) with bromoacetic acid bromide, followed by bimolecular nucleophilic substitution with a long chain tertiary alkylamine. The structures of products were confirmed by spectral (1H-NMR, 13C-NMR, and FT-IR) analysis, mass spectrometry and PM5 semiempirical methods. The pharmacotherapeutic potential of synthesized compounds has been estimated on the basis of Prediction of Activity Spectra for Substances (PASS).


Introduction
The steroids are modified triterpenoids with the tetracyclic ring system of lanosterol. However, the compounds do not have methyl groups at the C(4) and C (14) position and they have differently modified side chains [1,2] (Figure 1). The compounds of this type are of natural origin and play important biological functions in plant and animal cells. They are also the main sex hormones in mammals (e.g., testosterone and estrogens) and plants (e.g., brassinosteroids). They also regulate metabolism (e.g., glycocholic and taurocholic acid or vitamin D) and are important cardioactive glycosides (e.g., digoxin, gitoxin and scillaren A) [3][4][5][6]. Exceptionally interesting group of steroids are the sterols, e.g., cholesterol, cholestanol, ergosterol and stigmasterol [7][8][9]. Sterols are crystalline compounds which have a secondary hydroxyl group in the C(3) position of the steroid skeleton, one or two double bonds and differently modified side chains. Rings A/B of the steroid skeleton may have trans geometry (the allo series) or cis (the normal series). Sterols have a hydroxy group in the average plane of the ring, and can form a number of β-sterols [10,11].
One of the most important sterol is ergosterol (1, provitamin D 2 ), which performs analogous functions like cholesterol (2). but in the cells of fungi ( Figure 2). Ergosterol is vital for fungal survival. It serves two purposes: a bulk membrane function and a vigorous function [1,2]. Furthermore ergosterol is a biological precursor to vitamin D 2 [12,13]. Another important compound of this group is cholesterol (and its metabolite cholestanol (3)), which is fundamental component of the cell membranes of animal cells. Cholesterol in the ester form stabilizes and stiffens a protein-lipid membrane. Cholesterol in mammals regulates the cell membrane's permeability and fluidity, growth rate and membrane-bound enzyme activity. Modifications of functional groups in the molecules of sterols such as cholesterol or ergosterol provide compounds with high pharmacological activity. Connecting steroid compound molecules with natural products such as pyrimidines, purines, alkaloids or polyamines allows one to obtain new compounds with high biological activity as well as complexing or gelator agents. All compounds of this type may be classified as steroid conjugates [14].
Quaternary alkylammonium salts are very wide class of compounds which have many applications. Some of them are used as antiseptics and preservation agents [15]. It is proved that the derivatives which contain from 8 to 14 carbon atoms in the alkyl chain group show the greatest biocidal activity [16][17][18]. The mechanism of biocidal activity of quaternary alkylammonium salts is based on adsorption of the alkylammonium cation on the bacterial cell surface, diffusion through the cell wall and then binding and disruption of the cytoplasmatic membrane. Damage of the membrane results in a release of potassium ions and other cytoplasmatic constituents, finally leading to cell death [19][20][21][22][23][24]. A frequently used microbiocide, especially in sublethal concentrations, can result in an increasing resistance of microorganisms. One of the ways to overcome this serious negative side effect is the periodic application of new microbiocides with modified structures.
This work reports the synthesis and physicochemical properties of new quaternary alkylammonium conjugates of ergosteryl 3β-bromoacetate (4), cholesteryl 3β-bromoacetate (5) and dihydrocholesteryl 3β-bromoacetate (6) with N,N-dimethyl-N-octylamine (7,11,15), N,N-dimethyl-N-decylamine (8,12,16), N,N-dimethyl-N-dodecylamine (9,13,17), N,N-dimethyl-N-tetradecylamine (10,14,18) in acetonitrile. The potential pharmacological activities of the synthesized compounds have been studied using a computer-aided drug discovery approach with the in silico Prediction of Activity Spectra for Substances (PASSs) program. It is based on a robust analysis of the structure-activity relationships in a heterogeneous training set currently including about 60,000 biologically active compounds from different chemical series with about 4,500 types of biological activities. Since only the structural formula of the chemical compound is necessary to obtain a PASS prediction, this approach can be used at the earliest stages of investigation. There are many examples of the successful use of the PASS approach leading to new pharmacological agents [40][41][42][43][44]. The PASS software is useful for the study of biological activity of secondary metabolites. We have selected the types of activities that were predicted for a potential compound with the highest probability (focal activities). If predicted activity (PA) > 0.7, the substance is very likely to exhibit experimental activity and the chance of the substance being the analogue of a known pharmaceutical agent is also high. If 0.5 < PA < 0.7, the substance is unlikely to exhibit the activity in experiment, the probability is less, and the substance is unlike any known pharmaceutical agent.

Results and Discussion
The new quaternary alkylammonium conjugates of steroids were obtained by reaction of sterols (ergosterol, cholesterol, dihydrocholesterol) with bromoacetic acid bromide to give intermediates 4-6. The 3β-bromoacetates of sterols were prepared according to the literature procedures [45]. The structure of ergosteryl 3β-bromoacetate (4) was confirmed by 1 H-NMR, 13 C-NMR, and FT-IR analysis, as well as ESI-MS. The syntheses of conjugates 7-18 are shown in Scheme 1.
The structures of all synthesized compounds were determined from their 1 H-and 13 C-NMR, FT-IR and ESI-MS spectra. Moreover, PM5 calculations were performed on all compounds [46][47][48]. Additionally, analyses of the biological prediction activity spectra for the new esters prepared herein are good examples of in silico studies of chemical compounds.

5'
We also selected the types of activity that were predicted for a potential compound with the highest probability (focal activities, Table 1). According to these data the most frequently predicted types of biological activity are: cholesterol antagonist, antihypercholesterolemic, adenomatous polyposis treatment and glyceryl-ether monooxygenase, acylcarnitine hydrolase, alcohol O-acetyltransferase, oxidoreductase, prostaglandin-E2 9-reductase, alkylacetylglycerophosphatase, alkenylglycerophosphocholine hydrolase or dextranase inhibitors, respectively.     The ESI-MS spectra were recorded in methanol. In all cases, the molecular ion [M] + is present, which is associated with the presence of a quaternary ammonium ion. In Figure 6 we present the ESI-MS spectrum of conjugate 13. In the spectrum of this conjugate, the [M] + molecular ion peak is observed at   Table 2. Representative compounds 9, 13 and 17 are shown in Figure 7.    This fact can be explained by the increase in the number of possible conformers. In turn the length of the hydrocarbon chain is not without significance for the antimicrobial activity of the obtained conjugates. The spatial arrangement and interaction of the conjugate 10 is shown in Figure 8. The final heat of formation is −1249.429 kcal/mol and the distances between the quaternary nitrogen and the anion bromide are 4.19 Å. Compensation charges occur only through intermolecular electrostatic interaction. This is a very good confirmation of the conclusion that interactions reduce HOF.

General
The NMR spectra were measured with a Spectrometer NMR Varian Mercury 300 (Oxford, UK), operating at 300.07 MHz and 75.4614 MHz 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 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, 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.