Quaternary Alkylammonium Conjugates of Steroids: Synthesis, Molecular Structure, and Biological Studies

The methods of synthesis as well as physical, spectroscopic (1H-NMR, 13C-NMR, and FT-IR, ESI-MS), and biological properties of quaternary and dimeric quaternary alkylammonium conjugates of steroids are presented. The results were contrasted with theoretical calculations (PM5 methods) and potential pharmacological properties (PASS). Alkylammonium sterols exhibit a broad spectrum of antimicrobial activity comparable to squalamine.


Introduction
Steroids are an enormous group of very important natural products. The most significant compounds of this group are sterols (cholesterol, ergosterol, stigmasterol), bile acids (lithocholic, deoxycholic, cholic), and hormones (testosterone, estrogens, progesterone) [1][2][3][4][5]. Sterols are crucial constituents of the cell membrane of eukaryotes. Bile acids are amphipathic molecules with large, curved and rigid skeletons; chirality as well as the specific orientation of their chemically different polar hydroxy groups play an important role in metabolic processes. In turn, hormones determine the characteristics of sex and regulate pregnancy in animals, while plant hormones (brassinosteroids) cause elongation of stems and stimulate cell division (e.g., brassinolide) [6].

Quaternary Alkylammonium Conjugates of Steroids
The basic criteria for the synthesis of biologically active conjugates of steroids and polyamines have been given by Salunke et al. [11]. Firstly, the structure must have a rigid extensive hydrophobic part and a flexible hydrophilic chain with a polar head group attached to a hydrophobic part. Secondly, the sulfate groups can be removed or replaced by a hydroxyl or carboxylate group. In turn, the structure of the polyamine is not important, and parts of steroids can be modified in various ways.
On this basis, Kim et al. described the synthesis of a squalamine analogue from bisnoralcohol (2) (Scheme 1) [16]. The structure of the product was confirmed by 1 H-NMR, 13 C-NMR, DEPT, COSY, HETCOR, and FT-IR, as well as low-and high-resolution mass spectra. Additionally, the biological activity of (4) has been determined. The squalamine analogue shows biocidal activity against M. luteus 9341, S. aureus 6538P, K. pneumoniae 10031, S. equi 6580C, and B. subtilis 6633. However, E. coli 25922, P. aeruginosa 27853, P. mirabilis 25933, S. marcescens 27117, and S. typhimurium 14028 are not sensitive to (4). In general, the antimicrobial activity of compound (4) is weaker in comparison to the antibacterial activity of squalamine.

Quaternary Alkylammonium Conjugates of Steroids
The basic criteria for the synthesis of biologically active conjugates of steroids and polyamines have been given by Salunke et al. [11]. Firstly, the structure must have a rigid extensive hydrophobic part and a flexible hydrophilic chain with a polar head group attached to a hydrophobic part. Secondly, the sulfate groups can be removed or replaced by a hydroxyl or carboxylate group. In turn, the structure of the polyamine is not important, and parts of steroids can be modified in various ways.
On this basis, Kim et al. described the synthesis of a squalamine analogue from bisnoralcohol (2) (Scheme 1) [16]. The structure of the product was confirmed by 1 H-NMR, 13 C-NMR, DEPT, COSY, HETCOR, and FT-IR, as well as low-and high-resolution mass spectra. Additionally, the biological activity of (4) has been determined. The squalamine analogue shows biocidal activity against M. luteus 9341, S. aureus 6538P, K. pneumoniae 10031, S. equi 6580C, and B. subtilis 6633. However, E. coli 25922, P. aeruginosa 27853, P. mirabilis 25933, S. marcescens 27117, and S. typhimurium 14028 are not sensitive to (4). In general, the antimicrobial activity of compound (4) is weaker in comparison to the antibacterial activity of squalamine.

Quaternary Alkylammonium Conjugates of Steroids
The basic criteria for the synthesis of biologically active conjugates of steroids and polyamines have been given by Salunke et al. [11]. Firstly, the structure must have a rigid extensive hydrophobic part and a flexible hydrophilic chain with a polar head group attached to a hydrophobic part. Secondly, the sulfate groups can be removed or replaced by a hydroxyl or carboxylate group. In turn, the structure of the polyamine is not important, and parts of steroids can be modified in various ways.
On this basis, Kim et al. described the synthesis of a squalamine analogue from bisnoralcohol (2) (Scheme 1) [16]. The structure of the product was confirmed by 1 H-NMR, 13 C-NMR, DEPT, COSY, HETCOR, and FT-IR, as well as low-and high-resolution mass spectra. Additionally, the biological activity of (4) has been determined. The squalamine analogue shows biocidal activity against M. luteus 9341, S. aureus 6538P, K. pneumoniae 10031, S. equi 6580C, and B. subtilis 6633. However, E. coli 25922, P. aeruginosa 27853, P. mirabilis 25933, S. marcescens 27117, and S. typhimurium 14028 are not sensitive to (4). In general, the antimicrobial activity of compound (4) is weaker in comparison to the antibacterial activity of squalamine.
Other analogs (6-15) of MSI-1436 (5) have been synthesized from stigmasterol by Shu et al. (Figure 2) [33]. The multistep reactions gave final products with very good yields. All analogs exhibit a broad spectrum of antimicrobial activity, which strongly depend on the stereochemistry of C(7) and C(3). By contrast, the stereochemistry at the C(24) has a negligible effect on the antibacterial activity.
In turn, Rao and co-workers isolated six other aminosterols (43)(44)(45)(46)(47)(48) from the liver of the dogfish shark ( Figure 4) [15]. The authors presented a very accurate spectral analysis based on 2D NMR (COSY, HETCOR, HMBC) as well as low-and high-resolution mass spectra (FAB, ESI, MALDI). The antimicrobial activity of aminosterols (43)(44)(45)(46)(47)(48) and squalamine (1)     Synthesis of 6β-hydroxy-3-α-(or β-)aminosterols (53-58) from hyodeoxycholic acid (49) has been presented by Jones et al. (Scheme 3) [44]. The modification of hyodeoxycholic acid was carried out by the esterification of the carboxyl group and oxidation of both hydroxyl groups to ketones, followed by a conversion of the A/B ring system from cis to trans by acid-catalyzed isomerization. Then various polyamines were added and the corresponding stereoconjugates were obtained.  Synthesis of 6β-hydroxy-3-α-(or β-)aminosterols (53-58) from hyodeoxycholic acid (49) has been presented by Jones et al. (Scheme 3) [44]. The modification of hyodeoxycholic acid was carried out by the esterification of the carboxyl group and oxidation of both hydroxyl groups to ketones, followed by a conversion of the A/B ring system from cis to trans by acid-catalyzed isomerization. Then various polyamines were added and the corresponding stereoconjugates were obtained.  The synthesized aminosterol conjugates (53-58) exhibit a broad spectrum of antimicrobial activity, similar to other aminosterols (Table 3). The presented data show that the β-analogs (54, 56) are slightly more active against microorganisms than the α-analogs (53, 55). Moreover, the biocidal efficacy against S. aureus is higher for methyl esters (54,56) in comparison to free acids (57,58). The chain length of the polyamine has no significant effect on biocidal activity. However, for acid derivatives, a conjugate with spermine chain (58) was much more active than a conjugate with an ethylene diamine chain (57). The synthesized aminosterol conjugates (53-58) exhibit a broad spectrum of antimicrobial activity, similar to other aminosterols (Table 3). The presented data show that the β-analogs (54, 56) are slightly more active against microorganisms than the α-analogs (53, 55). Moreover, the biocidal efficacy against S. aureus is higher for methyl esters (54,56) in comparison to free acids (57,58). The chain length of the polyamine has no significant effect on biocidal activity. However, for acid derivatives, a conjugate with spermine chain (58) was much more active than a conjugate with an ethylene diamine chain (57).
Maitra et al. used their own method to modify the side chain of bile acids [45,46]. The synthesis of quaternary alkylammonium conjugates of bile acids (63-75) is shown in Scheme 4. Bile acids (59, 60) were transformed to the 24-nor-23-iodo (61, 62) derivatives by a Hunsdiecker reaction followed by a reaction with secondary or tertiary amines, respectively. All conjugates (63-75) were obtained with good yields 65%-75% and were characterized by 1 H-NMR, 13 C-NMR, and FT-IR, as well as mass spectrometry. These quaternary ammonium conjugates were found to be good gelators. Some of the quaternary ammonium bile salts gelled water and many of them gelled aqueous salt solutions even in the presence of organic solvents such as alcohol (methanol, ethanol) as well as DMF or DMSO. These gels form fibrous networks [46].
Lopushanskii and Udovitskaya described the method to prepare cholesteryl 3β-bromoacetate and 3β-chloroacetate, which were used in the synthesis of quaternary ammonium derivatives of cholesterol and its 5α,6β-dibromo derivatives (81-91) (Scheme 5) [47].  Bile acids (59, 60) were transformed to the 24-nor-23-iodo (61, 62) derivatives by a Hunsdiecker reaction followed by a reaction with secondary or tertiary amines, respectively. All conjugates (63-75) were obtained with good yields 65%-75% and were characterized by 1 H-NMR, 13 C-NMR, and FT-IR, as well as mass spectrometry. These quaternary ammonium conjugates were found to be good gelators. Some of the quaternary ammonium bile salts gelled water and many of them gelled aqueous salt solutions even in the presence of organic solvents such as alcohol (methanol, ethanol) as well as DMF or DMSO. These gels form fibrous networks [46].
Lopushanskii and Udovitskaya described the method to prepare cholesteryl 3β-bromoacetate and 3β-chloroacetate, which were used in the synthesis of quaternary ammonium derivatives of cholesterol and its 5α,6β-dibromo derivatives (81-91) (Scheme 5) [47]. Bile acids (59, 60) were transformed to the 24-nor-23-iodo (61, 62) derivatives by a Hunsdiecker reaction followed by a reaction with secondary or tertiary amines, respectively. All conjugates (63-75) were obtained with good yields 65%-75% and were characterized by 1 H-NMR, 13 C-NMR, and FT-IR, as well as mass spectrometry. These quaternary ammonium conjugates were found to be good gelators. Some of the quaternary ammonium bile salts gelled water and many of them gelled aqueous salt solutions even in the presence of organic solvents such as alcohol (methanol, ethanol) as well as DMF or DMSO. These gels form fibrous networks [46].
Molecules 2015, 20, page-page 9 (106-109, 118, 124-126) and cholesterol (110-113, 114, 127-129), where the double bonds increase the reactivity of the molecule, thereby increasing values of HOF (Figure 9). In turn, the HOF of conjugates of methyl esters of bile acids (121-123) can be explained in a similar manner. For these compounds the number of hydroxyl groups in the steroid skeleton lowers the value of HOF. 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 4500 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 [55][56][57][58][59]. The PASS software is useful for the study of the biological activity of secondary metabolites. The types of activities that were predicted for a potential compound with the highest probability (focal activities) have been selected. If predicted activity (PA) > 70, 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 50 < PA < 70, the substance is unlikely to exhibit the activity in experiment, the probability is less, and the substance is unlike any known pharmaceutical agent. A research group led by Brycki selected the types of activity that were predicted for a potential compound with the highest probability (Table 4). 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 4500 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 [55][56][57][58][59]. The PASS software is useful for the study of the biological activity of secondary metabolites. The types of activities that were predicted for a potential compound with the highest probability (focal activities) have been selected. If predicted activity (PA) > 70, 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 50 < PA < 70, the substance is unlikely to exhibit the activity in experiment, the probability is less, and the substance is unlike any known pharmaceutical agent. A research group led by Brycki selected the types of activity that were predicted for a potential compound with the highest probability (Table 4). Table 4. Probability "to be Active" (PA) values for predicted biological activity of compounds (106-132).

Conclusions
The design and preparation of new steroid conjugates allow us to develop the fields of supramolecular chemistry, material chemistry, and nanotechnology. In this paper we described the synthesis and physicochemical properties of quaternary alkylammonium conjugates of steroids. Most of the described compounds are characterized by high biological activity with a broad spectrum of antimicrobial and antifungal activity. Moreover, these compounds can actively participate in transport across biological membranes, which offers tremendous possibilities in biochemistry, pharmacology, and medicine. The spectroscopic data, semiempirical calculations, and potential pharmacological properties (PASS) obtained in this work significantly extend the library of new steroid conjugates.