Synthesis, Spectroscopic and Theoretical Studies of New Quasi-Podands from Bile Acid Derivatives Linked by 1,2,3-Triazole Rings

A novel method for the synthesis of bile acid derivatives has been developed using “click chemistry”. Intermolecular 1,3-dipolar cycloaddition of the propargyl ester of bile acids and azide groups of 1,3,5-tris(azidomethyl)benzene gave a new quasi-podands with 1,2,3-triazole rings. The structures of the products were confirmed by spectral (1H-NMR, 13C-NMR, and FT-IR) analysis, mass spectrometry and PM5 semiempirical methods. Estimation of the pharmacotherapeutic potential has been accomplished for synthesized compounds on the basis of Prediction of Activity Spectra for Substances (PASS).


Introduction
Bile acids were isolated from the bile of mammals in 1828 by L. Gmelin. They are produced from cholesterol in the liver and are stored in the gallbladder [1][2][3][4][5][6], from where contraction with feeding releases bile acids into the intestine. The terminal carboxylic acid group at C (17) in the side chain can be conjugated with taurine or glycine. The specific structure of bile acids with a large rigid and curved skeleton, chirality, the orientation of their chemically different polar hydroxy groups (3α; 3α,7α and 3α,7α,12α) toward the center of a concave face, as well as their amphiphilic properties make bile acids
Synthetic or natural podands have acyclic structures where polyether chains are linked to the same binding centre, which can be different heteroatoms, e.g., nitrogen, phosphorus and sulphur. In view of their specific properties, these compounds are so-called open-chain simple analogues of crown ethers and cryptands [26]. Like the above compounds, podand hosts are generally able to form stable complexes with monovalent cations [27]. Synthetic podands have advantages over biological ones, in terms of facile synthesis and molecular structure versatility, therefore the synthesis of compounds containing a steroid skeleton is very interesting and useful. In our previous work, we reported the synthesis and physicochemical properties of new bile acid esters of 1,3,5-tris(bromomethyl)benzene or 1,2,4,5-tetrakis(bromomethyl)benzene and 3α-acetoxy-5β-cholanic acid, 3α,12α-diacetoxy-5β-cholanic acid and 3α,7α,12α-triacetoxy-5β-cholanic acid [17]. To get new quasi-podands we decided to modify the structure of bile acids by introduction of additional 1,2,3-triazole rings using "click chemistry" methods.
"Click chemistry" is a relatively new and very attractive trend in modern organic synthesis. It includes a broad spectrum of carbon-heteroatom bond forming reactions that fulfil specified requirements such as high efficiency and selectivity, simple reaction conditions and easy product isolation [28]. Moreover the products of "click chemistry" are stable in various solvents, including water [28,29]. A very important and effective method is the copper(I)-catalyzed 1,3-dipolar cycloaddition (the Huisgen reaction) between azides and terminal alkynes which is regarded as an important example of the "click" reaction. This is a very convenient and simple method of 1,2,3-triazole synthesis. Regioselective cycloaddition reactions lead to the formation of various substituted triazoles. Depending on the catalyst used 1,4-disubstituted triazoles, 1,5-disubstituted triazoles or a mixture of both are formed (Scheme 1) [30,31]. Scheme 1. The possible "click" reaction pathways occurring between terminal alkynes and azides.  Compounds of this type are very resistant to the hydrolysis, oxidation and reduction conditions of metabolic degradation. Moreover, they can participate in the formation of hydrogen bonds which are crucial interactions in biological systems. In particular, 1,4-disubstitued 1,2,3-triazoles show the ability to participate in hydrogen bonds and dipole interactions [32]. The Cu(I)-catalyzed "click" reaction is thus an extremely useful method to obtain new 1,2,3-triazole derivatives of bile acids [33][34][35][36][37][38][39].
The syntheses of compounds 9-13 are shown in Scheme 3. We have obtained trisubstituted products 11-13 and we have also isolated and characterized two disubstituted products 9-10. These conjugates, in contrast to molecular pockets or umbrellas have a rigid benzyl platform. This allows the easier creation of conformers with an appropriate geometry. The aromatic ring is flat, so it can easily interact with the surface of biopolymers, for example. It performs the function of a specific anchor. Ramírez-López et al. have described this type of connection obtained from hormones such as estrone and estradiol and the corresponding azide. These compounds demonstrate differently gelling properties [43,44]. Scheme 3. Synthesis of quasi podands of bile acids derivatives 9-13 linked by 1,2,3-triazole rings.
(4) or (5) or (6) sodium ascorbate,  (8) The potential pharmacological activity of synthesized compounds has been determined on the basis of computer-aided drug discovery approaches with the Prediction of Activity Spectra for Substances (PASSs) program. It is based on a robust analysis of 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 activity. Since only the structural formula of the chemical compound is necessary to obtain a PASS prediction, this approach can be used as the earliest stage of an investigation. There are many examples of the successful use of the PASS approach leading to new pharmacological agents [45][46][47][48][49].
Additionally, analyses of the biological prediction activity spectra for the new esters prepared herein are good examples of in silico studies of chemical compounds. The biological activity spectra were predicted with PASS for only two synthesized compounds (9 and 10). 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, hypolipemic and inhibitors of acylcarnitine hydrolase, glyceryl-ether monooxygenase, alkenylglycerophosphocholine hydrolase or alkylacetylglycerophosphatase. We could not determine the potential biological properties of the compounds 11-13 because their molecular weight was over 1.200 g/mol. 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 [50][51][52].   Moreover, the 1 H-NMR spectra of compounds 9 and 10 showed characteristic, diagnostic singlets at 7.62-7.61 ppm assigned to the two protons of the triazole rings. In the spectra of compounds 11-13 were three triazole ring protons in the 8.17-7.58 ppm range were seen. In the 1 H-NMR spectra of compounds 9-13 the most characteristic signals were observed for the aromatic protons of the 1,3,5-trisubstituted benzene. These signals appeared as singlets at 7.21-7.19 and 7.26-7.16 ppm for 9-10 and 11-13, respectively.
The 13 C-NMR spectra of compounds 9-13 show characteristic signals at 12.4-12.0 ppm, 23.4-22.8 ppm, and 18.3-17.5 ppm, which are assigned to CH 3 -18, CH 3 -19, and CH 3 -21, respectively. The carbon atoms of the CO 2 -CH 2 -triazole ring unit resonate in the range of 173.9-173.6 ppm (CO 2 ) and 65.7-63.1 ppm (CH 2 ). In the 13 C-NMR spectra of compounds 9 and 10 the signals due to CH 2 in N 3 -CH 2 Table 2. Representative compounds 10 and 13 are shown in Figure 4. The lowest HOF value is observed for cholic acid derivative 13, where an increasing number of hydroxyl groups facilitate the formation of intramolecular hydrogen bonds. Monosubstituted derivatives of bile acids linked to 1,2,3-triazole rings are not formed because the heat of formation is very high (approx. −66 kcal/mol).
In the all quasi-podands π-π stacking sandwich type interactions between two triazole rings were observed. The calculated interplanar separation is about 5.5 Å. These distances are greater about 1.7 Å in a comparison to the classical π-π stacking interactions because the triazole ring is attached to a rigid aromatic ring which imposes an increasing distance. This is confirmed by the size of the angle between N(28)-C(31)-C(32) atoms (Table 2). Moreover this spatial arrangement of bile acids and 1,2,3-triazole rings can facilitate the formation of stable host-guest complexes.

General
The NMR spectra were measured with a Varian Mercury 300 MHz NMR spectrometer (Oxford, UK), operating at 300.07 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 or DMSO-d 6 (compound 13) 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.

Conclusions
In conclusion, five new bile acid esters 9-13 were prepared from propargyl esters of lithocholic, deoxycholic and cholic acid and 1,3,5-tris(azidomethyl)benzene t-butanol/water mixture in the presence of sodium ascorbate and CuSO 4 ·5H 2 O at 65 °C. These new compounds were characterized by spectroscopic and molecular structure methods. These bile acid esters may find applications in molecular recognition, supramolecular chemistry, and in pharmacology.