Design, synthesis, spectral and theoretical study of new bile acid–sterol conjugates linked via 1,2,3-triazole ring

s New four steroid conjugates have been prepared from bile acids and sterol derivatives using click chemistry method. The azide-alkyne Huisgen cycloaddition (intermolecular 1,3-dipolar cycloaddition) of the propargyl ester of lithocholic, deoxycholic, cholic acid as well as dehydrocholic acids and azide derivatives of cholesterol gave a new bile acid-sterol conjugates linked with a 1,2,3-triazole ring. Previously, bile acids were converted into bromoacetyl substituted derivatives by the reaction of propargyl esters of lithocholic, deoxycholic, cholic with bromoacetic acid bromide in toluene with TEBA and sodium hydride. All conjugates were obtained in good yields using an efficient synthesis method. The structures of all products were confirmed by spectral (1H- and 13C-NMR, and FT-IR) analysis, mass spectrometry (ESI-MS), as well as PM5 semiempirical methods. Estimation of the pharmacotherapeutic potential has been accomplished for the synthesized compounds on the basis of Prediction of Activity Spectra for Substances (PASS).


Introduction
Among the vast number of compounds of natural origin one of the most important groups are steroids. This group are a large class of very important compounds witch display very significant roles in different organisms. The most famous steroid is cholesterol. It is constituent of the cell membrane and it's present in large concentrations in the brain and nervous tissue [1][2][3][4]. This sterol is the biosynthetic precursor of vitamin D, bile acids, steroid hormones and lipoproteins [5][6][7]. All sterols are crystalline with a secondary hydroxyl group in the position C(3) of the steroid skeleton. This group located in the plane of the A/B rings forms β-sterols. Ring A/B of the steroid skeleton may form an allo (trans geometry) or normal series (cis geometry). Moreover this class of compounds has differently modified side chains and one or two double bonds [8][9][10]. In turn, bile acids have preferred hydroxyl groups on the C(3) position in the α orientation. These compounds and their derivatives have a large, inflexible, and curved skeleton. Additionally, bile acids have chemically different polar hydroxyl groups 3α or 3α,7α and 3α,12α as well as 3α,7α,12α which are responsible to some extent for their amphiphilic properties. This specific structure makes that bile acids are common in the study of biomimetic chemistry, host-guest chemistry or molecular recognition as well as in supramolecular chemistry and as drugs in pharmacology [11,12]. Bile acids themselves have been used as building blocks for the design and construction of new molecular receptors that are able to recognize guest molecules of various chemical character. Simultaneously bile acid dimers can be used for the synthesis of different macrocyclic molecules as artificial receptors [13][14][15][16][17]. On the other hand some derivatives of bile acids are very good organogelators [11,[18][19][20].
Due to the specific and amazing properties of bile acids and the triazole ring, various conjugates containing amino acid [21,22], nucleosides [23][24][25] sugar [26], as well as β-lactam [27] in the structure were synthesized. Whereas the connection of molecules of bile acids with cholesterol by the 1,2,3-triazole ring allows the synthesis of novel conjugates with a multitude of applications [28]. Synthesis of new steroid conjugates entails the possibility of receiving more compounds with high biological activity [29].
The "click" chemistry is very beautiful part of modern organic synthesis. It includes a general field of carbon-heteroatom bond forming reactions that fulfill specified requirements such as very simple reaction conditions, high efficiency and selectivity as well as simple product isolation [30]. Another extremely important argument in favor of such a synthesis is fact, that products are stable in various solvents, including water [30,31]. One of the more important methods of preparing a 1,2,3-triazole ring is Huisgen 1,3-dipolar cycloaddition. The reaction occurs between azides and terminal alkynes and it is the copper(I) catalyzed [32,33].
Compounds of this type are very resistant to the hydrolysis, oxidation and reduction conditions of metabolic degradation. The Cu(I)-catalyzed "click" reaction is thus an extremely useful method to obtain new 1,2,3triazole derivatives of bile acids [34][35][36][37][38][39]. These five-membered heterocyclic rings can form intra-or intermolecular hydrogen bonds together with dipole-dipole interactions with an increase in the solubility of conjugates and facilitate binding of molecular targets [40].

Instrumentation and chemicals
All of the synthesis reagents lithocholic, deoxycholic, cholic and dehydrocholic acids, cholesterol, propargyl alcohol, bromoacetic acid bromide, N,N'-Dicyclohexylcarbodiimide, 4-dimethylaminopyridine, sodium azide, sodium hydride, benzyltriethylammonium chloride, sodium ascorbate were purchased from Sigma-Aldrich Corporation. Solvents chloroform, dichloromethane, toluene, hexane, t-butanol, methanol were obtained from common com-mercial sources (Merck, Fisher) and used with-out purification. General. IR Spectra: FT/IR-4600 type A in solid state or oil; v in cm − 1 . 1 H and 13 C NMR spectra: Varian Mercury 300 MHz spectrometer (Oxford, UK), operating at 300.07 and 75.4614 for 1 H and 13 C, resp.; δ in ppm rel. to Me 4 Si as internal standard, J in Hz. Typical conditions for the H-atom spectra: pulse width 32 • , acquisition time 5 s, FT size 32 K and digital resolution 0.3 Hz per point; and for the C-atom 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. ESI-MS: Waters/Micromass (Manchester, UK) ZQ mass spectrometer equipped with a Harvard Apparatus (Saint Laurent, Canada), syringe pump; in m/z. The sample solns. were prepared in MeOH at the concentration of ca. 10 -5 M. The standard ESI-MS mass spectra were recorded at the cone voltage 90 V.

Synthesis
Molar ratios of the respective reactants are shown in Table 1. Procedure for propargyl esters of bile acids: the bile acids (lithocholic 1, deoxycholic 2, cholic 3, dehydrocholic 4) was dissolved in 15 mL of dichloromethane. Then, propargyl alcohol, DCC and DMAP was added and the reaction was carried out for 24 h at room temperature. Next the mixture washed with cool water, extracted ethyl acetate and washed with water, brine, and dried (Na 2 SO 4 ). The solvent was evaporated under reduced pressure to give the products: (85%) of (5), (92%) of (6), (73%) of (7) and (92%) of (8).
Procedure for 3-bromoacetoxy derivatives of bile acids (9-11) and cholesterol (13): the propargyl esters of bile acids (5-7), as well as cholesterol (12) was dissolved in 5 mL of anhydrous toluene. Then sodium hydride and TEBA were added and the reaction was carried out for 1 h at room temperature. Subsequently, bromoacetic acid bromide was added dropwise and the reaction mixture was kept at room temperature for 24 h. Then the excess of sodium hydride was filtered (then neutralize it with anhydrous methanol), and the filtrate was washed with NaHCO 3 (5%, 20 mL), brine (200 mL) and finally dried over Na 2 CO 3 . The solvent was evaporated under reduced pressure to give the crude product. Products were purified by chromatography on silica gel (Merck, type 60, 70-230 mesh) with chloroform/hexane as eluent and to give the products 85% of (9), (10-11) and 66% of (13). Procedure for cholester-3β-yl 2-azidoacetate (14): cholester-3β-yl 2bromoacetate (13) was dissolved in 15 mL of DMF. Then, NaN 3 was added, the mixture was heated at 50 • C for 4 h. DMF was evaporated, extracted with toluene, washed with brine, and dried (Na 2 SO 4 ) and to give the product (70%).

Computational detail
The PM5 semiempirical calculations were performed using the WinMopac 2003 program. The final heat of formation (HOF) for dimers of bile acids and cholesterol derivatives (15-18) linked 1,2,3-triazole ring is presented in Table 4. The molecular models of compounds all compounds are shown in Figs. 3 and 4.
In the 1 H NMR spectrum of dehydrocholic propargyl ester (8) showed characteristic two hydrogen singlets in the range 1.07 and 1.40 ppm and doublet at 0.85 ppm assigned to CH 3 -18, CH 3 -19, and CH 3 -21, respectively. The protons of the CO 2 CH 2 group gave signals in the range 4.68 ppm. Whereas the terminal proton in propargyl group gave characteristic signal at 2.47 ppm. The 1 H NMR spectra of compounds (10) and (11) show characteristic multiplets in the range 4.81-4.63 ppm assigned to axial positions of the C3β-H protons in steroid skeleton. In the spectrum of compound (11) additionally is present doublet of the C7β-H proton at 5.01 ppm. However, in the case of these bromoacetates derivatives protons of C12β-H appear as triplets in the range of 5.17-5.16 ppm. The 1 H NMR spectra of these compounds show characteristic singlets in the range 3.80-3.79 ppm for the protons of the 3α-CO 2 CH 2 Br group, whereas for compound (11) characteristic doublets at 3.85 ppm for the protons of the 7α-CO 2 CH 2 Br group are observed. However, protons of 12α-CO 2 CH 2 Br group for both compounds appear as a singlet at 3.89 ppm. The doublet of doublets of CO 2 CH 2 protons at 4.67 ppm for both discussed derivatives were observed in 1 H NMR spectra. The protons of C ---CH group gave signals in the range 2.48-2.47 ppm. Two hydrogen singlets in the range of 0.76-0.75 and 0.94-0.92, as well as characteristic doublets at 0.85-0.83 ppm are assigned to CH 3 -18, CH 3 -19, and CH 3 -21, respectively.
The diagnostics proton signals of the triazole ring C27-H of all conjugates with 1,2,3-triazole ring (15)(16)(17)(18) in CDCl 3 arise as a singlet at about 7.76-7.75 ppm. In turns, the protons of the methylene groups C25-H as well as C28-H linked directly to the triazole ring give signals at 5.24 ppm and 5.14 ppm, respectively. The 1 H NMR spectra of (15)(16)(17)(18) showed characteristic multiplets of protons of C3 ′ α-H of sterol skeleton in the range of 4.89-4.59 ppm. In the same range there are also multiplets from the protons of C3β-H group of bile acid skeleton.
In the spectra of compounds (16) and (17) characteristic broad singlets in the range 5.14 ppm are observed which are due to the C12β-H protons. 1 H NMR spectra of conjugate (17) show singlet at 5.01 ppm assigned to the C7β-H protons of the bile acid skeleton.
In the bile acids skeleton was observed two hydrogen singlets ranking from 1.05 to 0.62 and 1.40-0.90 ppm and characteristic doublet at 0.85-0.79 ppm assigned to CH 3 -18, CH 3 -19 and CH 3 -21, respectively. On the other hand in the cholesterol part the characteristic hydrogen singlets at 0.68 assigned to CH 3 -18 ′ . The second sets of singlets at 1.02 ppm were assigned to CH 3 -19 ′ . The characteristic doublet of CH 3 -21 ′ are at 0.93-0.91 in the all conjugates. The 1 H NMR spectra of (15)(16)(17)(18) showed a doublet at 0.86 ppm for the protons of the CH 3 -26 ′   (15) in the corresponding 13 C NMR spectrum. and CH 3 -27 ′ methyl groups. For cholesterol derivatives diagnostics is doublet for C6 ′ -H at 5.39-5.38 ppm. Additionally in the 1 H NMR spectra of compounds (15)(16)(17)(18) show diagnostics singlets in the range 3.80-3.79 ppm for the protons of the 3α-CO 2 CH 2 Br group, whereas for compound (17) characteristic singlet at 3.85 ppm for the protons of the 7α-CO 2 CH 2 Br group are observed. However, protons of 12α-CO 2 CH 2 Br group for the compounds 16 and 17 appear as a singlet at 3.86 and 3.89 ppm, respectively. The characteristic protons shifts for compounds (15)(16)(17)(18) are collected in Table 2.
The 13 C NMR spectra of compounds (8)

Infrared spectroscopy
The most characteristic feature of the FT-IR spectra of compounds (8), (10) and (11)

T. Pospieszny and H. Koenig
It should be noted, that the most characteristic in the FT-IR spectra of (15)(16)(17)(18)  respectively. In turn, the C--C strong bands were rather weak for all conjugated steroids, and they were observed at 1650, 1653, 1664 and 1662 cm − 1 , respectively.
In all spectra of conjugates there are also visible bands related to vibrations of C -H bonds in the region 2935-2863 cm − 1 .

PM5 calculations
PM5 semiempirical calculations were performed using the  Table 4. The molecular models of all conjugates are shown in Fig. 3. In many works that describe the application of computational methods, one can find information on the comparison of theoretical results with crystallographic structures. Not without significance is the use of computational methods in determining the properties of the docking [48][49][50]. We were able to obtain a very good picture of molecular modeling using semiempirical calculations [51]. The lowest HOF values were observed for cholic acid derivatives (17), where the OH groups facilitate the formation of intramolecular Hbonds and stable host-guest complexes. These complexes may be stabilized by H-bonding or electrostatic interactions that arise from the OH groups in the bile acid molecule. The HOF value decreases with the increasing number of OH groups in the steroid skeleton. It should be noted that in the case of the dehydrocholic acid derivative, the heat of formation is similar to that of the deoxycholic acid derivative. The lower HOF value for derivative (18) can be explained by the greater stability of the carbonyl group, which is also noticed for free acids (2) and (4), respectively. Derivative (18) cannot be hydrogen atoms donor in the formation of hydrogen bonds, therefore such HOF values were observed. (1)(2)(3)(4), bromoacetyl substituted derivatives of propargyl esters of bile acids (9-11) and bromoacetyl substituted derivative of cholesterol (13) as well as its azidoacetyl substituted derivative (14).

Prediction of activity spectra for substances
Potential pharmacological activities of the synthesized conjugates have been determined on the basis of a computer-aided drug discovery approach with in silico Prediction of Activity Spectra for Substances (PASSs) program [52][53][54][55]. In previous works were presented and discussed in silico studies of steroid conjugates with different long chain amines, as well as linked triazole rings [39,44,[56][57][58][59]. In this work, the biological activity spectra were predicted for all four synthesized conjugates (15)(16)(17)(18) with PASS. Furthermore, the types of activity, which were predicted for a potential compound with the highest probability (focal activities), have also been selected. They are presented in Fig. 5. According to these data, the most frequently predicted types of biological activity are the cholesterol antagonist, antineoplastic and glycerylether monooxygenase inhibitor beyond conjugate (18). On the other hand conjugate (15) is only antieczematic activity, (18) dermatologic, antiinfertility female, UGT1A4 substrate and (17) only to hypolipemic and biliary tract disorders treatment, respectively.