DOI: 10.17344/acsi.2014.608 Acta Chim. Slov. 2015, 62, 15-27 15 Scientific paper Spectroscopic Methods and Theoretical Studies of Bromoacetyl Substituted Derivatives of Bile Acids Tomasz Pospieszny,* Hanna Koenig, Iwona Kowalczyk and Bogumil Brycki Laboratory of Microbiocides Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland * Corresponding author: E-mail: tposp@amu.edu.pl Received: 08-05-2014 Abstract The structure of seven bromoacetyl substituted derivatives of bile acids have been characterized by 1H MMR, 13C NMR, 2D NMR, FT-IR and mass spectrometry (ESI-MS) as well as PM5 semiempirical and B3LYP ab initio methods. Estimation of the pharmacotherapeutic potential has been accomplished for the synthesized compounds on the basis of Prediction of Activity Spectra for Substances (PASS). Keywords: Bile acids, bromoacetyl substituted derivatives, Prediction of Activity Spectra for Substances, spectroscopic methods, PM5 and B3LYP calculations. 1. Introduction Steroids are beside carbohydrates, amino acids, peptides and nucleobases a large class of natural compounds. Compounds of this type display a very important role in plant and animal organisms. Steroids are not only constituents of the cell membrane in eukaryotes (e.g., cholesterol, cholestanol, ergosterol), but they are also the main sex hormones in mammals (e.g., testosterone, estrogens, progesterone) and plants (e.g. brassinosteroids). Steroids play also important functions in the regulation of metabolism (e.g., bile acids and vitamin D).1-4 Especially important compounds are bile acids (e.g., lithocholic, deoxycholic and cholic) and their derivatives. The cholic acid was first isolated from the bile of mammals in 1828 by L. Gmelin. The bile acids are produced from cholesterol in the liver and are stored in the gallbladder.5-10 However, gallbladder contraction with feeding releases bile acids into the intestine. Additionally, the terminal carboxylic acid group in C(17) side chain may be conjugated with taurine or glycine. The amphiphilic properties of bile acids together with their specific structure composed of a large, rigid and curved skeleton, as well as chirality and orientation of their chemically different polar hydroxy groups (3 a; 3a,7a and 3a,7a,12a) toward the center of a concave face make them interesting starting materials for the synthesis of macrocyclic compounds as molecular dimers, molecular tweezers, cholaphanes or quasi podands.11-18 Moreover, the unique structural elements of bile acids are very important in the study of molecular recognition, host-guest chemistry and biomimetic chemistry. They also play a very significant role in supramolecular chemistry and as drugs in pharmacology. Bile acids themselves have been used as building blocks for the design and construction of new molecular receptors that are capable to recognize guest molecules of diverse chemical nature.13-16 Bile acid dimers can be used for the synthesis of macrocyclic compounds as artificial recep-tors.19-23 Some derivatives of bile acids are very good or-ganogelators.24-26 The above mentioned applications of bile acids make them very interesting and promising materials. Halogenoacetyl (chloro- or bromo-) substituted derivatives of bile acids play an extremely useful role in various organic syntheses. Compounds of this type can be prepared in many different ways. Bile acids can react with halogenoacetic acid halides and potassium carbonate in chloroform or dichloromethane, with calcium or sodium hydride and tetrabutylammonium bromide (TEBA) in toluene, as well as pyridine or 4-(dimethylamino)pyridine (DMAP) in toluene.27-35 These compounds were used in nucleophilic reactions with N-, S- or O-nucleophiles. In many cases bromo- or chloroacetyl substituted derivatives of bile acids react with pyrimidines (e.g. selective N-1-alkylation of uracil),27 purines (e. g. selective N-9-alkyla-tion of adenine),28 thio analogs of pyrimidine bases (e. g. selective N-1-alkylation of 2-thiouracil),29 ammonium morpholinyl dithiocarbamate (selective S-alkylation),30 N-1-alkylation of imidazole,31 sodium azide32-34 or tamoxifen (antagonist of the estrogen receptor in breast tissue).35 In the case of sodium azide, the resulting products are used as substrates for click chemistry reactions. Singh et al. employed a series of chloro substituted derivatives of bile acids that were used in the synthesis of cationic bile acid-based facial amphiphiles featuring trimethyl ammonium head groups. The authors evaluated the role of these amphiphile compounds for cytotoxic activity against colon cancer cells.36 However, to the best of our knowledge, no work has been published on the spectroscopic (1H MMR, 13C NMR, 2D NMR, FT-IR), semiempirical (PM5) and ab initio (B3LYP) methods, as well as in silico (PASS) and mass spectrometry (ESI-MS) studies of bromoacetyl substituted bile acids. 2. Experimental Procedure 2. 1. Instrumentation The NMR spectra were measured with a Spectrometer NMR Varian Mercury 300 MHz (Oxford, UK), operating at 300.07 and 75.4614 for 1H and 13C, 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 13C and 1H chemical shifts were measured in CDQ3 relative to TMS as the internal standard. The 2D 1H-1H (COSY) and Heteronuclear Multiple-Bond Connectivity (HMBC, HSQC) spectra were recorded on a Bruker Avance DRX spectrometer operating at 599.93 and 150.85 MHz for 1H and 13C, respectively. Infrared spectra were recorded as KBr pellets (1.5 mg/300 mg KBr) using a FT-IR Bruker IFS 66 spectrometer (Karlsruhe, Germany) at 295 K. The ESI (electron spray ionization) mass spectra were recorded on a Wa-ters/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 90 V. 2. 2. Computational Details The PM5 semiempirical calculations were performed using the WinMopac 2003 program.37-39 The calculations were performed using the GAUSSIAN 03 program package40 at the B3LYP41-43 levels of theory with the 6-31G(d,p) basis set.44 The NMR isotropic shielding constants were calculated using the standard GIAO (Gauge- Independent Atomic Orbital) approach of Gaussian 03.45,46 Potential pharmacological activities of the compounds synthesized have been evaluated on the basis of computer-aided drug discovery approach with in silico Prediction of Activity Spectra for Substances (PASSs) program. It is based on a robust analysis of the structure-activity relationship 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.47-51 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 the activity in experiment and the chance of the substance being the analogue of a known pharmaceutical agent is also high. And 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. 2. 3. Synthesis The methyl esters of bile acids (1 eq.) were dissolved in 6 mL anhydrous toluene. Then sodium hydride (3-5 eq.) and TEBA (0.1 eq.) were added and the reaction was carried out for 1 h at room temperature. Subsequently, bromoacetic acid bromide was added dropwise (1.1 eq. for each hydroxyl group at steroid skeleton) and the reaction mixture was kept at room temperature for 24 h. Then the excess of sodium hydride was filtered, and the filtrate was washed with NaHCO3 (5%, 20 mL), brine (20 mL) and finally dried over Na2CO3. 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). Methyl 3a-bromoacetoxy-5ß-cholan-24-oate (4) isolated yield 59%, mp 127-128 °C. Anal. Calcd for C27H43BrO4: C 63.40, H 8.47. Found: C 63.58, H 8.30; 1H NMR (CDCl3): 5 4.83-4.75 (m, 1H, 3ß-H), 3.80 (s, 2H, 3a-CH2Br), 3.67 (s, 3H, OCH3), 0.93 (s, 3H, CH3-19), 0.91 (d, J = 6.4 Hz, 3H, CH3-21), 0.65 (s, 3H, CHr18); 13C NMR (CDCl3): 5 174.7 (C-24), 166.8 (3a-CO2), 76.6 (C-3), 56.4, 55.9, 51.5 (C-25), 42.7, 41.9, 40.4, 40.1, 35.8, 35.3, 34.9, 34.6, 31.9, 31.0, 30.9, 28.2 (3a-CH2Br), 26.9, 26.4, 26.4, 26.3, 24.2, 23.3 (C-19), 20.8, 18.3 (C-21), 12.0 (C-18). ESI-MS (MeOH): m/z (%) 533 (100) [M+Na]+, 549 (25) [M+K]+; FT-IR: v 1730 (C=O), 1292 (C-O) cm-1. Methyl 3a-bromoacetoxy-12a-hydroxy-5ß-cholan-24-oate (5), isolated yield 50%, oil. Anal. Calcd for C27H43BrO5: C 61.47, H 8.22. Found: C 61.63, H 8.40; 1H NMR (CDCl3): 5 4.81-4.76 (m, 1H, 3ß-H), 3.99 (bs,1H, 12ß-H), 3.80 (s, 2H, 3a-CH2Br), 3.67 (s, 3H, OCH3), 0.97 (d, J = 6.4 Hz, 3H, CH3-21), 0.93 (s, 3H, CH3-19), 0.68 (s, 3H, CH3-18); 13C NMR (CDCl3): 5 174.6 (C-24), 166.8 (3a-CO2), 76.5 (C-3), 73.1 (C312), 51.5 (C-25), 48.25, 47.37, 426.47, 41.82, 35.94, 35.02, 34.73, 34.09, 33.65, 31.82, 31.03, 30.86, 28.7 (3a-CH2Br), 27.40, 26.88, 26.40, 26.24, 25.96, 23.56, 23.1 (C-19), 17.3 (C-21), 12.7 (C-18); ESI-MS (MeOH): m/z (%) 551 (100) [M+Na]+, 567 (30) [M+K]+; FT-IR: v 3543 (OH), 1734 (C=O), 1287 (C-O) cm-1. Methyl 3a,12a-dibromoacetoxy-5ß-cholan-24-oate (6) isolated yield 70%, oil. Anal. Calcd for C27H43Br2O6: C 53.71, H 6.84. Found: C 53.49, H 6.52; 1H NMR (CDCl3): 5 5.16 (t, J = 2.8 Hz, 1H, 12ß-H), 4.81-4.73 (m, 1H, 3ß-H), 3.85 (s, 2H, 12a-CH2Br), 3.79 (s, 2H, 3a-CH2Br), 3.66 (s, 3H, OCH3), 0.92 (s, 3H, CH3-19), 0.83 (d, J = 6.3 Hz, 3H, CH3-21), 0.75 (s, 3H, CH3-18); 13C NMR (CDCl3): 5 174.6 (C-24), 166.7 (3a-CO2), 166.6 (12a-CO2), 77.9 (C-12), 76.3 (C-3), 51.5 (C-25), 49.2, 47.3, 45.1, 41.7, 35.6, 34.8, 34.6, 34.3, 34.1, 31.8, 30.9, 30.8, 27.4 (3a-CH2Br), 26.8 (12a-CH2Br), 26.4, 26.2, 25.9, 25.9, 25.3, 23.42, 22.9 (C-19), 17.4 (C-21), 12.3 (C-18); ESI-MS (MeOH): m/z (%) 671 (100) [M+Na]+, 687 (43) [M+K]+; FT-IR: v 1733 (C=O), 1286 (C-O) cm-1. Methyl 3a-bromoacetoxy-7a,12a-dihydroxy-5ß-cho-lan-24-oate (7), isolated yield 57%, oil. Anal. Calcd for C27H43BrO6: C 59.66, H 7.97. Found: C 59.34, H 7.82; 1H NMR (CDCl3): 5 4.69-4.61 (m, 1H, 3ß-H), 4.00 (bs, 1H, 12ß-H), 3.98-3.86 (m, 1H, 7ß-H), 3.79 (s, 2H, 3a-CH2Br), 3.67 (s, 3H, OCH3), 0.99 (d, J = 6.3 Hz, 3H, CH3-21), 0.91 (s, 3H, CH3-19), 0.80 (s, 3H, CH3-18); 13C NMR (CDCl3): 5 174.7 (C-24), 166.8 (3a-CO2), 76.5 (C-3), 72.8 (C-12), 68.2 (C-7), 51.5 (C-25), 47.2, 46.5, 42.0, 41.1, 39.5, 35.1, 34.8, 34.7, 34.6, 34.2, 31.9, 31.0, 30.8, 29.4, 28.4 (3a-CH2Br), 27.40, 26.7, 26.5, 26.4, 23.1, 22.7 (C-19), 17.3 (C-21), 14.12, 12.6 (C-18); ESI-MS (MeOH): m/z (%) 567 (100) [M+Na]+, 583 (55) [M+K]+; 579 (100) [M+Cl]-, 623 (35) [M+Br]-; FT-IR: v 3433 (OH), 1734 (C=O), 1286 (C-O) cm-1. Methyl 3a,12a-dibromoacetoxy-7a-hydroxy-5ß-cho-lan-24-oate (8) isolated yield 23%, oil. Anal. Calcd for C27H43Br2O7: C 52.42, H 6.67. Found: C 52.74, H 6.43; 1H NMR (CDCl3): 5 5.17 (t, J = 2.0 Hz, 1H, 12ß-H), 4.67-4.59 (m, 1H, 3ß-H), 3.91-3.88 (m, 1H, 7ß-H), 3.86 (s, 2H, 12a-CH2Br), 3.79 (s, 2H, 3a-CH2Br), 3.66 (s, 3H, OCH3), 0.91 (s2 3H, CH3-19), 0.85 (d, J = 6.4 Hz, 3H, CH3-21), 0.77 (s, 3H, CH3-18); 13C NMR (CDCl3): 5 174.6 (C-24), 166.7 (3a-CO2), 166.8 (12a-CO2), 77.5 (C-12), 76.3 (C-3), 67.9 (C-7), 51.5 (C-25), 47.1, 45.1, 43.2, 41.0, 39.6, 34.8, 34.6, 34.6, 34.3, 30.9, 30.7, 27.5 (3a-CH2Br), 27.3, 26.5 (12a-CH2Br), 26.3, 25.9, 25.1, 22.9 (C-19), 22.3, 17.4 (C-21), 12.2 (C-18); ESI-MS (MeOH): m/z (%) 687 (100) [M+Na]+; FT-IR: v 3541 (OH), 1731 (C=O), 1286 (C-O) cm-1. Methyl 3a,7a-dibromoacetoxy-12a-hydroxy-5ß-cho-lan-24-oate (9), isolated yield 24%, oil. Anal. Calcd for C27H43Br2O7: C 52.42, H 6.67. Found: C 52.59, H 6.58; 1H NMR (CDCl3): 5 4.99-4.96 (m, 1H, 7ß-H), 4.70-4.63 (m, 1H, 3ß-H), 4.01 (bs, 1H, 12ß-H), 3.83 (s, 2H, 7a-CH2Br), 3.80 (s, 2H, 3a-CH2Br), 3.66 (s, 3H, OCH3), 0.982 (d, J = 6.2 Hz, 3H, CH3-21), 0.94 (s, 3H, CH3-19), 0.69 (s, 3H, CH3-18); 13C NMR (CDCl3): 5 174.6 (C3-24), 166.8 (3a-CO2), 166.6 (7a-CO2), 76.1 (C-3), 73.3 (C-7), 72.6 (C-12), 51.5 (C-25), 47.2, 46.6, 41.9, 40.7, 38.2, 34.9, 34.6, 34.4, 34.3, 31.1, 31.0, 30.8, 29.7, 28.5, 28.1, 27.2 (3a-CH2Br), 26.3 (7a-CH2Br), 26.3, 22.9 (C-19), 22.4, 17.3 (C-21), 12.5 (C-18); ESI-MS (MeOH): m/z (%) 687 (100) [M+Na]+; 183 (100) [NaBr+Br]-; 699 (13) [M+Cl]-; 743 (75) [M+Br]-; FT-IR: v 3530 (OH), 1733 (C=O), 1287 (C-O) cm-1. Methyl 3a,7a,12a-tribromoacetoxy-5ß-cholan-24-oa- te (10), isolated yield 54%, oil. Anal. Calcd for C27H43Br3O8: C 47.41, H 5.78. Found: C 47.68, H 5.58; 1H NMR (CDCl3): 5 5.17 (t, J = 2.9 Hz, 1H, 12ß-H), 5.02-5.00 (m, 1H, 7ß-H), 4.68-4.60 (m, 1H, 3ß-H), 3.89 (s, 2H, 12a-CH2Br), 3.85 (s, 2H, 7a-CH2Br), 3.79 (s, 2H, 3a-CH2Br), 3.66 (s, 3H, OCH3), 0.94 (s, 3H, CH3-19), 0.84 (d, J = 6.4 Hz, 3H, CH3-21), 0.76 (s, 3H, CHr18); 13C NMR (CDCl3): 5 174.4 (C-24), 166.7 (3a-CO2), 166.5 (12a-CO2), 166.3 (7a-CO2), 77.2 (C-12), 75.8 (C-3), 73.1 (C-7), 51.5 (C-25), 47.2, 45.1, 42.7, 40.6, 37.9, 34.7, 34.5, 34.3, 34.3, 31.1, 30.9, 30.7, 29.7, 28.4, 27.2, 26.3, 26.4 (3a-CH2Br), 26.2 (7a-CH2Br), 26.1 (12a-CH2Br), 24.9, 22.9 (C-19), 22.2, 17.5 (C-21), 11.9 (C-18)2 ESI-MS (MeOH): m/z (%) 808 (100) [M+Na]+; FT-IR: v 1732 (C=O), 1286 (C-O) cm-1. 3. Results and Discussion 3. 1. Synthesis Bromoacetyl substituted derivatives of lithocholic, deoxycholic and cholic acid were obtained by reaction of methyl esters of bile acids with bromoacetic acid bromide in toluene with TEBA and sodium hydride to give compounds 4-10.33 The syntheses of compounds 4-10 are depicted in Scheme 1. In the chemical literature there is no report on the compounds 6-10. Some data about compounds 4 and 5, which were synthesized with potassium carbonate in chloroform, are given by Chattopadhyay.27 Products were obtained with very good yields, however no information about purification method is available. Among the com- Scheme 1. Synthesis of bromoacetyl substituted derivatives of bile acids 4-10. Figure 1. Molecular models of compounds 4-10 calculated by PM5 method. pounds prepared only methyl 3a-bromoacetoxy-5ß-cho-lan-24-oate (4) was a solid, the rest of the conjugates were oils. We are the first to offer a full spectroscopic characterization of bromoacetyl substituted derivatives of lithoc-holic, deoxycholic and cholic acid. 3. 2. PM5 and B3LYP Calculations The PM5 semiempirical calculations were performed using the WinMopac 2003 program. The final heat of formation (HOF) for the methyl esters of bile acids 1-3 and its bromoacetyl substituted derivatives 4-10 is presented in Table 1. The molecular models of compounds 4-10 are shown in Figure 1. The lowest HOF value is observed for methyl 3a,7a,12a-tribromoacetoxy-5ß-cholan-24-oate (10). This fact can be explained by the greater stability by ester groups in isolated molecule. The HOF of methyl Table 1. Heat of formation (HOF) [kcal/mol] of compounds 4-10. Compound HOF AHOF HOF of five molecules 1 -226.5896 - - 2 -268.6761 - - 3 -309.3719 - - 4 -266.3263 -39.7367 -1348.8740 5 -308.5947 -39.9186 -1558.2560 6 -345.9485 -77.2724 -1744.5095 7 -348.0126 -38.6407 -1756.1661 8 -386.0620 -76.6901 -1941.6084 9 -388.6102 -79.2383 -1957.0453 10 -425.4859 -116.1140 -2108.7200 — HOF - HOF — nwr (compounds 4-10) nv7r (methyl ester of bile acids 1-3) 3a,12a-dibromoacetoxy-7a-hydroxy-5ß-cholan-24-oa-te (8) and methyl 3a,7a-dibromoacetoxy-12a-hydroxy-5ß-cholan-24-oate (9) are comparable. The increase of the number of free hydroxyl groups causes the increase of the HOF values. It can be also caused by difficulty to form intramolecular hydrogen bonds. The spatial arrangement and interaction of the conjugate 8 is shown in Figure 2. The final heat of formation is -1941.6084 kcal/mol and the distances between the carbonyl group of 3a-CO2 and 12a-CO2 are 8.03 À and 7.45 À, respectively. In turn, the distance between the C(24)O2 groups is 7.68 À. Compensation charges occur only through in-termolecular electrostatic interaction. This is a very good confirmation of the conclusion that interactions reduce HOF. 3. 3. The Predicted Biological Activity The biological activity spectra were predicted for all synthesized compounds using PASS. We selected the types of activity that were predicted for a potential compound with the highest probability (focal activities, Table 2). According to these data the most frequently predicted types of biological activity, e.g. inhibitors of acylcarnitine hydrolase (PA > 95%), alkenylglycerophosphocholine hydrolase (PA > 90%), alkylacetylglycerophosphatase (PA > 90%), D-lactaldehyde dehydrogenase, glucan endo-1,3-ß-D-glucosidase, glyceryl-ether monooxygenase as well as choleretic, cholesterol antagonist, CYP3A substrate and CYP3A4 substrate. 3. 4. 1H and 13C NMR Spectra The structures of all synthesized compounds were determined from their 1H and 13C, as well as two-dimen- Figure 2. Molecular models of compound 8 calculated by PM5 method. Table 2. Probability »to be Active« (PA) values for predicted biological activity of compounds 4-10. Focal predicted activity (PA > 80%) 4 5 6 Compounds 7 8 9 10 Acylcarnitine hydrolase inhibitor 96 98 97 99 98 98 97 Adenomatous polyposis treatment 81 - - 82 82 - - Alkenylglycerophosphocholine hydrolase inhibitor 94 97 95 96 94 96 92 Alkenylglycerophosphoethanolamine hydrolase inhibitor 81 89 82 85 81 85 - Alkylacetylglycerophosphatase inhibitor 94 97 95 95 94 95 92 Choleretic - 86 - 90 87 86 - Cholesterol antagonist 89 85 81 88 88 82 - CYP3A substrate - 82 - 83 - 83 - CYP3A4 substrate 81 84 - 85 82 85 - Dextranase inhibitor 88 - 89 91 88 91 83 D-lactaldehyde dehydrogenase inhibitor - - - 86 81 81 - Glucan endo-1,3-ß-D-glucosidase inhibitor 87 95 89 91 88 91 83 Glyceryl-ether monooxygenase inhibitor - 86 84 89 88 88 86 Peptidoglycan glycosyltransferase inhibitor 83 91 85 87 84 87 - Plasmanylethanolamine desaturase inhibitor - - - 84 81 81 - Protein-disulfide reductase (glutathione) inhibitor - 88 81 81 - 81 - Vitamin-K-epoxide reductase inhibitor - 81 - 87 83 83 - sional NMR spectra (COSY, HSQC, HMBC). The assignments of proton and carbon-13 chemical shifts for 4-9 are listed in Table 3 and are based on 2D 1H-1H, 1H-13C NMR experiments. Additionally the 1H-13C HSQC spectrum shows correlations over three bonds between the C3ß-H, C7ß-H, C12ß-H protons and C=O carbon atoms in 10.52 The HSQC spectrum of 10 is shown in Figure 3. Cross-peaks between H(21)-C(18), H(11)-C(12), H(18)-C(12) confirmed its structure. The 1H and 13C NMR spectra were measured in chloroform (Table 3). The 1H NMR spectra of compounds 4-10 show characteristic multiplets in the range Table 3. The most characteristic chemical shifts (ppm) in !H and 13C NMR spectra of compounds 4-9. No of atom 4 5 6 7 8 9 1H NMR 18 0.65 0.68 0.75 0.80 0.77 0.69 19 0.93 0.93 0.92 0.91 0.91 0.94 21 0.91 0.97 0.83 0.99 0.85 0.98 25 3.67 3.67 3.66 3.67 3.66 3.66 3ß-H 4.83-4.75 4.81-4.76 4.81-4.73 4.69-4.61 4.67-4.59 4.70-4.63 7ß-H - - - 3.98-3.86 3.91-3.88 4.99-4.96 12ß-H - 3.99 5.16 4.00 5.17 4.01 3a-CH2Br 3.80 3.80 3.79 3.79 3.79 3.80 7a-CH2Br - - - - - 3.83 12a-CH2Br - - 3.85 - 3.86 - 13C NMR 18 12.0 12.7 12.3 12.6 12.2 12.5 19 23.3 23.1 22.9 22.7 22.9 22.9 21 18.3 17.3 17.4 17.3 17.4 17.3 24 174.7 174.6 174.6 174.7 174.6 174.6 25 51.5 51.5 51.5 51.5 51.5 51.5 3ß-H 76.6 76.5 76.3 76.5 76.3 76.1 7ß-H - - - 68.2 67.9 73.3 12ß-H - 73.1 77.9 72.8 77.5 72.6 3a-CO2 166.8 166.8 166.7 166.8 166.7 166.8 7a-CO2 - - - - - 166.6 12a-CO2 - - 166.6 - 166.8 - 3a-CH2Br 28.2 28.7 27.4 28.4 27.5 27.2 7a-CH2Br - - - - - 26.3 12a-CH2Br - - 26.8 - v26.5 - 4.83-4.59 ppm assigned to the C3ß-H protons (in axial positions) of the steroid skeleton. In the spectra of compounds 5, 7 and 9 where unsub-stituted hydroxy group in position C(12) is present, characteristic broad singlets in the range 4.01-3.99 ppm are observed which are due to the C12ß-H protons (in equatorial positions). However, in the case of the bromoacetates (compounds 6, 8 and 10) protons C12ß-H appear as triplets in the range of 5.17-5.16 ppm. 1H NMR spectra of derivatives of cholic acid 7, 8 and 9, 10 show characteristic multiplets in the ranges of 3.98-3.86 and 5.02-4.96 ppm assigned to the C(7ß)-H protons (in equatorial positions) of the steroid skeleton, respectively. The position of the signals at lower chemical shift values is related to the presence of the unsubstituted hydroxyl group in 7 and 8, while the higher chemical shift values correspond to the OOCCH2Br group in 9 and 10 (Figure 4). Two hydrogen singlets in the range of 0.80-0.65 and 0.94-0.91, as well as characteristic doublets at 0.99-0.83 ppm are assigned to CH3-18, CH3-19, and CH3-21, respectively. The characteristic singlets of CO2CH3 protons in the range 3.67-3.66 ppm for all discussed compounds were observed in 1H NMR spectra. The 1H NMR spectra of compounds 4-10 show characteristic singlets in the range 3.80-3.79 ppm for the protons of the 3a-OCOCH2Br group, whereas for compounds 9 and 10 characteristic doublets at 3.83 and 3.85 ppm for the protons of the 7a-OCOCH2Br group are observed. However, protons of 12a-OCOCH2Br group for the compound 6 appear as a doublet and for compounds 8 and 10 as a singlet at 3.85, 3.86 and 3.89 ppm, respectively. The characteristic protons shifts for compounds 4-10 are collected in Table 3. The 13C NMR spectra of compounds 4-10 show characteristic signals at 12.7-11.9, 23.3-22.7 and 18.3-17.3 ppm which are assigned to CH3-18, CH3-19 and CH3-21, respectively. The carbon atoms of the CO2CH3 group are observed in the range 174.4-174.7 ppm and at 51.5 ppm and are assigned to CO2 and CH3, respectively. On the other hand, carbon atoms of bromoa-cetoxy groups in positions 3a, 7a or 12a resonate in the range of 166.8-166.3 ppm. Unusually, a relationship was observed between the signals of carbon atoms C(3) and C(12) of the steroid skeleton. The carbon atoms of the C(12) steroid skeleton gave signals in the range of 73.1-72.6 and 77.9-77.2 ppm assigned to C(12)OH and C(12)OCOCH2Br, respectively. However, carbons of the C(7) gave signals in the range 68.2-67.9 and 73.1-73.3 Figure 3. HSQC NMR spectrum for methyl 3a,7a,12a-tribromoacetoxy-5ß-cholan-24-oate (10). Figure 4.1H NMR spectra in the 5.20-3.80 ppm region showing the most characteristic signals of compounds 7 (red), 8 (black) and 9 (green). Figure 5. Plots of the experimental chemical shifts (8 vs. the magnetic isotropic shielding constants (o) from the GIAO/B3LYP/6-31G(d,p) approach calculated for 10 in CDCl3; 8exp = a + b ffcalc; (a) carbon-13, (b) proton. ppm assigned to C(7)OH and C(7)OCOCH2Br, respectively. The presence of bromoacetoxy group at C(7) shift the signals of carbon atoms C(3) to lower chemical shift in comparison to bromoacetoxy group at C(12). The diagnostic signal for BrCH2 groups is observed at 28.7-26.1 ppm. The relation between the experimental 13C and 1H chemical shifts (5exp) and the Gauge Including Atomic Orbitals (GIAO) magnetic isotropic shielding constants (acalc), which are widely used,45'46'53-55 are usually linear and described by the equation 5exp = a + b acalc. The slope and intercept of the least-square correlation lines are used to scale the GIAO isotropic absolute shielding constants a, and to predict chemical shifts in CDCl3 for 10 (Figure 5, Table 4). Table 4. Chemical shifts (5, ppm) in CDCl3 calculating GIAO nuclear magnetic shielding tensors (acalc) for 10. The predicted GIAO chemical shifts were computed from the linear equation 5 = a + b• acalc with a and b determined from the fit of the experimental data. carbon-13 S exp. Salc ^alc proton S exp. Scalc aalc C(1) 38.00 30.24 197.52 H(1) 1.72 1.49 31.44 C(2) 27.15 20.66 203.68 H(1) 1.11 1.14 31.69 C(3) 75.83 77.78 166.92 H(2) 1.50 1.46 31.46 C(4) 34.30 26.49 199.93 H(2) 1.47 0.86 31.89 C(5) 31.14 39.19 191.76 H(3) 4.64 4.09 29.58 C(6) 34.32 24.94 200.93 H(4) 1.69 1.43 31.48 C(7) 73.05 75.00 168.71 H(4) 2.11 1.88 31.16 C(8) 38.00 36.81 193.29 H(5) 1.33 1.35 31.54 C(9) 34.71 33.44 195.46 H(6) 1.72 1.50 31.43 C(10) 34.50 40.60 190.85 H(6) 2.02 1.83 31.20 C(11) 25.91 22.36 202.59 H(7) 5.02 4.49 29.29 C(12) 77.22 79.62 165.74 H(8) 1.73 1.50 31.43 C(13) 45.11 52.36 183.28 H(9) 2.11 1.76 31.25 C(14) 42.70 45.82 187.49 H(11) 1.55 1.35 31.54 C(15) 24.87 28.98 198.33 H(11) 1.81 1.63 31.34 C(16) 30.67 33.83 195.21 H(12) 5.17 4.63 29.19 C(17) 47.18 49.29 185.26 H(14) 2.05 3.08 30.30 C(18) 11.92 10.39 210.29 H(15) 1.52 1.41 31.50 C(19) 22.86 16.39 206.43 H(15) 1.57 1.63 31.34 C(20) 34.71 38.86 191.97 H(16) 1.79 2.41 30.78 C(21) 17.46 11.26 209.73 H(16) 1.81 3.08 30.30 C(22) 30.88 27.56 199.24 H(17) 1.71 2.72 30.56 C(23) 30.88 29.92 197.72 H(18) 0.76 0.85 31.90 C(24) 174.40 170.54 107.23 H(19) 0.94 1.04 31.76 CH3O 51.48 54.54 181.88 H(20) 1.40 1.24 31.62 3a-CO2 166.70 165.25 110.63 H(21) 0.84 0.86 31.89 7a-CO2 166.25 164.34 111.22 H(22) 1.72 1.95 31.11 12a-CO2 166.51 164.79 110.93 H(22) 1.33 1.88 31.16 3a-CH2 26.36 33.41 195.48 H(23) 2.20 2.63 30.62 7a-CH2 26.19 31.06 196.99 H(23) 2.23 2.79 30.51 12a-CH2 26.06 31.00 197.03 CH3O 3.66 3.90 29.71 3a-CH2 3.79 3.15 30.25 7a-CH2 3.85 3.36 30.10 12a-CH2 3.89 3.37 30.09 aa 337.1619 45.3617 bb -1.5539 -1.3954 0.9895 0.8322 a intercept; b slope; c correlation coefficient As can be seen from Figure 5 the agreement between the experimental and the calculated data for protons is worse than for carbons-13.55 The protons are located on the periphery of the molecule, thus their interactions with solvent molecules are much stronger than the interactions of the more hidden carbon atoms. The differences between the calculated and experimental shifts for protons are probably due to the fact that the shifts are calculated for single molecules in a gas phase. 3. 5. FT-IR Spectra The FT-IR spectra of the representative conjugates 7, 9 and 10 are shown in Figure 6. The most characteristic in the FT-IR spectra are the bands at 3543 cm-1 (5), 3433 Wave riu m be rs [cm" Figure 6. FT-IR spectra (film) of compounds 7 (black), 9 (red) and 10 (blue) in the 3600-400 cm-1 region. cm-1 (7), 3541 cm-1 (8) and 3530 cm-1 (9) assigned to the stretching vibrations v(OH) of the O(7)H, O(12)H or O(7)H and O(12)H. The FT-IR spectra of all compounds revealed two strong characteristic signals in the regions 1734-1730 cm-1 and 1287-1284 cm-1, assigned to v(C=O) and v(C-O), respectively. On the other hand, the presence of chloroacetoxy groups in disubstituted derivatives of methyl ester of deoxycholic acid at position C(3) and C(12) or trisubstituted derivatives of methyl ester of cholic acid at position C(3), C(7) and C(12) slightly shift the carbonyl groups v(C=O) to higher wavenumbers, 1737-1731 cm-1, which is due to the inductive influence of chlorine.33 Furthermore, the comparison of carbonyl groups v(C=O) bands for methyl and ethyl derivatives show that carbonyl band of ethyl esters are shifted by about 20 cm-1 to higher wavenumbers.56 3. 5. ESI-MS Spectra The ESI-MS spectra were recorded in methanol. In Figure 7 we present the ESI-MS spectra of compounds 7 and 9. In all cases, the molecular ion is present as a [M+Na]+. Synthesized compounds showed a higher affinity for sodium cation than potassium ion. In all spectra the molecular ion peak is 100% relative abundance, furthermore the elimination of BrCH2CO2H is observed from which the fragmented ions [M - BrCH2CO2H+Na]+ come. Because these ions are seen in all the discussed compounds, it can be concluded that the neutral molecule of BrCH2CO2H is eliminated from the C(3) position of the steroid skeleton. Furthermore, we observed for compounds 4-7 in the ESI mass spectra ions [M+K]+. These ions have a relative abundances of 30-35%, only for compound 7 it amounts to 55%. In this case, there was no elimination of the BrCH2CO2H molecule. For compounds 4, 5 and 7-9 ions [M+Br]- (100%) in the negative ion mode were observed. In the spectra of the compounds 7 and 9 in the negative ion mode the presence of ion [M+Cl]- was also observed. For 3a-bromoacetoxy-7a,12a-dihydroxy-5ß-cholan-24-oate (7) the relative abundance of this ion was 100% (Figure 7). 4. Conclusions Seven bromoacetyl substituted derivatives 4-10 of lithocholic, deoxycholic and cholic acid were obtained by the reaction of methyl esters of bile acids with bromoace-tic acid bromide in toluene with TEBA and sodium hydride. The structures of all synthesized compounds 4-10 were determined from their 1H and 13C NMR, 2D NMR (COSY, HSQC, HMBC), FT-IR as well as ESI-MS spectra. Moreover, PM5 calculations were performed on all compounds. Additionally, analyses of the biological prediction activity spectra for bromoacetyl substituted derivatives of bile acids prepared herein are examples of in si-lico studies of chemical compounds. Linear correlations between the experimental 1H and 13C chemical shifts and the computed screening constants confirm the optimized geometry. Estimation of the pharmacotherapeutic potential has been accomplished for the synthesized compounds on the basis of Prediction of Activity Spectra for Substances (PASS). The obtained compounds may find applications as subtrates in organic synthesis. Figure 7. ESI-MS spectra in the negative and positive ion mode of compounds 7 (a) and 9 (b). 5. Acknowledgements This work was supported by the funds from Adam Mickiewicz University, Faculty of Chemistry. 6. References 1. I. Kirson, E. Glotter, J. Nat. Prod. 1981, 44, 633-647. http://dx.doi.org/10.1021/np50018a001 2. H. Gao, J. R. Dias, Org. Prep. Proced. Int. 1999, 32, 145-166. http://dx.doi.org/10.1080/00304949909355705 3. M. Fetizon, F. J. Kakis, V. Ignatiadou-Ragoussis, J. Org. Chem. 1973, 38, 4308-4311. http://dx.doi.org/10.1021/jo00964a022 4. W. H. Okamura, M. M. Midland, M. W. Hammond, N. A. Rahman, M. C. Dormanen, I. Nemere, A. W. J. Norman, Steroid Biochem. Mol. Biol. 1995, 53, 603-613. 5. K. C. Nicolaou, T. Montagnon, Molecules that Changed the World, John Wiley & Sons, Ltd., UK, 2008, pp 79-90. 6. P. M. Dewick, Medicinal Natural Products A Biosynthetic Approach 3rd Ed., John Wiley & Sons, Ltd., UK, 2009, pp 275-277. http://dx.doi.org/10.1002/9780470742761 7. L. Lack, F. O. Donity, T. Walker, G. D. Singletary, J. Lipid Res. 1973, 14, 367-370. 8. K.-Y. Tserng, D. L. Hachey, P. D. Klein, J. Lipid Res. 1977, 18, 404-407. 9. A. K. Batta, G. Salen, S. Shefer, J. Bio. Chem. 1984, 259, 15035-15039. 10. S. M. Huijghebaert, A. F. Hofmann, J. Lipid Res. 1986, 27, 742-752. 11. L. Yuexian, J. R. Dias, Chem. Rev. 1997, 97, 283-304. http://dx.doi.org/10.1021/cr9600565 12. A. P. Davis, Chem. Soc. Rev. 1993, 22, 243-253. http://dx.doi.org/10.1039/cs9932200243 13. P. Willimann, T. Marti, A. Fürer, F. Diederich, Chem. Rev. 1997, 97, 1567-1608. http://dx.doi.org/10.1021/cr960373b 14. A. P. Davis, R. P. Bonar-Law, J. K. M. Sanders, In Comprehensive Supramolecular Chemistry, J. L. Atwood, J. E. D. Davis, D. D. Macnicol, F. Vögtle, Eds., Elsevier Oxford, 1996, 257-286. 15. Y. Li, R. Dias, Chem. Rev. 1997, 97, 283-304. http://dx.doi.org/10.1021/cr9600565 16. J. Tamminen, E. Kolehmainen, Molecules 2001, 6, 21-46. http://dx.doi.org/10.3390/60100021 17. T. Pospieszny, H. Koenig, B. Brycki, Tetrahedron Lett. 2013, 54, 4700-4704. http://dx.doi.org/10.1016/j.tetlet.2013.06.096 18. L. Yuexian, J. R. Dias, Chem. Rev. 1997, 97, 283-304. http://dx.doi.org/10.1021/cr9600565 19. H. Gao, J. R. Dias, J. Prakt. Chem. 1997, 339, 187-190. http://dx.doi.org/10.1002/prac.19973390135 20. Y. X. Li, J. R. Dias, Org. Prep. Proced. Int. 1996, 28, 203209. http://dx.doi.org/10.1080/00304949609356522 21. H. P. Hsieh, J. G. Muller, C. J. Burrows, J. Am. Chem. Soc. 1994, 116, 12077-12078. http://dx.doi.org/10.1021/ja00105a068 22. J. P. Guthrie, P. A. Cullimore, R. S. McDonald, S. O'Leary, Can. J. Chem. 1982, 60, 747-764. http://dx.doi.org/10.1139/v82-111 23. Z. Paryzek, R. Joachimiak, M. Piasecka, T. Pospieszny, Tetrahedron Lett. 2012, 46, 6212-6215. http://dx.doi.org/10.1016/j.tetlet.2012.08.151 24. H. M. Willemen, T. Vermonden, A. T. M. Marcelis, E. J. R. Sudhölter, Eur. J. Org. Chem. 2001, 2329-2335. http://dx. doi.org/10.1002/1099-0690(200106)2001:12<2329::AID-EJOC2329>3.0.CO;2-N 25. H. M. Willemen, T. Vermonden, A. T. M. Marcelis, E. J. R. Sudhölter, Langmuir 2002, 18, 7102-7106. http://dx.doi.org/10.1021/la025514l 26. A. Valkonen, M. Lahtinen, E. Virtanen, S. Kaikkonen, E. Kolehmainen, Biosens. Bioelectron. 2004, 20, 1233-1241. http://dx.doi.org/10.1016/j.bios.2004.06.029 27. P. Chattopadhyay, P. S. Pandey, Bioorg. Med. Chem. Lett. 2007, 17, 1553-1557. http://dx.doi.org/10.1016/j.bmcl.2006.12.115 28. R. Rai, P. S. Pandey, Bioorg. Med. Chem. Lett. 2005, 15, 2923-2925. http://dx.doi.org/10.1016/j.bmcl.2005.03.097 29. T. Pospieszny, I. Malecka, Z. Paryzek, Tetrahedron Lett. 2010, 51, 4166-4169. http://dx.doi.org/10.1016/j.tetlet.2010.05.094 30. H. Wang, W.-H. Chan, Tetrahedron 2007, 63, 8825-8830. http://dx.doi.org/10.1016/j.tet.2007.06.026 31. V. K. Khatri, S. Upreti, P. S. Pandey, Org. Lett. 2006, 8, 1755-1758. http://dx.doi.org/10.1021/ol060168a 32. A. Kumar, R. K. Chhatra, P. S. Pandey, Org. Lett. 2010, 12, 24-27. http://dx.doi.org/10.1021/ol902351g 33. N. G. Aher, V. S. Pore, S. P. Patil, Tetrahedron 2007, 63, 12927-12934. http://dx.doi.org/10.1016/j.tet.2007.10.042 34. T. Pospieszny, I. Malecka, Z. Paryzek, Tetrahedron Lett. 2012, 53, 301-305. http://dx.doi.org/10.1016/j.tetlet.2011.11.027 35. V. Sreekanth, S. Bansal, R. K. Motiani, S. Kundu, S. K. Muppu, T. D. Majumdar, K. Panjamurthy, S. Sengupta, A. Bajaj, Bioconjugate Chem. 2013, 24, 1468-1484. http://dx.doi.org/10.1021/bc300664k 36. M. Singh, A. Singh, S. Kundu, S. Bansal, A. Bajaj, Biochimica et Biophysica Acta 2013, 1828, 1926-1937. 37. CAChe 5.04 UserGuide, Fujitsu: Chiba, Japan, 2003. 38. J. J. P. Stewart, J. Comput. Chem. 1991, 12, 320-341. http://dx.doi.org/10.1002/jcc.540120306 39. J. J. P. Stewart, J. Comput. Chem. 1989, 10, 209-220. http://dx.doi.org/10.1002/jcc.540100208 40. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, T. Vreven, Jr., K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gom-perts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Ma-lick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian 03, Revision C.01; Gaussian, Inc.: Wallingford, CT, USA, 2004. 41. A. D. Becke, J. Chem. Phys. 1993, 98, 5648-5652. http://dx.doi.org/10.1063/L464913 42. A. D. Becke, J. Chem. Phys. 1997, 107, 8554. http://dx.doi.org/10.1063/L475007 43. C. Lee, W. Yang, G. R. Parr, Phys. Rev. B 1988, 57, 785-789. http://dx.doi.org/10.1103/PhysRevB.37.785 44. W. J. Hehre, L. Random, P. V. R. Schleyer, J. A. Pople, Ab Initio Molecular Orbital Theory. Wiley: New York, 1986. 45. R. Dichfield, Mol. Phys. 1974, 27, 789-807. http://dx.doi.org/10.1080/00268977400100711 46. K. Wolinski, J. F. Hilton, P. Pulay, J. Am. Chem. Soc. 1990, 112, 8251-8260. http://dx.doi.org/10.1021/ja00179a005 47. www.pharmaexpert.ru/PASSOnline/. 48. V. V. Poroikov, D. A. Filimonov, Y. V. Borodina, A. A. Lagunin, A. Kos, J. Chem. Inf. Comput. Sci. 2000, 40, 1349-1355. http://dx.doi.org/10.1021/ci000383k 49. V. V. Poroikov, D. A. Filimonov, J. Comput. Aided Mol. Des. 2002, 16, 819-824. http://dx.doi.org/10.1023/A:1023836829456 50. V. V. Poroikov, D. A. Filimonov, In Predictive Toxicology; Helma, Christopher, Eds.; Taylor and Francis, 2005. 51. A. V. Stepanchikova, A. A. Lagunin, D. A. Filimonov, V. V. Poroikov, Curr. Med. Chem. 2003, 10, 225-233. http://dx.doi.org/10.2174/0929867033368510 52. H. Simpson, Organic Structure Determination Using 2-D NMR Spectroscopy, Academic Press Elsevier, Amsterdam, 2008. 53. A. Forsyth, A. B. Sebag, J. Am. Chem. Soc. 1997, 119, 9483-9494. http://dx.doi.org/10.1021/ja970112z 54. B. Osmialowski, E. Kolehmainen, R. Gawinecki, Magn. Res. Chem. 2001, 39, 334-340 (and references cited therein). http://dx.doi.org/10.1002/mrc.856 55. A. R. Katritzky, N. G. Akhmedov, A. Güven, E. F. V. Scriven, S. Majumder, R. G. Akhmedova, C. D. Hall, J. Mol. Struct. 2006, 783, 191-203. http://dx.doi.org/10.1016/j.molstruc.2005.07.003 56. K. N. Kuhajda, S. M. Cvjeticanin, E. A. Djurendic, M. N. Sakač, K. M. P Gasi, V. V. Kojic, G. M. Bogdanowic, Hem. Ind. 2009, 63, 313-318. http://dx.doi.org/10.2298/HEMIND0904313K Povzetek Z uporabo 1H MMR, 13C NMR, 2D NMR, FT-IR in masne spektrometrije (ESI-MS) smo določili strukturo sedmih bro-moacetil substituiranih derivatov žolčnih kislin. Ob tem smo uporabili tudi PM5 semiempirične izračune in B3LYP ab initio metode. Oceno morebitnega farmakoterapevtskega potenciala sintetiziranih spojin smo izvedli s pomočjo programa »napovedovanje aktivnostnega spektra spojin« (Prediction of Activity Spectra for Substances oz. PASS).