Spectroscopic Methods and Theoretical Studies of Bromoacetyl Substituted Derivatives of Bile Acids

The structure of seven bromoacetyl substituted derivatives of bile acids have been characterized by H MMR, C 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).


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][2][3][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.][7][8][9][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.
[13][14][15][16][17][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.5][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.8][29][30][31][32][33][34][35] These compounds were used in nucleophilic reactions with N-, Sor O-nucleophiles.In many cases bromo-or chloroacetyl substituted derivatives of bile acids react with pyrimidines (e.g.selective N-1alkylation of uracil), 27 purines (e. g. selective N-9-alkylation of adenine), 28  selective N-1-alkylation of 2-thiouracil), 29 ammonium morpholinyl dithiocarbamate (selective S-alkylation), 30 N-1-alkylation of imidazole, 31 sodium azide [32][33][34] or tamoxifen (antagonist of the estrogen receptor in breast tissue). 35n 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. 36owever, to the best of our knowledge, no work has been published on the spectroscopic ( 1 H MMR, 13 C 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.

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 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 TMS as the internal standard.The 2D 1 H-1 H (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 1 H and 13 C, 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 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 90 V.

Computational Details
8][39] The calculations were performed using the GAUSSIAN 03 program package 40 at the B3LYP 41-43 levels of theory with the 6-31G(d,p) basis set. 44The NMR isotropic shielding constants were calculated using the standard GIAO (Gauge-Independent Atomic Orbital) approach of Gaussian 03. 45,46otential 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.8][49][50][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.

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. 33The 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. 27Products were obtained with very good yields, however no information about purification method is available.Among the com-Pospieszny et al.: Spectroscopic Methods and Theoretical Studies ...  pounds prepared only methyl 3α-bromoacetoxy-5β-cholan-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 lithocholic, deoxycholic and cholic acid.

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 3α,7α,12α-tribromoacetoxy-5β-cholan-24-oate (10).This fact can be explained by the greater stability by ester groups in isolated molecule.The HOF of methyl

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-β-D-glucosidase, glyceryl-ether monooxygenase as well as choleretic, cholesterol antagonist, CYP3A substrate and CYP3A4 substrate.

4. 1 H and 13 C NMR Spectra
The structures of all synthesized compounds were determined from their 1 H and 13 C, as well as two-dimen-Pospieszny et al.: Spectroscopic Methods and Theoretical Studies ... 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 1 H-1 H, 1 H- 13 C NMR experiments.Additionally the 1 H- 13 C HSQC spectrum shows correlations over three bonds between the C3β-H, C7β-H, C12β-H protons and C=O carbon atoms in 10. 52 The HSQC spectrum of 10 is shown in Figure 3. Crosspeaks between H(21)-C( 18), H(11)-C( 12), H(18)-C( 12) confirmed its structure.
The 1 H and 13 C NMR spectra were measured in chloroform (Table 3).The 1 H NMR spectra of compounds 4-10 show characteristic multiplets in the range  In the spectra of compounds 5, 7 and 9 where unsubstituted hydroxy group in position C( 12) is present, characteristic broad singlets in the range 4.01-3.99ppm are observed which are due to the C12β-H protons (in equatorial positions).However, in the case of the bromoacetates (compounds 6, 8 and 10) protons C12β-H appear as triplets in the range of 5.17-5.16ppm.
1 H NMR spectra of derivatives of cholic acid 7, 8 and 9, 10 show characteristic multiplets in the ranges of 3.98-3.86and 5.02-4.96ppm assigned to the C(7β)-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 OOCCH 2 Br group in 9 and 10 (Figure 4).
The characteristic singlets of CO 2 CH 3 protons in the range 3.67-3.66ppm for all discussed compounds were observed in 1 H NMR spectra.
The 1 H NMR spectra of compounds 4-10 show characteristic singlets in the range 3.80-3.79ppm for the protons of the 3α-OCOCH 2 Br group, whereas for compounds 9 and 10 characteristic doublets at 3.83 and 3.85 ppm for the protons of the 7α-OCOCH 2 Br group are observed.However, protons of 12α-OCOCH 2 Br 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     ppm assigned to C(7)OH and C(7)OCOCH 2 Br, 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 BrCH 2 groups is observed at 28.7-26.1 ppm.
The relation between the experimental 13 C and 1 H chemical shifts (δ exp ) and the Gauge Including Atomic Orbitals (GIAO) magnetic isotropic shielding constants (σ calc ), which are widely used, 45,46,[53][54][55] are usually linear and described by the equation δ exp = a + b σ calc .The slope and intercept of the least-square correlation lines are used to scale the GIAO isotropic absolute shielding constants σ, and to predict chemical shifts in CDCl 3 for 10 (Figure 5, Table 4).
As can be seen from Figure 5 the agreement between the experimental and the calculated data for protons is worse than for carbons-13. 55The 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.

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   12) slightly shift the carbonyl groups ν(C=O) to higher wavenumbers, 1737-1731 cm -1 , which is due to the inductive influence of chlorine. 33urthermore, the comparison of carbonyl groups ν(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

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 BrCH 2 CO 2 H is observed from which the fragmented ions [M -BrCH 2 CO 2 H+Na] + come.Because these ions are seen in all the discussed compounds, it can be concluded that the neutral molecule of BrCH 2 CO 2 H 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 BrCH 2 CO 2 H 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 3α-bromoacetoxy-7α,12α-dihydroxy-5β-cholan-24-oate (7) the relative abundance of this ion was 100% (Figure 7).

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 bromoacetic acid bromide in toluene with TEBA and sodium hydride.
The structures of all synthesized compounds 4-10 were determined from their 1 H and 13 C 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 silico studies of chemical compounds.
Linear correlations between the experimental 1 H and 13 C 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.
13 C NMR spectra of compounds 4-10 show characteristic signals at 12.7-11.9,23.3-22.7 and 18.3-17.3ppm which are assigned to CH 3 -18, CH 3 -19 and CH 3 -21, respectively.The carbon atoms of the CO 2 CH 3 group are observed in the range 174.4-174.7 ppm and at 51.5 ppm and are assigned to CO 2 and CH 3 , respectively.On the other hand, carbon atoms of bromoacetoxy groups in positions 3α, 7α or 12α resonate in the range of 166.8-166.3ppm.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 77.9-77.2ppm assigned to C(12)OH and C(12)OCOCH 2 Br, respectively.However, carbons of the C(7) gave signals in the range 68.2-67.9 and 73.1-73.

Figure 7 .
Figure 7. ESI-MS spectra in the negative and positive ion mode of compounds 7 (a) and 9 (b).

Table 2 .
Probability »to be Active« (PA) values for predicted biological activity of compounds 4-10.
4.83-4.59ppm assigned to the C3β-H protons (in axial positions) of the steroid skeleton.

Table 4 .
Chemical shifts (δ, ppm) in CDCl 3 calculating GIAO nuclear magnetic shielding tensors (σ calc ) for 10.The predicted GIAO chemical shifts were computed from the linear equation δ exp.= a + b•σ calc with a and b determined from the fit of the experimental data.
a intercept; b slope; c correlation coefficient