Spectroscopic methods and theoretical studies of bromoacetyl substituted derivatives of bile acids

Article abstract of DOI:10.17344/acsi.2014.608

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). Bile acids, bromoacetyl substituted derivatives, Prediction of Activity Spectra for Substances, spectroscopic methods, PM5 and B3LYP calculations.

Full text of DOI:10.17344/acsi.2014.608

DOI: 10.17344/acsi.2014.608
Acta Chim. Slov. 2015, 62, 15–27
1
5
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 Poznañ, Poland
*
Corresponding author: E-mail: tposp@amu.edu.pl
Received: 08-05-2014
Abstract
1
13
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. Esti-
mation of the pharmacotherapeutic potential has been accomplished for the synthesized compounds on the basis of Pre-
diction of Activity Spectra for Substances (PASS).
Keywords: Bile acids, bromoacetyl substituted derivatives, Prediction of Activity Spectra for Substances, spectroscopic
methods, PM5 and B3LYP calculations.
Moreover, the unique structural elements of bile
1
. Introduction
acids are very important in the study of molecular recog-
nition, 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 de-
sign and construction of new molecular receptors that
are capable to recognize guest molecules of diverse che-
Steroids are beside carbohydrates, amino acids, pepti-
des 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 consti-
tuents of the cell membrane in eukaryotes (e.g., cholesterol,
cholestanol, ergosterol), but they are also the main sex hor-
mones in mammals (e.g., testosterone, estrogens, progeste-
rone) and plants (e.g. brassinosteroids). Steroids play also
important functions in the regulation of metabolism (e.g.,
bile acids and vitamin D). Especially important com-
pounds are bile acids (e.g., lithocholic, deoxycholic and
cholic) and their derivatives. The cholic acid was first isola-
ted from the bile of mammals in 1828 by L. Gmelin. The
bile acids are produced from cholesterol in the liver and are
1
3–16
mical nature.
synthesis of macrocyclic compounds as artificial recep-
Bile acid dimers can be used for the
1
9–23
tors.
Some derivatives of bile acids are very good or-
1
–4
24–26
ganogelators.
The above mentioned applications of
bile acids make them very interesting and promising ma-
terials.
Halogenoacetyl (chloro- or bromo-) substituted de-
rivatives of bile acids play an extremely useful role in va-
rious 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 to-
luene, as well as pyridine or 4-(dimethylamino)pyridine
5–10
stored in the gallbladder.
However, gallbladder contrac-
tion with feeding releases bile acids into the intestine. Addi-
tionally, 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 (3α;
α,7α and 3α,7α,12α) toward the center of a concave fa-
ce make them interesting starting materials for the synthe-
sis of macrocyclic compounds as molecular dimers, mole-
2
7–35
(DMAP) in toluene.
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-
3
2
7
alkylation of uracil), purines (e. g. selective N-9-alkyla-
1
1–18
28
cular tweezers, cholaphanes or quasi podands.
tion of adenine), thio analogs of pyrimidine bases (e. g.
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1
6
2
9
selective N-1-alkylation of 2-thiouracil), ammonium
Independent Atomic Orbital) approach of Gaussian
03.
3
0
45,46
morpholinyl dithiocarbamate (selective S-alkylation), N-
3
1
32–34
1
-alkylation of imidazole, sodium azide
or tamoxi-
Potential pharmacological activities of the com-
pounds 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 structu-
re–activity relationship in a heterogeneous training set
currently including about 60,000 biologically active com-
pounds from different chemical series with about 4,500
types of biological activities. Since only the structural for-
mula 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 phar-
3
5
fen (antagonist of the estrogen receptor in breast tissue).
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 ammo-
nium head groups. The authors evaluated the role of these
amphiphile compounds for cytotoxic activity against co-
lon cancer cells.
However, to the best of our knowledge, no work has
been published on the spectroscopic ( H MMR, C NMR,
D NMR, FT-IR), semiempirical (PM5) and ab initio
B3LYP) methods, as well as in silico (PASS) and mass
3
6
1
13
2
(
4
7–51
macological agents.
The PASS software is useful for
spectrometry (ESI-MS) studies of bromoacetyl substitu-
ted bile acids.
the study of biological activity of secondary metabolites.
We have selected the types of activities that were predic-
ted for a potential compound with the highest probability
(focal activities). If predicted activity (PA) > 0.7, the sub-
2
. Experimental Procedure
stance 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
. 1. Instrumentation
The NMR spectra were measured with a Spectro-
meter NMR Varian Mercury 300 MHz (Oxford, UK),
operating at 300.07 and 75.4614 for H and C, respecti-
vely. Typical conditions for the proton spectra were: pul-
se width 32°, acquisition time 5 s, FT size 32 K and digi-
tal 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
0,000 per spectrum. The C and H chemical shifts we-
re measured in CDCl relative to TMS as the internal
standard. The 2D H- H (COSY) and Heteronuclear Mul-
tiple-Bond Connectivity (HMBC, HSQC) spectra were
recorded on a Bruker Avance DRX spectrometer opera-
ting at 599.93 and 150.85 MHz for H and C, respecti-
vely. Infrared spectra were recorded as KBr pellets (1.5
mg/300 mg KBr) using a FT-IR Bruker IFS 66 spectro-
meter (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, Cana-
da) syringe pump. The sample solutions were prepared in
1
13
2. 3. Synthesis
The methyl esters of bile acids (1 eq.) were dissol-
ved 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. Subse-
quently, 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
1
3
1
1
3
1
1
1
13
filtrate was washed with NaHCO (5%, 20 mL), brine (20
3
mL) and finally dried over Na CO . The solvent was eva-
2
3
porated under reduced pressure to give the crude product.
Products were purified by chromatography on silica gel
(Merck, type 60, 70–230 mesh).
Methyl 3α-bromoacetoxy-5β-cholan-24-oate (4) isola-
-
5
methanol at the concentration of approximately 10 M.
The standard ESI-MS mass spectra were recorded at the
cone voltage 90 V.
ted yield 59%, mp 127–128 °C. Anal. Calcd for
1
C H BrO : C 63.40, H 8.47. Found: C 63.58, H 8.30; H
2
7
43
4
NMR (CDCl ): δ 4.83–4.75 (m, 1H, 3β-H), 3.80 (s, 2H,
3
3
0
α-CH Br), 3.67 (s, 3H, OCH ), 0.93 (s, 3H, CH -19),
2 3 3
.91 (d, J = 6.4 Hz, 3H, CH -21), 0.65 (s, 3H, CH -18);
3
3
2
. 2. Computational Details
1
3
C NMR (CDCl ): δ 174.7 (C-24), 166.8 (3α-CO ), 76.6
3
2
The PM5 semiempirical calculations were perfor-
med using the WinMopac 2003 program.
(C-3), 56.4, 55.9, 51.5 (C-25), 42.7, 41.9, 40.4, 40.1,
3
7–39
The calcula-
35.8, 35.3, 34.9, 34.6, 31.9, 31.0, 30.9, 28.2 (3α-CH Br),
2
tions were performed using the GAUSSIAN 03 program
26.9, 26.4, 26.4, 26.3, 24.2, 23.3 (C-19), 20.8, 18.3
4
0
41–43
package at the B3LYP
levels of theory with the 6-
1G(d,p) basis set. The NMR isotropic shielding con-
stants were calculated using the standard GIAO (Gauge-
(C-21), 12.0 (C-18). ESI-MS (MeOH): m/z (%) 533 (100)
4
4
+
+
3
[M+Na] , 549 (25) [M+K] ; FT-IR: ν 1730 (C=O), 1292
–1
(C–O) cm .
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Acta Chim. Slov. 2015, 62, 15–27
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7
Methyl 3α-bromoacetoxy-12α-hydroxy-5β-cholan-24-
41.0, 39.6, 34.8, 34.6, 34.6, 34.3, 30.9, 30.7, 27.5 (3α-
CH Br), 27.3, 26.5 (12α-CH Br), 26.3, 25.9, 25.1, 22.9
oate (5), isolated yield 50%, oil. Anal. Calcd for
2
2
1
C H BrO : C 61.47, H 8.22. Found: C 61.63, H 8.40; H
(C-19), 22.3, 17.4 (C-21), 12.2 (C-18); ESI-MS (MeOH):
2
7
43
5
+
NMR (CDCl ): δ 4.81–4.76 (m, 1H, 3β-H), 3.99 (bs,1H,
m/z (%) 687 (100) [M+Na] ; FT-IR: ν 3541 (OH), 1731
3
–
1
1
2β-H), 3.80 (s, 2H, 3α-CH Br), 3.67 (s, 3H, OCH ), 0.97
(C=O), 1286 (C–O) cm .
2
3
(d, J = 6.4 Hz, 3H, CH -21), 0.93 (s, 3H, CH -19), 0.68 (s,
3
3
1
3
3
H, CH -18); C NMR (CDCl ): δ 174.6 (C-24), 166.8
Methyl 3α,7α-dibromoacetoxy-12α-hydroxy-5β-cho-
lan-24-oate (9), isolated yield 24%, oil. Anal. Calcd for
C H Br O : C 52.42, H 6.67. Found: C 52.59, H 6.58;
3
3
(
3α-CO ), 76.5 (C-3), 73.1 (C-12), 51.5 (C-25), 48.25,
2
4
3
2
7.37, 46.47, 41.82, 35.94, 35.02, 34.73, 34.09, 33.65,
2
7
43
2
7
1
1.82, 31.03, 30.86, 28.7 (3α-CH Br), 27.40, 26.88,
H NMR (CDCl ): δ 4.99–4.96 (m, 1H, 7β-H), 4.70–4.63
2
3
6.40, 26.24, 25.96, 23.56, 23.1 (C-19), 17.3 (C-21), 12.7
(m, 1H, 3β-H), 4.01 (bs, 1H, 12β-H), 3.83 (s, 2H, 7α-
CH Br), 3.80 (s, 2H, 3α-CH Br), 3.66 (s, 3H, OCH ),
+
(
5
(
C-18); ESI-MS (MeOH): m/z (%) 551 (100) [M+Na] ,
2
2
3
+
67 (30) [M+K] ; FT-IR: ν 3543 (OH), 1734 (C=O), 1287
0.98 (d, J = 6.2 Hz, 3H, CH -21), 0.94 (s, 3H, CH -19),
3
3
–
1
13
C–O) cm .
0.69 (s, 3H, CH -18); C NMR (CDCl ): δ 174.6 (C-24),
3 3
1
66.8 (3α-CO ), 166.6 (7α-CO ), 76.1 (C-3), 73.3 (C-7),
2 2
Methyl 3α,12α-dibromoacetoxy-5β-cholan-24-oate (6)
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 (3α-CH Br), 26.3 (7α-CH Br), 26.3, 22.9 (C-19),
isolated yield 70%, oil. Anal. Calcd for C H Br O : C
2
7
43
2
6
1
5
3.71, H 6.84. Found: C 53.49, H 6.52; H NMR (CDCl ):
3
2
2
δ 5.16 (t, J = 2.8 Hz, 1H, 12β-H), 4.81–4.73 (m, 1H, 3β-
H), 3.85 (s, 2H, 12α-CH Br), 3.79 (s, 2H, 3α-CH Br),
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)
2
2
–
–
3
.66 (s, 3H, OCH ), 0.92 (s, 3H, CH -19), 0.83 (d, J = 6.3
[M+Cl] ; 743 (75) [M+Br] ; FT-IR: ν 3530 (OH), 1733
(C=O), 1287 (C–O) cm .
3
3
13
–1
Hz, 3H, CH -21), 0.75 (s, 3H, CH -18); C NMR (CDCl ):
3
3
3
δ 174.6 (C-24), 166.7 (3α-CO ), 166.6 (12α-CO ), 77.9
2
2
(
C-12), 76.3 (C-3), 51.5 (C-25), 49.2, 47.3, 45.1, 41.7,
5.6, 34.8, 34.6, 34.3, 34.1, 31.8, 30.9, 30.8, 27.4 (3α-
CH Br), 26.8 (12α-CH Br), 26.4, 26.2, 25.9, 25.9, 25.3,
Methyl 3α,7α,12α-tribromoacetoxy-5β-cholan-24-oa-
te (10), isolated yield 54%, oil. Anal. Calcd for
C H Br O : C 47.41, H 5.78. Found: C 47.68, H 5.58;
3
2
2
27 43
3
8
1
2
3.4, 22.9 (C-19), 17.4 (C-21), 12.3 (C-18); ESI-MS (Me-
H NMR (CDCl ): δ 5.17 (t, J = 2.9 Hz, 1H, 12β-H),
3
+
+
OH): m/z (%) 671 (100) [M+Na] , 687 (43) [M+K] ; FT-
IR: ν 1733 (C=O), 1286 (C–O) cm .
5.02–5.00 (m, 1H, 7β-H), 4.68–4.60 (m, 1H, 3β-H), 3.89
(s, 2H, 12α-CH Br), 3.85 (s, 2H, 7α-CH Br), 3.79 (s, 2H,
–
1
2
2
3
α-CH Br), 3.66 (s, 3H, OCH ), 0.94 (s, 3H, CH -19),
2 3 3
Methyl 3α-bromoacetoxy-7α,12α-dihydroxy-5β-cho-
lan-24-oate (7), isolated yield 57%, oil. Anal. Calcd for
0.84 (d, J = 6.4 Hz, 3H, CH -21), 0.76 (s, 3H, CH -18);
3
3
1
3
C NMR (CDCl ): δ 174.4 (C-24), 166.7 (3α-CO ),
3
2
1
C H BrO : C 59.66, H 7.97. Found: C 59.34, H 7.82; H
166.5 (12α-CO ), 166.3 (7α-CO ), 77.2 (C-12), 75.8 (C-
2
7
43
6
2 2
NMR (CDCl ): δ 4.69–4.61 (m, 1H, 3β-H), 4.00 (bs, 1H,
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 (3α-CH Br), 26.2 (7α-CH Br), 26.1 (12α-
3
1
2β-H), 3.98-3.86 (m, 1H, 7β-H), 3.79 (s, 2H, 3α-
CH Br), 3.67 (s, 3H, OCH ), 0.99 (d, J = 6.3 Hz, 3H, CH -
2
2
3
3
2
2
1
3
1), 0.91 (s, 3H, CH -19), 0.80 (s, 3H, CH -18); C NMR
CH Br), 24.9, 22.9 (C-19), 22.2, 17.5 (C-21), 11.9 (C-
3
3
2
+
(
CDCl ): δ 174.7 (C-24), 166.8 (3α-CO ), 76.5 (C-3),
18); ESI-MS (MeOH): m/z (%) 808 (100) [M+Na] ; FT-
IR: ν 1732 (C=O), 1286 (C–O) cm .
3
2
–
1
7
4
2
2.8 (C-12), 68.2 (C-7), 51.5 (C-25), 47.2, 46.5, 42.0,
1.1, 39.5, 35.1, 34.8, 34.7, 34.6, 34.2, 31.9, 31.0, 30.8,
9.4, 28.4 (3α-CH Br), 27.40, 26.7, 26.5, 26.4, 23.1, 22.7
2
(
C-19), 17.3 (C-21), 14.12, 12.6 (C-18); ESI-MS (Me-
3. Results and Discussion
+
+
OH): m/z (%) 567 (100) [M+Na] , 583 (55) [M+K] ; 579
–
–
3
. 1. Synthesis
(
1
100) [M+Cl] , 623 (35) [M+Br] ; FT-IR: ν 3433 (OH),
–
1
734 (C=O), 1286 (C–O) cm .
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 com-
pounds 4–10. The syntheses of compounds 4–10 are de-
picted in Scheme 1.
Methyl 3α,12α-dibromoacetoxy-7α-hydroxy-5β-cho-
lan-24-oate (8) isolated yield 23%, oil. Anal. Calcd for
C H Br O : C 52.42, H 6.67. Found: C 52.74, H 6.43;
3
3
2
7
43
2
7
1
H NMR (CDCl ): δ 5.17 (t, J = 2.0 Hz, 1H, 12β-H),
3
4
.67–4.59 (m, 1H, 3β-H), 3.91-3.88 (m, 1H, 7β-H), 3.86
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 chlo-
(
s, 2H, 12α-CH Br), 3.79 (s, 2H, 3α-CH Br), 3.66 (s, 3H,
2
2
OCH ), 0.91 (s, 3H, CH -19), 0.85 (d, J = 6.4 Hz, 3H,
3
3
1
3
27
CH -21), 0.77 (s, 3H, CH -18); C NMR (CDCl ): δ
roform, are given by Chattopadhyay. Products were ob-
3
3
3
1
1
74.6 (C-24), 166.7 (3α-CO ), 166.8 (12α-CO ), 77.5 (C-
tained with very good yields, however no information
about purification method is available. Among the com-
2
2
2), 76.3 (C-3), 67.9 (C-7), 51.5 (C-25), 47.1, 45.1, 43.2,
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Acta Chim. Slov. 2015, 62, 15–27
1
8
Scheme 1. Synthesis of bromoacetyl substituted derivatives of bile acids 4–10.
Figure 1. Molecular models of compounds 4–10 calculated by PM5 method.
Pospieszny et al.: Spectroscopic Methods and Theoretical Studies ...

Acta Chim. Slov. 2015, 62, 15–27
pounds prepared only methyl 3α-bromoacetoxy-5β-cho-
lan-24-oate (4) was a solid, the rest of the conjugates we-
re oils. We are the first to offer a full spectroscopic charac-
terization of bromoacetyl substituted derivatives of lithoc-
holic, deoxycholic and cholic acid.
1
9
3α,12α-dibromoacetoxy-7α-hydroxy-5β-cholan-24-oa-
te (8) and methyl 3α,7α-dibromoacetoxy-12α-hydroxy-
5β-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 arran-
gement and interaction of the conjugate 8 is shown in Fi-
gure 2. The final heat of formation is –1941.6084
kcal/mol and the distances between the carbonyl group
of 3α-CO and 12α-CO are 8.03 Å and 7.45 Å, respec-
3
. 2. PM5 and B3LYP Calculations
The PM5 semiempirical calculations were perfor-
med 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 pre-
sented in Table 1. The molecular models of compounds
2
2
tively. In turn, the distance between the C(24)O groups
2
is 7.68 Å. Compensation charges occur only through in-
termolecular electrostatic interaction. This is a very
good confirmation of the conclusion that interactions re-
duce HOF.
4
–10 are shown in Figure 1.
The lowest HOF value is observed for methyl
3
α,7α,12α-tribromoacetoxy-5β-cholan-24-oate (10).
This fact can be explained by the greater stability by es-
ter groups in isolated molecule. The HOF of methyl
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 com-
pound with the highest probability (focal activities, Table
Table 1. Heat of formation (HOF) [kcal/mol] of compounds 4–10.
HOF of
five molecules
2). According to these data the most frequently predicted
Compound
HOF
ΔHOF
types of biological activity, e.g. inhibitors of acylcarnitine
hydrolase (PA > 95%), alkenylglycerophosphocholine
hydrolase (PA > 90%), alkylacetylglycerophosphatase
1
2
3
4
5
6
7
8
9
–226.5896
–268.6761
–309.3719
–266.3263
–308.5947
–345.9485
–348.0126
–386.0620
–388.6102
–425.4859
–
–
–
–
–
–
(PA > 90%), D-lactaldehyde dehydrogenase, glucan endo-
–39.7367
–39.9186
–77.2724
–38.6407
–76.6901
–79.2383
–116.1140
–1348.8740
–1558.2560
–1744.5095
–1756.1661
–1941.6084
–1957.0453
–2108.7200
1
,3-β-D-glucosidase, glyceryl-ether monooxygenase as
well as choleretic, cholesterol antagonist, CYP3A substra-
te and CYP3A4 substrate.
1
13
3
. 4. H and C NMR Spectra
1
0
The structures of all synthesized compounds were
determined from their H and C, as well as two-dimen-
1
13
ΔHOF = HOF(
compounds 4–10)
– HOF(methyl ester of bile acids 1–3)
Figure 2. Molecular models of compound 8 calculated by PM5 method.
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Acta Chim. Slov. 2015, 62, 15–27
Table 2. Probability »to be Active« (PA) values for predicted biological activity of compounds 4–10.
Focal predicted activity (PA > 80%)
2
0
Compounds
7
4
5
6
8
9
10
Acylcarnitine hydrolase inhibitor
Adenomatous polyposis treatment
Alkenylglycerophosphocholine hydrolase inhibitor
Alkenylglycerophosphoethanolamine hydrolase inhibitor
Alkylacetylglycerophosphatase inhibitor
Choleretic
Cholesterol antagonist
CYP3A substrate
CYP3A4 substrate
Dextranase inhibitor
D-lactaldehyde dehydrogenase inhibitor
Glucan endo-1,3-β-D-glucosidase inhibitor
Glyceryl-ether monooxygenase inhibitor
Peptidoglycan glycosyltransferase inhibitor
Plasmanylethanolamine desaturase inhibitor
Protein-disulfide reductase (glutathione) inhibitor
Vitamin-K-epoxide reductase inhibitor
96
81
94
81
94
–
89
–
81
88
–
87
–
83
–
–
–
98
–
97
–
95
82
95
–
81
–
–
89
–
89
84
85
–
99
82
96
85
95
90
88
83
85
91
86
91
89
87
84
81
87
98
82
94
81
94
87
88
–
82
88
81
88
88
84
81
–
98
–
97
–
92
–
92
–
–
97
89
97
86
85
82
84
–
96
85
95
86
82
83
85
91
81
91
88
87
81
81
83
–
–
83
–
83
86
–
–
–
–
–
95
86
91
–
88
81
81
–
83
sional NMR spectra (COSY, HSQC, HMBC). The assign-
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.
ments of proton and carbon-13 chemical shifts for 4–9 are
1
1
1
13
listed in Table 3 and are based on 2D H- H, H- C NMR
1
13
1
13
experiments. Additionally the H- C HSQC spectrum
The H and C NMR spectra were measured in
1
shows correlations over three bonds between the C3β-H,
chloroform (Table 3). The H NMR spectra of compounds
5
2
C7β-H, C12β-H protons and C=O carbon atoms in 10.
4–10 show characteristic multiplets in the range
1
13
Table 3. The most characteristic chemical shifts (ppm) in H and C NMR spectra of compounds 4–9.
No of atom
1H NMR
4
5
6
7
8
9
1
1
2
2
8
9
1
5
0.65
0.93
0.91
3.67
0.68
0.93
0.97
3.67
4.81–4.76
–
0.75
0.92
0.83
3.66
4.81–4.73
–
0.80
0.91
0.99
0.77
0.91
0.85
0.69
0.94
0.98
3.67
3.66
3.66
3
7
1
β-H
β-H
2β-H
4.83–4.75
4.69–4.61
3.98–3.86
4.00
4.67–4.59
3.91–3.88
5.17
4.70–4.63
4.99–4.96
4.01
–
–
3.80
–
3.99
3.80
–
5.16
3.79
–
3
7
α-CH Br
3.79
–
–
3.79
–
3.86
3.80
3.83
–
2
α-CH Br
2
1
1
2α-CH Br
–
–
3.85
2
3
C NMR
1
1
2
2
2
8
9
1
4
5
12.0
23.3
18.3
174.7
51.5
76.6
–
12.7
23.1
17.3
174.6
51.5
76.5
–
73.1
166.8
–
12.3
22.9
17.4
174.6
51.5
76.3
–
77.9
166.7
–
12.6
22.7
17.3
174.7
51.5
76.5
68.2
72.8
166.8
–
12.2
22.9
17.4
174.6
51.5
76.3
67.9
77.5
166.7
–
12.5
22.9
17.3
174.6
51.5
76.1
73.3
72.6
166.8
166.6
–
3
7
1
β-H
β-H
2β-H
–
3
7
1
3
α-CO2
α-CO2
166.8
–
–
28.2
–
–
2α-CO2
–
28.7
–
166.6
27.4
–
–
28.4
–
166.8
27.5
–
α-CH Br
27.2
26.3
–
2
7
α-CH Br
2
1
2α-CH Br
–
26.8
–
v26.5
2
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1
1
4
.83–4.59 ppm assigned to the C3β-H protons (in axial
The H NMR spectra of compounds 4–10 show cha-
positions) of the steroid skeleton.
racteristic singlets in the range 3.80–3.79 ppm for the pro-
In the spectra of compounds 5, 7 and 9 where unsub-
stituted hydroxy group in position C(12) is present, cha-
racteristic broad singlets in the range 4.01–3.99 ppm are
observed which are due to the C12β-H protons (in equato-
rial positions). However, in the case of the bromoacetates
tons of the 3α-OCOCH Br group, whereas for com-
2
pounds 9 and 10 characteristic doublets at 3.83 and 3.85
ppm for the protons of the 7α-OCOCH Br group are ob-
2
served. However, protons of 12α-OCOCH Br group for
2
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, respecti-
vely. The characteristic protons shifts for compounds
4–10 are collected in Table 3.
(
compounds 6, 8 and 10) protons C12β-H appear as tri-
plets in the range of 5.17–5.16 ppm.
1
H NMR spectra of derivatives of cholic acid 7, 8
1
3
and 9, 10 show characteristic multiplets in the ranges of
The C 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 CH -18, CH -19
3
.98–3.86 and 5.02–4.96 ppm 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
3
3
and CH -21, respectively. The carbon atoms of the
3
CO CH group are observed in the range 174.4–174.7
2
3
ppm and at 51.5 ppm and are assigned to CO and CH ,
2
3
values correspond to the OOCCH Br group in 9 and 10
respectively. On the other hand, carbon atoms of bromoa-
cetoxy groups in positions 3α, 7α or 12α 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
2
(
Figure 4).
Two hydrogen singlets in the range of 0.80–0.65 and
.94–0.91, as well as characteristic doublets at 0.99–0.83
ppm are assigned to CH -18, CH -19, and CH -21, res-
0
3
3
3
pectively.
The characteristic singlets of CO CH protons in the
2
3
range 3.67–3.66 ppm for all discussed compounds were
observed in H NMR spectra.
C(12)OCOCH Br, respectively. However, carbons of the
C(7) gave signals in the range 68.2–67.9 and 73.1–73.3
2
1
Figure 3. HSQC NMR spectrum for methyl 3α,7α,12α-tribromoacetoxy-5β-cholan-24-oate (10).
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1
Figure 4. H 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 (δ) vs. the magnetic isotropic shielding constants (σ) from the GIAO/B3LYP/6-31G(d,p) ap-
proach calculated for 10 in CDCl ; δ = a + b σcalc; (a) carbon-13, (b) proton.
3
exp
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3
ppm assigned to C(7)OH and C(7)OCOCH Br, respecti-
vely. 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 diagno-
As can be seen from Figure 5 the agreement bet-
ween the experimental and the calculated data for protons
2
5
5
is worse than for carbons-13. 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 bet-
ween 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.
stic signal for BrCH groups is observed at 28.7–26.1
2
ppm.
1
3
1
The relation between the experimental C and H
chemical shifts (δ ) and the Gauge Including Atomic Or-
exp
bitals (GIAO) magnetic isotropic shielding constants
4
5,46,53–55
(σcalc), which are widely used,
are usually linear
and described by the equation δexp = a + b σcalc. The slope
and intercept of the least-square correlation lines are used
to scale the GIAO isotropic absolute shielding constants
3
. 5. FT-IR Spectra
The FT-IR spectra of the representative conjugates
σ, and to predict chemical shifts in CDCl for 10 (Figure
7, 9 and 10 are shown in Figure 6. The most characteristic
in the FT-IR spectra are the bands at 3543 cm (5), 3433
3
–
1
5, Table 4).
Table 4. Chemical shifts (δ, ppm) in CDCl calculating GIAO nuclear magnetic shielding tensors (σcalc) for 10. The predicted GIAO chemical shifts
3
were computed from the linear equation δexp. = a + b·σcalc with a and b determined from the fit of the experimental data.
carbon-13
δexp.
δcalc
σcalc
proton
δexp.
δcalc
σcalc
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
38.00
27.15
75.83
34.30
31.14
34.32
73.05
38.00
34.71
34.50
25.91
77.22
45.11
42.70
24.87
30.67
47.18
11.92
22.86
34.71
17.46
30.88
30.88
174.40
51.48
166.70
166.25
166.51
26.36
26.19
26.06
30.24
20.66
77.78
26.49
39.19
24.94
75.00
36.81
33.44
40.60
22.36
79.62
52.36
45.82
28.98
33.83
49.29
10.39
16.39
38.86
11.26
27.56
29.92
170.54
54.54
165.25
164.34
164.79
33.41
31.06
31.00
197.52
203.68
166.92
199.93
191.76
200.93
168.71
193.29
195.46
190.85
202.59
165.74
183.28
187.49
198.33
195.21
185.26
210.29
206.43
191.97
209.73
199.24
197.72
107.23
181.88
110.63
111.22
110.93
195.48
196.99
197.03
H(1)
H(1)
H(2)
H(2)
H(3)
H(4)
H(4)
H(5)
H(6)
H(6)
H(7)
H(8)
H(9)
H(11)
H(11)
H(12)
H(14)
H(15)
H(15)
H(16)
H(16)
H(17)
H(18)
H(19)
H(20)
H(21)
H(22)
H(22)
H(23)
H(23)
1.72
1.11
1.50
1.47
4.64
1.69
2.11
1.33
1.72
2.02
5.02
1.73
2.11
1.55
1.81
5.17
2.05
1.52
1.57
1.79
1.81
1.71
0.76
0.94
1.40
0.84
1.72
1.33
2.20
2.23
3.66
3.79
3.85
3.89
1.49
1.14
1.46
0.86
4.09
1.43
1.88
1.35
1.50
1.83
4.49
1.50
1.76
1.35
1.63
4.63
3.08
1.41
1.63
2.41
3.08
2.72
0.85
1.04
1.24
0.86
1.95
1.88
2.63
2.79
3.90
3.15
3.36
3.37
45.3617
–1.3954
0.8322
31.44
31.69
31.46
31.89
29.58
31.48
31.16
31.54
31.43
31.20
29.29
31.43
31.25
31.54
31.34
29.19
30.30
31.50
31.34
30.78
30.30
30.56
31.90
31.76
31.62
31.89
31.11
31.16
30.62
30.51
29.71
30.25
30.10
30.09
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
C(22)
C(23)
C(24)
CH O
3
3α-CO2
7α-CO2
12α-CO2
3α-CH2
7α-CH2
12α-CH2
CH O
3
3
7
1
α-CH2
α-CH2
2α-CH2
a
b
a
b
r
337.1619
–1.5539
0.9895
2c
a
b
c
intercept; slope; correlation coefficient
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–
1
Figure 6. FT-IR spectra (film) of compounds 7 (black), 9 (red) and 10 (blue) in the 3600–400 cm region.
–
1
–1
–1
cm (7), 3541 cm (8) and 3530 cm (9) assigned to the
stretching vibrations ν(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
ions have a relative abundances of 30–35%, only for com-
pound 7 it amounts to 55%. In this case, there was no eli-
mination of the BrCH CO H molecule. For compounds 4,
2
2
–
5 and 7–9 ions [M+Br] (100%) in the negative ion mode
–
1
–1
1
734–1730 cm and 1287–1284 cm , assigned to
were observed. In the spectra of the compounds 7 and 9 in
–
ν(C=O) and ν(C–O), respectively. On the other hand, the
presence of chloroacetoxy groups in disubstituted deriva-
tives 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 ν(C=O) to higher wavenumbers,
the negative ion mode the presence of ion [M+Cl] was al-
so observed. For 3α-bromoacetoxy-7α,12α-dihydroxy-
5β-cholan-24-oate (7) the relative abundance of this ion
was 100% (Figure 7).
–
1
1
737–1731 cm , which is due to the inductive influence
of chlorine.
4. Conclusions
3
3
Furthermore, the comparison of carbonyl groups
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 hydri-
de.
ν(C=O) bands for methyl and ethyl derivatives show that
–
1
carbonyl band of ethyl esters are shifted by about 20 cm
to higher wavenumbers.
5
6
The structures of all synthesized compounds 4–10
3
. 5. ESI-MS Spectra
1
13
were determined from their H and C NMR, 2D NMR
(COSY, HSQC, HMBC), FT-IR as well as ESI-MS spec-
tra. Moreover, PM5 calculations were performed on all
compounds. Additionally, analyses of the biological pre-
diction activity spectra for bromoacetyl substituted deri-
vatives of bile acids prepared herein are examples of in si-
lico studies of chemical compounds.
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 affi-
nity for sodium cation than potassium ion. In all spectra
the molecular ion peak is 100% relative abundance, furt-
hermore the elimination of BrCH CO H is observed from
1
Linear correlations between the experimental H
1
3
and C chemical shifts and the computed screening con-
stants 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.
2
2
+
which the fragmented ions [M – BrCH CO H+Na] come.
2
2
Because these ions are seen in all the discussed com-
pounds, it can be concluded that the neutral molecule of
BrCH CO H is eliminated from the C(3) position of the
2
2
steroid skeleton. Furthermore, we observed for com-
pounds 4–7 in the ESI mass spectra ions [M+K] . These
+
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Figure 7. ESI-MS spectra in the negative and positive ion mode of compounds 7 (a) and 9 (b).
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6
5
. Acknowledgements
http://dx.doi.org/10.1080/00304949609356522
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This work was supported by the funds from Adam
Mickiewicz University, Faculty of Chemistry.
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Povzetek
1
13
Z uporabo H MMR, C NMR, 2D NMR, FT-IR in masne spektrometrije (ESI-MS) smo dolo~ili strukturo sedmih bro-
moacetil substituiranih derivatov `ol~nih kislin. Ob tem smo uporabili tudi PM5 semiempiri~ne izra~une in B3LYP ab
initio metode. Oceno morebitnega farmakoterapevtskega potenciala sintetiziranih spojin smo izvedli s pomo~jo progra-
ma »napovedovanje aktivnostnega spektra spojin« (Prediction of Activity Spectra for Substances oz. PASS).
Pospieszny et al.: Spectroscopic Methods and Theoretical Studies ...

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