Lithocholic acid and derivatives: Antibacterial activity

Article abstract of DOI:10.1016/j.steroids.2015.07.007

In order to develop bioactive lithocholic acid derivatives, we prepared fifteen semi-synthetic compounds through modification at C-3 and/or C-24. The reactions showed yields ranging from 37% to 100%. The structures of all compounds obtained were identified on the basis of their spectral data (IR, MS, 1D- and 2D-NMR). The activity of lithocholic acid and derivatives was evaluated against the growth of Escherichia coli, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa. The derivative 3α-formyloxy-5β-cholan-24-oic acid (LA-06) showed the best activity, with MIC values of 0.0790 mM against E. coli (Ec 27) and B. cereus in both cases, and 0.0395 mM against S. aureus (ATCC 12692). Lithocholic acid and the derivatives with MIC ≤ 1.2 mM were evaluated on the susceptibility of some bacterial pathogens to the aminoglycoside antibiotics neomycin, amikacin and gentamicin was evaluated. There are no previously reported studies about these compounds as modifiers of the action of antibiotics or any other drugs.

Full text of DOI:10.1016/j.steroids.2015.07.007

Accepted Manuscript
Lithocholic Acid and Derivatives: Antibacterial Activity
Patrícia G.G. do Nascimento, Telma L.G. Lemos, Macia C.S. Almeida, Juliana
M.O. de Souza, Ayla M.C. Bizerra, Gilvandete M.P. Santiago, José G.M. da
Costa, Henrique D.M. Coutinho
PII:
S0039-128X(15)00212-3
http://dx.doi.org/10.1016/j.steroids.2015.07.007
STE 7816
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 April 2015
18 July 2015
23 July 2015
Please cite this article as: do Nascimento, P.G.G., Lemos, T.L.G., Almeida, M.C.S., de Souza, J.M.O., Bizerra,
A.M.C., Santiago, G.M.P., da Costa, J.G.M., Coutinho, H.D.M., Lithocholic Acid and Derivatives: Antibacterial
Activity, Steroids (2015), doi: http://dx.doi.org/10.1016/j.steroids.2015.07.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ꢀ
Lithocholic Acid and Derivatives: Antibacterial Activity
Patrícia G.G. do Nascimentoa, Telma L.G. Lemosa,*, Macia C.S. Almeidaa, Juliana M.O. de
Souzaa, Ayla M.C. Bizerraa, Gilvandete M.P. Santiagob, José G.M. da Costac, Henrique D.M.
Coutinhoc
aDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Campus
do Pici, 60021-940, Fortaleza-CE, Brazil
bDepartamento de Farmácia, Universidade Federal do Ceará, Rua Capitão Francisco Pedro
No 1210, Campus do Porangabussu, 60430-370, Fortaleza-CE, Brazil
cDepartamento de Química Biológica, Universidade Regional do Cariri, 63105-000, Crato-
CE, Brazil
*Corresponding author at: Departamento de Química Orgânica e Inorgânica, Universidade
Federal do Ceará, Campus do Pici, 60021-940, Fortaleza-CE, Brazil. Tel.: +55 85 33669366;
fax: +55 85 33669782
E-mail address: tlemos@dqoi.ufc.br
Sꢀ
ꢀ

ꢀ
ABSTRACT
In order to develop bioactive lithocholic acid derivatives, we prepared fifteen semi-
synthetic compounds through modification at C-3 and/or C-24. The reactions showed yields
ranging from 37 to 100%. The structures of all compounds obtained were identified on the
basis of their spectral data (IR, MS, 1D- and 2D-NMR). The activity of lithocholic acid and
derivatives was evaluated against the growth of Escherichia coli, Staphylococcus aureus,
Bacillus cereus and Pseudomonas aeruginosa. The derivative 3ꢀ-formyloxy-5!-cholan-24-oic
acid (LA-06) showed the best activity, with MIC values of 0.0790 mM against E. coli (Ec 27)
and B. cereus in both cases, and 0.0395 mM against S. aureus (ATCC 12692). Lithocholic
acid and the derivatives with MIC 0 1.2 mM were evaluated on the susceptibility of some
bacterial pathogens to the aminoglycoside antibiotics neomycin, amikacin and gentamicin was
evaluated. There are no previously reported studies about these compounds as modifiers of the
action of antibiotics or any other drugs.
Keywords: bile acids, lithocholic acid derivatives, antibacterial activity
ꢀ
ꢀ
Tꢀ
ꢀ

ꢀ
1. Introduction
The bile acids are formed from cholesterol in the liver of mammals [1]. They have a
physiological function to help in the digestion of lipids and lipophilic vitamins reabsorption
[2], and also are used in the coupling with drugs used in conventional cancer treatment via
covalent bonds and, for this reason, various synthetic derivatives of bile acids have been
developed [1].
Lithocholic acid (LA), one of major bile acids excreted by mammals, is formed in the
+#2ꢁ -*'1+ by the bacterial 7-α-dehydroxylation of the primary bile acid, chenodeoxycholic
acid, in the colon [1]. The biological properties of this compound and derivatives have been
extensively studied, among them antimicrobial [3], membrane probe [4], tumor promotion or
inhibition [5], vitamin D receptor modulation [6], antiproliferative and pro-apoptotic effect on
human cancer cell lines [1] and proteasome inhibitors [7]. ꢂ,4#12'%ꢁ2#"ꢀ ꢁ,"ꢀ 3,.3 *'1&#"ꢀ
0#13*21ꢀ 1&-5#"ꢀ 2&ꢁ2ꢀ ꢁ+-,%ꢀ 2&#ꢀ '*#ꢀ ꢁ!'"1Qꢀ *'2&-!&-*'!ꢀ ꢁ!'"ꢀ 1&-5#"ꢀ 1'%,'$'!ꢁ,2ꢀ
ꢁ,2' ꢁ!2#0'ꢁ*ꢀꢁ!2'4'27T
Microbial resistance against antibiotics is a serious health problem. This happens by
the indiscriminate use of such chemotherapeutic agents, making it difficult to control species
of bacteria of medical sanitary interest [8]. Pathogenic bacteria such as Staphylococcus
aureus, Staphylococcus epidermis, Salmonella sp., Salmonella enteritidis and Salmonella
enteritidis, already have strains resistant to conventional antibiotics, making their presence in
food establishments and commercial potential health threat [8,9].
The aim of this study was to 17,2&#1'8# derivatives of this acid (LA), and to
investigate their antibacterial activity, and also to evaluate the influence of the derivatives with
Uꢀ
ꢀ

ꢀ
MIC 0 1.2 mM on the aminoglycosides antibiotics neomycin, amikacin, kanamycin and
gentamicin susceptibility of several Gram-positive and Gram-negative bacteria.
2. Experimental
2.1. General methods
Lithocholic acid (LA) was purchased from Sigma-Aldrich (St. Louis, MO). Melting
points were determined on a digital Mettler Toledo FP82HT apparatus and are uncorrected.
The IR spectra were measured in KBr pellets using a Perkin-Elmer FT-IR Spectrum 1000. A
Bruker® Avance DPX 300 spectrometer, operating at 300 MHz for 1H-NMR, and 75 MHz for
13C-NMR was used for experiments 1D and 2D with chemical shifts given in ppm. The
spectra were run using CDCl3 as the solvent. Chemical shifts, measured on the ꢁ scale. The
HRESIMS spectra were acquired using an LCMSIT-TOF spectrometer (Shimadzu, Japan).
The positive ion mass spectra were recorded in the range m/z 300-700 Da by using a potential
of 4.5 V on the capillary and He as collision gas. For the MS/MS scanning mode, the
percentage of collision energy was 50%. Optical rotations were measured on a Perkin Elmer
341 digital polarimeter (USA). Silica gel 60 (70–230 mesh) was used for column
chromatography, and thin layer chromatography (TLC) was performed on precoated silica gel
G60 F254 by detection by spraying with vanillin in perchloric acid/ethanol. All solvents used
for chromatography were from Synth. The microbiological culture media were purchased
from Fundação Oswaldo Cruz-FIOCRUZ (Rio de Janeiro, Brazil).
Vꢀ
ꢀ

ꢀ
2.2. Chemical modifications
2.2.1. General procedure for the preparation of LA(a), LA(b) and LA(c)
Lithocholic acid (LA, 1.0 g, 2.65 mmol) was refluxed with 150 mL of methanol,
ethanol or isopropanol in presence of sulphuric acid (1 mL) for 24 h. After this period the
solvent was evaporated, followed by addition of water (100 mL) and extraction with
dichloromethane (3 x 30 mL). The combined extracts were washed with H2O (3 x 60 mL),
Na2CO3 solution 20% (3 x 60 mL) and brine, dried over anhydrous Na2SO4, and evaporated
under reduced pressure. The residues were purified 7ꢀ!-*3+,ꢀ!&0-+ꢁ2-%0ꢁ.&7ꢀ-,ꢀ1'*'!ꢁꢀ%#*ꢀ
(30 g) with n-hexane: ethyl acetate (80:20) (v/v).
2.2.1.1. Methyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(a)). Amorphous white powder; 0,95 g;
20
yield 92%; m.p. 110-112 °C (literature [10] m.p. 116-117 °C); [ꢀ]D +25.5 (c 0.01; CHCl3)
(literature [11] [ꢀ]D +34.45 (c 0.72; CHCl3)); IR (KBr, cm-1): 3518, 2932, 2861, 1712, 1444,
1
1384, 1239; H NMR (CDCl3; 300 MHz): δ 3.58 - 3.63 (m, 1H, H-3!), 2.18 - 2.26 (m, 1H,
CH-23), 2.30 - 2.40 (m, 1H, CH-23). 3.65 (s, 3H, OCH3), 0.63 (s, 3H, CH3-18); 0.91 (s, 3H,
13
CH3-19); 0.89 (d, J = 5.4, 3H, CH3-21); C NMR (CDCl3, 75 MHz): δ 36.70 (CH2-1), 30.78
(CH2-2), 72.08 (CH-3), 36.70 (CH2-4), 42.34 (CH-5), 27.41 (CH2-6), 26.63 (CH2-7), 36.08
(CH-8), 40.68 (CH-9), 34.79 (C-10), 21.04 (CH2-11), 40.40 (CH2-12), 42.96 (C-13), 56.72
(CH-14), 24.41 (CH2-15), 28.38 (CH2-16), 56.20 (CH-17), 12.24 (CH3-18), 23.57 (CH3-19),
35.58 (CH-20), 18.47 (CH3-21), 31.29 (CH2-22), 31.23 (CH2-23), 174.95 (C-24), 51.63
(OCH3). The NMR data are in agreement with the literature values [12]; positive HRESIMS
m/z 373.3112 [M - H2O]+ (calcd for C25H42O3, 390.3134).
Wꢀ
ꢀ

ꢀ
2.2.1.2. Ethyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(b)). Amorphous white powder; 0.87 g; yield
81%; m.p. 81-83 °C; [ꢀ]D +25.7 (c 0.01; CHCl3); IR (KBr, cm-1): 3302 (OH), 2925, 2863,
20
1732, 1446, 1366, 123;. 1H NMR (CDCl3; 300 MHz): δ 3.57 - 3.65 (m, 1H, H-3!), 2.14 - 2.24
(m, 1H, CH-23), 2.28 - 2.38 (m, 1H, CH-23), 4.07 (q, J = 7.1 Hz, 2H, OCH2), 1.22 (t, J = 7.1
Hz, 3H, CH3), 0.63 (s, 3H, CH3-18), 0.91 (s, 3H, CH3-19, 0.89 (d, J = 4.6 Hz, 3H, CH3-21);
13C NMR (CDCl3,75 MHz): δ 35.58 (CH2-1), 30.75 (CH2-2), 72.03 (CH-3), 36.66 (CH2-4),
42.33 (CH-5), 27.41 (CH2-6), 26.63 (CH2-7), 36.07 (CH-8), 40.67 (CH-9), 34.79 (C-10),
21.04 (CH2-11), 40.39 (CH2-12), 42.95 (C-13), 56.72 (CH-14), 24.41 (CH2-15), 28.37 (CH2-
16), 56.20 (CH-17), 12.24 (CH3-18), 23.58 (CH3-19), 35.55 (CH-20), 18.48 (CH3-21), 31.54
(CH2-22), 31.21 (CH2-23), 174.54 (C-24), 60.36 (OCH2), 14,45 (CH3); positive HRESIMS
m/z 387.3266 [M - H2O]+ (calcd for C26H44O3, 404.3290).
2.2.1.3. Isopropyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(c)). Amorphous white powder; 0.90 g;
yield 81%; m.p. 78-80 °C; [ꢀ]D +23.8 (c 0.01; CHCl3); IR (KBr, cm-1): 3300, 2927, 2864,
20
1729, 1447, 1373, 1252; 1H NMR (CDCl3, 300 MHz): δ 3.56 - 3.67 (m, 1H, H-3!), 2.11 - 2.22
(m, 1H, CH-23), 2.25 - 2.33 (m, 1H, CH-23), 4.94 - 5.03 (m, 1H, OCH), 1.21 (d, J = 6.2 Hz,
6H, 2CH3), 0.63 (s, 3H, CH3-18), 0.91 (s, 3H, CH3-19), 0.89 (d, J = 5.0 Hz, 3H, CH3-21); 13C
NMR (CDCl3, 75 MHz): δ 35.55 (CH2-1), 30.71 (CH2-2), 72.01 (CH-3), 36.63 (CH2-4), 42.30
(CH-5), 27.39 (CH2-6), 26.61 (CH2-7), 36.04 (CH-8), 40.63 (CH-9), 34.76 (C-10), 21.01
(CH2-11), 40.37 (CH2-12), 42.92 (C-13), 56.69 (CH-14), 24.38 (CH2-15), 28.36 (CH2-16),
56.19 (CH-17), 12.21 (CH3-18), 23.56 (CH3-19), 35.50 (CH-20), 18.45 (CH3-21), 31.85 (CH2-
22), 31.21 (CH2-23), 174.04 (C-24), 67.49 (OCH), 22.04 (2CH3); positive HRESIMS m/z
401.3435 [M - H2O]+ (calcd for C27H46O3, 418.3447).
Xꢀ
ꢀ

ꢀ
2.2.2. General procedure for the preparation of LA-04, LA(a)-04, LA(b)-04 and LA(c)-04
To a solution of lithocholic acid (LA), methyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(a)),
ethyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(b)) or isopropyl 3ꢀ-hydroxy-5!-cholan-24-oate
(LA(c)) (1 mmol) in pyridine (2 mL) was added Ac2O (4 mL, 2.10 mmol) and catalytic
amount of 4-("'+#2&7*ꢁ+',-ꢃ.70'"',#ꢀꢄDMAP) (60 mg). After stirring at room temperature
for 24 h, the reaction mixture was quenched with saturated CuSO4 (20 mL) and extracted with
EtOAc (3 x 30 mL). The combined extracts were washed with H2O (3 x 20 mL) and brine (1 x
20 mL), dried over anhydrous Na2SO4, and evaporated under reduced pressure. The residues
were purified by column chromatography on silica gel (10 g) with n-hexane: ethyl acetate
(80:20) (v/v).
2.2.2.1. 3ꢀ-Acetoxy-5!-cholan-24-oic acid (LA-04). Amorphous white powder; 0.40 g; yield
96%; m.p. 156-158 °C (literature [1] m.p. 167 °C); [ꢀ]D20 +41.6 (c 0.01; CHCl3); IR (KBr, cm-
1): 2923, 2868, 1731, 1707, 1449, 1375, 1244; 1H NMR (CDCl3, 300 MHz): δ 4.67 - 4.77 (m,
1H, H-3!), 2.20 - 2.31 (m, 1H, CH-23), 2.35 - 2.45 (m, 1H, CH-23), 2.03 (s, 3H, CH3CO),
13
0.65 (s, 3H, CH3-18), 0.93 (s, 3H, CH3-19), 0.91 (d, J = 4.3 Hz, 3H, CH3-21); C NMR
(CDCl3, 75 MHz): δ 30.99 (CH2-1), 26.54 (CH2-2), 74.66 (CH-3), 35.26 (CH2-4), 42.12 (CH-
5), 27.24 (CH2-6), 26.85 (CH2-7), 36.02 (CH-8), 40.65 (CH-9), 34.80 (C-10), 21.05 (CH2-11),
40.37 (CH2-12), 42.97 (C-13), 56.71 (CH-14), 24.39 (CH2-15), 28.37 (CH2-16), 56.22 (CH-
17), 12.26 (CH3-18), 23.54 (CH3-19), 35.52 (CH-20), 18.46 (CH3-21), 31.22 (CH2-22), 32.47
(CH2-23), 180.32 (C-24), 170.94 (C=O), 21.66 (CH3CO). The NMR data are in agreement
with the literature values [1]; negative HRESIMS m/z 417.3083 [M - H]- (calcd for C26H42O4,
418.3083).
Yꢀ
ꢀ

ꢀ
2.2.2.2. Methyl 3ꢀ-acetoxy-5!-cholan-24-oate (LA(a)-04). Crystal white powder; 0.42 g; yield
97%; m.p. 122-123 °C; [ꢀ]D20 +41.3 (c 0.01; CHCl3); IR (KBr, cm-1): 2929, 2865, 1730, 1435,
1
1376, 1244; H NMR (CDCl3, 300 MHz): δ 4.65 - 4.76 (m, 1H, H-3!), 2.15 - 2.25 (m, 1H,
CH-23), 2.29 - 2.39 (m, 1H, CH-23), 2.01 (s, 3H, CH3CO), 3.65 (s, 3H, OCH3), 0.63 (s, 3H,
CH3-18), 0.91 (s, 3H, CH3-19), 0.89 (d, J = 6.0 Hz, 3H, CH3-21); 13C NMR (CDCl3, 75 MHz):
δ 31.21 (CH2-1), 26.52 (CH2-2), 74.59 (CH-3), 35.24 (CH2-4), 42.10 (CH-5), 27.22 (CH2-6),
26.83 (CH2-7), 36.00 (CH-8), 40.62 (CH-9), 34.78 (C-10), 21.03 (CH2-11), 40.35 (CH2-12),
42.94 (C-13), 56.70 (CH-14), 24.38 (CH2-15), 28.37 (CH2-16), 56.21 (CH-17), 12.23 (CH3-
18), 23.52 (CH3-19), 35.56 (CH-20), 18.46 (CH3-21), 31.25 (CH2-22), 32.45 (CH2-23), 174.92
(C-24), 170.82 (C=O), 21.64 (CH3CO), 51.64 (OCH3); positive HRESIMS m/z 455.3144 [M +
Na]+ (calcd for C27H44O4, 432.3240).
2.2.2.3. Ethyl 3ꢀ-acetoxy-5!-cholan-24-oate (LA(b)-04). Crystal white powder; 0.37 g; yield
91%; m.p. 93-95 °C; [ꢀ]D +37.06 (c 0.00623; CHCl3); IR (KBr, cm-1): 2930, 2865, 1735,
20
1449, 1376, 1241; 1H NMR (CDCl3, 300 MHz): δ 4.66 - 4.77 (m, 1H, H-3!), 2.14 - 2.25 (m,
1H, CH-23), 2.28 - 2.38 (m, 1H, CH-23), 4.08 (q, J = 6.0 Hz, 2H, OCH2), 1.22 (t, J = 6.0 Hz,
3H, CH3), 2.02 (s, 3H, CH3CO), 0.64 (s, 3H, CH3-18), 0.92 (s, 3H, CH3-19), 0.90 (d, J = 6.0
Hz, 3H, CH3-21); 13C NMR (CDCl3, 75 MHz): δ 31.20 (CH2-1), 26.51 (CH2-2), 74.59 (CH-3),
35.23 (CH2-4), 42.10 (CH-5), 27.22 (CH2-6), 26.82 (CH2-7), 36.00 (CH-8), 40.61 (CH-9),
34.77 (C-10), 21.03 (CH2-11), 40.34 (CH2-12), 42.93 (C-13), 56.69 (CH-14), 24.37 (CH2-15),
28.36 (CH2-16), 56.22 (CH-17), 12.22 (CH3-18), 23.51 (CH3-19), 35.53 (CH-20), 18.46 (CH3-
21), 32.45 (CH2-22), 32.45 (CH2-23), 174.49 (C-24), 170.81 (C=O), 60.34 (OCH2), 14.44
Zꢀ
ꢀ

ꢀ
(CH3), 21.63 (CH3CO); positive HRESIMS m/z 469.3312 [M + Na]+ (calcd for C28H46O4,
446.3396).
2.2.2.4. Isopropyl 3ꢀ-acetoxy-5!-cholan-24-oate (LA(c)-04). Crystal white powder; 0.46 g;
yield 100%; m.p. 96-97 °C; [ꢀ]D +35 (c 0.01, CHCl3); IR (KBr; cm-1): 2928, 2869, 1733,
20
1453, 1378, 1240; 1H NMR (CDCl3, 300 MHz): δ 4.66 - 4.77 (m, 1H, H-3!), 2.12 - 2.22 (m,
1H, CH-23), 2.25 - 2.36 (m, 1H, CH-23), 4.95 - 5.04 (m, 1H, OCH), 1.21 (d, J = 6.2 Hz, 6H,
2CH3), 2.02 (s, 3H, CH3CO), 0.64 (s, 3H, CH3-18), 0.92 (s, 3H, CH3-19), 0.90 (d, J = 7.2 Hz,
13
3H, CH3-21); C NMR (CDCl3,75 MHz): δ 31.23 (CH2-1), 26.53 (CH2-2), 74.62 (CH-3),
34.78 (CH2-4), 42.10 (CH-5), 27.22 (CH2-6), 26.83 (CH2-7), 36.00 (CH-8), 40.62 (CH-9),
34.78 (C-10), 21.03 (CH2-11), 40.36 (CH2-12), 42.94 (C-13), 56.70 (CH-14), 24.38 (CH2-15),
28.37 (CH2-16), 56.25 (CH-17), 12.22 (CH3-18), 23.52 (CH3-19), 35.52 (CH-20), 18.47 (CH3-
21), 31.88 (CH2-22), 32.46 (CH2-23), 174.06 (C-24), 170.87 (C=O), 67.52 (OCH), 21.65
(CH3CO), 22.04 (2CH3); positive HRESIMS m/z 483.3450 [M + Na]+ (calcd for C29H48O4,
460.3553).
2.2.3. General procedure for the preparation of LA-05, LA(a)-05, LA(b)-05 and LA(c)-05
To a solution of lithocholic acid (LA), methyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(a)),
ethyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(b)) or isopropyl 3ꢀ-hydroxy-5!-cholan-24-oate
(LA(c)) (1 mmol) in acetone/CH2Cl2 (6/4 mL) was added .70'"','3+ꢀ !&*-0-!&0-+ꢁ2#ꢀ
ꢄPCC) (600 mg, 2.78 mmol). After stirring at room temperature for 24 h, the mixture was
concentrated and partitioned with H2O (10 mL) and CH2Cl2 (3 x 10 mL). The organic layer
[ꢀ
ꢀ

ꢀ
was concentrated and purified by silica gel column chromatography (13 g) eluted with hexane:
ethyl acetate (50:50) (v/v).
2.2.3.1. 3-Oxo-5!-cholan-24-oic acid (LA-05). Amorphous white powder; 0.14 g; yield 37%;
m.p. 121-123 °C (literature [10] m.p. 121-122 °C); [ꢀ]D20 +28.1 (c 0.01; CHCl3); IR (KBr, cm-
1): 2926 , 2879, 1698, 1447, 1376; 1H NMR (CDCl3, 300 MHz,): δ 2.21 - 2.30 (m, 1H, H-23),
2.32 - 2.44 (m, 1H, CH-23), 0.67 (s, 3H, CH-18), 1.00 (s, 3H, CH3-19), 0.91 (d, J = 6.3 Hz,
13
3H, CH3-21); C NMR (CDCl3, 75 MHz): δ 37.38 (CH2-1), 37.20 (CH2-2), 213.88 (C-3),
42.53 (CH2-4), 44.51 (CH-5), 25.96 (CH2-6), 26.81 (CH2-7), 35.49 (CH-8), 40.94 (CH-9),
35.07 (C-10), 21.39 (CH2-11), 40.25 (CH2-12), 42.99 (C-13), 56.62 (CH-14), 24.35 (CH2-15),
28.33 (CH2-16), 56.16 (CH-17), 12.28 (CH3-18), 22.83 (CH3-19), 35.73 (CH-20), 18.45 (CH3-
21), 31.22 (CH2-22), 30.94 (CH2-23), 180.26 (C-24). The NMR data are in agreement with the
literature values [10]; positive HRESIMS m/z 469.3312 [M + Na]+ (calcd for C28H46O4,
446.3396).
2.2.3.2. Methyl 3-oxo-5!-cholan-24-oate (LA(a)-05). Crystal white powder; 0.33 g; yield
85%; m.p. 107-109 °C; [ꢀ]D +9.9 (c 0.0017; CHCl3); IR (KBr, cm-1): 2926, 2869, 1735,
20
1710, 1452, 1379, 1256; 1H NMR (CDCl3, 300 MHz): δ 2.16 - 2.28 (m, 1H, H-23), 2.30 - 2.38
(m, 1H, CH-23), 3.65 (s, 3H, OCH3), 0.67 (s, 3H, CH3-18), 1.01 (s, 3H, CH3-19), 0.90 (d, J =
13
6.3 Hz, 3H, CH3-21); C NMR (CDCl3, 75 MHz): δ 37.37 (CH2-1), 37.19 (CH2-2), 213.46
(C-3), 42.53 (CH2-4), 44.49 (CH-5), 25.94 (CH2-6), 26.80 (CH2-7), 35.51 (CH-8), 40.92 (CH-
9), 35.06 (C-10), 21.37 (CH2-11), 40.22 (CH2-12), 42.95 (C-13), 56.61 (CH-14), 24.32 (CH2-
15), 28.31 (CH2-16), 56.15 (CH-17), 12.23 (CH3-18), 22.82 (CH3-19), 35.71 (CH-20), 18.45
SRꢀ
ꢀ

ꢀ
(CH3-21), 31.21 (CH2-22), 31.15 (CH2-23), 174.85 (C-24), 51.63 (OCH3); positive HRESIMS
m/z 411.2886 [M + Na]+ (calcd for C25H40O3, 388.2977).
2.2.3.3. Ethyl 3-oxo-5!-cholan-24-oate (LA(b)-05). Amorphous white powder; 0.38 g; yield
84%; m.p. 88-90 °C (literature [3f] m.p. 85-88 °C); [ꢀ]D20 +27.5 (c 0.0017; CHCl3); (literature
1
[13] [ꢀ]D +28.9); IR (KBr, cm-1): 2937, 2865, 1734, 1712, 1446, 1380, 1243; H NMR
(CDCl3, 300 MHz): δ 4.08 (q, J = 7.1 Hz, 2H, OCH2), 1.22 (t, J = 7.1 Hz, 3H, CH3), 0.67 (s,
13
3H, CH3-18), 1.01 (s, 3H, CH3-19), 0.90 (d, J = 6.3 Hz, 3H, CH3-21); C NMR (CDCl3, 75
MHz): δ 37.38 (CH2-1), 37.19 (CH2-2), 213.51 (C-3), 42.53 (CH2-4), 44.50 (CH-5), 25.95
(CH2-6), 26.80 (CH2-7), 35.50 (CH-8), 40.92 (CH-9), 35.06 (C-10), 21.37 (CH2-11), 40.23
(CH2-12), 42.95 (C-13), 56.62 (CH-14), 24.33 (CH2-15), 28.32 (CH2-16), 56.17 (CH-17),
12.24 (CH3-18), 22.83 (CH3-19), 35.71 (CH-20), 18.46 (CH3-21), 31.49 (CH2-22), 31.15
(CH2-23), 174.43 (C-24), 60.34 (OCH2), 14.43 (CH3); positive HRESIMS m/z 425.3052 [M +
Na]+ (calcd for C26H42O3, 402.3134).
20
2.2.3.4. Isopropyl 3-oxo-5!-cholan-24-oate (LA(c)-05). Yellow oil; 0.38 g; yield 91%; [ꢀ]D
1
+26.3 (c 0.0017; CHCl3); IR (KBr, cm-1): 2932, 2865, 1715, 1446, 1374, 1256; H NMR
(CDCl3, 300 MHz): δ 4.95 - 5.01 (m, 1H, OCH), 1.21 (d, J = 6.3 Hz, 6H, 2CH3), 0.67 (s, 3H,
CH3-18), 1.01 (s, 3H, CH3-19), 0.90 (d, J = 6.3 Hz, 3H, CH3-21); 13C NMR (CDCl3, 75 MHz):
δ 37.39 (CH2-1), 37.20 (CH2-2), 213.53 (C-3), 42.55 (CH2-4), 44.51 (CH-5), 25.96 (CH2-6),
26.82 (CH2-7), 35.49 (CH-8), 40.94 (CH-9), 35.08 (C-10), 21.38 (CH2-11), 40.25 (CH2-12),
42.97 (C-13), 56.64 (CH-14), 24.34 (CH2-15), 28.33 (CH2-16), 56.21 (CH-17), 12.24 (CH3-
18), 22.84 (CH3-19), 35.73 (CH-20), 18.47 (CH3-21), 31.84 (CH2-22), 31.19 (CH2-23), 173.97
SSꢀ
ꢀ

ꢀ
(C-24), 67.48 (OCH), 22.05 (2CH3); positive HRESIMS m/z 439.3204 [M + Na]+ (calcd for
C27H44O3, 416.3290).
2.2.4. General procedure for the preparation of LA-06, LA(a)-06, LA(b)-06 and LA(c)-06
A solution of of lithocholic acid (LA), methyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(a)),
ethyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(b)) or isopropyl 3ꢀ-hydroxy-5!-cholan-24-oate
(LA(c)) (1 mmol) in of 195% HCO2H (2 mL) and 70% perchloric acid (20 drops) was heated
in an H2O bath at 60° for 4 h. The solution was removed from the bath and allowed to cool to
about 40°. Ac2O was then added dropwise while the temperature was maintained between 55
and 60° until a large quantity of bubbles appeared (1 mL of Ac2O was required). The solution
was then cooled to room temperature and poured into 10 mL of H2O, with stirring [14]. The
precipitate was filtered under vacuum, washed with H2O.
2.2.4.1. 3ꢀ-Formyloxy-5!-cholan-24-oic acid (LA-06). Amorphous white powder; 0.37 g;
20
yield 92%; m.p. 128-130 °C (literature [15] m.p. 127-128 °C); [ꢀ]D +38.5 (c 0.01; CHCl3);
(literature [14] [ꢀ]D +38.5 (c 10; CHCl3)); IR (KBr, cm-1): 2936, 2866, 1718, 1704, 1448,
23
1
1379, 1249; H NMR (CDCl3, 300 MHz): δ 8.03 (s, 1H, HC=O), 4.81 - 4.86 (m, 1H, H-3!),
2.20 - 2.30 (m, 1H, CH-23), 2.34 - 2.43 (m, 1H, CH-23), 0.65 (s, 3H, CH3-18), 0.93 (s, 3H,
13
CH3-19), 0.91 (d, J = 7.4 Hz, 3H, CH3-21); C NMR (CDCl3, 75 MHz): δ 30.96 (CH2-1),
26.51 (CH2-2), 74.64 (CH-3), 35.17 (CH2-4), 42.12 (CH-5), 27.19 (CH2-6), 26.84 (CH2-7),
36.00 (CH-8), 40.67 (CH-9), 34.78 (C-10), 21.05 (CH2-11), 40.33 (CH2-12), 42.96 (C-13),
56.67 (CH-14), 24.38 (CH2-15), 28.35 (CH2-16), 56.17 (CH-17), 12.26 (CH3-18), 23.52 (CH3-
19), 35.50 (CH-20), 18.45 (CH3-21), 31.24 (CH2-22), 32.43 (CH2-23), 180.60 (C-24), 161.05
STꢀ
ꢀ

ꢀ
(HC=O). The NMR data are in agreement with the literature values [15]; negative HRESIMS
m/z 403.2885 [M - H]- (calcd for C25H40O4, 404.2927).
2.2.4.2. Methyl 3ꢀ-formyloxy-5!-cholan-24-oate (LA(a)-06). Amorphous white powder; 0.38
g; yield 91%; m.p 106-108 °C; [ꢀ]D +43.1 (c 0.0051; CHCl3); IR (KBr, cm-1): 2926, 2866,
20
1719, 1444, 1378, 1251; 1H NMR (CDCl3, 300 MHz): δ 8.04 (s, 1H, HC=O), 4.85 (m, 1H, H-
3!), 2.18 - 2.27 (m, 1H, CH-23), 2.30 - 2.39 (m, 1H, CH-23), 3.66 (s, 3H, OCH3), 0.65 (s, 3H,
CH3-18), 0.92 (s, 3H, CH3-19), 0.90 (d, J = 4.9 Hz, 3H, CH3-21); 13C NMR (CDCl3, 75 MHz):
δ 31.21 (CH2-1), 26.51 (CH2-2), 74.61 (CH-3), 35.17 (CH2-4), 42.13 (CH-5), 27.18 (CH2-6),
26.84 (CH2-7), 36.00 (CH-8), 40.67 (CH-9), 34.78 (C-10), 21.04 (CH2-11), 40.33 (CH2-12),
42.94 (C-13), 56.67 (CH-14), 24.37 (CH2-15), 28.36 (CH2-16), 56.19 (CH-17), 12.24 (CH3-
18), 23.52 (CH3-19), 35.55 (CH-20), 18.47 (CH3-21), 31.22 (CH2-22), 32.43 (CH2-23), 174.93
(C-24), 160.97 (HC=O), 51.65 (OCH3); positive HRESIMS m/z 441.2999 [M + Na]+ (calcd
for C26H42O4, 418.3083).
2.2.4.3. Ethyl 3ꢀ-formyloxy-5!-cholan-24-oate (LA(b)-06). Crystal white powder; 0.25 g;
yield 57%; m.p. 65-66 °C; [ꢀ]D +37.6 (c 0.0029; CHCl3); IR (KBr, cm-1): 2925, 2864,
20
1737, 1720, 1449, 1376, 1245; 1H NMR (CDCl3, 300 MHz): δ 8.03 (s, 1H, HC=O), 4.85 (m,
1H, H-3!), 2.15 - 2.25 (m, 1H, CH-23), 2.29 - 2.34 (m, 1H, CH-23), 4.08 (q, J = 7.1 Hz, 2H,
OCH2), 1.23 (t, J = 7.6 Hz, 3H, CH3), 0.64 (s, 3H, CH3-18), 0.92 (s, 3H, CH3-19), 0.91 (d, J =
13
3.9 Hz, 3H, CH3-21); C NMR (CDCl3, 75 MHz): δ 31.22 (CH2-1), 26.53 (CH2-2), 74.62
(CH-3), 35.19 (CH2-4), 42.14 (CH-5), 27.20 (CH2-6), 26.86 (CH2-7), 36.02 (CH-8), 40.68
(CH-9), 34.80 (C-10), 21.06 (CH2-11), 40.35 (CH2-12), 42.96 (C-13), 56.70 (CH-14), 24.39
SUꢀ
ꢀ

ꢀ
(CH2-15), 28.38 (CH2-16), 56.23 (CH-17), 12.25 (CH3-18), 23.53 (CH3-19), 35.55 (CH-20),
18.49 (CH3-21), 31.55 (CH2-22), 32.45 (CH2-23), 174.50 (C-24), 160.96 (HC=O), 60.36
(OCH2), 14.47 (CH3); positive HRESIMS m/z 451.2591 [M + K]+ (calcd for C27H44O4,
432.3240).
2.2.4.4. Isopropyl 3ꢀ-formyloxy-5!-cholan-24-oate (LA(c)-06). Crystal white powder; 0.21 g;
yield 46%; m.p. 45-47 °C; [ꢀ]D20 +31.5 (c 0.002; CHCl3); 1H NMR (CDCl3, 300 MHz): δ 8.04
(s, 1H, HC=O), 4.80 - 4.89 (m, 1H, H-3!), 2.12 - 2.23 (m, 1H, CH-23), 2.26 - 2.36 (m, 1H,
CH-23), 4.96 - 5.04 (m, 1H, OCH), 1.22 (t, J = 6.3 Hz, 3H, 2CH3), 0.65 (s, 3H, CH3-18), 0.94
(s, 3H, CH3-19), 0.90 (d, J = 6.5 Hz, 3H, CH3-21); 13C NMR (CDCl3, 75 MHz): δ 31.25 (CH2-
1), 26.54 (CH2-2), 74.63 (CH-3), 35.20 (CH2-4), 42.16 (CH-5), 27.22 (CH2-6), 26.87 (CH2-7),
36.03 (CH-8), 40.69 (CH-9), 34.81 (C-10), 21.07 (CH2-11), 40.36 (CH2-12), 42.97 (C-13),
56.71 (CH-14), 24.40 (CH2-15), 28.39 (CH2-16), 56.26 (CH-17), 12.26 (CH3-18), 23.54 (CH3-
19), 35.55 (CH-20), 18.50 (CH3-21), 31.90 (CH2-22), 32.46 (CH2-23), 174.03 (C-24), 160.98
(HC=O), 67.52 (OCH), 22.07, 22.08 (2CH3).
2.3. Antibacterial Activity and Minimal Inhibitory Concentration
The antibacterial activity of lithocholic acid and its derivatives was investigated
employing a microdilution method, recommended by National Committee for Clinical and
Laboratory Standards M7-A6 [16]. In tests were used three standard strains of Gram (-) and
three Gram (+), and two clinical isolates of multidrug-resistant Escherichia coli (Ec 27) (from
sputum) and Staphylococcus aureus (Sa 358) of the surgical wound. The brain heart infusion
(BHI 3.8%) broth was used for the bacterial growth (24 h, 35 2°C). The inoculum was an
SVꢀ
ꢀ

ꢀ
overnight culture of each bacterial species in the BHI broth diluted in the same medium to a
final concentration of approximately 1 × 108 CFU/mL (0.5 NTU – McFarland scale). After
this, the suspension was diluted to 1 × 106 CFU/mL in 10% BHI. A total of 100 )L of each
dilution was distributed in 96-well plates plus substance, achieving 5 × 105 CFU/mL as the
final concentration of the inoculums [17-19].
The initial solutions of the lithocholic acid and its derivatives were prepared using
dimethyl sulfoxide (DMSO). From this concentration, several dilutions were made in distilled
water in order to obtain a stock solution of each compound corresponding with 1024 )g/mL
(varying between 2.22 and 2.63 mM for each compound). Further serial dilutions were
performed by the addition of the BHI broth. All experiments were performed in triplicate and
the most common result was used. The microdilution trays were incubated at 35 2°C for 24
h. The antibacterial activity was detected using a colorimetric method by adding 25 )L of the
resauzurin staining (0.01%) aqueous solution in each well at the end of the incubation period
[20]. The minimal inhibitory concentration (MIC) was defined as the lowest extract
concentration able to inhibit the bacteria growth, as indicated by resauzurin staining (dead
bacterial cells are not able to change the staining color by visual observation – blue to red).
2.4. Evaluation of the modulatory activity by direct contact
In the investigation of the lithocholic acid and derivatives as modulators of antibiotic
resistance, the MICs of aminoglycosides antibiotics neomycin, amikacin, and gentamicin
against the analyzed strains were determined in the presence or absence of the samples using
the microdutiltion test. Subinhibitory concentrations (MIC 1/8) in 10% BHI were used. The
antibiotic solutions corresponding with 1024 )g/mL (varying between 1.66 and 2.14 mM for
SWꢀ
ꢀ

ꢀ
each antibiotic) were prepared in distilled water for use on the same day. A total of 100 )L of
the antibiotic solution, using serial dilutions (1:2), was added to the wells containing 10% BHI
and the diluted bacterial suspension (1:10). Microplates were incubated at 35 2°C for 24 h
and the antibacterial activity was determined as described before [21]. All experiments were
performed in triplicate and the most common result was used.
3. Results and Discussion
3.1. Chemistry
The lithocholic acid derivatives were synthesized through modification at the
oxygenated carbon C-3 and/or at the carboxyl carbon C-24. Initially, lithocholic acid (LA)
was transformed into its methyl, ethyl and isopropyl esters under general esterification
conditions, with the appropriate alcohol in presence of sulphuric acid (H2SO4). The synthesis
of others derivatives was accomplished by treating lithocholic acid (LA), methyl 3ꢀ-hydroxy-
5!-cholan-24-oate (LA(a)), ethyl 3ꢀ-hydroxy-5!-cholan-24-oate (LA(b)) or isopropyl 3ꢀ-
hydroxy-5!-cholan-24-oate with acetic anhydride in the presence of pyridine and 4-
(dimethylamino)pyridine (DMAP); pyridinium chlorochromate (PCC) and HCO2H in the
presence of perchloric acid (Figure 1). The structures of all compounds were established by
1D 1H and 13C (and DEPT) and 2D HSQC and HMBC NMR spectral data.
3.2. Biological evaluations
Lithocholic acid (LA) and its derivatives were tested for antibacterial activity against
six bacterial strains (two strains of Staphylococcus aureus, two strains of Escherichia coli,
SXꢀ
ꢀ

ꢀ
Bacillus cereus and Pseudomonas aeruginosa), by employing the microdilution method. The
values of minimum inhibitory concentration (MIC) of lithocholic acid (LA) and its derivatives
are shown in Table 1. According to the results (Table 1), it was found that the derivative LA-
06 (3ꢀ-formyloxy-5!-cholan-24-oic acid) showed the best results, with MIC values 0 0.3162
mM against all tested bacterial strains. The results found against E. coli (Ec 27) and B. cereus
showed MIC value of 0.0790 mM in both cases, and against S. aureus (ATCC 12692)
representing the most significant result, with a MIC value of 0.0395 mM. Interestingly,
among all evaluated compounds in this study, the derivative LA-04 (3ꢀ-acetoxy-5!-cholan-
24-oic acid) was inactive against all bacterial strains evaluated, with MIC 1 2.44 mM.
The association results of the lithocholic acid (LA) LA(a), LA(b), LA(c), LA(a)-04,
LA(b)-04, LA-05 and LA-06 with aminoglycosides antibiotics amikacin, gentamicin and
neomycin on MIC values are shown in Table 2. In these assays were selected the compounds
that showed MIC 0 1.2 mM.
Modifier of antibiotic activity is a term used for substances that modulate or even
reverse bacterial resistance to certain antibiotics, where it can alter the microbial susceptibility
to antibiotics by inhibition of the resistance mechanisms [22]. When the substance utilized in
combination intervenes in a positive way, it is considered increasing the activity of the
antibiotic, is considered to have a synergistic effect. On the contrary, when there is a decrease
in the action or inactivation of the antibiotic, the added substance has an antagonistic effect
[23].
The association of lithocholic acid (LA) with aminoglycosides antibiotics increased the
activity of all antibiotics tested against E. coli (ATCC 25922), while in the effect against S.
aureus (ATCC 12692), LA showed a synergistic effect on the activity of amikacin. LA(a)
SYꢀ
ꢀ

ꢀ
(methyl 3ꢀ-hydroxy-5!-cholan-24-oate) showed the potentiation of the activity of antibiotics
when tested against E. coli (Ec 27). Already in the effect on B. cereus (ATCC 33018), LA(a)
showed an antagonistic action on the activity of amikacin and neomycin. The association of
LA(b) (ethyl 3ꢀ-hydroxy-5!-cholan-24-oate) with the aminoglycosides antibiotics increased
the effect of amikacin and neomycin against E. coli (Ec 27). LA(c) (isopropyl 3ꢀ-hydroxy-5!-
cholan-24-oate) associated to the ꢁ+',-%*7!-1'"#1ꢀ ꢁ,2' '-2'!1 enhanced the activity of
amikacin and neomycin against S. aureus (ATCC 12692), and showed an antagonistic action
on the activity of amikacin against E. coli (ATCC 25922).
Compound LA(a)-04 (methyl 3ꢀ-acetoxy-5!-cholan-24-oate), having a acetyl group at
C-3, showed potentiation of the activity of amikacin and neomycin against the strain S. aureus
(ATCC 12692). It was observed that LA(a)-04 showed an antagonistic action on the activity
of gentamicin against the strain E. coli (ATCC 25922). A little change at carbon size in the
ester derivative LA(b)-04 (ethyl 3ꢀ-acetoxy-5!-cholan-24-oate) potentiated the activity of
neomycin against S. aureus (ATCC 12692) and E. coli (ATCC 25922).
The results of the association of the derivative oxidized at C-3 LA-05 (3-oxo-5!-
cholan-24-oic acid) with the ꢁ+',-%*7!-1'"#1ꢀꢁ,2' '-2'!1 showed the increase of the activity
of antibiotics neomycin and amikacin against the strain S. aureus (ATCC 12692). On the
strain P. aeruginosa (ATCC 15442), LA-05 also demonstrated a synergistic action on the
activity of amikacin. Finally, it was found that the association of the compound having a
formyl group at C-3 LA-06 (3ꢀ-formyloxy-5!-cholan-24-oic acid) with the ꢁ+',-%*7!-1'"#1ꢀ
ꢁ,2' '-2'!1 increases the activity of antibiotics against P. aeruginosa (ATCC 15442) with
significant results when compared to the MIC of the antibiotic in the absence of the substance,
and may highlight the neomycin reduction in MIC of 0.0129 to 0.008 mM (94%) and
SZꢀ
ꢀ

ꢀ
reduction with amikacin a MIC of 0.0543 to 0.0135 mM. Already in interaction with S. aureus
strain (ATCC 12692), LA-06 showed synergistic action on the activity of aminoglycosides
antibiotics.
The gram-negative bacteria E. coli and P. aeruginosa have higher amounts of lipids in
their structure, which can explain the favorable results seen in modulation [24,25],
demonstrating the greater affinity between the derivatives of the lithocholic acid and the own
acid with the cell membrane.
The Figure 1 shows the formation of the lithocholic acid and its derivatives, and all
these compounds demonstrating a nonpolar behavior. Lipophilic substances such as the
lithocholic acid and its derivatives allow perturbations in the bacterial membrane, resulting in
damage to essential elements for the integrity of the membrane such as: reduction in
membrane potential and loss of ions, cytochrome C, proteins and radicals, followed by the
collapse of proton pumps and decrease in ATP [26,27].
Besides that lithocolic acid and its derivatives can interact with the lipid bilayer of the
cell membrane and affect the respiratory chain and energy production of bacteria [28], it can
also make the cell more permeable to antibiotics, leading to interruption of vital cell activity
and interference with the enzyme systems of bacteria, which is also a potential mechanism of
action [29,30]. In the Table 2, we can see that the main compound with modulatory activity is
the own lithocholic acid. Beside the other compounds be lipophilic, none of them demonstrate
the same activity of the original compound, which suggest that the modifications in C3 and in
carboxyl group in C17 have not increased this effect.
The bacterial resistance modifying effect of lithocholic acid and its derivatives is due
to these compounds being capable of acting through a lipophilic action in the cell envelope,
S[ꢀ
ꢀ

ꢀ
resulting in an imbalance in the fluid mosaic nature of the bacterial membrane [31] and makes
the modulation more effective. The outer and inner membranes can be subject to
permeabilization, facilitating the entrance of antibiotics, besides causing cell lysis and necrosis
[32,33]
Cholesterol is an important component of biological membranes of eukaryotes, and is
absent from the membrane of bacteria. They are responsible for maintaining the permeability
of membranes. The cell can control its fluidity through the regulation of cholesterol levels or
the degree of saturation of the hydrocarbon chains of phospholipids [34]. Due to the lipophilic
behavior, compounds analogous to the cholesterol, such as lithocolic acid and its derivatives
demonstrated an effect over the bacterial membrane.
There are no previously reported studies of the utilization of bile acid and 15
derivatives as modifiers of the action of antibiotics or any other drugs, where this is the first
study to be conducted with this aim.
4. Conclusions
The results show that these compounds exhibit significant antibacterial activity and
some of them potentiate the effect of antibiotics such as amikacin, gentamicin and neomycin.
Since the parent compound lithocholic acid (LA) is part of the mammalian metabolism, and
therefore without toxicity, these compounds are promising antibiotic agents.
Acknowledgments
The authors thank the Brazilian agencies CAPES and CNPq for fellowships and financial
support.
TRꢀ
ꢀ

ꢀ
References
[1] El Kihel L, Clément M, Bazin MA, Descamps G, Khalid M, Rault S. New lithocholic and
chenodeoxycholic piperazinylcarboxamides with antiproliferative and pro-apoptotic
effects on human cancer cell lines. Bioorgan Med Chem 2008; 16: 8737-44.
[2] Valkonen A, Lahtinen M, Tamminen J, Kolehmainen E. Solid state structural studies of
five bile acid derivatives. J Mol Struct 2008; 886: 197-206.
[3] (a) Bellini AM, Quaglio MP, Guarneri M, Cavazzini G. Antimicrobial activity of basic
cholane derivatives. Parts I and II. Eur J Med Chem 198; 18: 185-95;
(b) Bellini AM, Quaglio MP, Cavazzini G, Ceccherini R. Antimicrobial activity of basic
cholane derivatives. Part IV. Farmaco 1984; 39: 305-10;
(c) Bellini AM, Quaglio MP, Guarneri M, Cavazzini G. Antimicrobial activity of basic
cholane derivatives: amino and amido derivatives (VI). Boll Chim Farm 1986; l25: 362-
65;
(d) Bellini AM, Quaglio MP, Mencini E, Guarneri M, Cavazzini G, Fini A. Antimicrobial
activity of basic cholane derivatives. Part VIII. Arch Pharm 1989; 322: 879-83;
(e) Bellini AM, Quaglio MP, Mencini E, Guarneri M, Fini A. Antimicrobial activity of
basic cholane derivatives. Part IX. Arch Pharm 1990; 323: 201-05;
(f) Bellini AM, Mencini E, Quaglio MP, Guarneri M, Fini A. Antimicrobial activity of
basic cholane derivatives. X. Synthesis of 3α- and 3β-amino-5-β-cholan-24-oic acids.
Steroids 1991; 56: 395-98.
[4] Banerjee S, Trivedi GK, Srivastava S, Phadke RS. Proxyl nitroxide of lithocholic acid: a
potential spin probe for model membranes. Bioorg Med Chem1993; 1: 341-47.
TSꢀ
ꢀ

ꢀ
[5] (a) Schneider H, Fiander H, Harrison KA, Watson M, Burton GW, Arya P. Inhibitory
potency of lithocholic acid analogs and other bile acids on glucuronosyltransferase
activity in a colon cancer cell line. Bioorg Med Chem Lett 1996; 6: 637-42;
(b) Halvorsen B, Staff AC, Ligaarden S, Prydz K, Kolset SO. Lithocholic acid and
sulphated lithocholic acid differ in the ability to promote matrix metalloproteinase
secretion in the human colon cancer cell line CaCo-2. Biochem J 2000; 349: 189-93.
[6] (a) Adachi R, Honma Y, Masuno H, Kawana K, Shimomura I, Yamada S, Makishima M.
Selective activation of vitamin D receptor by lithocholic acid acetate, a bile acid
derivative. J Lipid Res 2005; 46: 46-57;
(b) Jurutka PW, Thompson PD, Whitfield GK, Eichhorst KR, Hall N, Dominguez CE,
Hsieh J-C, Haussler CA, Haussler MR. Molecular and functional comparison of 1,25-
dihydroxyvitamin D3 and the novel vitamin D receptor ligand, lithocholic acid, in
activating transcription of cytochrome P450 3A4. J Cell Biochem 2005; 94: 917-43;
(c) Ishizawa M, Matsunawa M, Adachi R, Uno S, Ikeda K, Masuno H, Shimizu M,
Iwasaki K.-I, Yamada S, Makishima M. Lithocholic acid derivatives act as selective
vitamin D receptor modulators without inducing hypercalcemia. J Lipid Res 2008; 49:
763-72.
[7] Dang Z, Lin A, Ho P, Soroka D, Lee KH, Huang L, Chen CH. Synthesis and proteasome
inhibition of lithocholic acid derivatives. Bioorg Med Chem Lett 2011; 21: 1926-28.
[8] Baccaro MR, Moreno AM, Corrêa A, Ferreira AJP, Calderaro FF. Resistência
antimicrobiana de amostras de Escherichia coli isoladas de fezes de leitões com diarréia.
Arq Inst Biol 2002; 69: 15-8.
TTꢀ
ꢀ

ꢀ
ꢅ[ꢆꢀ ꢄꢁꢃꢀ Ribeiro AR, Kellermann A, Santos LR, Fittél AP, Nascimento VP. Resistência
antimicrobiana em Salmonella enterica subsp enterica sorovar hadar isoladas de carcaças
de frango. Arq Inst Biol 2006; 73: 357-60.
(b) Mantilla SPS, Franco RM, Oliveira Lat, Santos EB, Gouvêa R. Resistência
antimicrobiana de bactérias do gênero Listeria spp. isoladas de carne moída bovina. Braz
J Vet Res Anim Sci 2008; 45: 116-21.
[10] Nahar L, Turner AB. Synthesis of ester-linked lithocholic acid dimmers. Steroids 2003;
68: 1157-61.
[11] Mizushina Y, Kasai N, Miura K, Hanashima S, Takemura M, Yoshida H, Sugawara F,
Sakaguchi K. Structural relationship of lithocholic acid derivatives binding to the N-
terminal 8-kDA domain of DNA polymerase β. Biochemistry 2004; 43: 10669-77.
[12] Aranda G, Fetizon M, Tayeb N. Synthese d’antibiotiques triterpeniques a partir d’acides
biliaires. Tetrahedron 1987; 43: 4147-57.
[13] Chang FC, Blickenstaff RT, Feldstein A, Gray JR, McCaleb GS, Sprunt DH.
Seroflocculating steroids III chloro and other bile acid derivatives. J Am Chem Soc
1957; 79: 2164-67.
[14] Lemos TLG, Mcchesney JD. Utilization of common natural products as synthons:
Preparation of progesterone from lithocholic acid. J Nat Prod 1990; 53: 152–56.
[15] Chattopadhyay P, Pandey PS. Synthesis and binding ability of bile acid-based receptors
for recognition of flavin analogues. Tetrahedron 2006; 62: 8620-24
[16] National Committee for Clinical Laboratory Standards. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved
Standard—Sixth Edition; CLSI document M7-A6; NCCLS: Wayne, PA, USA, 2003.
TUꢀ
ꢀ

ꢀ
[17] Hadacek F, Greger H. Testing of antifungal natural products: methodologies,
comparability of results and assay choice. Phytochem Analysis 2000; 11: 137–47.
[18] National Committee for Clinical Laboratory Standards. Performance Standards for
Antimicrobial Susceptibility Testing: Twelfth Informational Supplement; CLSI
document M100-S12; NCCLS: Wayne, PA, USA, 2002; Volume 22; Number 1.
[19] Viljoen A, Vuuren AV, Ernst E, Klepser M, Demirci B, Baser H, Vanwyk BE.
Osmitopsis asteriscoides (Asteraceae) the antimicrobial activity and essential oil
composition of a Cape-Dutch remedy. J Ethnopharmacol 2003; 88: 137–43.
[20] Salvat A, Antonnacci L, Fortunato RH, Suarez EY, Godoy HM. Screening of some plants
from Northern Argentina for their antimicrobial activity. Lett Appl Microbiol 2001; 32:
293-97.
[21] Sagdiç O. Sensitivity of four pathogens pathogenic bacteria to Turkish thyme and
oregano hydrosols. Lebensm Wiss Technol 2003; 36: 467–73.
[22] Costa VCO, Tavares JF, Agra MF, Falcão-silva VS, Facanali, R, Vieira MAR, Marques
MOM, Siqueira-Júnior JP, Silva MS. Composição química e modulação da resistência
bacteriana a drogas do óleo essencial das folhas de Rollinia leptopetala R. E. Fries. Rev
Bras Farmacogn 2008; 18: 245-8.
[23] Canton M, Onofre SB. Interferência de extratos da Baccharis dracunculifolia DC.,
Asteraceae, sobre a atividade de antibióticos usados na clínica. Rev Bras Farmacogn
2010; 20: 348-54.
[24] Vargas AC, Loguercio AP, Witt NM, Costa MM, Silva MS, Viana LR. Atividade
antimicrobiana “in vitro” de extrato alcoólico de própolis. Ci Rural 2004; 34: 159-63.
TVꢀ
ꢀ

ꢀ
[25] Sartori MRK. Atividade antimicrobiana de frações e extratos e compostos puros obtidos
das flores de Acmela brasilensis Spreng (Wedelia paludosa) (Asteraceae). 81f. MSc.
Thesis - Universidade Vale do Itajaí, Itajaí, 2005.
[26] Sikkema J, Bont JAM, Poolman B. Interaction of cyclic hydrocarbons with biological
membranes. J Biol Chem 1994; 269: 8022-8.
[27] Turina AV, Nolan MV, Zygadlo JA, Perillo MA. Natural terpenes: self-assembly and
membrane partitioning. Biophys Chem 2006; 122: 101-13.
[28] Nicolson K, Evans G, Otoole PW. Potentiation of methicillin. Activity against
methicillin-resistant Staphylococcus aureus by diterpenes. FEMS Microbiol Lett 1999;
179: 233–9.
[29] Köhler T, Pechére JC, Plésiat P. Bacterial antibiotic efflux systems of medical
importance. Cell. Assoc. Mol. Life Sci 1999; 56: 771-8.
[30] Burt S. Essential oils: their antibacterial properties and potential applications in foods – a
review. Int J Food Microbiol 2004; 94: 223-53.
[31] Nostro A, Blanco AR, Cannatelli MA, Enea V, Flamini G, Morelli I, Roccaro AS,
Alonzo V. Susceptibility of methicillin-resistant staphylococci to oregano essential oil,
carvacrol and thymol. FEMS Microbiol Lett 2004; 230: 191-5.
[32] Knowles JR, Roller S, Murray DB, Naidu AS. Antimicrobial action of carvacrol at
different stages of dual-species biofilm development by Staphylococcus aureus and
Salmonella enterica Serovar Typhimurium. Appl Environm Microbiol 2005; 71: 797–
803.
[33] Armstrong, JS. Mitochondria: a target for cancer therapy. Brit J Pharmacol 2006; 147:
239-48.
TWꢀ
ꢀ

ꢀ
[34] Frézard F, Schettini DA. Lipossomas: propriedades físico-químicas e farmacológicas,
aplicações na quimioterapia à base de antimônio. Quim Nova 2005; 28: 511-8.
TXꢀ
ꢀ

ꢀ
COOH
O
O
H
LA(a)-04: R= CH3
LA(b)-04: R= CH2CH3
LA(c)-04: R= CH(CH)3
a
COOR
COOR
COOR
c
b
O
H
O
HO
O
H
H
H
LA(a)-05: R= CH3
LA(b)-05: R= CH2CH3
LA(c)-05: R= CH(CH)3
LA(a): R= CH3
LA(b): R= CH2CH3
LA(c): R= CH(CH)3
LA(a)-06: R= CH3
LA(b)-06: R= CH2CH3
LA(c)-06: R= CH(CH)3
d
COOH
COOH
COOH
c
b
O
H
O
HO
O
H
H
H
LA-05
LA-06
LA
a
COOH
O
O
H
LA-04
ꢀ
Reagents and conditions: (a) Ac2O/pyridine, DMAP (4-(dimethylamino)pyridine), rt, 24 h; (b) PCC (pyridinium
chlorochromate), rt, 24 h; (c) HCO2H, perchloric acid, 60 °C, 4 h (d) Methanol, ethanol or isopropanol, H2SO4,
reflux, 24 h
ꢀ
TYꢀ
ꢀ

Table
Table 1. Values of the minimal inhibitory concentration (MIC) of lithocholic acid and
derivatives.
Bacterial strains
Compounds
MIC (mM)
P. aeruginosa
(ATCC
15442)
S. aureus
(ATCC 12692) (Sa 358) (ATCC 25922) (Ec 27) (ATCC 33018)
S. aureus
E. coli
E. coli
B. cereus
1.36
LA
1.36
1.31
1.36
≥ 2.62
≥ 2.53
≥ 2.44
≥≥ 2.44
≥ 2.37
≥ 2.29
≥ 2.22
1.145
1.36
1.31
1.36
0.655
0.6325
1.22
1.36
0.655
1.265
≥ 2.44
≥2.44
1.185
1.145
1.11
LA (a)
1.31
1.265
LA (b)
1.265
1.22
0.6325
0.61
≥ 2.44
LA (c)
LA -04
≥ 2.44
1.185
0.2862
1.11
≥2.44
1.185
0.5725
1.11
≥2.44
1.185
0.5725
1.11
≥2.44
1.185
LA (a)-04
LA (b)-04
LA (c)-04
LA -05
1.145
≥ 2.22
0.5725
1.315
0.635
1.23
1.145
≥ 2.63
1.27
0.5725
≥ 2.63
1.27
1.145
1.315
≥ 2.54
≥ 2.46
0.0790
1.22
0.5725
≥ 2.63
≥ 2.54
≥ 2.46
0.1581
≥ 2.44
≥ 2.37
≥ 2.29
LA (a)-05
LA (b)-05
LA (c)-05
LA -06
0.6575
≥ 2.54
≥ 2.46
0.6325
1.22
≥ 2.46
0.3162
256
1.23
0.0395
1.22
0.0790
≥ 2.44
1.185
1.145
LA (a)-06
LA (b)-06
LA (c)-06
1.185
≥ 2.29
≥ 2.37
≥ 2.29
1.185
1.145
≥ 2.37
≥ 2.29

ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢁꢂ *#ꢀTTꢀ *','+ꢁ*ꢀ ',&' '2-07ꢀ !-,!#,20ꢁ2'-,ꢀ ꢄ*ꢂ+ꢃꢀ 4ꢁ*3#1ꢀ $-0ꢀ 2&#ꢀ ꢁ+',-%*7!-1'"#ꢀ ',ꢀ 2&#ꢀ
ꢁ 1#,!#ꢀꢁ,"ꢀ.0#1#,!#ꢀ-$ꢀ2&#ꢀ*'2&-!&-*'!ꢀꢁ!'"ꢀꢁ,"ꢀ"#0'4ꢁ2'4#1Tꢀ
ꢄꢅ ꢀꢆ+ꢄꢇꢀ
ꢃꢂ!2#0'ꢂ*ꢀ120ꢂ',1ꢀ
-,2' '-2'!ꢀ
&"'ꢀ
-,2' '-2'!ꢀꢀ&ꢀ1ꢁ+.*#ꢀ
ꢈꢄꢅꢀ &"'ꢀ '"(ꢀ
ꢈꢄꢅꢀ
'"(ꢀ
ꢀTꢁꢂ30#31ꢀꢄ-2++ꢀSTX[Tꢃꢀ RTTSYWꢀ RTTXYWꢀ RTSRUYꢀ RTSRZYꢀ RTTXYWꢀ RTSRUYꢀ
Tꢁ!-*'ꢀꢄ-2++ꢀTW[TTꢃꢀ
RTSRZYꢀ RTTXYWꢀ
RTZUꢀ
RTRTYSꢀ RTSUUYꢀ RTTRYWꢀ
,-ꢄꢁꢃꢀ
ꢁ
Tꢁ!-*'ꢀꢄ3!ꢀTYꢃꢀ
ꢀ
RTWUWꢀ
RTSRZYꢀ
RTRWSZꢀ RTRWVUꢀ RTTXYWꢀ RTRST[ꢀ
ꢃTꢁ!#0#31ꢀꢄ-2++ꢀUURSZꢃꢀ RTRWVUꢀ RTTXYWꢀ RTRTW[ꢀ RTSRZYꢀ RTTXYWꢀ RTRWSZꢀ
ꢁ
ꢀ
,-ꢄ ꢃꢀ
Tꢁ!-*'ꢀꢄ-2++ꢀTW[TTꢃꢀ
RTRWVUꢀ RTTXYWꢀ
RTZUꢀ
RTSRZYꢀ RTTXYWꢀ RTVSWꢀ
Tꢁ!-*'ꢀꢄ3!ꢀTYꢃꢀ
RTVUWꢀ
RTTXYWꢀ RTTRYWꢀ RTTSYWꢀ RTTXYWꢀ RTSRUYꢀ
ꢁ
ꢀ
,-ꢄ!ꢃꢀ
ꢀTꢁꢂ30#31ꢀꢄ-2++ꢀSTX[Tꢃꢀ
RTVUWꢀ
RTTXYWꢀ
RTVSWꢀ
RTVSWꢀ
RTTSYWꢀ RTTXYWꢀ RTSRUYꢀ
RTSRZYꢀ RTSUUYꢀ RTVSWꢀ
,-ꢄꢁꢃVRVꢀ
Tꢁ!-*'ꢀꢄ-2++ꢀTW[TTꢃꢀ
RTRWVUꢀ RTTXYWꢀ
ꢁ
ꢀ
ꢀTꢁꢂ30#31ꢀꢄ-2++ꢀSTX[Tꢃꢀ
Tꢁ!-*'ꢀꢄ-2++ꢀTW[TTꢃꢀ
ꢁ
RTVUWꢀ
RTTXYWꢀ
RTVSWꢀ
RTVSWꢀ
RTTSYWꢀ RTTXYWꢀ RTTRYWꢀ
RTSRZYꢀ RTTXYWꢀ RTTRYWꢀ
,-ꢄ ꢃVRVꢀ
RTSRZYꢀ RTSUUYꢀ
ꢀ
ꢀTꢁꢂ30#31ꢀꢄ-2++ꢀSTX[Tꢃꢀ RTTSYWꢀ RTTXYWꢀ RTTRYWꢀ RTTSYWꢀ RTTXYWꢀ RTSRUYꢀ
Tꢁ!-*'ꢀꢄ-2++ꢀTW[TTꢃꢀ
RTRWVUꢀ RTTXYWꢀ
RTVSWꢀ
RTRWVUꢀ RTTXYWꢀ RTTRYWꢀ
,-VRWꢀ
ꢁ
ꢀ
ꢀTꢁꢂ30#31ꢀꢄ-2++ꢀSTX[Tꢃꢀ RTSRZYꢀ RTTXYWꢀ
ꢄTꢁꢂ#03%',-1ꢂꢀꢄ-2++ꢀ
SWVVTꢃꢀ
RTVSWꢀ
RTRWVUꢀ RTTXYWꢀ RTRST[ꢀ
RTTSYWꢀ RTTXYWꢀ RTRST[ꢀ RTRWVUꢀ RTTXYWꢀ RTRWSZꢀ
ꢁ
ꢀ
,-VRXꢀ
ꢀTꢁꢂ30#31ꢀꢄ-2++ꢀSTX[Tꢃꢀ RTSRZYꢀ RTTXYWꢀ RTRST[ꢀ RTSRZYꢀ RTTXYWꢀ RTRST[ꢀ
ꢄTꢁꢂ#03%',-1ꢂꢀꢄ-2++ꢀ
SWVVTꢃꢀ
AMI: Amikacin; GEN: Gentamicin; NEO: Neomycin
RTRWVUꢀ RTTXYWꢀ RTRST[ꢀ RTRSUWꢀ RTSUUYꢀ RTRRZꢀ
T[ꢀ
ꢀ