Bile acid toxicity structure-activity relationships: Correlations between cell viability and lipophilicity in a panel of new and known bile acids using an oesophageal cell line (HET-1A)

Article abstract of DOI:10.1016/j.bmc.2010.07.030

The molecular mechanisms and interactions underlying bile acid cytotoxicity are important to understand for intestinal and hepatic disease treatment and prevention and the design of bile acid-based therapeutics. Bile acid lipophilicity is believed to be an important cytotoxicity determinant but the relationship is not well characterized. In this study we prepared new azido and other lipophilic BAs and altogether assembled a panel of 37 BAs with good dispersion in lipophilicity as reflected in RPTLC R_Mw. The MTT cell viability assay was used to assess cytotoxicity over 24 h in the HET-1A cell line (oesophageal). R_Mw values inversely correlated with cell viability for the whole set (r² = 0.6) but this became more significant when non-acid compounds were excluded (r² = 0.82, n = 29). The association in more homologous subgroups was stronger still (r ² >0.96). None of the polar compounds were cytotoxic at 500 μM, however, not all lipophilic BAs were cytotoxic. Notably, apart from the UDCA primary amide, lipophilic neutral derivatives of UDCA were not cytotoxic. Finally, CDCA, DCA and LagoDCA were prominent outliers being more toxic than predicted by R_Mw. In a hepatic carcinoma line, lipophilicity did not correlate with toxicity except for the common naturally occurring bile acids and their conjugates. There were other significant differences in toxicity between the two cell lines that suggest a possible basis for selective cytotoxicity. The study shows: (i) azido substitution in BAs imparts lipophilicity and toxicity depending on orientation and ionizability; (ii) there is an inverse correlation between R_Mw and toxicity that has good predictive value in homologous sets; (iii) lipophilicity is a necessary but apparently not sufficient characteristic for BA cytocidal activity to which it appears to be indirectly related.

Full text of DOI:10.1016/j.bmc.2010.07.030

Bioorganic & Medicinal Chemistry 18 (2010) 6886–6895
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bile acid toxicity structure–activity relationships: Correlations between cell
viability and lipophilicity in a panel of new and known bile acids using an
oesophageal cell line (HET-1A)
a
a
a
a
b
Ruchika Sharma a,b, , Ferenc Majer , Vijaya Kumar Peta , Jun Wang , Ray Keaveney , Dermot Kelleher ,
*
Aideen Long b, John F. Gilmer a,
*
a School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland
b Cell Signalling, Institute of Molecular Medicine, Trinity Centre for Health Science, St. James’s Hospital, Dublin 8, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The molecular mechanisms and interactions underlying bile acid cytotoxicity are important to under-
stand for intestinal and hepatic disease treatment and prevention and the design of bile acid-based ther-
apeutics.
Bile acid lipophilicity is believed to be an important cytotoxicity determinant but the relationship is
not well characterized. In this study we prepared new azido and other lipophilic BAs and altogether
assembled a panel of 37 BAs with good dispersion in lipophilicity as reflected in RPTLC RMw. The
MTT cell viability assay was used to assess cytotoxicity over 24 h in the HET-1A cell line (oesopha-
geal). RMw values inversely correlated with cell viability for the whole set (r2 = 0.6) but this became
more significant when non-acid compounds were excluded (r2 = 0.82, n = 29). The association in more
homologous subgroups was stronger still (r2 >0.96). None of the polar compounds were cytotoxic at
Received 25 March 2010
Revised 12 July 2010
Accepted 13 July 2010
Available online 19 July 2010
Keywords:
Bile acids
Lipophilicity
Physicochemical characteristics
Cell viability
500 lM, however, not all lipophilic BAs were cytotoxic. Notably, apart from the UDCA primary amide,
Cytotoxicity
Azido steroid
lipophilic neutral derivatives of UDCA were not cytotoxic. Finally, CDCA, DCA and LagoDCA were
prominent outliers being more toxic than predicted by RMw. In a hepatic carcinoma line, lipophilicity
did not correlate with toxicity except for the common naturally occurring bile acids and their conju-
gates. There were other significant differences in toxicity between the two cell lines that suggest a
possible basis for selective cytotoxicity. The study shows: (i) azido substitution in BAs imparts lipo-
philicity and toxicity depending on orientation and ionizability; (ii) there is an inverse correlation
between RMw and toxicity that has good predictive value in homologous sets; (iii) lipophilicity is a
necessary but apparently not sufficient characteristic for BA cytocidal activity to which it appears to
be indirectly related.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
nogenesis.4–7 An understanding of the structural basis for the
apoptotic effects of BAs is important to ongoing efforts to harness
Bile acids (BAs) exert multiple biological effects, non-specifi-
cally on organic solutes and membranes and specifically as signal-
ling agents by binding to membrane and nuclear receptors.1–3
Their peculiar effects on cell proliferation (negative and positive)
attract attention because of the relevance of these phenomena to
the pathogenesis of cholestatic liver diseases and intestinal carci-
this property pharmacologically in the design of selective cytocidal
and cytoprotective agents.8–12
A specific binding target(s) for BAs in triggering apoptotic pro-
cesses remains elusive but there is substantial evidence for the
involvement of membrane interactions.13 The ability to induce
necrosis through detergent effects is one manifestation of this.14
At lower concentration, BAs can induce apoptosis through extrinsic
and intrinsic pathways, and through ER stress.7 Membrane effects
are important in this context too. BAs can modify membrane fluid-
ity and composition15–17 as well as protein mobilization and acti-
vation18 without directly affecting barrier function.19 Some of
these effects resemble those observed with non-BA hydrophobic
solutes such as cholesterol myristate and detergents n-octylglyco-
side and Triton-X, at sub-lytic concentrations.20
Abbreviations: GCA, glcyocholic acid; GCDCA, glycochenodeoxycholic acid;
GDCA, glycodeoxycholic acid; GUDCA, glycoursodeoxycholic acid; HSA, hydropho-
bic surface area; HCA, hyocholic acid; NCA, nutriacholic acid; UCA, ursocholic acid;
PSA, polar surface area; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid;
TDCA, taurodeoxycholic acid; TUDCA, tauroursodeoxycholic acid.
* Corresponding authors. Tel.: +353 18963350/8962844; fax: +353 18962810
(R.S.); tel.: +353 18962795 (J.F.G.).
E-mail addresses: rsharma@tcd.ie (R. Sharma), gilmerjf@tcd.ie (J.F. Gilmer).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.030

R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
6887
An influential early observation about the SAR of BA toxicity was
that deoxycholate (DCA) causes more membrane damage than cho-
lic acid (CA) and its conjugates.21 Numerous studies have since
shown that DCA, chenodeoxycholic acid (CDCA) and lithocholic acid
(LCA) have greater effects on cell viability7 and mitochondrial func-
tion than other BAs.22 They are also more toxic than their own glyco-
and tauro-conjugates.7,23 In partitioning experiments these BAs are
lipophilic relative to other less toxic physiological BAs suggesting
that lipophilicity and toxicity are associated or even correlated.
The activation of PKC in vitro in constituted micelles correlated with
chromatographic measures of hydrophobicity for DCA, ursodoexy-
cholic acid (UDCA), CA and CDCA but especially with their taurine
conjugates.24 In a HCT116 cell line, growth arrest and apoptosis
roughly paralleled a RPHPLC hydrophobicity ranking for a range of
16 BAs, though interestingly the behaviour of DCA and CDCA was
not contiguous with the rest of the series.25 LCA, DCA and CDCA oc-
cupy the toxic/hydrophobic end of the spectrum of common BAs but
their toxicity may not be attributable to their hydrophobicity alone.
In a recent study, natural bile acids LCA, CDCA and DCA were re-
ported to initiate more apoptosis than their enantiomers ent-LCA,
ent-CDCA and ent-DCA in HT-29 and HCT-116 cell lines. However,
hydrophobic interactions are predicted to be identical in enantio-
meric pairs unless the environment is itself chiral.26,27
In order to further explore the requirements for BA toxicity we
constructed a panel of 37 naturally occurring and synthetic bile
acids (Fig. 1). We sought to enrich the panel with new hydrophobic
BAs. We focused especially on compounds related to UDCA and
DCA being the prototypical cytoprotective and cytotoxic BAs,
respectively. We were drawn in this regard towards azido ana-
logues since the azide group is well known to enhance lipophilic-
ity. We evaluated the relative polarity and effect on cell viability
in the human oesophageal HET-1A cell line. We chose this line be-
cause of our ongoing investigation into the role of BAs in provoking
oesophageal cancer. The objective of the present study was to
X
OH
X
CDCA
CA
1
2
3
X=CH2CH2COOH
X=CH2CH2CONH(CH2)2SO3H
X=CH2CH2CONHCH2COOH
4
5
6
HO
OH
HO
OH
H
H
UDCA panel
X
17 X=(CH2)2COOH, Y=H
18 X=CH2COOH, Y=αOH
19 X=COOH, Y=αOH
20 X=CH2CH2CN, Y=αOH
21 X=CN, Y=αOH
22 X=(CH2)3OH, Y=αOH
23 X=(CH2)3N3, Y=αOH
7 X=(CH2)2COOH, Y=αOH
8 X=(CH2)2CONH(CH2)2SO3H, Y=αOH
9 X=(CH2)2CONHCH2COOH, Y=αOH
13 X=(CH2)2COOMe, Y=αOH
14 X=CH2CH2CONH2, Y=αOH
15 X=(CH2)2COOH, Y=αN3
Y
OH
H
16 X=(CH2)2COOH, Y=βN3
DCA panel OH
X
10 X=CH2CH2COOH, Y=αOH
OH
(CH2)2CO2H
11 X=CH2CH2CONH(CH2)2SO3H, Y=αOH
12 X=CH2CH2CONHCH2COOH, Y=αOH
24 X=(CH2)2COOMe, Y=αOH
25 X=CH2CH2CONH2 , Y=αOH
26 X=CH2CH2COOH, Y= αN3
27 X=CH2CH2COOH, Y= βN3
28
LagoDCA
Y
HO
H
H
(CH2)2CO2H
Y
(CH2)2CO2H
Ketones
29 X=αOH, Y=O
30 X=αOH, Z=O
31 X=αOAc, Z=O
32 X=O, Y=O, Z=O
33 X=O, Z=O
34
HO
N3
X
z
H
H
OH
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
LCA
35
HCA
37
HO
OH
H
HO
HO
H
H
36
UCA
OH
Figure 1. Structures of BAs.

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R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
characterize the relationship between cytotoxicity and polarity
and to investigate if toxicity is related to functional group type
and arrangement in synthetic and natural BAs.
in pure form after which azide substitution with NaN3 in DMPU
afforded the 24-azide. By performing various conventional side
chain (Scheme 2) and 3-OH transformations (including the
3-deoxy compound 17), we assembled a panel of 15 UDCA analogs
in which the 7-bOH group was conserved (Fig. 1). Notably, eight of
these retained an ionizable side chain (7–9, 15–19). For compari-
son, seven DCA analogs were assembled along with LagoDCA (28)
the epimeric 12-bOH compound (Fig. 1). A third panel consisted
of five oxosteroid analogues (29–33).
2. Results and discussion
2.1. Synthesis
A group of 16 BAs was assembled from gifts and commercially
available products. This was augmented with 21 compounds previ-
ously reported and synthesised for this study or newly prepared
and characterised (compound identity synthesised using conven-
tional methods was confirmed from references listed in Table 1).
UDCA and DCA 3-azides were synthesised as outlined in Scheme
1 from the respective protected BAs 38 and 41, which were pre-
pared in three steps from the parent BAs. Introduction of 3a-azides
(15, 26) (i.e., with retention of configuration) was accomplished by
generating first the b-bromides (39, 42). The b-azides (16, 27)
2.2. Reverse phase thin layer chromatography (RPTLC)
RPTLC has been used widely for lipophilicity determination
including for BAs.28,29 Relative to LOGP/D experiments it has the
usual chromatographic advantages; relative to HPLC it has higher
throughput and, for generally UV transparent analytes such as
BAs, it permits simple post-chromatographic derivatization. We
developed the RPTLC approach here using commercially available
BAs UDCA, CDCA, CA and DCA along with their glycine and taurine
conjugates (compounds 1–12). The relationship between retention
and pH was assessed in the pH range 5.4ꢀ8.4 for this group. The
approach was optimised with respect to pH and solvent composi-
tion before application to the wider panel of compounds. Over four
concentration levels in the organic modifier concentration range
(/) 50–80% the relationship with RM was linear (r2 >0.99) which al-
lowed the RMw to be estimated by extrapolation:
could be obtained by direct SN2 substitution on the
a-mesylates
(40, 43). Similarly, the 7-b azido analog of UDCA (34) was gener-
ated by treating the appropriately protected CDCA-derived mesy-
late (45) with NaN3 in DMPU followed by deprotection in
aqueous base. The 24-azido UDCA (23) was prepared by LiAlH4
reduction of UDCA to alcohol 22 followed by regioselective mesy-
lation of the primary alcohol in cold solvent. Chromatographic iso-
lation yielded the 24-mesylate (46) (and 3a, 24 dimesyloxy UDCA)
Table 1
BAs physicochemical properties (RMw, total hydrophobic surface area (HSA), total polar surface area (PSA))
Compound number and trivial name
RMw
HSA (Å2)
PSA (Å2)
Cell viability at24 h
500 ( M) (HET-1A)
CC50
(
lM) (95% CI)
Cell viability at
24 h 500 (lM) (Huh7)
l
1 CDCA
2 GCDCA
3 TCDCA
4 CA
5 GCA
6 TCA
7 UDCA
8 GUDCA
9 TUDCA
10 DCA
11 GDCA
12 TDCA
13 MeUDCA37
4.193
2.926
2.597
3.25
2.243
1.908
3.35
458
450
521
433
430
493
452
452
520
454
455
517
514
447
445
446
475
427
416
449
413
480
476
512
451
441
452
457
457
452
480
425
447
440
479
429
428
157
230
233
183
237
254
164
234
231
156
222
229
146
185
205
194
145
158
147
162.3
155
140
181
137
176
196
193
158
157
155
172
175
152
203
130
186
188
0.422
1.116
1.300
1.016
1.121
1.052
0.788
1.073
1.092
0.412
1.148
1.113
0.943
0.397
0.368
0.303
0.399
0.888
0.805
0.940
0.805
0.975
0.759
0.281
0.386
0.307
0.354
0.590
0.818
0.769
0.472
1.109
0.935
0.290
0.417
0.808
0.882
216 (134–346)
nd
729
1075 (910–1270)
nd
nd
1313 (997–1727)
1002 (753–1333)
nd
257 (199–332)
nd
1237
nd
161 (115–225)
45 (28–70)
37 (30–44)
30 (22–40)
nd
799
nd
nd
nd
688 (206–1000)
46 (30–69)
39 (32–49)
71 (59–84)
97 (72–129)(
389 (251–601)
nd
0.723
1.146
1.053
0.908
0.879
0.877
0.740
1.049
1.038
0.570
1.062
1.050
0.812
0.500
0.266
1.009
0.237
0.718
0.748
0.852
0.873
0.445
0.451
0.488
0.252
0.371
1.085
0.890
0.806
0.756
0.777
0.544
0.546
0.641
0.637
0.823
0.738
2.124
1.717
4.023
2.926
2.529
4.776
4.54
5.412
5.452
5.304
3.395
3.623
4.938
3.623
5.032
5.299
4.697
3.895
5.402
5.454
3.89
38
14 UDCA-24NH2
15 UDCA3
16 UDCA3bN3
aN3
17 3deoxyUDCA39
18 Nor UDCA40
19 BisNorUDCA41
20 UDCA24CN
21 BisNorUDCACN
22 UDCA-24OH42
23 UDCA-24N3
24 MeDCA43
38
25 DCA-24NH2
26 DCA3aN3
27 DCA3bN344
28 LagoDCA45
29 12-KetoLCA43
30 NCA46
3.66
4.468
5.638
1.925
3.266
5.466
5.27
nd
31 NCA3-acetate47
32 3,7,12-ketone48
33 3,7-diketone48
34 3OH,7bN3
35 LCA
366 (260–516)
nd
nd
99 (65–147)
25 (17–36)
nd
36 UCA49
3.011
4.407
37 HCA50
nd
The effect on cell viability relative to control in the HET-1A cell line after 24 h incubation is shown (n = 6) along with an estimated CC50 for this. The effect in the hepatic Huh7
line was also evaluated. Compounds were introduced in DMSO and cell viability was normalised to control DMSO at the same concentration.

R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
6889
CO2Me
CO2H
i
ii,iii
91%
71%
Br
OAc
N3
OH
H
H
39
CO2Me
15
CO2Me
CO2H
97%
HO
OAc
H
v, iii
iv
38
96%
MsO
OAc
16
N3
OH
H
H
40
OAc
OH
CO2Me
CO2H
i
ii,iii
79%
65%
Br
CO2Me
N3
26
H
42
OAc
H
OAc
OH
CO2Me
CO2H
HO
H
iv
41
v,iii
MsO
43
27
N3
H
H
CO2Me
CO2Me
CO2H
44
v, iii
iv
34
AcO
OMs
AcO
OH
HO
OMs
HO
N3
45
H
H
H
OH
22
N3
v, iii
iv
23
46
HO
OH
HO
OH
OH
H
H
H
Scheme 1. Synthesis of azidobile acids. Reagents and conditions: P(Ph3)3, NBS, THF, ꢀ18 °C to rt, 1.5 h; (ii) NaN3, DMF, 60 °C, 3 d; (iii) 2 M NaOH/MeOH (pH ꢁ14), reflux, 24 h;
(iv) MsCl, NEt3, DCM, 0 °C, 20 min; (v) NaN3, DMPU, 50 °C.
RM ¼ ꢀS/ þ RMw where RM ¼ Logð1=Rf ꢀ 1Þ
higher at pH 5.4 than at the latter two pH values, which were sim-
ilar. It was decided therefore to assess the retention for the whole
group of 37 compounds at pH 7.4 since this was the pH at which
toxicity was to be determined in the cell-based assay and varia-
The slope S which is a measure of the degree of responsiveness
tions due to shifts in pKa in methanol mixtures had been shown
to changes in mobile phase composition was found to be linear
to be marginal. The rank order at pH 7.4 for the initial test set of
with RMw for the set. For the set of 12 development compounds
12 was: CDCAꢁDCAꢂUDCA>CA>GDCA>GCDCA>TCDCA>TDCA
RMw values were estimated at pH 5.4, 7.4, 8.4. In most cases,
>GCA>GUDCA>TCA>TUDCA. This is roughly as expected and in
accordance with numerous studies on the relative chromato-
including, unexpectedly, the taurine conjugates, RMw values were

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R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
O
OH
O
N
NH2
i
ii, iii
FO
OF
FO
OF
HO
OH
H
H
H
48
47
20
OH
OH
O
O
N
iv
v, iii
HO
OH
FO
OF
HO
OH
H
H
H
49
18
21
Scheme 2. Synthesis of nitriles: (i) (BOC)2O, pyridine, NH4HCO3, rt, 24 h; (ii) trifluoroacetic anhydride/pyridine (1:1); (iii) NaOMe/MeOH reflux, 1 h; (iv) formic acid,
perchloric acid 47 °C, 3 h; (v) THF, rt, 3 h. TFA, trifluoroacetic anhydride, NaNO2 0–5 °C (1 h), 38–40 °C (2 h).
graphic retention of these bile acids and their conjugates.30,31
Retention in RP chromatographic systems is a function of the num-
ber of hydroxyl groups, their topological arrangement and extent
of ionisation, bearing in mind the differences between glyco- and
tauro-conjugates.30 Relative retention of the BAs is also influenced
by organic modifier and stationary phase identity.31 The relative
retention of unconjugated CA to UDCA was found to be pH depen-
dent in our system but at pH 7.4 CA (3.25) was slightly more polar
than UDCA (3.35) which accords with its greater hydrophilicity in
other measures such as Log D and water solubility.32 On the other
hand the UDCA conjugates GUDCA and TUDCA were more polar
than the corresponding CA conjugates. There was a significant in-
verse correlation between RMw and estimated polar surface area
(r2 = 0.81) in this set, little correlation with hydrophobic surface
area (r2 = 0.15) and a moderate positive correlation with the ratio
of HSA to PSA (r2 = 0.65). Associations with these global properties
are strongly influenced by variation in molecular size and substitu-
tion pattern. For example, tauro- and glyco-conjugates have in-
creased HSA and PSA relative to the unconjugated compounds
toxic. Where we observed a significant effect on cell viability at
500 M, the experiment was repeated at successively lower con-
l
centration in order to estimate a CC50 value (half maximal cyto-
toxic concentration). In a number of cases the experiment was
repeated at increasing concentration up to 10 mM. Concentration
response curves were not calculated in cases where there were
poor convergence using a monophasic sigmoid function. The
500 lM cell viability values were found to be most useful therefore
for comparisons and they were consistent with CC50 values in cases
where these were estimated. It is also worth noting that while the
results here for classical bile acids are consistent with reported ef-
fects on cell viability using other measures of apoptosis, the MTT
assay is a reflection of mitochondrial function which could be
attenuated in cells that are nonetheless alive. On the other hand
BA induction of apoptosis is to a significant extent due to mitroc-
hondrial interactions directly and indirectly.
In this context the following general structure-cytotoxicity
observations can be made: (i) glycine and taurine conjugates of
CA, CDCA, DCA and UDCA were not cytotoxic at 500 lM. There
and hence the HSA:PSA ratio does not reflect the decreased RMw
RMw data for the whole set at pH 7.4 is presented in Table 1.
.
was in these cases a trend towards increased cell proliferation
which only achieved statistical significance in the case of TDCA.
Three of these compounds (3, 8, 12) did trigger cell death when
the concentration was raised to >1 mM. The conjugates and least
toxic unconjugated BAs caused cell death at a similar concentra-
tion suggesting that for these compounds the mechanism of cell
death was not structure specific and at such high concentration
attributable to detergency and necrosis. The HET-1A cell line is
not known to express a BA transporter and therefore considered
impermeable to the BA conjugates; (ii) CDCA, DCA and particularly
LCA were potently cytotoxic as expected (Table 1); (iii) all of the
ionisable azido compounds were potently cytotoxic. The UDCA
and DCA 3-azido analogues were significantly more toxic than
the parent compounds. Indeed the UDCA 3-azides were more toxic
than the corresponding DCA compounds. The 7b-azido compound
The panel as a whole (1–37) exhibited satisfactory dispersion
and range in lipophilicity as reflected in RMw (1.717(9)–
5.466(34)) (Table 1). The six azido-analogs were highly lipophilic
as expected. Indeed the azides with ionizable side chains were
more highly retained than some of the non-azido neutral com-
pounds. However, on the whole the neutral compounds such as
the esters and amides yielded RMw values above the median. Intro-
duction of keto (oxo) groups at the 3, 7 and 12 positions has the ef-
fect of depressing hydrophobicity by promoting water access to
both faces of the steroid28,29 and this had the expected effect on
RMw through compounds 29–33.
2.3. Effects on cell viability
(34) was also cytotoxic (CC50 99
24-azide had not effect on cell viability at 500
lipophilicity; (iv) the DCA and UDCA primary amides (25, 14)
markedly reduced viability (CC50 39, 161 M, respectively); (v) in
l
M) however the UDCA-based
Cell viability in the oesophageal HET-1A line was determined at
an initial BA test concentration of 500 lM for all compounds in the
set over 24 h (Fig. 2). This high concentration (pharmacologically)
lM despite its high
l
has been widely used to evaluate BA toxicity. It has been reported
contrast, the DCA methyl ester (24) was more toxic that DCA itself
but the UDCA analogue 13 was not more toxic than UDCA. DCA
methyl ester 24 was not acting as a prodrug for DCA in this context
because it could be recovered unchanged from the medium at
24 h; (v) the 3-deoxy UDCA compound (17) (an isomer of LCA)
that 500 lM DCA causes cell death through apoptosis rather than
necrosis.19 Moreover, BAs can achieve this concentration in vivo
and during UDCA treatment.33,34 In the present case it allowed rel-
ative toxicity assessment of compounds that are moderately cyto-

R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
6891
was highly toxic; (vi) apart from the amide 14, the UDCA side chain
2.4. Relationship between lipophilicity and cell viability
in the HET-1A cell line
analogs (18–23) which are neutral and hydrophobic were not cyto-
toxic; (vii) oxidation of the steroid secondary alcohols to ketone le-
vel was associated with a reduction in cytotoxicity. This is
consistent with the reported haemolytic potential of this
series.28,29
Overall, the most polar compounds were least cytotoxic and
there was an association between lipophilicity and toxicity with
some important exceptions, which are discussed below. The azido
and amide analogs were stable over the time course of the exper-
iments as evidenced by TLC/HPLC.
A relationship between lipophilicity and toxicity of BAs has
been speculated to exist for some time based mainly on the con-
trasting behaviour of LCA, DCA and CDCA and more polar di- and
tri-hydroxy BAs such as UDCA and CA. In the present set of 37 com-
pounds there was a significant negative correlation between RMw
and cell viability at 24 h (r2 = 0.6) (Fig. 3). A similar significance
level was achieved using the sparser CC50 values. The strength of
the association was significantly increased when the neutral
1.5
udca series
dca series
azides
other natural bile acids
*
1.0
0.5
0.0
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Figure 2. Effect of compounds 1–37 on HET-1A viability at 500 lM: n = 6, p <0.05.
1.5
1.0
0.5
0.0
1.5
B
A
Cpds 1-37
Ionizable compounds
1.0
0.5
r2=0.82
r2=0.60
0.0
1.5
2
3
4
5
6
2
3
4
5
6
RMw
RMw
1.2
1.0
0.8
0.6
0.4
C
D
Ketones
1.0
0.5
UDCA acid analogs
r2=0.96
r2=0.97
2
3
4
5
6
2
3
4
5
6
RMw
RMw
Figure 3. Linear regression lines showing 95% confidence bands for cell viability at 24 h in the HET-1A cell line and RMw. The panels show: (A) linear regression for
compounds 1–37; (B) linear regression for the ionisable (acidic) compounds 1–12, 15–19, 26–37; CDCA (j) DCA (N) and LagoDCA (d) are highlighted; (C) linear regression for
the ketones 29–33; (D) linear regression for the UDCA analogues with an acidic side chain.

6892
R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
compounds were excluded (Fig. 3B: r2 = 0.82, n = 29). This might be
explained by differences in ionization between the neutral and acid
compounds that overwhelm more subtle retention interactions.
However, in the group of neutral compounds (n = 8) there was a
very weak association between RMw and cell viability (r2 = 0.11).
The correlation between RMw and toxicity may therefore be contin-
gent on BA amphilicity.
selective cytocidal agents. Simple DCA and UDCA amides also hold
promise in this regard. RPTLC retention extrapolated to zero organ-
ic modifier was predictive of BA toxicity in the HET-1A cell line and
it could be a useful high throughput tool for cytotoxic BA discovery
and development in this context. This is the first direct linear cor-
relation between BA toxicity and a lipophilicity parameter that we
are aware of.
It is notable that DCA, CDCA and LagoDCA are out of trend
(Fig. 3B) being significantly more toxic than predicted on the basis
of RMw. This may be evidence for an additional more specific mech-
anism for cell death induction in these cases. Interestingly, these
three BAs are reported to depart from the norm in prosecretory
behaviour and toxicity towards colon cells.19,35
Excellent correlations were obtained for the ionisable UDCA
group (r2 = 0.96, n = 8) and the ketone panel (r2 = 0.97, n = 5)
(Fig. 3C and D); focussing in this way on relatively homologous
groups minimizes confounding structural factors. It would be
interesting to assess correlations in even more homologous BA ser-
ies with diverse lipophilicity such as fluoro- or methyl-substituted
BAs. There were no significant correlations between the in silico
bulk hydophobicity/polarity indices (HSA, PSA) and cell viability,
consistent with the idea that (relative) BA hydrophobicity/lipophil-
icity is a shape dependent property36 that is better predicted
chromatographically.28
The lipophilicity–toxicity correlation was strongest with acidic
BAs and it was especially strong in structurally homologous sub-
groups. We are unable to say whether the association is due to a
hidden common factor, membrane-perturbation effects or whether
it is a reflection of a capacity to bind specifically to unidentified BA
target proteins: correlations between lipophilicity and affinity/po-
tency are well known. Significantly, the correlation broke down for
non-ionizable lipophilic BAs, and it does not fully account for the
toxicity associated with DCA, CDCA and LagoDCA. Apart from the
naturally occurring bile acids and their conjugates, the correlation
between lipophilicity and cytotoxicity did not hold in a hepatic
carcinoma cell line (Huh7). Collectively, the results indicate that
the relationship between lipophilicity and toxicity is complex,
and probably indirect.
4. Experimental
4.1. Synthesis
2.5. Relationship between lipophilicity and cell viability
in the Huh7 cell line
Uncorrected melting points were obtained using a StuartÒ melt-
ing point SMP11 melting point apparatus. Spectra were obtained
using a Perking Elmer 205 FT Infrared Paragon 1000 spectrometer.
Band positions are given in cmꢀ1. Solid samples were obtained by
KBr disk; oils were analyzed as neat films on NaCl plates. 1H and
13C spectra were recorded at 27 °C on a Bruker Advance II
600 MHz spectrometer (600.13 MHz 1H, 150.91 MHz 13C) and Bru-
ker DPX 400 MHz FT NMR spectrometer (400.13 MHz 1H,
100.16 MHz 13C), in either CDCl3 or CD3OD, (tetramethylsilane as
internal standard). For CDCl3, 1H NMR spectra were assigned rela-
tive to the TMS peak at 0.00 d and 13C NMR spectra were assigned
relative to the middle CDCl3 triplet at 77.00 ppm. For CD3OD, 1H
and 13C NMR spectra were assigned relative to the centre peaks
of the CD3OD multiplets at 3.30 d and 49.00 ppm, respectively.
High resolution mass spectrometry (HRMS) was performed on a
Micromass mass spectrophotometer (EI mode) at the Department
of Chemistry, Trinity College. HPLC was performed on a reverse
In order to assess the generality of the findings made in the
oesophageal cell line, we evaluated the effect of the compounds
on the MTT signal in the Huh7 cell line at 24 h (500 lM, n = 3;
Table 1). This cell line is a human hepatic carcinoma cell line with
significantly different characteristics to the HET-1A line (hepatic
versus oesophageal, cancer versus normal). Nevertheless we were
able to make some interesting observations. The mean MTT signal
was similar in both sets (0.75). Overall there was a weak correla-
tion between MTT values between the two sets (r2 = 0.28). There
was also a weak correlation in the Huh7 set between cell viability
and lipophilicity (r2 = 0.22). This residual correlation appeared to
be dependent on the contribution from the development set of nat-
urally occurring BAs CA, CDCA, DCA, UDCA, their glyco- and tauro-
conjugates and LCA (r2 = 0.52, n = 13). The results for these com-
pounds correlated well between the two cell lines (r2 = 0.8).
Amongst the rest of the compounds (n = 24), mainly synthetic
BAs, there was no relationship between lipophilicity and cytotoxic-
ity in the hepatic cell line. The cytotoxicity in the hepatic cell line
seemed in general to be more structure dependent and less on lipo-
phase 250 mm ꢃ 4.6 mm Waters Spherisorb ODS-2, 5
lm column
using a Waters Alliance 2695 chromatograph equipped with an
autosampler, column oven and dual wavelength detector. The flow
rate was 1 ml/min with a mobile phase consisting of 40% phos-
phate buffer pH 2.5 and 60% acetonitrile at time 0 and grading to
philicity. For example, whereas in the HET-1A cell line, the 3
3b-azides of UDCA and DCA were all cytotoxic, in the Huh7 line,
the - and b-compounds diverged: the -azides of UDCA and
a- and
85% acetonitrile at 4 min. Injection volume was 20 ll, and areas
a
a
determined at 254 nm. The isocratic HPLC method was aqueous
phosphate buffer solution pH 2.5 40% and acetonitrile 60%. Flow
rate was 1 ml/min. Flash chromatography was performed on
Merck Kieselgel 60 particle size 0.040–0.063 mm. TLC was per-
formed on silica gel Merck F-254 plates. Compounds were visually
detected by absorbance at 254 nm and/or vanillin staining.
BAs 1–12 and 35 (Table 1) were obtained from Sigma–Aldrich
Chemical Co. (St. Louis, MO, USA). UCA and HCA (36, 37) were a gift
from Professor B. Natalini (University of Perugia). Identity of all BAs
was confirmed with 1H NMR, 13C NMR and HRMS. Purity was con-
firmed by HPLC and TLC.
DCA (15, 26) were toxic whereas the b-azides were not. This had
a significant effect on the strength of the correlation in the UDCA
set in the Huh7 cell line which was otherwise strong. The 24-azide
and -alcohol compounds (23, 22) were significantly more toxic in
the Huh7 cell line than in the HET1-A line. Whether these observa-
tions are attributable to differences in metabolic competency (and
detoxification) or to intrinsic cytocidal effect they suggest pros-
pects for selective toxicity.
3. Conclusions
In BAs, azido substitution is associated with enhanced toxicity
and the azide group may therefore be a useful design tool for
BA-based cytotoxic agents. Azide orientation may be important
in determining toxicity which could be useful in the design of
4.1.1. 24-Methyl 3b-bromo, 7bꢀacetoxy-5b-cholanoate (39)
Triphenylphosphine (0.170 g) was added to a stirred solution of
38 (0.145 g) in anhydrous THF (15 ml) and the mixture was cooled
to ꢀ18 °C. N-Bromosuccinimide (0.115 g) was added dropwise and

R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
6893
the mixture allowed to warm to room temperature. After 1.5 h,
NMR d (400 MHz, CDCl3): 4.74 (m, 1H, 7a-H), 3.95 (s, 1H, 3a-H),
when TLC analysis showed no more starting material, the reaction
mixture was poured into water (50 ml) and extracted with ethyl
acetate (3 ꢃ 50 ml). The organic phase was dried over MgSO4, fil-
tered and the solvent was evaporated under reduced pressure.
The product was separated by flash chromatography, using hex-
ane/ethyl acetate 3:1 as mobile phase to yield colourless oil
3.68 (s, 3H, –O–CH3), 2.00 (s, 3H, 7-C@OCH3), 1.01 (s, 3H,
19-CH3), 0.93 (d, 3H, J = 6.53 Hz, 21-CH3), 0.69 (s, 3H, 18-CH3).
13C NMR ppm (CDCl3): 174.52 (C@O, 24-C), 170.52 (C@O,
7-OC@OCH3), 73.47 (CH, 7-C), 57.94 (CH, 3-C), 51.33 (CH3, OCH3),
21.65 (CH3, 7-C@OCH3). IRvmax (DCM): 3445.10, 2946.85,
2871.37, 2102.43, 1736.79 and 1248.22 cmꢀ1. HRMS: Found:
(MꢀNa)+ = 496.3164. The crude ester azide was hydrolysed as de-
scribed above (4.1.2) yielding the azide (16) as a white solid. 1H
(0.151 g, 91%). 1H NMR d (CDCl3): 4.76 (s, 1H, 3
-H), 3.67 (s, 3H, –O–CH3), 2.00 (s, 3H, 7-C@OCH3), 1.06 (s, 3H,
a-H), 4.69 (m, 1H,
7a
19-CH3), 0.93 (d, 3H, J = 6.53 Hz, 21-CH3), 0.69 (s, 3H, 18-CH3).
13C NMR ppm (CDCl3): 174.83 (C@O, 24-C), 170.81 (C@O,
7-OC@OCH3), 74.01 (CH, 7-C), 51.64 (CH3, OCH3), 21.97 (CH3, 7-
C@OCH3). IRvmax (DCM): 3435.54, 2948.89, 1729.41, 1646.49 and
1243.23 cmꢀ1. HRMS: Found: (MꢀNa)+ = 533.2249.
NMR d (CDCl3): 3.93 (s, 1H, 3a-H), 3.55 (m, 1H, 7a-H), 1.00 (s,
3H, 19-CH3), 0.96 (d, 3H, J = 6.02 Hz, 21-CH3), 0.70 (s, 3H, 18-
CH3). 13C NMR ppm (CDCl3): 179.81 (C@O, 24-C), 71.54 (CH, 7-C),
58.42 (CH, 3-C). IRvmax (KBr): 3330.87, 2929.30, 2867.30, 2103.92,
1687.49 and 1243.05 cmꢀ1. HRMS: Found: (M)ꢀ = 416.2913.
4.1.2. 3
a
-Azido, 7bꢀhydroxy-5b-cholanoate (15)
4.1.5. 3
Formyl UDCA amide (48) (0.42 g) was dissolved in dry THF
(10 ml) at 0 °C. Pyridine (150 l) and trifluoroacetic anhydride
(270 l) were added to this mixture. After completion of the reac-
a,7b-Dihydroxy-5 b-cholan-24-nitrile (20)
To a stirred solution of 39 (1.876 g) in anhydrous N,N-DMF
(40 ml) was added 5 equiv NaN3 (1.192 g) at 60 °C. The mixture
was stirred overnight then poured into saturated aqueous NaHCO3
(150 ml) and extracted with ethyl acetate (3 ꢃ 100 ml). The organ-
ic phase was washed with brine (200 ml), dried over MgSO4, fil-
tered and the solvent was evaporated in vacuum. The product
was separated on a flash column (hexane/ethyl acetate 9:1) to
yield orange foam (1.258 g, 72%). The azide (0.150 g) was dissolved
in methanol (15 ml) to which was added 2 M sodium hydroxide
solution to pH ꢁ14 and stirred at reflux for 1 day, when TLC anal-
ysis showed the hydrolysis was complete. Then the reaction mix-
ture was poured into 1 M HCl solution (50 ml) and extracted
with ethyl acetate (3 ꢃ 50 ml). The organic layer was washed with
water (2 ꢃ 100 ml) and brine (1 ꢃ 100 ml), dried over Na2SO4, fil-
tered and the solvent was removed under reduced pressure to give
the product as a light yellow solid (0.131 g, 99%). 1H NMR d
l
l
tion as monitored by TLC (10 h) the solvent was removed in vacuo
and the residue redissolved in ethyl acetate (20 ml) which was
then washed with HCl (3 ꢃ 20 ml) and water to neutrality. Chro-
matographic elution with ethyl acetate/hexane (1:1) afforded a
white solid. Sodium (0.2 g) was added to methanol (10 ml) to form
an excess of sodium methoxide. The formyl nitrile obtained in the
above reaction was added to this solution which was then refluxed
for 2 h. After 2 h the reaction was cooled to room temperature and
added to water (20 ml). The nitrile product (20) was extracted with
ethyl acetate. The organic layer was then washed with water
(3 ꢃ 20 ml) and dried (MgSO4). The solvent was removed in vacuo
to yield a white solid (0.3 g, 78%). 1H NMR d (MeOD) 3.50 (m, 2-H,
3-b H, 7-
a H), 2.8 (m, 1-H, 20-CH), 1.00 (d, 3-H, J = 6.02 Hz, 21-
(CDCl3): 3.60 (m, 1H, 7
a
-H), 3.30 (m, 1H, 3b-H), 0.98 (s, 3H, 19-
CH3), 0.98 (s, 3-H, 19-CH3), 0.75 (s, 3-H, 18-CH3). 13C NMR ppm
(CDCl3): 124 (CN, 22-C), 71 (3-C, 7-C). HRMS: Found: (MꢀNa)+
396.2867.
CH3), 0.96 (d, 3H, J = 6.53 Hz, 21-CH3), 0.69 (s, 3H, 18-CH3). 13C
NMR ppm (CDCl3): 180.19 (C@O, 24-C), 71.68 (CH, 7-C), 61.30
(CH, 3-C). IRvmax (KBr): 3375.06, 2934.00, 2867.03, 2093.18,
1690.57 and 1255.27 cmꢀ1. HRMS: Found: (MꢀNa)+ = 440.2896.
4.1.6. 3a,7b-Dihydroxy-24-bisnor-5b-cholane-22-nitrile (21)
Formyl protected norUDCA (3.5 g) (18) was stirred in trifluoro-
acetic acid (4 ml) and trifluoroacetic anhydride (1 ml) at 0–5 °C un-
til dissolution was complete. Sodium nitrite (0.786 g) was then
added in small portions, waiting for the salt to react between addi-
tions. After addition the mixture was stirred at 0–5 °C for 1 h. The
mixture was then warmed to 38–40 °C and left to stir for another
2 h. The brown solution was cooled to room temperature and
added to a mixture of water/1 M NaOH (1:1, 50 ml). The nitrile
was extracted with ethyl acetate and washed with NaOH
(4 ꢃ 20 ml) and water to neutrality. The ethyl acetate was dried
(MgSO4) and removed in vacuo to yield an off white solid. Sodium
(1 g) was added to MeOH (50 ml) to form an excess of sodium
methoxide. The formyl bisnornitrile obtained in the above reaction
was added to this solution and refluxed for 2 h. After 2 h the reac-
tion was cooled to room temperature and added to water (100 ml).
The title compound was extracted with ethyl acetate. The organic
layer was then washed with water (3 ꢃ 20 ml) and dried (MgSO4).
The solvent was removed in vacuo to yield a white solid (2.6 g,
4.1.3. 24-Methyl 3
cholanoate (40)
a-(methylsulfonyl)oxy, 7b-acetoxy-5b-
To a solution of 38 (1 g) and triethylamine (0.34 ml) in anhy-
drous dichloromethane (30 ml) was added methanesulfonylchlo-
ride (0.26 ml in 10 ml anhydrous DCM) dropwise at 0 °C and
stirred for 20 min. Then, cooled water (50 ml) was added to the
mixture, which was separated and the aqueous phase was ex-
tracted with DCM (2 ꢃ 40 ml). The organic phase was washed with
brine (100 ml), dried over MgSO4, filtered and the solvent was re-
moved under reduced pressure to give colourless oil as product
(1.115 g, 95%). 1H NMR d (CDCl3): 4.77 (m, 1H, 7
a-H), 4.62 (m,
1H, 3b-H), 3.68 (s, 3H, –O–CH3), 3.02 (s, 3H, –OSO2CH3), 2.00 (s,
3H, 7-C@OCH3), 0.99 (s, 3H, 19-CH3), 0.94 (d, 3H, J = 6.02 Hz, 21-
CH3), 0.69 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3): 174.82 (C@O,
24-C), 170.70 (C@O, 7-OC@OCH3), 81.72 (CH, 3-C), 73.42 (CH,
7-C), 52.68 (CH3, –OSO2CH3), 51.64 (CH3, OCH3), 21.92 (CH3, 7-
C@OCH3). IRvmax (DCM): 3436.82, 2950.45, 2873.89, 1729.45,
1646.30 and 1173.83 cmꢀ1. HRMS: Found: (MꢀNa)+ = 549.2858.
94%). 1H NMR d (CDCl3) 3.48 (m, 2-H, 3-b H, 7-
a H), 2.8 (m, 1-H,
20-CH), 1.34 (d, 3-H, J = 7.02 Hz, 21-CH3), 1.00 (s, 3-H, 19-CH3),
0.76 (s, 3-H, 18-CH3). 13C NMR ppm (CDCl3): 124 (CN, 22-C), 71
(3-C, 7-C). HRMS: Found: (M)ꢀ = 344.2590.
4.1.4. 3b-Azido, 7b-hydroxy-5b-cholanoate (16)
Compound 40 (0.705 g) and sodium azide (0.870 g) in DMPU
(20 ml) were stirred at 50 °C for 11 days (TLC hexane/ethyl acetate
3:1) then poured into water (50 ml) and extracted with ethyl ace-
tate (3 ꢃ 50 ml). The organic phase was washed with brine
(100 ml) dried over MgSO4, filtered and the solvent was removed
under reduced pressure to give the crude product as yellow oil.
This was flash columned using 5% then 10% ethyl acetate in hexane
as mobile phase to yield white solid as product (0.537 g, 85%). 1H
4.1.7. 3
,7b,24-Trihydroxy-5 b-cholane (0.2 g) (22) was dissolved in
dry pyridine (4 ml) at 0 °C followed by the addition of methane sul-
fonyl chloride (63 l). After 20 min the reaction was quenched by
a,7b-Dihydroxy-5 b-cholan-24-azide (23)
3a
l
adding crushed ice and the compound was extracted with ethyl
acetate (2 ꢃ 10 ml). The organic layer was washed with cold water

6894
R. Sharma et al. / Bioorg. Med. Chem. 18 (2010) 6886–6895
(2 ꢃ 20 ml), cold HCl (2 ꢃ 20 ml) and dried (MgSO4) to afford a
white solid (46). TLC of this product revealed starting material
and a new product spot. Chromatographic elution with ethyl ace-
tate afforded a white solid (0.13 g, 53%). The product was dissolved
in DMPU and treated with an excess of NaN3 at 50 °C for two days.
The title compound was obtained by partitioning between ethyl
acetate and water, followed by successive acid and base wash,
evaporation and flash chromatography. 1H NMR d (MeOD) 3.5
1745.61, 1722.74, 1256.74 and 1164.61 cmꢀ1. HRMS: Found:
(MꢀNa)+ = 496.3159.
4.1.11. 3
a-Hydroxy, 7b-azido-5b-cholanoate (34)
1H NMR d (MeOD) 3.6369 (m, 1-H, 3 b-H), 3.0519 (m, 1-H, 7
a
-
H), 0.9583 (s, 3-H, 19-CH3), 0.9407 (d, 3-H, J = 6.02, 21-CH3), 0.6986
(s, 3H, 18-CH3). 13C NMR ppm (MeOD): 180 (C@O, 24-C), 71 (3-C),
61 (7-C). IRvmax (KBr) 3340.06, 2915.00, 2105.01, 1695.34 and
1235.21 cmꢀ1. HRMS: Found: (MꢀNa)+ = 440.2889.
(m, 2-H, 3-b H, 7-
a H), 3.26 (m, 2-H, 23-CH2), 0.99 (d, 3-H,
J = 6.02, 21-CH3), 0.95 (s, 3-H, 19-CH3), 0.70 (3-H, s, 18-CH3). 13C
NMR ppm (MeOD): 71 (3-C, 7-C), 51 (23-C), IRvmax (KBr) 3341.06.
2091, HRMS: Found: (MꢀNa)+ = 426.3096.
4.2. Reverse phase TLC
TLC was performed on pre-coated C18 reversed-phase HPTLC
(20x10, F254) plates (Merck, Darmstadt, Germany). Test solutions
were applied in DMSO. The plates were then dried at 40 °C for
1 h. Plates were developed in a closed chamber at room tempera-
ture across a development distance of 15 cm. Methanol: aqueous
ammonium acetate (15 mM) adjusted to the required pH with ace-
tic acid was used as the mobile phase. After development, the
plates were dried under ambient conditions and stained with a
vanillin solution to visualize spots.
4.1.8. 24-Methyl 3b-bromo, 12a-acetoxy-5b-cholanoate (42)
Triphenylphosphine (0.821 g) was added to a stirred solution of
41 (1.035 g) in anhydrous THF (40 ml) and the mixture was cooled
to ꢀ18 °C. N-Bromosuccinimide (1.210 g) was added in three parts
to the reaction mixture over 1 h and it was allowed to warm to
room temperature. After 1.5 h, when TLC analysis showed no more
starting material, the reaction mixture was poured into 1 M HCl
solution (150 ml) and extracted with ethyl acetate (3 ꢃ 100 ml).
The organic phase was washed with water (2 ꢃ 100 ml) and brine
(1 ꢃ 100 ml), dried over MgSO4, filtered and the solvent was evap-
orated under reduced pressure. The product was separated by flash
chromatography twice, using 30% ethyl acetate first then 0–12%
ethyl acetate in hexane as mobile phase to yield colourless semi-
solid (0.766 g, 65%). 1H NMR d (CDCl3): 5.10 (s, 1H, 12b-H), 4.80
4.3. Cell culture
The human oesophageal squamous epithelial cell line HET-1A
was obtained from American Type Culture Collection (ATCC, Rock-
ville, MD). Cells were cultured in bronchial epithelial cell basal
medium (Lonza Group Ltd, Switzerland) supplemented with triio-
dothyronine, insulin, transferrin, retinoic acid, hydrocortisone, hu-
man recombinant epidermal growth factor, epinephrine and
bovine pituitary extract.
The human hepatoma cell line, Huh7 was a kind gift from
Dr. Ralf Bartenschlager (Department of Molecular Virology, Univer-
sity of Heidelberg, Germany). Cells were cultured in Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% foetal
calf serum and 100 U/ml penicillin/streptomycin. Cultures were
maintained at 37 °C in a humidified atmosphere containing 5% CO2.
(s, 1H, 3a-H), 3.68 (s, 3H, –O–CH3), 2.09 (s, 3H, 12-C@OCH3), 1.00
(s, 3H, 19-CH3), 0.83 (d, 3H, J = 6.28 Hz, 21-CH3), 0.75 (s, 3H, 18-
CH3). 13C NMR ppm (CDCl3): 175.07 (C@O, 24-C), 170.85 (C@O,
12-OC@OCH3), 76.41 (CH, 12-C), 51.97 (CH3, OCH3), 21.78 (CH3,
12-C@OCH3). IRvmax (KBr): 3444.74, 2940.49, 2873.22, 1736.69
and 1245.03 cmꢀ1. HRMS: Found: (MꢀNa)+ = 533.2252.
4.1.9. 3a-Azido, 12a-hydroxy-5b-cholanoate (26)
To a solution of 22 (0.150 g) in methanol (15 ml) was added 2 M
sodium hydroxide solution to pH ꢁ14 and stirred at reflux for
1 day, when TLC analysis showed the hydrolysis was complete.
Then the reaction mixture was poured into 1 M HCl solution
(50 ml) and extracted with ethyl acetate (3 ꢃ 50 ml). The organic
layer was washed with water (2 ꢃ 100 ml) and brine
(1 ꢃ 100 ml), dried over Na2SO4, filtered and the solvent was re-
moved under reduced pressure to give the product as a light yellow
solid (0.131 g, 99%) mp 88 °C. 1H NMR d (CDCl3): 4.01 (s, 1H, 12b-
H), 3.35 (m, 1H, 3b-H), 1.01 (d, 3H, J = 6.03 Hz, 21-CH3), 0.94 (s, 3H,
19-CH3), 0.70 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3): 179.80 (C@O,
24-C), 73.29 (CH, 12-C), 61.40 (CH, 3-C). IRvmax (KBr): 3440.00,
4.4. Calculation of physicochemical descriptors
UDCA was extracted from the X-ray crystal structure bound to
AKR1C2 (PDB entry 1IHI) and visualized in molecular operating
environment (MOE 2007.09, Chemical Computing Group, Mon-
treal, Canada). Energy minima for the other test bile acids were
generated from this by modification in MOE using sequential SD
(100 steps) and TN (1000 steps or to an RMS gradient of
*
<0.01 kcal/(mol A)) using the PM3 algorithm, Stochastic searches
2941.49, 2866.14, 2090.46, 1709.85, 1679.00 and 1246.00 cmꢀ1
.
were performed for the conjugates using PM3 over 1000 steps.
Physicochemical descriptor parameters including hydrophobic
surface area (ASAH) and polar surface area (ASAP) were calculated
based on the minimised steroid molecules following a stochastic
conformational search procedure using the database functions of
MOE.
HRMS: Found: (MꢀNa)+ = 440.2878.
4.1.10. 24-Methyl 3a-azido, 7bꢀacetoxy-5b-cholanoate (22)
To a stirred solution of 19 (1.876 g) in anhydrous N,N-DMF
(40 ml) was added 5 equiv sodium azide (1.192 g) at 60 °C. The
mixture was stirred overnight then poured into std. NaHCO3
(150 ml) and extracted with ethyl acetate (3 ꢃ 100 ml). The organ-
ic phase was washed with brine (200 ml), dried over MgSO4, fil-
tered and the solvent was evaporated in vacuo. The product was
separated on a flash column (hexane/ethyl acetate 9:1) to yield or-
4.5. MTT assay
Cell viability was measured using the 3-[4,5-dimethylthiazol-2-
yl] 2,5-diphenyltetrazolium bromide (MTT) assay. Cells were pla-
ted into 96 well plates at a concentration of 8 ꢃ 104 cells/ml
ange foam (1.258 g, 72%). 1H NMR
d
(CDCl3): 4.76 (6, 1H,
(100 ll/well). After 24 h the cells were treated with various bile
J1 = 5.52 Hz, J2 = 5.53 Hz, J3 = 5.01 Hz, 7
a-H), 3.68 (s, 3H, –O–CH3),
acids at concentrations ranging from 4 lM to 10 mM for a further
3.28 (m, 1H, 3b-H), 2.00 (s, 3H, 7-C@OCH3), 0.99 (s, 3H, 19-CH3),
0.94 (d, 3H, J = 6.53 Hz, 21-CH3), 0.69 (s, 3H, 18-CH3). 13C NMR
ppm (CDCl3): 174.52 (C@O, 24-C), 170.40 (C@O, 7-OC@OCH3),
73.31 (CH, 7-C), 60.58 (CH, 3-C), 51.32 (CH3, OCH3), 21.63 (CH3,
7-C@OCH3). IRvmax (KBr): 3430.05, 2929.21, 2871.64, 2099.67,
24 h in supplement free medium. Test compounds were main-
tained as 200 mM stock solutions in DMSO. The compounds were
diluted to the required concentrations with medium. No change
in pH was observed on addition of bile acids to the medium.
Control wells were treated with BEBM without supplements or

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Funding: This work was supported by the Health Research
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