Putative irreversible inhibitors of the human sodiumdependent bile acid transporter (hASBT; SLC10A2)
support the role of transmembrane domain 7 in substrate
binding/translocation
Supplementary Material
Pharmaceutical Research
Pablo M. González*, Naissan Hussainzada, Peter W. Swaan, Alexander D. MacKerell
Jr., and James E. Polli
*Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de
Chile, Santiago, Chile
pmgonzal@uc.cl
1
Table of Contents
1. Synthesis of Electrophilic CDCA Derivatives [including Scheme 1 (Synthetic route to
obtain 3β-Cl-CDCA) and Scheme 2 (Synthetic route for the preparation of 7α-MsCDCA]
page 3
Fig. S1.A. 13C-NMR 3-Cl-CDCA
Fig. S1.B. 1H-NMR 3-Cl-CDCA
page 5
page 5
Fig. S2.A. 13C-NMR 7-Ms-CDCA
Fig. S2.B. 1H-NMR 7-Ms-CDCA
page 6
page 6
Fig S3. hASBT-inhibition profile of 7-Ms-CDCA
Fig S4. hASBT-inhibition profile of 3-Cl-CDCA
page 7
page 7
Fig. S5.A. Remaining hASBT activity after pre-incubation with 3-Cl-CDCA
Fig. S5.B. Kitz and Wilson plot for 3-Cl-CDCA
page 8
page 8
Fig. S6. Remaining hOCTN2 activity after pre-incubation with 3-Cl-CDCA
page 9
Table S1.
Appendix 1
page 10
REFERENCES
page 12
2
1. Synthesis of Electrophilic CDCA Derivatives.
The putatively irreversible hASBT inhibitors 3-chloro-7-hydroxy-5-cholan-24-oic
acid (5) and 3-hydroxy-7-mesyloxy-5-cholan-24-oic acid (9) (3-Cl-CDCA, and 7Ms-CDCA, respectively) were synthesized from 3,7-dihydroxy-5-cholan-24-oic acid
(chenodeoxycholic acid, 1) in several steps. First, the benzyl ester of 1 was prepared
using DMF as solvent. Chenodeoxycholic acid benzyl ester (2) was obtained in almost
quantitative yield. Ester 2 was then reacted with p-toluenesulfonyl chloride (TsCl) in
pyridine (1), obtaining benzyl 3-(tosyloxy)-7-hydroxy-5-cholanoate (3) in 82% yield.
Chlorination of the 3-position was achieved by reacting 3 with an excess of pyridine
hydrochloride in pyridine at 90oC as described previously (2). The substitution product 4
was purified by column chromatography in mixture ethyl acetate/hexanes and obtained in
57% yield. Catalytic hydrogenation of benzyl ester 4 produced 3-Cl-CDCA (5) as a
white fluffy solid (Scheme S1).
Scheme 1. Synthetic route to obtain 3-Cl-CDCA
3
7-Ms-CDCA (9) was obtained from chenodeoxycholate benzyl ester 2 after several
steps (Scheme 2). First, 3-oxo-7-hydroxy-5-cholanoate benzyl ester (6) was obtained
by refluxing 2 and 50% Ag2CO3/celite in toluene as described previously (3). Compound
6 was obtained in 89% yield after chromatographic purification. 3-oxo-7-mesyloxy-5cholanoate benzyl ester (7) was prepared as described for 3 using an excess of mesyl
chloride (MsCl) and refluxing in pyridine (68% yield). The 3-oxo-precursor 7 was
reduced to the 3-hydroxy derivative using NaBH4 in methanol (4) (75% yield after
chromatography). 7-Ms-CDCA (9) was obtained as an off-white solid after catalytic
hydrogenolysis.
1D 13C-NMR and 1H-NMR spectra of final products 5 and 9 are shown in Figs S1 and
S2 panes A and B, respectively.
Scheme 2. Synthetic route for the preparation of 7-Ms-CDCA.
4
Fig. S1.A. 13C-NMR 3-Cl-CDCA
Fig. S1.B. 1H-NMR 3-Cl-CDCA
5
Fig. S2.A. 13C-NMR 7-Ms-CDCA
Fig. S2.B. 1H-NMR 7-Ms-CDCA
6
Fig S3. hASBT-inhibition profile of 7-Ms-CDCA
Taurocholate uptake (pmol/cm 2/s)
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
5
10
15
20
25
30
35
40
45
50
35
40
45
50
7-Ms-CDCA (mM)
Fig. S4. hASBT-inhibition profile of 3-Cl-CDCA
Taurocholate uptake (pmol/cm 2/s)
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
5
10
15
20
25
30
3-Cl-CDCA (mM)
7
Fig. S5.A. Remaining hASBT activity after pre-incubation with 3-Cl-CDCA
hASBT activity after pre-incubation
(pmol/cm 2/s)
0.6
0.55
3 min
0.5
5 min
0.45
7 min
0.4
10 min
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
5
10
15
20
25
30
35
40
45
50
3-Cl-CDCA (mM)
Fig. S5.B. Kitz and Wilson plot for 3-Cl-CDCA
16
14
1/Kapp (min -1)
12
10
8
6
4
2
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
-1
3-Cl-CDCA (mM )
8
Fig. S6. Remaining hOCTN2 activity after pre-incubation with 3-Cl-CDCA
OCTN2 activity after pre-incubation
(pmol/cm 2/s)
0.6
0.5
0.4
0.3
0.2
3 min
7 min
0.1
10 min
0
0
5
10
15
20
25
30
35
40
45
50
3-Cl-CDCA (mM)
Table S1.
hASBT inactivation rate and inhibitory affinity of 3-Cl-CDCA and 7-Ms-CDCA
obtained from Eqn 2.
Parameter
3-Cl-CDCA
7-Ms-CDCA
k3 (min-1)
0.399
0.136
Ki (mM)
2.17
3.59
9
Appendix 1
The objective of this appendix is to show the derivation of the irreversible inhibition
model. Kitz and Wilson provided the model in the following form:
Ln( J / J 0 )  
k3
 t   K app  t
Ki
1
[I ]
(A1)
where J is remaining TCA flux after pre-incubation (i.e. remaining hASBT activity), J0 is
TCA flux without pre-incubation, k3 is inactivation rate, Ki is binding affinity, I is the
irreversible inhibitor concentration, and t is pre-incubation time (i.e. duration of preincubation). Eqn A1 is the same as eqn 1. However, Kitz and Wilson did not show the
derivation in great detail, perhaps since eqn A1 was not directly used in regression
analysis. A detailed derivation is provided here, since the present manuscript directly
employs a form of the equation for regression analysis.
In the model for irreversible inhibition, the amount of active transporter is:
E  Eu  EI
(A2)
where E is the amount of transporter that is not irreversibly inactivated, Eu is the amount
of unbound transporter, and EI is the amount of transporter-inhibitor complex prior to
inactivation.
Ki 
[ Eu ]  [ I ]
[ EI ]
(A3)
Substituting eqn A2 into eqn A3 and solving for EI yields
[ EI ] 
[E] [ I ]
Ki  [ I ]
(A4)
10
The rate of loss of amount of transporter due to inactivation is
dE
 k3  [ EI ]
dt
(A5)
Substituting eqn A4 into eqn A5 yields
k [ E]
dE k3  [ E ]  [ I ]

 3
K
dt
Ki  [ I ]
1 i
[I ]
(A6)
Solving eqn A6 yields
E  E0  e


 k3


t
K
 1 i

[I ]

(A7)
where E0 is the initial amount of transporter.
The amount of transporter is assessed functionally by measuring active TCA flux.
Hence, eqn A7 yields
J  J0  e


 k3


t
Ki
 1

[I ]

(A8)
Eqn A8 is Eqn 1 in the main text.
11
REFERENCES
1.
Iida T, Chang F. Potential bile acid metabolites. 7. 3,7,12-trihydroxy-5betacholanic acids and related compounds. Journal of Organic Chemistry. 1982 January,
1982;47:2972-8.
2.
Chang F, Blickenstaff R, Feldstein A, Gray J, McCaleb G, Sprunt D.
Seroflocculating Steroids. III. Chloro and other bile acid derivatives. Journal of the
American Chemical Society. 1957;79:2164-7.
3.
Jones S, Selinsky B. Efficient route to 7alpha-(benzyloxy)-3-(dioxolaneCholestane-24(R)-ol, a key intermediate in the synthesis of squalamine. Journal of
Organic Chemistry. 1998;63:3786-9.
4.
Uekawa T, Ishigami K, Kithara T. Short-step synthesis of chenodiol from
sigmasterol. Bioscience, Biothechnology and Biochemistry. 2004;68(6):1332-7.
12