Glyco-deoxycholic acid, sodium salt (5d)18 was obtained in
73% isolated yield as white solid, mp: 203–209 °C. RP-HPLC
Rt = 13.07 min. δH (400 MHz; d6-DMSO) 0.6 (3 H, s, C(18)
H3), 0.8 (3 H, s, C(19)H3), 0.9 (3 H, d, C(21)H3), 2.6 (2 H, dd,
C(3)H), 3.3 (1 H, m, C(3)H), 3.7 (2 H, d, J = 7 Hz, C(25)H2),
3.8 (1 H, s, C(12)H), 4.2 (1 H, bs s, OH), 4.4 (1 H, bs s, OH),
8.1 (1 H, bs pst, NH). δC (100 MHz; d6-DMSO) 12.5, 17.1,
23.1, 23.5, 26.1, 27.0, 27.2, 28.6, 30.2, 32.2, 33.0, 33.8, 35.0,
35.2, 35.7, 36.3, 40.5, 41.6, 46.0, 46.3, 47.5, 70.0, 71.1, 171.5,
173.0.
entered in a 10 ml PTFE coil reactor warmed at 80 °C, fitted
with back pressure regulator (100 psi), and then directed in a
Michel-Miller chromatography column (L × I.D. 300 mm ×
21 mm) packed with sulphonic acid resin (20 g Amberlyst
A-15). The output was collected, partially concentrated in
vacuum and acidified at pH 2 with HCl 3 N. The white solid was
collected, washed with acetone to obtain 16.28 g (36 mmol,
91%) of pure glyco-UDCA (5a), mp: 225–229 °C. RP-HPLC Rt
= 6.31 min. δH (400 MHz; d6-DMSO) 0.6 (3 H, s, C(18)H3), 0.9
(3 H, s, C(19)H3), 0.9 (3 H, d, C(21)H3), 3.3 (1 H, m, C(3)H),
3.3 (1 H, s, C(7)H), 3.7 (2 H, d, J = 7 Hz, C(25)H2), 8.1 (1 H,
bs pst, NH), 12.2 (1 H, bs s, COOH). δC (100 MHz; d6-DMSO)
12.1, 18.5, 20.9, 23.3, 26.7, 28.2, 30.2, 31.6, 32.1, 33.8, 34.9,
34.9, 37.3, 37.7, 38.8, 40.5, 42.2, 43.0, 43.1, 54.8, 55.9, 69.5,
69.8, 171.4, 173.0.
Tauro-ursodeoxycholic acid, sodium salt (6a)18 was obtained
in 95% isolated yield as white solid, mp: 294–201 °C.
RP-HPLC Rt = 4.04 min. δH (400 MHz; d6-DMSO) 0.6 (3 H, s,
C(18)H3), 0.9 (3 H, s, C(19)H3), 0.9 (3 H, d, C(21)H3), 2.6 (2
H, dd, J1 = 6 Hz, J1 = 7.5 Hz, C(26)H2), 3.3–3.4 (4 H, m, C(3)
H, C(25)H2, C(7)H). δC (100 MHz; d6-DMSO) 12.1, 18.5, 20.9,
23.3, 36.7, 28.2, 30.3, 31.6, 32.6, 33.8, 34.9, 35.0, 35.5, 37.3,
37.7, 38.7, 39.9, 42.2, 43.0, 43.1, 50.6, 54.7, 55.9, 69.5, 69.8,
172.2.
RP-HPLC isocratic analysis
The chromatographic analyses were directly executed on each of
the reaction mixtures. The mobile phase was prepared by dissol-
ving 100 mM HCO2NH4 in a H2O : MeCN – 60 : 40 (v/v)
mobile phase; then, the apparent pH [swpH, that is the one
measured in the employed hydro-organic mobile phase (s), while
the calibration of the pH system was done in water (w)] was
adjusted to 3.5 with HCO2H. The analyses were carried out at a
1.0 ml min−1 flow rate after previous conditioning by passing
through the column the selected mobile phase for at least 30 min
at the same eluent velocity. Before being used, all the mobile
phases were always filtered through a 0.22 μm Millipore filter
(Bedford, MA, USA) and then degassed with 20 min sonication.
All the analyses were conducted at a 25 °C column temperature.
Tauro-chenodeoxycholic acid, sodium salt (6b)18 was
obtained in 93% isolated yield as white solid, mp: 163–165 °C.
RP-HPLC Rt = 6.48 min. δH (400 MHz; d6-DMSO) 0.6 (3 H, s,
C(18)H3), 0.8 (3 H, s, C(19)H3), 0.9 (3 H, d, C(21)H3), 2.6 (2
H, dd, J1 = 6 Hz, J1 = 7.5 Hz, C(26)H2), 3.2 (1 H, m, C(3)H),
3.3 (2 H, dd, J1 = 6 Hz, J1 = 8.2 Hz, C(25)H2), 3.6 (1 H, s, C(7)
H), 4.1 (1 H, bs s, OH), 4.4 (1 H, bs s, OH), 7.7 (1 H, bs pst,
NH). δC (100 MHz; d6-DMSO) 11.6, 18.4, 20.3, 22.7, 23.2,
27.8, 30.7, 31.5, 32.3, 32.6, 34.7, 34.8, 35.0, 35.3, 35.4, 39.1,
39.6, 41.5, 41.9, 50.0, 50.6, 55.6, 66.2, 70.4, 172.2.
Tauro-cholic acid, sodium salt (6c)18 was obtained in 88% iso-
lated yield as white solid, mp: 228–230 °C. RP-HPLC Rt =
4.21 min. δH (400 MHz; d6-DMSO) 0.6 (3 H, s, C(18)H3), 0.8
(3 H, s, C(19)H3), 0.9 (3 H, d, C(21)H3), 2.6 (2 H, dd, J1 = 6
Hz, J1 = 7.5 Hz, C(26)H2), 3.2 (1 H, m, C(3)H), 3.3 (2 H, dd, J1
= 6 Hz, J1 = 8.2 Hz, C(25)H2), 3.6 (1 H, m, C(7)H), 3.8 (1 H,
m, C(12)H), 3.9 (1 H, bs d, OH), 4.0 (1 H, bs d, OH), 4.3 (1 H,
bs d, OH), 7.7 (1 H, bs pst, NH). δC (100 MHz; d6-DMSO)
12.4, 17.1, 22.6, 22.8, 26.2, 27.3, 28.6, 30.4, 31.6, 32.7, 34.4,
34.9, 35.2, 35.3, 35.5, 41.4, 41.6, 45.8, 46.1, 50.6, 66.3, 70.5,
71.0, 172.3.
Notes and references
1 For review see: (a) P. Lefebvre, B. Cariou, F. Lien, F. Kuipers and
B. Staels, Physiol. Rev., 2009, 89, 147; (b) C. Thomas, R. Pellicciari,
M. Pruzanski, J. Auwerx and K. Schoonjans, Nat. Rev. Drug Discovery,
2008, 7, 1; (c) A. F. Hofmann and L. R. Hagey, Cell. Mol. Life Sci.,
2008, 65, 2461.
2 (a) R. Pellicciari, A. Gioiello, P. Sabbatini, F. Venturoni, R. Nuti,
C. Colliva, G. Rizzo, L. Adorini, M. Pruzanski, A. Roda and
A. Macchiarulo, ACS Med. Chem. Lett., 2012, DOI: 10.1021/ml200256d;
(b) R. Sharma, A. Long and J. F. Gilmer, Curr. Med. Chem., 2011, 18,
4029; (c) A. Gioiello, A. Macchiarulo, A. Carotti, P. Filipponi,
G. Costantino, G. Rizzo, L. Adorini and R. Pellicciari, Bioorg. Med.
Chem., 2011, 19, 2650; (d) Y. Iguchi, M. Yamaguchi, H. Sato, K. Kihira,
T. Nishimaki-Mogami and M. Une, J. Lipid Res., 2010, 51, 1432;
(e) R. Pellicciari, A. Gioiello, A. Macchiarulo, C. Thomas, E. Rosatelli,
B. Natalini, R. Sardella, M. Pruzanski, A. Roda, E. Pastorini,
K. Schoonjans and J. Auwerx, J. Med. Chem., 2009, 52, 7958;
(f) K. Kuhajda, S. Kevresan, J. Kandrac, J. P. Fawcett and M. Mikov,
Eur. J. Drug Metab. Pharmacokinet., 2006, 31, 179; (g) R. Pellicciari,
G. Costantino, E. Camaioni, B. M. Sadeghpour, A. Entrena, T.
M. Willson, S. Fiorucci, C. Clerici and A. Gioiello, J. Med. Chem., 2004,
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G. Costantino, P. R. Maloney, A. Morelli, D. J. Parks and T. M. Willson,
J. Med. Chem., 2002, 45, 3569.
3 (a) T. W. H. Pols, M. Nomura, T. Harach, G. Lo Sasso, M. H. Oosterveer,
C. Thomas, G. Rizzo, A. Gioiello, L. Adorini, R. Pellicciari, J. Auwerx
and K. Schoonjans, Cell Metab., 2011, 14, 747; (b) T. Mizuochi,
A. Kimura, I. Ueki, T. Takahashi, T. Hashimoto, A. Takao, Y. Seki,
H. Takei, H. Nittono, T. Kurosawa and T. Matsuishi, Pediatr. Res., 2010,
68, 258; (c) C. Thomas, A. Gioiello, L. Noriega, A. Strehle, J. Oury,
G. Rizzo, A. Macchiarulo, H. Yamamoto, C. Mataki, M. Pruzanski,
R. Pellicciari, J. Auwerx and K. Schoonjans, Cell Metab., 2009, 10, 167;
Tauro-deoxycholic acid, sodium salt (6d)18 was obtained in
95% isolated yield as white solid, mp: 170–175 °C. RP-HPLC
Rt = 7.40 min. δH (400 MHz; d6-DMSO) 0.6 (3 H, s, C(18)H3),
0.8 (3 H, s, C(19)H3), 0.9 (3 H, d, C(21)H3), 2.6 (2 H, dd, J1 = 6
Hz, J1 = 7.5 Hz, C(26)H2), 3.3 (1 H, m, C(3)H), 3.3 (2 H, dd, J1
= 6 Hz, J1 = 8.2 Hz, C(25)H2), 3.8 (1 H, s, C(12)H), 4.2 (1 H,
bs d, OH), 4.4 (1 H, bs d, OH) 7.7 (1 H, bs pst, NH). δC
(100 MHz; d6-DMSO) 12.5, 17.1, 23.1, 23.5, 26.1, 27.0, 27.2,
28.6, 30.3, 31.6, 32.7, 33.0, 33.8, 35.1, 35.2, 35.5, 35.7, 36.3,
41.7, 46.0, 46.2, 47.5, 50.6, 70.0, 71.0, 172.2.
General procedures for the scaled-up synthesis of glyco-
UDCA (5a).19 UDCA (15.7 g, 40 mmol) and EEDQ (80 mmol)
were dissolved in a solution of CH3CN–t-BuOH (3 : 1, v/v) and
the volume was made up to 200 ml. The resulting mixture was
premixed at 50 °C for 10 min, and pumped at 0.500 ml min−1
.
An aqueous solution (200 ml) of glycine (100 mmol) and NaOH
(80 mmol) was premixed at 50 °C for 10 min and pumped at
0.5 ml min−1. The two solutions were joined in a T-piece and
4114 | Org. Biomol. Chem., 2012, 10, 4109–4115
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