A uronic acid analogue of isofagomine lactam as a nanomolar glucuronidase inhibitor

Article abstract of DOI:10.1016/j.tetlet.2012.02.009

The synthesis of (3S,4R,5R)-3,4-dihydroxypiperidin-2-one-5-carboxylic acid ('isofagomine lactam uronate') from d-arabinose is reported. The product is a potent inhibitor in the low nanomolar range (K_i 36 nM) for bovine liver β-glucuronidase.

Full text of DOI:10.1016/j.tetlet.2012.02.009

Tetrahedron Letters 53 (2012) 2045–2047
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A uronic acid analogue of isofagomine lactam as a nanomolar
glucuronidase inhibitor
Emil Lindbäck a, You Zhou a, Óscar López b, José G. Fernández-Bolaños b, Christian Marcus Pedersen a,
Mikael Bols a,
⇑
a Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Kbh Ø, Denmark
b Department of Organic Chemistry, University of Seville, Profesor García González 1, 41012 Seville, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of (3S,4R,5R)-3,4-dihydroxypiperidin-2-one-5-carboxylic acid (‘isofagomine lactam uro-
Received 10 December 2011
Revised 23 January 2012
Accepted 3 February 2012
Available online 9 February 2012
nate’) from
36 nM) for bovine liver b-glucuronidase.
D-arabinose is reported. The product is a potent inhibitor in the low nanomolar range (Ki
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
b-Glucuronidase
b-Glucuronidase inhibitor
(2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl
Bisacetoxyiodobenzene
Selective oxidation
The polar monosaccharide
D
-glucuronic acid (1) plays an impor-
of bovine liver b-glucuronidase (Fig. 1, Table 1).11 A more potent
inhibitor is the uronic acid derivative of isofagomine (3, Ki
79 nM),12 while the uronic azafagomine 4 has intermediary inhibi-
tant role in human biochemistry by forming glucuronide conjugates
with xenobiotics and other undesired and lipophilic substances to
allow them to be excreted (Fig. 1).1 A large number of toxic com-
pounds including many drug metabolites are eliminated safely from
the body as glucuronides before they can cause harm. b-Glucuroni-
dase, a glycosidase, catalyses the hydrolysis of glucuronides back to
1 and aglycone, and this enzyme is therefore potentially counter-
productive to the natural catabolic protection system against xeno-
biotics.2 Indeed the inhibition of b-glucuronidases has recently been
shown to be useful in alleviating drug toxicity in cancer chemother-
apy.3 Therefore potent and selective b-glucuronidase inhibitors
could become important supplement drugs in chemotherapy.
Iminosugars are among the most potent and selective glycosi-
dase inhibitors.4,5 They are monosaccharide analogues in which
the endocyclic oxygen6,7 and/or anomeric carbon8,9 has been re-
placed by nitrogen. Their inhibitory activity stems from their amine
functional group either through their ionic interactions in conjugate
acid form, with carboxylate groups in the enzyme active site or, and
hotly debated, as mimics of a transition state or intermediate. Yet
the neutral lactam analogues of iminosugars are also occasionally
surprisingly potent inhibitors.10 Several iminosugars mimicking 1
have been prepared and found to be b-glucuronidase inhibitors.
tory potency (Ki 1 l
M).13 Recently, an analogue of 3, compound 5
Uronic-1-deoxynojirimycin (2) is a moderate inhibitor (Ki 6.5 lM)
⇑
Corresponding author. Tel.: +45 35320160; fax: +45 35320214.
E-mail address: bols@kemi.ku.dk (M. Bols).
Figure 1.
D-Glucuronic acid (1) and the known nitrogen-containing b-glucuronidase
inhibitors 2–6, amidine 7 and the target compounds 8 and 9.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.02.009

2046
E. Lindbäck et al. / Tetrahedron Letters 53 (2012) 2045–2047
Table 1
Inhibition constants (Ki, nM) of iminosugars 2–6 and 9
Compound
Structure
Bovine liver
b-glucuronidase
2
650011
3
4
5
6
9
7912
100013
Scheme 1. Synthesis of uronic-isofagomine lactam (9).
230014
3215
36
(Fig. 1), with the 2-OH intact and a calystegin-like structure was re-
ported.14 This compound is less potent versus the mammalian en-
zyme (Ki 2.3
enzyme (Ki 60 nM).
Surprisingly
-glucurono-1,5-lactam (6)15 was reported to be a
lM), but very potent against the Escherichia coli
D
much more potent bovine liver b-glucuronidase inhibitor than 2,
which suggests that for this mammalian enzyme, lactams are actu-
ally better inhibitors than amines. We recently prepared the ami-
dine 7,16 and our interest in glucuronidase inhibitors led us to
target the synthesis of amidine 8 and lactam 9. The synthesis of
8 ultimately failed as the product was too unstable, but we present
here the synthesis and inhibitory testing of uronic-isofagomine
lactam 9 which showed it to be a very potent b-glucuronidase
inhibitor.
Scheme 2. Toward the synthesis of uronic-isofagomidine 8.
oxidation of 14 gave 15 in 52–88% yield, after TBDPS protection.
The unpredictable yield of the silyl ester 15 was caused by its
hydrolytic instability during purification by flash chromatography.
Compound 15 was then subjected to TFA-mediated selective
deprotection of the Boc group and subsequently CuCl-mediated
ring-closure in methanol. The copper salt complexes strongly to
the amidine, but treatment with H2S gas precipitated the copper
salt as the sulfide. Subsequent column chromatography gave 16
as its acetate salt in a 51% yield. Removal of the protecting group
from 16 turned out to be more difficult than anticipated. Treatment
of 16 with concentrated hydrochloric acid in 1,4-dioxane or using
other deprotection methods did not give 8 as an analytically pure
compound.
The synthesis of uronic-isofagomine lactam 9 started from
building block 10, itself prepared from D
-arabinose (Scheme 1).16
Treatment of 10 with tetrabutylammonium fluoride (TBAF)
gave the alcohol 11 in an 86% yield.17 Alcohol 11 was then sub-
jected to a Dess–Martin periodinane (DMP)/sodium chlorite oxida-
tion sequence to furnish carboxylic acid 12 in low yield (13%). The
yield was significantly improved when 11 was reacted with
(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) and bisacetoxyi-
odobenzene (BAIB)18 in aqueous acetonitrile (MeCN); this fur-
nished 12 in a 92% yield. Finally, the trans-diacetal and Boc
protecting groups of 12 were removed by treatment with aqueous
trifluoroacetic acid (TFA) to give uronic-isofagomine lactam 9 in a
79% yield (Scheme 1).
Uronic-isofagomine lactam 9 showed inhibition in the low nano-
molar range (with a Ki value of 36 nM) for bovine liver b-glucuron-
idase (Table 1).19 This inhibitor was 181-fold more potent than
As part of this b-glucuronidase inhibitor project, we also tried to
synthesize uronic-isofagomidine 8 (Fig. 1). The synthesis started
from the reported building block 13 (Scheme 2),16 treatment of
which with TBAF gave 14 in high yield (91%). Ruthenium-catalyzed
uronic-1-deoxynojirimycin (2, Ki 6.5
lM), and more than two-fold
as potent as uronic-isofagomine (3, Ki 79 nM). Compound 9 and

E. Lindbäck et al. / Tetrahedron Letters 53 (2012) 2045–2047
2047
D
-glucolactam (6, Ki 32 nM) had almost identical Ki values for bovine
temperature for 30 min, and then the temperature was increased to room
temperature and the mixture stirred for 5.5 h. The solvent was removed under
reduced pressure. Purification of the concentrate by dry column
liver b-glucuronidase.
In summary, we have synthesized a uronic acid derivative of
chromatography (for
a description of this technique see Pedersen, D. S.;
isofagomine lactam 9 from D-arabinose. Compound 9 was found
to be a very potent b-glucuronidase inhibitor in the nanomolar
range (with a Ki value of 36 nM).
Rosenbohm, C. Synthesis 2001, 2431 and references therein, heptane–EtOAc,
1:0 ? 1:1) gave alcohol 11 (315 mg, 86%) as a white foam. Rf: 0.27 (EtOAc–
heptane, 1:1); [a]
D +97 (c 0.3, CH2Cl2); 1H NMR (500 MHz, CDCl3) d 4.28 (d, 1H,
J3,4 = 10.7 Hz, H-3), 3.85 (dd, 1H, J4,5 = 9.3 Hz, H-4), 3.80–3.76 (m, 1H, CHaOH),
3.72–3.66 (m, 3H, CHbOH, H-6a, H-6b), 3.29 (s, 3H, OCH3), 3.22 (s, 3H, OCH3),
2.25–2.20 (m, 2H, H-5, OH), 1.49 (s, 9H, (CH3)3C, Boc), 1.37 (s, 3H, CH3), 1.29 (s,
3H, CH3); 13C NMR (125 MHz, CDCl3) d 167.3 (C-2), 152.8 (C@O), 100.5, 99.3 (C-
20, C-30), 83.9 (C(CH3)3, Boc), 70.2 (C-3), 67.4 (C-4), 62.1 (CH2OH), 48.6, 48.1
(OCH3), 43.9 (C-6), 39.0 (C-5), 28.1 ((CH3)3C, Boc), 17.7, 17.6 (CH3); HRMS (ESP)
m/z calcd for C17H29NNaO8 ([M+Na]+): 398.1791, found: 398.1781.
Acknowledgments
We thank the Danish national research council (FNU), the Direc-
ción General de Investigación of Spain (CTQ 2008 02813), and Junta
of Andalucía (FQM134) for financial support.
(3S,4R,5R,20S,30S)-5-Carboxylic
dioxy)-1-(tert-butyloxycarbonyl)piperidin-2-one (12). To
acid-3.4-(20,30-dimethoxybutylene-20,30-
solution of
a
alcohol 11 (111 mg, 0.296 mmol) in MeCN–H2O (3:2, 2 mL) at room
temperature was added TEMPO (13.6 mg, 0.0887 mmol) and BAIB (238.4 mg,
0.74 mmol). The reaction mixture was left stirring for 4.5 h, and then the
solvent was removed under reduced pressure. Purification of the concentrate
by dry column chromatography (CHCl3–MeCN, 1:0 ? 0:1) furnished carboxylic
Supplementary data
Supplementary data (copies of 13C and 1H NMR spectra of 9 as
well as synthetic procedures for compounds 14, 15 and 16) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2012.02.009.
acid 12 (106 mg, 92%) as a white solid. Rf: 0.52 (MeCN); [a] +188 (c 0.15,
D
CH2Cl2); 1H NMR (500 MHz, CDCl3) d 4.35-4.31 (m, 2H, H-3, H-4), 4.27 (dd, 1H,
J6a,5 = 5.4 Hz, J6a,6b = 14.1 Hz, H-6a), 3.78 (dd, 1H, J6b,5 = 6.7 Hz, H-6b), 3.31 (s,
3H, OCH3), 3.27 (s, 3H, OCH3), 3.02–2.98 (m, 1H, H-5), 1.51 (s, 9H, (CH3)3C, Boc),
1.40 (s, 3H, CH3), 1.32 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3) d 174.7, 166.6
(C-2, CO2H), 151.7 (C@O), 100.7, 99.8 (C-20, C-30), 84.2 (C(CH3)3, Boc), 69.4, 66.5
(C-3, C-4), 48.7, 48.3 (OCH3), 43.0 (C-5), 42.3 (C-6), 28.1 ((CH3)3C, Boc), 17.6
(2ꢀCH3); HRMS (ESP) m/z calcd for C17H27NNaO9 ([M+Na]+): 412.1584, found
412.1606.
References and notes
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(3S,4R,5R)-5-Carboxylic acid-3,4-dihydroxypiperidin-2-one (9). A solution of
compound 13 (58 mg, 0.149 mml) in TFA–H2O (19:1, 4 mL) was stirred at room
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and the residue was purified by column chromatography (50 mN aqueous HCl–
MeCN, 1:199 ? 1:19 ? 1:4) to furnish the title compound (20.5 mg, 79%) as a
colorless oil. Rf: 0.49 (50 mN aqueous HCl–MeCN, 7:13); 1H NMR (500 MHz,
D2O) d 4.00–3.95 (m, 2H, H-3, H-4), 4.43 (dd, 1H, J6a,5 = 5.8 Hz, J6a,6b = 12.9 Hz,
H-6a), 3.34 (dd, 1H, J6b,5 = 9.9 Hz, H-6b), 2.96–2.91 (m, 1H, H-5); 13C NMR
(125 MHz, D2O) d 174.9, 173.2 (C-2, CO2H), 72.1, 71.0 (C-3, C-4), 46.0 (C-5),
39.8 (C-6); HRMS (ES) m/z calcd for C6H9NNaO5 ([M+Na]+): 198.0378, found:
198.0374.
18. van den Bos, L. J.; Litjens, R. E. J. N.; van den Berg, R. J. B. H. N.; Overkleeft, H. S.;
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19. The enzyme inhibition constant Ki was determined in the following manner:
Measurement of inhibition of bovine liver b-glucuronidase was carried out in
50 mM acetate buffer (pH 5.0) at 37 °C. The substrate used was
12. Igarashi, Y.; Ichikawa, M.; Ichikawa, Y. Tetrahedron Lett. 1996, 37, 2707.
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phenolphthalein b-
reaction mixture was transferred to
D
-glucuronide. Each second minute, 0.100 mL of the
cuvette consisting of 0.900 mL of
a
0.100 M glycine–NaOH buffer (pH 12.0). The formation of phenolphthalein was
monitored by measurement of the absorbance at 553 nm. Initial velocities
were calculated from the slopes from each reaction and used to construct two
Hanes plots ([S]/v vs [S]), one with and one without inhibitor. From the
Michaelis–Menten constants K0M and KM the Ki value was calculated.
15. Niwa, T.; Tsuruoka, T.; Inouye, S.; Koeda, T.; Naito, Y.; Koeda, T.; Niida, T. J.
Biochem. 1972, 72, 207.
9
16. Lindbäck, E.; Lopez, O.; Fernández-Bolaños, J. G.; Sauer, S. P. A.; Bols, M. Org.
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17. Compounds were prepared using the following procedures:
(3S,4R,5R,20S,30S)-3.4-(20,30-Dimethoxybutylene-20,30-dioxy)-5-
hydroxymethyl-1-(tert-butyloxycarbonyl)piperidin-2-one
(11).
To
a
solution of silyl ether 10 (600 mg, 0.978 mmol) in THF (9.5 mL) at 0 °C was
slowly added TBAF (1.08 mL, 1 M in THF). The mixture was stirred at this