Synthesis of a biotin-tagged photoaffinity probe of 2-azetidinone cholesterol absorption inhibitors

Article abstract of DOI:10.1016/S0968-0896(03)00047-6

The design and synthesis of a biotin-tagged photoreactive analogue C-4 of the cholesterol absorption inhibitor Ezetimibe is described. Photoaffinity labeling of intestinal brush border membrane vesicles with C-4 and subsequent streptavidin-biotin chromato

Full text of DOI:10.1016/S0968-0896(03)00047-6

Bioorganic & Medicinal Chemistry 11 (2003) 1639–1642
Synthesis of a Biotin-Tagged Photoaffinity Probe of
2-Azetidinone Cholesterol Absorption Inhibitors
Wendelin Frick, Andrea Bauer-Schafer, Jochen Bauer, Frank Girbig,
Daniel Corsiero, Hubert Heuer and Werner Kramer*
Aventis Pharma Deutschland GmbH, Disease Group Metabolic Diseases Industriepark Hochst,
¨
Building G 879D-65926, Frankfurt am Main, Germany
Received 5 November 2002; accepted 17 January 2003
Abstract—The design and synthesis of a biotin-tagged photoreactive analogue C-4 of the cholesterol absorption inhibitor Ezetimibe
is described. Photoaffinity labeling of intestinal brush border membrane vesicles with C-4 and subsequent streptavidin–biotin
chromatography leads to selective extraction of a 145 kDa integral membrane protein as the molecular target for cholesterol
absorption inhibitors.
# 2003 Elsevier Science Ltd. All rights reserved.
Intestinal cholesterol absorption is a major regulator of
1
serumcholesterol levels. Consequently, inhibition of
intestinal cholesterol absorption is a novel pharmacolo-
gical mechanism to regulate serum cholesterol levels.
Specific inhibitors for cholesterol absorption found
accidentally have proven their clinical efficacy.2,3 The
known cholesterol absorption inhibitors belong to two
different chemical classes, the 2-azetidinones 1,2 and the
sterol glycosides 3,4 (Fig. 1), both classes showing pro-
found structure–activity relationships.4,5 Despite their
pharmacological efficacy, the molecular mode of action
of these compounds and the mechanism of intestinal
cholesterol absorption is still unknown. For the dis-
covery of novel cholesterol absorption inhibitors and
optimisation of their structure–activity relationships the
availability of the molecular target for cholesterol
absorption inhibitors is a prerequisite. By photoaffinity
labelling with radioactively labelled photoreactive
2-azetidinone cholesterol absorption inhibitors6,7 we
succeeded in the identification of an integral 145-kDa
membrane protein as the molecular target for choles-
terol absorption inhibitors localized in the brush border
membrane of absorptive enterocytes from the rabbit
small intestine.6 An efficacious purification procedure
for the 145-kDa protein is necessary to determine its
amino acid sequence and to clone the protein. We
Figure 1. Structures of known cholesterol absorption inhibitors (1–4)
and structures of two photoreactive analogous 2-azetidiones (C-1 and
C-4).
*Corresponding author. Tel.: +49-69-305-3557; fax +49-69-305-
13333; e-mail: werner.kramer@aventis.com
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00047-6

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W. Frick et al. / Bioorg. Med. Chem. 11 (2003) 1639–1642
therefore aimed to extract the 145 kDa from solubilized
membrane proteins by streptavidin–biotin affinity chro-
matography using a photoreactive biotinylated deriva-
tive of the highly efficacious cholesterol absorption
inhibitor Sch 58235 (Ezetimibe) (Fig. 1). Structure–
activity relationships of 2-azetidinone cholesterol
absorption inhibitors4 revealed that in the para-position
of the N-phenylring rather bulky groups such as azido-
benzoyl6,7 or even fluorescent reporter groups8 can be
introduced conserving the pharmacological activity to
inhibit intestinal cholesterol absorption. We therefore
decided to use this position for the attachment of pho-
toreactive groups to allow a covalent crosslinking of the
probe to its binding protein as well as of the biotinyl
group for the subsequent streptavidin extraction of the
photocrosslinked target protein(s) (Scheme 1).
Synthesis of Biotinylated Photoaffinity Probe C-4
Our synthesis of the biotin-labeled photoaffinity com-
pound 7 (C-4) is based on an asymmetric synthesis of
ezetimibe (Sch 58235). The enatiomerically pure 50-(S)-t-
butyl-dimethyl-silyloxy-50-(phenyl) valeroyl-5-(S)-phe-
nyloxazolidinone 8 was treated with Hunig’s base, tri-
methylsilyl chloride and titanium tetrachloride to give
the intermediate b-amino acyloxazolidinone as an 2:1
mixture of diastereomers. This mixture of diastereomers
was used for the cyclization reaction with bis-tri-
methylsilyl acetamide (BSA) and catalytic amount of
tetrabutylammonium fluoride (TBAF) to get azetidi-
none 10. The azetidinone 10 was further used as a dia-
stereomeric mixture (it was not possible to separate this
mixture of diastereomers at any step, so the mixture was
used for the whole synthesis. In the reaction scheme 1
only the major diastereomer is shown). For reduction of
the nitrogroup of azetidinone 10, the compound was
hydrogenated with palladiumon charcoal (10% Pd).
Removal of the silylprotecting group was carried out in
an homogeneous mixture of tetrahydrofurane and aqu-
eous 2 N HCl at roomtemperature. The anilide 11 was
then coupled twice, first with Fmoc-protected 5-amino-
valerianic acid and then with Fmoc-p-azido-phenyl
alanine. For this reaction we have used O-((ethoxy-
carbonyl) cyanomethyleneamino),N,N,N,N-tetramethyl-
uronium-tetraflouroborate (TOTU) as coupling reagents.
Finally the photolabeled compound 13 was coupled
with d-biotin-N-succinimidyl ester which is commercial
available fromBachem(Germany) to get the double
labeled (with biotin and azido group) compound 7 (C-4)
as a 2:1 mixture of diastereomers.9,10
Scheme 1. Synthesis of biotin-labeled photoaffinity probe C-4: (a)
DIPEA, TMSCl, TiCl4, CH2Cl2 (74%); (b) BSA, MTB-ether, TBAF
(cat) (70%); (c) H2, Pd/C, 2 h, 5 bar, ethyl acetate (86%); (d) aqueous
2 N HCl, THF, 2 h, rt; (e) 5-(Fmoc-amino)-valerianic acid, TOTU,
ethyl (hydroxyimino)-cyanoacetate, NEM (all from Fluka), DMF, 1 h,
rt (41%); (f) NHEt2, DMF, 1 h, rt (56%); (g) Fmoc-p-azido-Phe-OH
(Bachem), TOTU, ethyl (hydroxyimino)-cyanoacetate, NEM (all
fromFluka), DMF, 1 h, rt (82%); (g) NHEt 2, DMF, 1 h, rt (19%); (i)
d-biotin-N-succinimidyl ester (Bachem), DMF, 1 h, rt (55%).
measured by an increased fecal excretion and a
decreased hepatic content of the administered radio-
actively labeled cholesterol was inhibited by the probe
(Table 1); its biological activity was, however, lower than
that of Ezetimibe indicating a negative influence of the
attached reporter groups on pharmacological efficacy.
Photoaffinity labeling of the 145 kDa protein by the
radiolabeled 2-azetidinone cholesterol absorption inhi-
bitor C-1 was concentration-dependently and specifi-
cally inhibited by the presence of C-4 indicating that the
biotin-tagged Ezetimibe photoaffinity probe C-4 specifi-
cally interacts with the 145 kDa binding protein for cho-
lesterol absorption inhibitors. Consequently, C-4 should
be suited for purification of the Ezetimibe target protein
by streptavidin–biotin chromatography. After photo-
affinity labeling of rabbit small intestinal brush border
membrane vesicles with C-4 membrane proteins were
solubilized and streptavidin–agarose beads were added.
After extensive washing, proteins retained by the strepta-
vidin matrix were eluted either with buffer containing
6 mM biotin or under denaturating conditions with SDS-
Biological Characteristics of the Biotinylated Photo-
affinity Probe for 2-Azetidinone Cholesterol Inhibitors
In order to evaluate whether the introduction of the
photolabile and the biotinyl reporter groups into the
Ezetimibe molecule would biologically be tolerated, the
compound was administered to NMRI mice and its
effect on intestinal cholesterol absorption was measured
using the Zilversmit/Hughes method11 as described.6 As
with Ezetimibe, intestinal cholesterol absorption as

W. Frick et al. / Bioorg. Med. Chem. 11 (2003) 1639–1642
1641
Table 1. In vivo activity of the biotin-tagged photoreactive choles-
terol absorption inhibitor C-4 in NMRI mice6,11
In conclusion, the biotinylated photoaffinity probe C-4
of 2-azetidinone cholesterol absorption inhibitors is well
suited to enable an efficient enrichment and purification
of the target protein for cholesterol absorption inhibi-
tors. Final purification, sequencing and cloning of the
145 kDa protein will improve our understanding of the
mechanism of intestinal cholesterol absorption and will
help to identify novel cholesterol absorption inhibitors.
Compd
14C-Cholesterol
excretion in
feces (nCi)
14C-Cholesterol
uptake by the
liver (nCi)
Control
280
78
Ezetimibe (0,1 mg/mouse)
Ezetimibe (1 mg/mouse)
C-4 (1 mg/mouse)
480 (+ 71%)
490 (+ 75%)
370 (+ 32%)
15 (À81%)
4 (À95%)
38 (À51%)
0.5 mL of a solution of the indicated amount of the cholesterol
absorption inhibitors in 0.5% methylcellulose/5% Solutol1 were
administered by gavage to male NMRI mice (four animals per group)
followed by 0.25 mL of Intralipid1 solution containing 0.34 mCi [14C]
cholesterol per animal. After 24 h, the amount of radioactivity in feces
and liver was determined. (The data represent the radioactivity found
in feces and liver of the four animals in each treatment group).
Acknowledgements
The authors greatly thank Meike Scharnagl, Susanne
Nicolaus and Klaus Bock for excellent assistance.
References and Notes
solutions.12 SDS-gel electrophoresis of the eluates
revealed that a 145 kDa protein was selectively extracted
by the streptavidin beads (Fig. 2, lane B). No 145 kDa
protein could be detected if ultraviolet irradiation was
omitted (Fig. 2, lane A) thereby preventing a covalent
attachment of the photolabile ligand analogue to its
specific binding protein(s). It is remarkable to detect
such a clear protein band after photoaffinity labelling
since only a small percentage of the activated nitrene of
the ligand crosslinks with amino acid moieties in the
binding site of the target protein. Furthermore, only
from brush border membranes isolated from the organs
where intestinal cholesterol absorption occurs, the small
intestine, the 145 kDa protein could be extracted,
whereas from stomach, cecum, colon, rectum, kidney,
liver or fat tissue plasma membranes no 145 kDa pro-
tein could be enriched.
1. Wilson, D.; Rudel, L. L. J. Lipid Res. 1994, 35, 943.
2. Bays, H. E.; Moore, P. B.; Drehobl, M. A.; Rosenblatt, S.;
Toth, P. D.; Dujovne, C. A.; Knopp, R. H.; Lipka, L. J.; Le
Beaut, A. P.; Yang, B.; Mellars, L. E.; Cuffie-Jackson, C.;
Vetri, M. D. Clin. Therapeutics 2001, 23, 1209.
3. Harris, W. S.; Windsor, S. L.; Newton, F. A.; Gelfand,
R. A. Clin. Pharmacol. Ther. 1997, 61, 385.
4. Clader, J. W.; Burnett, D. A.; Caplen, M. A.; Domalski,
M. S.; Dugar, S.; Vaccaro, W.; Sher, R.; Browne, M. E.; Zhao,
H.; Burrier, R. E.; Salisbury, B.; Davis, H. R., Jr. J. Med.
Chem. 1996, 39, 3684.
5. Harwood, H. J., Jr.; Chandler, C. E.; Pellarin, L. D.; Ban-
gerter, F. W.; Wilkins, R. W.; Long, C. A.; Cosgrove, P. G.;
Malinow, M. R.; Marzetta, C. A.; Pettini, J. L.; Savoy, Y. E.;
Mayne, J. T. J. Lipid Res. 1993, 34, 377.
6. Kramer, W.; Glombik, H.; Petry, S.; Heuer, H.; Schafer,
H. L.; Wendler, W.; Corsiero, D.; Girbig, F.; Weyland, C.
FEBS Lett. 2000, 487, 293.
7. Kramer, W., Glombik, H. International Patent Application
WO 02/18432, 2000.
8. Altmann, S. W.; Davis, H. R., Jr.; Yao, X.; Laverty, M.;
Compton, D. S.; Zhu, L.-J.; Crona, J. H.; Caplen, M. A.;
Hoss, L. M.; Tetzloff, G.; Priestley, T.; Burnett, D. A.; Stra-
der, C. D.; Graziano, M. P. Biochim. Biophys. Acta 2002,
1580, 77.
9. All compounds were characterized by 1H NMR, and the
purity by LC–MS.
10. Synthesis of Compound C-4(7). 3-{5-( tert-Butyl-dimethyl-
silanyloxy)-2-[(4-methoxy-phenyl)-(4-nitro-phenylamino)-methyl]-
5-phenyl-pentanoyl}-4-phenyl-oxazolidin-2-on (10). To 5.4 g
(12.0 mmol) of compound 8 and 6.2 g (24 mmol) of compound
9 in 135 mL of methylenchloride 8 mL of diisopropylethyla-
mine is added at 10 ꢀC and 4.8 mL of trimethylsilylchloridie is
added dropwise. After 1 h, 14 mL of a 1 molar solution of
titantetrachloride in methylenchloride is added dropwise at
À10 ꢀC. It is stirred for 3 h at À10 ꢀC and further 12 h at
À30 ꢀC stored without stirring. Afterwards 8 mL of acetic acid
and 140 mL of a 7% aqueous solution of tartaric acid is added
and stirred for further 2 h at roomtemperature. After addition
of 50 mL of a 20% aqueous solution of sodium hydro-
gensulfite it is stirred for another 1 h and extracted with
methylenchloride. The organic phase is dried by magne-
siumsulfate, evaporated and purified by chromatography on
silica/ethylacetate/heptane=1/3!1/1. 6.3g (74%) of titan
reaction product is obtained in formof a light yellow solid
compound: C40H47N3O7Si (709.92) MS (ESI+) 710 (M+H+).
A mixture comprising 6.1 g (8.6 mmol) of titan reaction product,
7.3 mL of bistrimethylsilylacetamide, 0.5 g of tetra-
butylammoniumfluorid and 100mL of tert-butylmethylether is
Figure 2. Photoaffinity labelling of rabbit ileal brush border mem-
brane vesicles with C-4 and following streptavidin–biotin extraction.
200 mg of rabbit ileal brush border membrane vesicles were incubated
with 200 mM of photoaffinity probe C-4 without (A) or with UV-irra-
diation (B). After solubilisation of membrane proteins, the proteins
containing the biotin-tagged cholesterol absorption inhibitor C-4 were
extracted with streptavidin–agarose beads. Bound proteins were
released with 6 mM biotin and subsequently analysed by SDS-gel
electrophoresis and staining with Coomassie-Blue R-250.

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W. Frick et al. / Bioorg. Med. Chem. 11 (2003) 1639–1642
stirred under an argon atmosphere for 10 h at room tempera-
ture. After finishing of the reaction 5 mL of acetic acid are
added slowly by cooling with ice and evaporated. The residue
is separated by chromatography on silica gel (ethylacetate/
heptan=1/2). 3.3 g (70%) of product 10 is obtained in formof
a light yellow compound in solid form: C31H38N2O5Si (546.74)
MS (ESI+) 547.3 (M+H+). 1-(4-Amino-phenyl)-3-(3-hydroxy-
Amino-3-(4-azido-phenyl)-propionylamino]-pentanoic acid{4-[3-
(3-hydroxy-3-phenyl-propyl)-2-(4-methoxy-phenyl)-4-oxo-azeti-
din-1-yl]-phenyl}-amid (13). 200 mg (0.40 mmol) of product 12
and 340 mg (0.79 mmol) of Fmoc-p-azido-Phe-OH (Bachem)
are solved in 4 mL DMF (dimethylformamide) and reacted
according to production of product 12. 42 mg of compound 13
is obtained in formof an amorphic solid copmound:
C39H43N7O5 (689.82) MS (ESI+) 690.3 (M+H+). 5-(2-Oxo-
3-phenyl-propyl)-4-(3-methoxy-phenyl)-azetidin-2-on (11).
A
reaction is performed with 3.0 g (5.5 mmol) of product 10 in
50 mL ethylacetate and 1.0 g of palladium charcoal 10% for
2 h at 5 bar of a hydrogen atmosphere using an autoclave. The
reaction solution is filtrated, evaporated and separated by
chromatography on silica gel (methylenchloride/metha-
nol=10/1). 2.4 g (86%) of the anilide is obtained in formof a
colourless compound in solid form: C31H40N2O3Si (516.76)
MS (ESI+) 517.4 (M+H+). 15 mL of 2 N aqueous hydro-
chloric acid are added to 2.3 g of the anilide in 20 mL of tet-
rahydrofuran and stirred for 2 h. An aqueous solution of
sodiumhydrogencarbonate is added to the reaction and
extracted by ethylacetate. The organic phase is dried by mag-
nesiumsulfate, evaporated and purified by chromatography
on silica gel (ethylacetate/heptan=1/1!1/0). 1.1 g of product
11 is obtained in form of a colorless compound in solid form:
C25H26N2O3 (402.50) MS (ESI+) 403.2 (M+H+). 5-Amino-
hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoic
acid-[2-(4-
azido-phenyl)-1-(4-{4-[3-(3-hydroxy-3-phenyl-propyl)-2-(4-meth-
oxy-phenyl)-4-oxo-azetidin-1-yl]-phenylcarbamoyl}-butylcarba-
moyl)-ethyl]-amid (7). 40 mg (0.058mmol) of compound 12 and
60 mg of d-biotinyl-N-hydroxysuccinimide (Bachem) are solved
in 0.5 mL DMF (dimethylformamide). It is evaporated after 1 h
at roomtemperature. The residue is purified by flash chroma-
tography (methylenchloride/methanol/concd ammonia 30:5:1).
29 mg (55%) of compound 7 is obtained in form of an amorphic
solid compound as
a
2:1 mixture of diastereomers:
C49H57N9O7S (916.12) MS (ESI+) 916.6 (M+H+).
11. Zilversmit, D. B.; Hughes, C. B. J. Lipid Res. 1974, 15,
465.
12. Photoaffinity labeling and streptavidin-biotin affinity chro-
matography. Rabbit ileal brush border membrane vesicles
(200 mg of protein) prepared as described13 were photolabelled
with 200 mM of C-4 in 10 mM Tris/Hepes buffer (pH 7.4)/
100 mM NaCl/100 mM mannitol by ultraviolet irradiation for
30 s at 254 nmin a Rayonet RPR 100 photochemical reactor
pentanoic
oxy-phenyl)-4-oxo-azetidin-1-yl]-phenyl}-amide
acid-{4-[3-(3-hydroxy-3-phenyl-propyl)-2-(4-meth-
(12). 0.8 g
(2,0 mmol) of product 11 and 1.35 g (4.0 mmol) of 5-(Fmoc-
amino)-valerianic acid (Fluka) are solved in 15 mL of DMF
(dimethylformamide). Stepwise is added the following: 4.8 g of
TOTU (Fluka), 1.6 g of oxime (hydroxyimino-cyanoacetic
acid-ethylester; Fluka) and 5.5 mL of NEM (4-ethyl-morpho-
line). After 1 h at roomtemperature, the reaction is diluted
with 100 mL of ethylacetat and washed for three times with
water. The organic phase is dried by MgSO4, filtrated and
evaporated. The residue is purified by flash chromatography
(ethylacetate/n-heptan 2:1). 0.58 g (41%) of coupling product
˚
equipped with 4 RPR 2537 A lamps. After washing membrane
proteins were solubilized with 250 mL of 10 mM Tris/Hepes
buffer (pH 7.4)/75 mM KCl/5 mM MgCl2/1 mM EGTA/1 mM
DTT/1% (w/v) n-octylglucoside/1% (w/v) Triton X-100/1 mM
Pefabloc and kept at 4 ꢀC for 60 min. After centrifugation the
supernatant containing the solubilized membrane proteins was
added to 50 mL of streptavidin-agarose beads and kept under
stirring at 4 ꢀC for 2 h. After centrifugation, the beads were
washed three times with 250 mL of 10 mM Tris/Hepes buffer
(pH 7.4)/300 mM mannitol/1% n-octylglucoside/4 mM PMSF/
4 mM iodoacetamide/4 mM EDTA at 4 ꢀC. Streptavidin-
bound proteins were eluted with 250 mL of the above buffer
containing 6 mM biotin and stirring for 60 min at 4 ꢀC. After
centrifugation, proteins were precipitated with chloroforme/
methanol and analysed by SDS gel electrophoresis..13
is obtained in form of
a amorphic solid compound:
C45H45N3O6 723.8) MS (ESI+) 724.4 (M+H+). 570 mg
(0.78 mmol) of the coupling product and 0.8 mL of diethyl-
amine are solved in 5 mL of DMF (dimethylformamide). It is
evaporated after 1 h at roomtemperature. The residue is pur-
ified by flash chromatography (methylenchloride/methanol/
concd ammonia 30:10:3) 220 mg (56%) of product 12 is
obtained in form of an amorphic solid compound:
C30H35N3O4 (501.63) MS (ESI+) 502.3 (M+H+). 5-[2-
13. Kramer, W.; Girbig, F.; Gutjahr, U.; Kowalewski, S.;
Jouvenal, K.; Muller, G.; Tripier, D.; Wess, G. J. Biol. Chem.
1993, 268, 18035.