Synthesis of iodinated biochemical tools related to the 2-azetidinone class of cholesterol absorption inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00750-8

The discoveries of Sch 48461 and Sch 58235 and their novel pharmacology of inhibition of cholesterol absorption have prompted efforts to determine their biological mechanism of action (MOA). To this end, a series of radioiodinated analogues with good to e

Full text of DOI:10.1016/S0960-894X(01)00750-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 311–314
Synthesis of Iodinated Biochemical Tools Related to the
2-Azetidinone Class of Cholesterol Absorption Inhibitors
Duane A. Burnett,* Mary Ann Caplen, Martin S. Domalski, Margaret E. Browne,
Harry R. Davis, Jr. and John W. Clader
Schering-Plough Research Institute, 2015 Galloping Hill Road MS 2800, Kenilworth, NJ 07033-0539, USA
Received 20 September 2001; accepted 1 November 2001
Abstract—The discoveries of Sch 48461 and Sch 58235 and their novel pharmacology of inhibition of cholesterol absorption have
prompted efforts to determine their biological mechanism of action (MOA). To this end, a series of radioiodinated analogues with
good to excellent in vivo activity have been designed and synthesized as single enantiomers. They are structurally consistent with the
allowable SAR of the 2-azetidinone class of cholesterol absorption inhibitors. # 2002 Elsevier Science Ltd. All rights reserved.
Recently, we described the discovery of a very potent
class of cholesterol absorption inhibitors (CAI) typified
by the original lead compound in this series, Sch 48461
in Figure 1.1 Our SAR development in this program
revealed the importance of side-chain metabolic hydroxyl-
discuss the design and preparation of high affinity radio-
labeled compounds, while the synthesis of related tools
will be presented elsewhere.8
Based on our extensive SAR development of the azeti-
dinone CAI series, we knew that the two best structural
sites for elaboration while maintaining in vivo activity
were the N-aryl ring and the pendant aryl ring. For
sensitivity reasons, our initial binding studies required
radioiodinated analogues and thus the targets 3 and 4
came to the fore. Compound 3 became our first target
because its preparation could serve to further our SAR
development in that region, proceeding through an
intermediate diazonium ion. The compound was prepared
in a straightforward manner as shown in Scheme 1.
ation to activity and led to the discovery of our current
2,3
clinical candidate, 2 (Sch58235, ezetimibe).
While
these 2-azetidinones are extremely potent inhibitors of
cholesterol absorption in vivo, the precise biological
mechanism by which this inhibition takes place has yet
to be discovered.4 Data presented previously suggest
that there is a molecular target for these compounds,
which we have designated the Cholesterol Absorption
Inhibitor Binding Protein (CAIBP). Relying on the
large SAR database that we subsequently developed in
this series,5 we have carefully designed and prepared a
number of biochemical tools for the investigation of this
mechanism.6 The scope of these tools include high
affinity radioactive or fluorescent analogues for binding/
affinity applications, crosslinking reagents for photo-
affinity labeling experiments, and biotinylated deriva-
tives for affinity chromatography applications. Our
initial approachwas to use radiolabeled binding com-
pounds for the identification of subcellular localization
sites in intestinal cells.7 Once we had some knowledge of
where the compounds were binding, we hoped to iden-
tify any specific proteins that had high affinity for this
class of CAIs using other techniques. This paper will
*Corresponding author. Fax:+1-908-740-7152; e-mail: duane.
burnett@spcorp.com
Figure 1. CAI derivatives.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00750-8

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D. A. Burnett et al. / Bioorg. Med. Chem. Lett. 12 (2002) 311–314
Phenyl valeric acid was nitrated predominately in the
para position. The resulting acid was converted to its
acid chloride and subjected to ketene-imine condensa-
tion conditions to generate the requisite trans b-lactam.9
Reduction of the nitro group and subsequent diazotiza-
tion gave the intermediate diazonium salt, which was
not isolated, but converted directly to aryl iodide 3.10,11
The regioisomeric aryl iodide 4 was prepared in a
somewhat simpler manner using analogous ketene-
imine chemistry and the corresponding p-methoxy-
benzylidene-4-iodoaniline (50% yield, 12:1 trans/cis).
Our synthesis of these iodinated analogues is based on
an asymmetric synthesis of Sch 58235 a0nd is shown in
Scheme 2.14 The enantiomerically pure 5 -(S)-acetoxy-50
-(4-fluorophenyl)valeroyl-5-(S)-phenyloxazolidinone, 11,
was treated withtitanium tetrachloride, Hu nig’s base, and
the acetoxy protected imine to give the intermediate b-
amino acyloxazolidinone. The major diastereomer was
purified to homogeneity by crystallization and then
cyclized in two steps by first silylation withbis-
trimethylsilylacetamide followed by treatment with a
catalytic amount of tetrabutylammonium fluoride. In
the workup the phenolic acetate group was lost resulting
in a single enantiomerically pure 2-azetidinone, 13, after
crystallization. Base hydrolysis revealed the benzylic
hydroxyl group to give the desired product 6. Analo-
gously, the pendant bromophenylpropyl analogue, 15,
was prepared from the bromophenyl oxazolidinone 12.
In order to generate the radioiodinated versions of these
compounds for binding studies, we next converted these
two analogues to their tributylstannyl derivatives by
palladium mediated stannylation in refluxing toluene.
Iodination was achieved by treatment with NaI and
Iodobeads1 or Iodogen1 as a mild oxidant in a pH 5.8
phosphate buffered ethanolic solution. Compounds 5
and 6 could both be prepared efficiently by this method.
Lacking an in vitro assay to gauge our success at designing
molecules withhighaffinity, our efforts were forced to rely
on an in vivo assay of cholesterol absorption to qualita-
tively assess the pharmacological summation of affinity,
absorption, distribution, metabolism, and excretion.12 We
felt confident that if an analogue displayed good in vivo
activity, then its affinity for the receptor must be high. The
in vivo data for these compounds in our cholesterol fed
hamster assay is shown in Table 1. Compounds 3 and 4
were bothactive in vivo, withthe N-iodophenyl ana-
logue 4 having slightly better potency.
The assumptions made in our assessment of the affinity
of these compounds for the putative CAIBP required
that any metabolites formed in vivo be less potent than
the parent. In fact, this assumption proved to be false.
In our studies leading to the discovery of Sch 58235, we
determined that metabolism of Sch 48461 primarily
occurred at the C-4 methoxyphenyl ether.3,13 Demethyl-
ation at this site generated the corresponding phenol,
which was about equipotent with the parent compound.
In addition, oxidation at the benzylic 30 position to
generate the analogous hydroxyl led to derivatives that
were as muchas two orders of magnitude more potent
than the parent. Although this extensive metabolism
occurred to only a small percentage in our animal
models, the enhanced activity of the metabolites could
potentially account for nearly all our in vivo activity.
Thus the design of our affinity ligands needed to reflect
this discovery. Our next generation of iodinated ligands
was therefore designed to resemble the more potent Sch
58235, while introducing the iodine atom at the two
previous sites of substitution.
Finally, we needed to address the issue of the bio-
relevant form of our tools. We had discovered that Sch
58235 exists predominately as its glucuronide in vivo.3
While this metabolite was not unexpected, in the
Table 1. In vivo activity of CAIs in a cholesterol-fed hamster assay
Compd
% Redn HCE @ dose
(mg/kg/day)
ED50
(mg/kg/day)
1 (À)
2 (À)
3 (ꢁ)
4 (ꢁ)
5
À93@10
À100@1
À56@50
À82@50
À54@1
À63@1
À54@1
2.2
0.04
na
na
na
6
22
0.49
na
na, not applicable.
Scheme 2. Synthesis of iodinated analogues of Sch 58235: (a) imine,
TiCl4, i-Pr2NEt, CH2Cl2 (51%); (b) BSA, toluene; (c) TBAF (cat)
(93%); (d) LiOH, THF, H2O (56%); (e) (Bu3Sn)2, Pd(Ph3P)4, toluene,
Á (47%); (f) NaI, iodobeads1, EtOH, pH 5.8 buffer (81%).
Scheme 1. Preparation of pendant aryliodide analogue, 3: (a) HNO3,
H2SO4, À10 ꢀC (28%); (b) (i) (COCl)2 (ii) ArCH¼NAr, Bu3N, tol, Á
(51%); (c) H2, Pd/C (97%); (d) (i) HOAc, NaNO2, H2O; (ii) KI (72%).

D. A. Burnett et al. / Bioorg. Med. Chem. Lett. 12 (2002) 311–314
313
absence of a clear understanding of the mechanism of
action, we could preclude neither the free phenol nor the
glucuronide as the bioactive species. We therefore
required the corresponding iodinated glucuronide of
our derivative 6 for evaluation in our studies. The
obvious choice to minimize the number of ‘hot’ steps in
our synthesis would be to prepare the corresponding
glucuronide of the N-arylstannane. Efforts along these
lines revealed a sensitivity of the arylstannane to the
acid moiety of the glucuronide. Thus we prepared the
analogous methyl ester 19 by glycosylation of the phe-
nol 13 using BF3-mediated trichloroimidate chemistry
on the fully protected sugar, 18, as shown in Scheme
3.13c Hydrolysis of all the acetate groups with KCN and
stannylation of the resulting methyl ester gave the
desired arylstannane 20. Iodination and subsequent
hydrolysis of the methyl ester proceeded to 22 without
problems. This longer route to the product was suffi-
cient to provide radioiodinated material in high yield
and purity, even though it requires the inclusion of a
second deprotection step in a ‘hot’ process.
localization studies by our biology group. Results from
those studies will be presented in due course.
Acknowledgements
The authors would like to thank Drs. Wayne Vaccaro,
William Greenlee, Michael Czarniecki, T. K. Thiru-
vengadam, and Michael Green for helpful discussions.
We would like to thank Doug Compton, Lizbeth Hoos,
Glen Tetzloff, and Pradip Das for technical assistance.
References and Notes
1. Burnett, D. A.; Caplen, M. A.; Davis, H. R., Jr.; Burrier,
R. E.; Clader, J. W. J. Med. Chem. 1994, 37, 1733.
2. Rosenblum, S. R.; Huynh, T.; Afonso, A.; Davis, H. R.,
Jr.; Yumibe, N.; Clader, J. W.; Burnett, D. A. J. Med. Chem.
1998, 41, 973.
3. Van Heek, M.; France, C. F.; Compton, D. S.; McLeod,
R. L.; Yumibe, N.; Alton, K. B.; Sybertz, E. J.; Davis, H. R.,
Jr. J. Pharmacol. Ext. Ther. 1997, 283, 157.
4. Salisbury, B. G.; Davis, H. R.; Burrier, R. E.; Burnett,
D. A.; Boykow, G.; Caplen, M. A.; Clemmons, A. L.; Comp-
ton, D. S.; Hoos, L. M.; McGregor, D. G.; Schnitzer-Polok-
off, R.; Smith, A. A.; Weig, B. C.; Zilli, D. L.; Clader, J. W.;
Sybertz, E. J. Atherosclerosis 1995, 115, 45.
5. 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.
6. Our initial targets and chemistry were first disclosed at the
Sixth Meeting of the French American Chemical Society,
Tucson, AZ, March1997.
7. Van Heek, M.; Farley, C.; Compton, D. S.; Hoos, L.;
Alton, K. B.; Sybertz, E. J.; Davis, H. R., Jr. Br. J. Pharmacol.
2000, 129, 1748.
8. The design and synthesis of related fluorescent analogues is
presented in the next paper in this journal: Burnett, D. A.;
Caplen, M. A.; Browne, M. E.; Zhau, H.; Altmann, S. W.;
Davis, H. R., Jr.; Carter, J. W. Bioorg. Med. Chem. Lett. 2002,
12, 315.
9. Browne, M. E.; Burnett, D. A.; Caplen, M. A.; Chen, L.-
Y.; Clader, J. W.; Domalski, M. S.; Dugar, S.; Pushpavanam,
P.; Sher, R.; Vaccaro, W.; Viziano, M.; Zhao, H. Tetrahedron
Lett. 1995, 36, 2555.
As can be seen from the data in Table 1, the derivatives
bearing the benzylic hydroxyl moiety have a significant
potency advantage over compounds 3 and 4. Consistent
withour previous results, the N-iodophenyl analogue 6
was slightly more potent than the pendent iodophenyl
derivative 5. Finally, the glucuronide showed significant
reduction of hepatic cholesterol esters at 1 mg/kg/day in
our cholesterol-fed hamster assay. Compounds 6 and 22
were chosen for radioiodination according to the pro-
cedures described above using carrier free sodium iodide
resulting in biochemical tools with specific activity of
ꢂ200 Ci/mmol.15 These compounds were suitable for
use in all our binding studies and MOA experiments.
We have described the evolution of the design of iodi-
nated analogues of Sch48461 and Sch58235 and the
synthesis of those analogues. Biological activity was
confirmed by in vivo testing of eachanalogue in order
to be assured of significant affinity for the target
CAIBP. The most active analogues, 6 and 22, were pre-
pared as ‘hot’ biochemical tools for use in binding and
10. The intermediate diazonium could also be trapped with
other nucleophiles to generate the substituted aryls. This
chemistry was exploited as part of our SAR studies. See ref 5.
1
11. All compounds were characterized by H NMR, MS and/
or HRMS where appropriate.
Analytical data: Compound 3: yellow oil, 1H NMR
(400 MHz, CDCl3) d 7.59 (d, 2H, J=8 Hz), 7.25 (d, 2H, J=9
Hz), 7.21 (d, 2H, J=9 Hz), 6.91 (d, 2H, J=9 Hz), 6.89 (d, 2H,
J=8 Hz), 6.77 (d, 2H, J=9 Hz), 4.54 (d, 1H, J=2 Hz, CHN),
3.80 (s, 3H, OMe), 3.74 (s, 3H, OMe), 3.05 (m, 1H, CHCO),
2.59 (t, 2H, J=7 Hz), 1.84–1.78 (m, 4H). MS (EI) m/z 527.1
(M+), Anal. calcd for C26H26INO3: C, 59.21, H, 4.97, N,
2.66%, found: C, 59.44, H, 4.86, N, 2.81%.
Compound 4: white crystalline solid, mp 96.5–97.5 ꢀC, H
1
NMR (400 MHz, CDCl3) d 7.52 (d, 2H, J=8.6 Hz), 7.31–7.14
(m, 7H), 7.03 (d, 2H, J=8.7 Hz), 6.89 (d, 2H, J=8.7 Hz), 4.56
(d, 1H, J=2.4 Hz, CHN), 3.80 (s, 3H, OMe), 3.08 (m, 1H,
CHCO), 2.64 (t, 2H, J=7 Hz), 1.97–1.79 (m, 4H). MS (CI) m/z
498 (M+H)+, Anal. calcd for C25H24INO2: C, 60.35, H, 4.87,
N, 2.82%, found: C, 60.41, H, 4.91, N, 3.09%.
Scheme 3. Preparation of iodinated glucuronide derivative 22: (a)
Cs2CO3, Cl3CCN, CH2Cl2 (55%); (b) 13, BF3–Et2O, CH2Cl2 (77%);
(c) KCN, MeOH (49%); (d) Bu3SnSnBu3, Pd(Ph3P)4, toluene, Á
(25%); (e) NaI, IodogenTM, 10:1 EtOAc/HOAc (74%); (f) MeOH,
Et3N, H2O 1:2:7 (quant).

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D. A. Burnett et al. / Bioorg. Med. Chem. Lett. 12 (2002) 311–314
1
Compound 5: H NMR (400 MHz, CDCl3) d 7.65 (d, 2H,
J=8 Hz), 7.25–7.16 (m, 4H), 7.08 (d, 2H, J=9 Hz), 6.92 (dd,
2H, J=8, 9 Hz), 6.83 (d, 2H, J=8 Hz), 5.30 (s, 1H, OH), 4.68
(m, 1H, CHOH), 4.55 (d, 1H, J=2 Hz, CHN), 3.08 (m, 1H,
CHCO), 2.40 (br s, 1H OH), 2.00–1.85 (m, 4H). MS (FAB) m/z
518 (M+H)+.
(m, 1H, CHOH), 3.87 (d, 1H, J=9 Hz, CHCO2), 3.53–3.43 (m,
1H), 3.39-3.34 (m, 1H), 3.10 (dd, 1H, J=7, 8 Hz), 3.01–2.93 (m,
1H, CHCO), 2.05 (br s, 1H), 1.80–1.69 (m, 1H). MS (FAB) m/
z 751 (M+NaCl)+, 717 (M+H +Na)+, 694 (M+H)+.
12. Schnitzer-Polokoff, R.; Compton, D.; Boykow, G.; Davis,
H.; Burrier, R. Comp. Biochem. Physiol. 1991, 99A, 665.
13. (a) Dugar, S.; Yumibe, N.; Clader, J. W.; Vizziano, M.;
Huie, K.; Van Heek, M.; Compton, D. S.; Davis, H. R., Jr.
Bioorg. Med. Chem. Lett. 1996, 6, 1271. (b) Vaccaro, W. D.;
Sher, R.; Davis, H. R., Jr. Bioorg. Med. Chem. Lett. 1998, 8,
319. (c) Vaccaro, W. D.; Davis, H. R., Jr. Bioorg. Med. Chem.
Lett. 1998, 8, 313.
14. (a) Thiruvengadam, T. K., McAllister, T, Tann, C.-H., US
5728827, March17, 1998. (b) Vaccaro, W. D.; Sher, R.;
Davis, H. R., Jr. Bioorg. Med. Chem. Lett. 1998, 8, 35.
15. Radioiodinations were performed by New England
Nuclear Corporation.
Compound 6: white solid, mp 169.5–170.0 ꢀC, 1H NMR
(400 MHz, CDCl3) d 7.52 (d, 2H, J=8.5 Hz), 7.28 (dd, 2H,
J=5.5, 8.8 Hz), 7.18 (d, 2H, J=8.5 Hz), 7.04–7.00 (m, 2H),
6.83 (d, 2H, J=8.5 Hz), 4.71 (t, 1H, J=6 Hz, CHOH), 4.56 (d,
1H, J=2 Hz, CHN), 3.06 (dt, 1H, Jd=2 Hz, Jt=7 Hz,
CHCO), 2.00–1.85 (m, 4H). MS (FAB) m/z 518 (M+H)+,
exact mass calcd for C24H22FINO3 m/z 518.0628, obsd m/z
518.0619.
Compound 22: beige solid, mp 135.0–137.0 ꢀC, 1H NMR
(300 MHz, CD3OD) d 7.45 (d, 2H, J=8 Hz), 7.22–6.86 (m,
10H), 4.87 (d, 1H, J=3 Hz, CHO2), 4.68 (br s, 1H, CHN), 4.48