318
D. A. Burnett et al. / Bioorg. Med. Chem. Lett. 12 (2002) 315–318
Smith, A. A.; Weig, B. C.; Zilli, D. L.; Clader, J. W.; Sybertz, E.
J. Atherosclerosis 1995, 115, 45.
6. This synthesis parallels a process route for the synthesis of
Sch 58235. See ref 1 and referenceswithin.
and then 1.2 mg (1 mmol) of palladium tetrakistriphenylphos-
phine, 0.6 mg (3 mmol) copper(I) iodide and 10 mL (7 mmol)
triethylamine were added. The reaction wasstirred overnight
under argon. The solvent was removed in vacuo and the pro-
duct purified via prep SiO2 TLC eluting with a 50:50:17:1
mixture of EtOAc/hexanes/MeOH/HOAc to give the desired
fluorescent methyl ester as an orange solid. MS (ES) m/z 909.2
(M+1)+.
7. Analytical data: Compound 7: yellow solid, 1H NMR
(400 MHz, CDCl3) d 8.03 (d, 1H, J=7 Hz), 7.70 (d, 1H, J=9
Hz), 7.57 (dd, 1H, J=7, 9 Hz), 7.34–7.25 (m, 4H), 7.03 (t, 2H,
J=7 Hz), 6.72 (d, 2H, J=7 Hz), 5.58 (s, 1H), 4.77 (m, 2H),
3.25 (m, 1H), 2.15–1.98 (m, 4H). UV absorbance lmax 370 nm
(62 mM in EtOH), emission lmax 495 nm (1.1 mM in EtOH) or
lmax 504 nm (1.1 mM in PBS, note: 30% intensity of EtOH,
PBS=15 mM aq Na2PO4, 0.138 M NaCl, pH 7.4). MS (FAB)
m/z 450 (M+H)+, HRMS (FAB) exact mass calcd for
C24H21N3FO3S m/z 450.1288, obsd m/z 450.1286.
Preparation of compound 16: 4.2 mg of the methyl ester 15
was dissolved in ꢀ1 mL of a 7:2:1 mixture of water/triethyl-
amine/methanol and stirred for 1 h. Evaporation to dryness
gave the desired fluorescent azetidinone glucuronide, 16 asan
1
orange solid isolated as (Et3NH+) salt, H NMR (400 MHz,
CD3OD) d 7.34–7.24 (m, 6H), 7.20 (d, 2H, J=8 Hz), 7.13 (d,
2H, J=8 Hz), 7.05–6.95 (m, 3H), 6.92–6.88 (m, 1H), 6.31 (d,
1H, J=4 Hz), 6.17 (s, 1H), 4.90 (m, 1H), 4.83 (m, 1H), 4.58
(m, 1H), 4.13 (s, 2H), 3.81 (m, 1H), 3.71 (d, 1H, J=9 Hz), 3.63
(m, 1H), 3.55–3.45 (m, 3H), 3.17–2.95 (m, 9H), 2.53 (s, 3H),
2.34 (t, 2H, J=7 Hz), 2.25 (br s, 3H), 1.90 (s, 1H), 1.94–1.85
(m, 2H), 1.63–1.55 (m, 2H), 1.28 (t, 9H, J=7 Hz), MS (ES):
m/z 875.3 (M+1ÀHF)+.
Compound 8:8 MS (FAB) m/z 648.2 (M+Na)+, HRMS
[FAB, (M+Na)+] exact mass calcd for C30H28N3O9SFNa m/z
648.1428, obsd m/z 648.1430.
Compound 14: orange solid isolated as a mixture of amide
1
rotamersof (Et NH+)2 salt, H NMR (400 MHz, CD3OD) d
3
8.42 (br s, 1H), 7.99–7.95 (m, 1H), 7.55 (d, 1H, J=8 Hz),
7.35–6.90 (m, 16H), 6.56 (m, 2H), 6.40 and 6.29 (2d, 1H, J=8
Hz) 4.93–4.78 (m, 2H), 4.58 (m, 1H), 4.13 and 4.08 (2s, 2H),
3.75–3.64 (m, 1H), 3.50–3.10 (m, 6H), 2.91 (q, 6H, J=7 Hz),
2.82 (q, 6H, J=7 Hz), 2.28–2.23 (m, 2H), 1.91 (s, 1H), 1.89 (s,
1H), 1.75–1.40 (m, 10H), 1.23 (t, 9H, J=7 Hz), 1.15 (t, 9H,
J=7 Hz). MS (ES) m/z 1092.3 (M+1)+, HRMS (FAB) exact
mass calcd for C60H55N3O16F m/z 1092.3566, obsd m/z
1092.3557.
8. Reiss, P.; Burnett, D. A.; Zaks, A. Bioorg. Med. Chem.
1999, 7, 2199.
9. (a) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 4467. (b) Robins, M. J.; Vinayak, R. S.; Wood,
S. G. Tetrahedron Lett. 1990, 31, 3731.
10. For an excellent overview of thistechnology, ese: Haug-
land, R. P. In Handbook of Fluorescent Probes and Research
Chemicals, 6th ed.; Spence, M. T. Z., Ed.; Molecular Probes:
Eugene, 1996; p 8.
Preparation of compound 11: To 8 mL (6 mg, 120 mmol) of
propargyl amine in 0.5 mL DMF and 1 drop of 0.1 M
NaHCO3, wasadded 10 mg (26 mmol) of the activated ester
BODIPY FL, SE (Molecular Probes, Inc.) in 0.1 mL DMF.
The reaction wasstirred at rt for 12 h. Concentration in vacuo
and chromatography over ꢀ5 g SiO2 eluting with 1% MeOH
in CH2Cl2 gave the desired alkyne, 11, asa yellow orange oil.
Rf 0.62 in 5% MeOH in CH2Cl2 on SiO2 TLC.
11. 16 Absorption lmax 502 nm, emission lmax 510 nm in PBS
(c 0.5 mM, e=20,000 MÀ1 cmÀ1), Note: under identical condi-
tions, fluorescein lmax 493 nm and the relative fluorescence
intensity was 2.5:1 (fluorescein/16).
12. Detailsof our studieswith 16 have been accepted for pub-
lication: Altmann, S. W.; Davis, H. R., Jr.; Yao, X.; Laverty,
M.; Compton, D. S.; Zhu, L.; Crona, J. H.; Caplen, M. A.;
Hoos, L. M.; Tetzloff, G.; Priestly, T.; Burnett, D. A.; Strader,
C. D.; Graziano, M. P. Biochim. et Biophys. Acta In press.
13. The biological activity of compound 7 wasmeausred in
our 7 day cholesterol fed hamster assay1 and found to lower
hepatic cholesterol esters 92% versus controls at 3 mg/kg/
day.
Preparation of compound 15: To 7.7 mg (10.9 mmol) of N-
iodophenylazetidinone glucuronide methyl ester 12 in 1 mL
DMF wasadded 7.2 mg (11 mmol) of the BODIPY alkyne 11.
Argon wasbubbled through the oslution for a few minutes