A. R. Schneekloth et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5904–5908
5907
6H). 13C NMR (100 MHz, CDCl3) d 164.1, 163.0, 157.3, 137.9, 133.0, 129.1,
128.8, 127.8, 127.4, 111.9, 105.5, 100.6, 77.2, 71.3, 55.6, 22.2. LRMS (ES+)
[M+H]+ 455.4, [M+Na]+ 477.4. HRMS (ES+) calcd for C25H24N2O2Cl2 [M+H]+
455.1287, found 455.1286. TLC (10% CH3OH in CHCl2), Rf 0.34 (UV).
proteasome dependent. These results are an important step in the
pursuit of small molecule PROTAC libraries, which could
potentially provide new insights into a number of important bio-
logical pathways through loss-of-function phenotypes. In addition,
these results lend support for potential PROTAC-based therapeutic
approaches in the treatment of various diseases.
Nutlin tert-butyl ester: To
isopropoxy-4-methoxy-phenyl)-4,5-dihydro-1H-imidazole
0.033 mmol) dissolved in THF (1 mL) at 0 °C were added Et3N (24
a
solution of 4,5-bis-(4-chloro-phenyl)-2-(2-
(15 mg,
L,
3
l
0.174 mmol) and triphosgene (76 mg, 0.257 mmol). After stirring at 0 °C for
2.5 h, the mixture was evaporated and evacuated for 0.5 h. To the residue
dissolved in CH2Cl2 (0.5 mL) at 0 °C was added dropwise a solution of amine 8
(110 mg, 0.515 mmol) in CH2Cl2 (0.5 mL) via cannula. The resulting mixture
was stirred at 0 °C for 1.5 h. The mixture was quenched with saturated NaHCO3
aqueous solution (5 mL). The mixture was extracted three times with CH2Cl2
and the extracts were washed with saturated NaCl, dried over Na2SO4, filtered,
and concentrated. The residue was purified by flash column chromatography
(1:99 methanol–dichloromethane initially, grading to 1:19 methanol–
dichloromethane) to give 22 mg (96%) of nutlin ester. 1H NMR (500 MHz,
CDCl3) d 7.58 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.5 Hz, 2H),
6.94 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 6.54 (dd, J = 8.4, 2.2 Hz, 1H), 6.47
(d, J = 2.2 Hz, 1H), 5.55 (d, J = 9.7 Hz, 1H), 5.47 (d, J = 9.7 Hz, 1H), 4.64–4.57 (m,
1H), 4.02 (d, J = 10.0 Hz, 1H), 3.94 (d, J = 10.0 Hz, 1H), 3.84 (s, 3H), 3.76 (d,
J = 9.1 Hz, 1H), 3.71 (d, J = 10.9 Hz, 1H), 3.52–3.45 (m, 1H), 3.33–3.28 (m, 1H),
3.07–3.05 (m, 2H), 1.42 (s, 9H), 1.38 (d,J = 6.0 Hz, 3H), 1.34 (d, J = 6.0 Hz, 3H).
LRMS (ES+) [M+H]+ 695.39, [M+Na]+ 717.14. TLC (10% CH3OH in CHCl3), Rf 0.54
(UV, CAM).
Acknowledgments
A.R.S. thanks the NSF for a predoctoral fellowship. M.P. thanks
the Human Frontier Science Program Organization for a cross-
disciplinary post-doctoral fellowship. This work was partially
supported by the Korea Research Foundation Grant (KRF-2005-
214-C00218) to H.S.T. This work was partially supported by
the National Institutes of Health (CA118631). We gratefully
acknowledge John Schneekloth and Nicholas Aberle for their
thorough review of the manuscript.
References and notes
Nutlin acid 4: To a solution of nutlin ester (14 mg, 0.020 mmol) dissolved in
CH2Cl2 (1 mL) at 0 °C was added TFA (0.2 mL). After stirring at rt for 17 h, the
resulting mixture was diluted with toluene (1 mL) and evaporated. The residue
was purified by flash column chromatography (3:99 methanol–
dichloromethane initially, grading to 1:9 methanol–dichloromethane) to give
12.3 mg (96%) of nutlin acid 4. 1H NMR (400 MHz, CD3OD) d 7.58 (d, J = 8.3 Hz,
1H), 7.13 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.94
(d, J = 8.3 Hz, 2H), 6.68–6.65 (m, 2H), 5.77 (d, J = 10.0 Hz, 1H), 5.57 (d,
J = 10.0 Hz, 1H), 4.76–4.70 (m, 1H), 3.87 (s, 3H), 3.83–3.72 (m, 4H), 3.55–3.48
(m, 1H), 3.41–3.35 (m, 1H), 3.08–3.04 (m, 2H), 1.38 (d, J = 6.0 Hz, 3H), 1.35 (d,
J = 6.0 Hz, 3H). 13C NMR (100 MHz, CD3OD) d 167.0, 165.1, 163.4, 158.6, 155.6,
137.3, 136.2, 134.2, 133.1, 130.7, 129.9, 129.1, 129.0, 113.4, 106.5, 101.2, 72.5,
71.7, 70.1, 56.1, 50.4, 47.8, 43.1, 42.9, 22.5, 22.4, 13.4. HRMS (ES+) calcd for
1. Mitchison, T. J. Chem. Biol. 1994, 1, 3.
2. Crews, C. M.; Splittgerber, U. Trends Biochem. Sci. 1999, 24, 317.
3. Sieber, S. A.; Cravatt, B. F. Chem. Commun. 2006, 2311.
4. Alaimo, P. J.; Shogren-Knaak, M. A.; Shokat, K. Curr. Opin. Chem. Biol. 2001, 5,
360.
5. Schneekloth, J. S., Jr.; Crews, C. M. ChemBioChem 2005, 6, 40.
6. Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews, C. M.; Deshaies, R.
J. Proc. Nat. Acad. Sci. U.S.A. 2001, 98, 8554.
7. Schneekloth, J. S., Jr.; Fonseca, F. N.; Koldobskiy, M.; Mandal, A.; Deshaies, R.;
Sakamoto, K.; Crews, C. M. J. Am. Chem. Soc. 2004, 126, 3748.
8. Ciechanover, A.; Orian, A.; Schwartz, A. L. BioEssays 2000, 22, 442.
9. Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.;
Rothbard, J. B. Proc. Nat. Acad. Sci. U.S.A. 2000, 97, 13003.
10. Debes, J. D.; Schmidt, L. J.; Huang, H.; Tindall, D. J. Cancer Res. 2002, 62, 5632.
11. Marhefka, C. A.; Gao, W.; Chung, K.; Kim, J.; He, Y.; Yin, D.; Bohl, C.; Dalton, J. T.;
Miller, D. D. J. Med. Chem. 2004, 47, 993.
12. Piette, J.; Neel, H.; Marechal, V. Oncogene 1997, 15, 1001.
13. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong,
N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303,
844.
C32H32N4O6Cl2 [M+H]+ 639.1771, found 639.1779. a 2D0
½ ꢀ +6.8° (c = 0.365,
MeOH). TLC (10% CH3OH in CHCl2), Rf 0.09 (UV, CAM).
16. Kong, N.; Liu, E. A.; Vu, B. T. Preparation of cis-2,4,5-triphenylimidazolines and
their use in the treatment of tumors. Patent WO2003051359, 2003.
17. Experimental.Azido alcohol 9: To
a solution of triethylene glycol (1.185 g,
7.892 mmol) in CH2Cl2 (20 mL) at 0 °C were added Ag2O (2.01 g, 8.681 mmol)
and MsCl (0.73 mL, 9.470 mmol). The black suspension was stirred at 0 °C for
10 min, allowed to warm to rt, and further stirred at rt for 26 h. The resulting
mixture was filtered through Celite with CH2Cl2. The filtrate was concentrated
in vacuo and the residue was purified by short column chromatography to give
14. Fujioka, H.; Murai, K.; Ohba, Y.; Hiramatsu, A.; Kita, Y. Tetrahedron Lett. 2005,
46, 2197.
15. Experimental.Ester 7: To a solution of 1-benzyloxycarbonyl-3-oxopiperazine
(234 mg, 1.0 mmol) in DMF (3 mL) at 0 °C was added NaH (60%, 48 mg,
1.8 g of mono-mesylated alcohol. To
a solution of the alcohol (1.8 g,
7.892 mmol) in DMF (25 mL) at rt was added NaN3 (770 mg, 11.838 mmol).
The reaction mixture was heated to 110 °C, stirred at 110 °C for 5.5 h, and
cooled to 0 °C. The resulting mixture was quenched with water (50 mL) and the
mixture was extracted three times with ethyl acetate. The extracts were
washed with saturated NaCl solution, dried over Na2SO4, filtered, and
concentrated. The concentrate was purified by flash column chromatography
(1:99 methanol–dichloromethane initially, grading to 1:19 methanol–
dichloromethane) to give 1.285 g (93%) of azido alcohol 9. 1H NMR
(400 MHz, CDCl3) d 3.73–3.71 (m, 2H), 3.67–3.66 (m, 6H), 3.60 (t, J = 4.2 Hz,
2H), 3.39 (t, J = 4.8 Hz, 2H), 2.32 (t, J = 6.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) d
72.5, 70.6, 70.4, 70.1, 61.8, 50.6. HRMS (ES+) calcd for C6H12N3O3Na [M+Na]+
198.0849, found 198.0846. TLC (10% CH3OH in EtOAc), Rf 0.58 (CAM).
Acid 10: To a solution of azido alcohol 9 (780 mg, 4.454 mmol) in DMF (4.5 mL)
at 0 °C was added NaH (60%, 178 mg, 4.454 mmol). After stirring at 0 °C for
30 min, sodium iodoacetate (926 mg, 4.454 mmol) was added to the mixture.
The reaction mixture was stirred at rt for 32 h and quenched with 1 N HCl in an
ice bath. The mixture was extracted three times with ethyl acetate. The
extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered,
and concentrated. The concentrate was purified by flash column
chromatography (1:99 methanol–dichloromethane initially, grading to 1:9
methanol–dichloromethane) to provide 860 mg (83%) of acid 10. 1H NMR
(500 MHz, CDCl3) d 8.02 (s, 1H), 4.16 (s, 2H), 3.77–3.75 (m, 2H), 3.71–3.70 (m,
4H), 3.68–3.66 (m, 4H), 3.39 (t, J = 4.9 Hz, 2H). 13C NMR (125 MHz, CDCl3) d
172.5, 71.4, 70.7, 70.4, 70.2, 70.0, 68.7, 50.6. HRMS (ES+) calcd for
C8H14N3O5Na[M+Na]+ 256.0904, found 256.0902. TLC (10% CH3OH in EtOAc),
Rf 0.12 (CAM).
Phenol 11: To a solution of acid 10 (250 mg, 1.072 mmol) and 4-aminophenol
hydrochloride (156 mg, 1.072 mmol) in CH2Cl2 (4 mL) at rt were added HOBt
(174 mg, 1.286 mmol) and DIPEA (0.47 mL, 2.680 mmol). The mixture was
cooled to 0 °C and EDCI (226 mg, 1.179 mmol) was added to the mixture. The
reaction mixture was brought to room temperature and further stirred at rt for
18 h. The resulting mixture was quenched with H2O in an ice bath. The mixture
was extracted three times with ethyl acetate. The extracts were washed with
saturated NaCl solution, dried over Na2SO4, filtered, and concentrated. The
concentrate was purified by flash column chromatography (1:99 methanol–
dichloromethane initially, grading to 1:9 methanol–dichloromethane) to afford
302 mg (87%) of phenol 11. 1H NMR (400 MHz, CDCl3) d 8.69 (s, 1H), 7.36 (d,
1.2 mmol). After stirring at 0 °C for 30 min, tert-butyl bromoacetate (160
lL,
1.1 mmol) was added to the mixture, which was then stirred at room
temperature (rt) for 3.0 h. The resulting mixture was quenched with H2O
(10 mL) and the aqueous solution was extracted twice with ethyl acetate. The
extracts were dried over Na2SO4, filtered, and concentrated. The concentrate
was purified by flash column chromatography (1:99 methanol–
dichloromethane initially, grading to 1:19 methanol–dichloromethane) to
furnish 338 mg (97%) of ester 7. 1H NMR (400 MHz, CD3OD) d 7.37–7.35 (m,
3H), 7.33–7.30 (m, 2H), 5.15 (s, 2H), 4.14 (s, 2H), 4.05 (s, 2H), 3.75 (s, 2H), 3.45
(t, J = 4.8 Hz, 2H), 1.45 (s, 9H). 13C NMR (100 MHz, CD3OD) d 169.3, 169.2,
156.1, 137.7, 129.6, 129.2, 129.0, 83.3 (2C), 68.7 (2C), 50.1, 48.2, 28.2. TLC (10%
CH3OH in CH2Cl2), Rf 0.58 (UV, CAM).
Amine 8: To a solution of ester 7 (300 mg, 0.861 mmol) in MeOH (8.5 mL) at rt
was added 10% Pd/C (26 mg, 30 mg/mmol). After introducing H2, the reaction
mixture was stirred vigorously for 16 h. The resulting mixture was filtered
through Celite and the filtrate was concentrated. The concentrate was purified
by flash column chromatography (1:99 methanol–dichloromethane initially,
grading to 1:19 methanol–dichloromethane) to provide 172 mg (93%) of amine
8. 1H NMR (400 MHz, CDCl3) d 4.02 (s, 2H), 3.55 (s, 2H), 3.37 (d, J = 5.4 Hz, 2H),
3.11 (d, J = 5.4 Hz, 2H), 1.72 (s, 1H), 1.46 (s, 9H). 13C NMR (100 MHz, CDCl3) d
168.4, 167.9, 82.0, 50.0, 49.2, 48.7, 43.0, 28.0. HRMS (ES+) calcd for
C10H17N2O3Na [M+Na]+ 237.1209, found 237.1204. TLC (10% CH3OH in
CH2Cl2), Rf 0.47 (ninhydrin).
Imidazoline 3. To a solution of 2-isopropoxy-4-methoxybenzaldehyde 1 (2.5 g,
12.88 mmol) in CH2Cl2 (130 mL) was added 1,2-bis(4-chlorophenyl)-1,2-
ethanediamine (3.65 g, 12.98 mmol). After stirring at 0 °C for 2.0 h, NBS
(2.5 g, 14.04 mmol) was added to the mixture. The reaction mixture was
warmed to rt and further stirred at rt for 16 h. The resulting mixture was
quenched with 10% KOH aqueous solution and the mixture was extracted three
times with CH2Cl2. The extracts were dried over Na2SO4, filtered, and
concentrated. The residue was purified by flash column chromatography
(1:99 methanol–dichloromethane initially, grading to 1:19 methanol–
dichloromethane) to give 5.14 g (88%) of imidazoline 3. 1H NMR (400 MHz,
CDCl3) d 8.27 (d, J = 8.7 Hz, 1H), 7.75 and 7.42 (d, J = 8.4 Hz, 1H), 7.04 (d,
J = 8.4 Hz, 4H), 6.90 (d, J = 8.4 Hz, 4H), 6.61 (dd, J = 8.8, 2.3 Hz, 1H), 6.54 (d,
J = 2.3 Hz, 1H), 5.36 (s, 2H), 4.74–4.68 (m, 1H), 3.86 (s, 3H), 1.37 (d, J = 6.1 Hz,