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979
the organic layer washed with brine, dried over Na2SO4, concentrated and
purified by silica-gel column chromatography (hexane/ethyl acetate = 5:1) to
give 1.0 g (97%) of isoxazole compound 4a: 1H NMR (300 MHz, CDCl3) d 7.33–
7.27 (m, 7H), 7.25 (dd, J = 7.2, 2.4 Hz, 1H), 7.20 (d, J = 1.8 Hz, 1H), 6.93 (dd,
J = 6.9, 2.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 1H), 5.00 (s, 2H), 3.90 (s, 3H), 3.85 (s, 3H);
13C NMR (75 MHz, CDCl3) d 163.4, 159.4, 151.9, 151.0, 148.1, 136.6, 130.0,
128.5, 127.9, 127.2, 122.5, 120.7, 120.4, 114.7, 114.4, 112.5, 111.6, 70.9, 55.9,
55.3; HRMS (FAB) m/z 340.1558 [(M+H)+, calcd for C24H22NO4 388.1549].
9. Compound 4b. To a solution of the isoxazole compound 4a (1.0 g, 2.58 mmol) in
ethyl acetate (10 mL) was added 10% palladium/c (40 mg). The reaction was
performed under 60 psi of hydrogen atmosphere for 14 h. The reaction solution
was passed through a short silica-gel pad by washing with ethyl acetate,
concentrated, and purified by silica-gel column chromatography (hexane/ethyl
acetate = 1:2) to give 0.7 g (90%) of 4b: 1H NMR (300 MHz, CDCl3) d 8.27 (s, 1H),
7.30 (dd, J = 6.3, 2.1 Hz, 2H), 7.21 (d, J = 1.8 Hz, 1H), 7.17 (dd, J = 8.7, 2.4 Hz, 1H),
6.92 (dd, J = 6.3, 2.1 Hz, 2H), 6.84 (d, J = 8.4 Hz, 1H), 5.67 (s, 1H), 3.92 (s, 3H),
3.84 (s, 3H); 13C NMR (75 MHz, CDCl3) d 163.4, 159.4, 152.0, 147.9, 145.6,
129.9, 122.4, 121.1, 119.7, 114.9, 114.4, 113.3, 110.6, 55.9, 55.2; HRMS (FAB) m/
z 297.1068 [(M+H)+, calcd for C17H16NO4 298.1079].
In summary, inhibitory activity was increased in methoxy moi-
ety of B-aryl group of KRIBB3 analogs than in hydroxyl moiety. The
effect of the number of phenolic hydrogens in A-aryl group of
KRIBB3 analogs on inhibitory activities of microtubule polymeriza-
tion was also examined for structure–activity relationships.
KRIBB3 analogs lacking phenolic hydrogen showed poor inhibitory
activity. The compound with two phenolic hydrogens in A-aryl
group of KRIBB3 analogs 4 exhibited more potent than the com-
pound with one phenolic hydrogen. Compound 4b possessing
one phenolic hydrogen and one methoxy group exhibited the
strongest inhibitory activity.
Acknowledgments
This research was supported by grants from KRIBB Research Ini-
tiative Program, the National Chemical Genomics Research Pro-
gram, and Center for Biological Modulators of the 21st Century
Frontier Research Program.
10. Compound 4c. Compound 4c was prepared from 3,4-dihydroxybenzaldehyde
according to the typical procedure: 1H NMR (300 MHz, CDCl3) d 8.26 (s, 1H),
7.45–7.26 (m, 13H), 7.20 (dd, J = 8.4, 2.4 Hz, 1H), 6.94 (dd, J = 6.9, 2.4 Hz, 2H),
6.91 (d, J = 7.8 Hz, 1H), 5.19 (s, 2H), 5.04 (s, 2H), 3.86 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 163.3, 159.4, 152.0, 150.2, 148.8, 136.7, 130.0, 128.5, 128.4, 127.9,
127.8, 127.2, 127.1, 122.5, 121.0, 120.7, 114.8, 114.4, 113.4, 71.1, 71.0, 55.3;
HRMS (FAB) m/z 464.1836 [(M+H)+, calcd for C30H26NO4 464.1862].
References and notes
Compound 4d. Compound 4d was prepared from compound 4c by hydro-
genation as described for synthesis of 4b: 1H NMR (300 MHz, DMSO-d6) d 9.40
(br s, 2H), 8.71 (s, 1H), 7.33 (dd, J = 6.9, 2.1 Hz, 2H), 6.98 (dd, J = 6.9, 2.1 Hz, 2H),
6.97 (s, 1H), 6.88 (dd, J = 7.8, 1.8 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H);
13C NMR (75 MHz, DMSO-d6) d 163.1, 158.7, 152.1, 147.4, 145.4, 129.5, 121.9,
118.7, 115.9, 114.3, 114.0, 113.8, 55.0; HRMS (FAB) m/z 284.0917 [(M+H)+,
calcd for C16H14NO4 284.0923].
Compound 4e. Compound 4e was prepared from 3,5-dimethyl-4-hydroxybenz-
aldehyde according to the typical procedure: 1H NMR (300 MHz, CDCl3) d 8.30
(s, 1H), 7.49–7.30 (m, 9H), 6.95 (m, 2H), 4.84 (s, 2H), 3.85 (s, 3H), 2.26 (s, 6H);
13C NMR (75 MHz, CDCl3) d 163.6, 159.3, 157.2, 151.8, 137.3, 131.7, 129.7,
128.5, 128.1, 127.8, 127.7, 123.5, 122.5, 115.1, 114.3, 70.0, 63.8, 55.2, 16.4;
HRMS (FAB) m/z 386.1748 [(M+H)+, calcd for C25H24NO3 386.1756].
Compound 4f. Compound 4f was prepared from compound 4e by hydro-
genation as described for synthesis of compound 4b: 1H NMR (300 MHz, CDCl3)
d 8.28 (s, 1H), 7.31 (dd, J = 6.6, 1.8 Hz, 2H), 7.28 (br s, 2H), 6.92 (dd, J = 6.6,
2.4 Hz, 2H), 4.91 (s, 1H), 3.85 (s, 3H), 2.21 (s, 6H); 13C NMR (75 MHz, CDCl3) d
163.9, 159.2, 153.7, 151.8, 129.7, 127.7, 123.3, 122.7, 119.9, 114.4, 114.3, 53.3,
15.8; HRMS (FAB) m/z 296.1284 [(M+H)+, calcd for C18H18NO3 296.1287].
Compound 4g. Compound 4g was prepared from 4-hydroxybenzaldehyde
according to the typical procedure and the subsequent hydrogenation: 1H NMR
(300 MHz, CD3OD) d 8.41 (s, 1H), 7.42 (dd, J = 6.6, 1.8 Hz, 2H), 7.29 (dd, J = 6.6,
1.8 Hz, 2H), 6.94 (dd, J = 6.6, 2.4 Hz, 2H), 6.78 (dd, J = 6.6, 2.4 Hz, 2H), 4.85 (s,
1H), 3.80 (s, 3H); 13C NMR (75 MHz, CD3OD) d 165.3, 160.9, 160.5, 153.0, 130.9,
129.8, 123.7, 120.2, 116.6, 115.8, 115.4, 55.7; HRMS (FAB) m/z 268.0968
[(M+H)+, calcd for C16H14NO3 268.0974].
Compound 4h. Compound 4h was prepared from 2-benzyloxy-4-methoxy-5-
ethylbenzaldehyde according to the typical procedure: 1H NMR (300 MHz,
CDCl3) d 8.42 (s, 1H), 7.25–7.00 (m, 8H), 6.78 (dd, J = 6.9, 2.4 Hz, 2H), 6.48 (s,
1H), 4.84 (s, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 2.56 (q, J = 7.8 Hz, 2H), 1.14 (t,
J = 7.8 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 162.3, 159.7, 158.7, 155.5, 150.5,
136.5, 130.8, 128.2, 127.6, 127.0, 125.3, 123.3, 116.6, 113.8, 109.3, 97.0, 70.7,
55.3, 55.2, 22.3, 14.0; HRMS (FAB) m/z 416.1859 [(M+H)+, calcd for C26H26NO4
416.1862].
1. Zhou, J.; Giannakakou, P. Curr. Med. Chem. Anticancer Agents 2005, 5, 65.
2. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253.
3. Mollinedo, F.; Gajate, C. Apoptosis 2003, 8, 413.
4. Jordan, M. A.; Thrower, D.; Wilson, L. Cancer Res. 1991, 51, 2212.
5. Shin, K. D.; Yoon, Y. J.; Kang, Y. R.; Son, K. H.; Kim, H. M.; Kwon, B. M.; Han, D. C.
Biochem. Pharmacol. 2008, 75, 383.
6. Sulikowski, M. M.; Ellis Davies, G. E. R.; Smith, A. B., III J. Chem. Soc., Perkin Trans.
1 1992, 979.
7. (a) SanMartin, R.; Marigorta, E. M.; Dominguez, E. Tetrahedron 1994, 50, 2255;
(b) Olivera, R.; SanMartin, R.; Dominguez, E.; Solans, X.; Urtiaga, M. K.;
Arriortua, M. I. J. Org. Chem. 2000, 65, 6398.
8. Typical procedure for synthesis of compound 4a. To a mixture of 3-hydroxy-4-
methoxybenzaldehyde (3.0 g, 19.7 mmol) and potassium carbonate (10.9 g,
78.8 mmol) in DMF (30 mL) was added benzyl bromide (4.7 mL, 39.4 mmol)
and the reaction mixture was stirred at room temperature for 16 h. The
reaction solution was filtered to remove inorganic salts, and the filtrate was
diluted with water (200 mL), extracted three times with ethyl acetate and
washed with brine. The combined organic layer was dried over anhydrous
magnesium sulfate, filtered, and concentrated under reduced pressure. The
concentrated residue was purified by silica-gel column chromatography
(hexane/ethyl acetate = 5:1) to give 4.7 g of benzylated compound. To
a
mixture of magnesium turnings (0.75 g, 0.031 mol) in THF (5 mL) at room
temperature was slowly added a solution of 4-methoxybenzylchloride (1.6 g,
10.2 mmol) in THF (10 mL). The reaction solution was refluxed with heating for
one hour and then cooled down in a 0 °C water bath. The ashy solution was
extracted by using a syringe, which was used as a Grignard reagent. To a
solution of the benzylated aldehyde (0.82 g, 3.4 mmol) in THF (15 mL) at 0 °C
was added the Grignard reagent slowly, and the reaction mixture was stirred at
room temperature for 1 h. Saturated ammonium chloride solution was added
to the reaction solution, and the mixture was extracted with ethyl acetate three
times and washed with brine. Combined organic layer was dried over
anhydrous magnesium sulfate, filtered, and concentrated under reduced
pressure. The concentrated residue was purified by silica-gel column
chromatography (hexane/ethyl acetate = 5:1), to give 1.2 g of alcoholic
compound. To a mixture of the alcoholic compound (1.2 g, 3.3 mmol), 4-
methylmorpholine N-oxide (0.57 g, 4.9 mmol) and anhydrous powdered 4 Å
molecular sieves (1.64 g) in dichloromethane (10 mL) was added
tetrapropylammonium perruthenate (57 mg). The reaction mixture was
stirred for 30 min, passed through a short silica-gel pad by washing with
ethyl acetate, and concentrated to give 1.1 g (88% in three-steps) of compound
2. To a solution of compound 2 (1.26 g, 3.5 mmol) in toluene (10 mL) was
added dimethylformamide dimethylacetal (DMFDMA) (1.2 g, 10 mmol). The
reaction mixture was refluxed for 16 h at 135 °C. The reaction solution was
cooled to 0 °C, concentrated and purified by silica-gel column chromatography
(hexane/ethyl acetate = 1:2) to give 1.35 g (93%) of compound 3. To a solution
of compound 3 (1.12 g, 2.7 mmol) in methanol (35 mL) was added sodium
carbonate (190 mg, 1.7 mmol) and NH2OH HCl (1.9 g, 27.0 mmol). The mixture
was adjusted to be pH 4–5 using acetic acid (1.0 mL) and then heated in a
heavy-wall screw capped tube at 115 °C for 2 h. The reaction mixture was
cooled to room temperature and methanol was removed under reduced
pressure. The residue was extracted with methylene chloride and water, and
11. Microtubule polymerization assay. For the detection of polymerization of
tubulin/microtubule, CytoDYNAMIX Screen 01 kits were purchased form
Cytoskeleton Inc. (Denver, CO) and polymerization assay was done as
described previously.4 Tubulin proteins (>97% purity) were suspended
(300 lg/sample) with 100 ll of G-PEM buffer (80 mM PIPES, 2 mg MgCl2,
0.5 mM EGTA, 1.0 mM GTP, pH 6.9) plus 5% glycerol in the 0.1% DMSO or test
compounds at 4 °C. Then the sample mixture was transferred to the pre-
warmed 96-well plate, and polymerization of tubulin was measured by the
change in absorbance at 340 nm every 1 min for 70 min (Wallac victor2;
PerkinElmer, Inc., Wellesley, MA) at 37 °C. Inhibitory activity of KRIBB3 or its
analogs was calculated using initial polymerization activity (from 0 to 10 min).
To compare each compound’s inhibitory activity, we used initial
polymerization reaction slop (from
0 to 10 min). Slop of polymerization
reaction was calculated using linear equation method with Sigma Plot Program
and slop of DMSO treated reaction was used as a control. Relative inhibition of
tube formation is percentage of slop for KRIBB3 analogs compared to the slop
for DMSO treated reaction.