Novobiocin
A R T I C L E S
dioxolo[4,5-c]pyran-4-yl 2,2,2-trichloroacetimidate (14, 588 mg, 1.62
mmol) and benzyl-7-hydroxy-8-methyl-2-oxo-2H-chromen-3-yl car-
bamate6 (13, 527 mg, 1.62 mmol) in DCM (16 mL). After the mixture
was stirred for 14 h, three drops of Et3N were added and the mixture
was concentrated. The residue was purified by chromatography (DCM
(2 mL) was stirred for 14 h. The solvent was removed and the residue
purified by preparative TLC (SiO2, 25:1, CH2Cl2:methanol, developed
seven times) to afford DHN2 (2.5 mg, 9%) and 4-deshydroxynovobiocin
(DHN1, 17.5 mg, 57%) as colorless solids. DHN1: [R]31 ) -20.3°
D
1
(c ) 0.300, 10% MeOH in CH2Cl2). H NMR (CDCl3, 400 MHz): δ
f 100:1, CH2Cl2:acetone) to afford 15 (670 mg, 81%) as a yellow
8.70 (s, 1H), 7.62 (d, J ) 2.3 Hz, 1H), 7.56 (dd, J ) 2.3, 8.4 Hz, 1H),
7.29 (d, J ) 8.8 Hz, 1H), 7.13 (d, J ) 8.8 Hz, 1H), 6.81 (d, J ) 8.4
Hz, 1H), 5.50 (d, J ) 2.3 Hz, 1H), 5.34-5.25 (m, 2H), 4.25 (t, J )
2.6 Hz, 1H), 3.53-3.51 (m, 1H), 3.50 (s, 3H), 3.34 (dd, J ) 3.2, 8.9
Hz, 2H), 2.99 (s, 1H), 2.94 (s, 1H), 2.27 (s, 3H), 1.74 (s, 3H), 1.71 (s,
3H), 1.33 (s, 3H), 1.13 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 166.3,
159.6, 158.9, 156.8, 155.9, 149.0, 133.9, 129.0, 128.6, 126.4, 125.6,
124.6, 124.1, 121.8, 121.4, 114.9, 114.4, 114.1, 111.2, 98.3, 81.4, 78.9,
72.2, 69.6, 61.5, 29.7, 29.3, 27.2, 22.6, 17.9, 8.2. IR (film): νmax 3400,
3379, 3360, 2978, 2928, 2853, 1709, 1659, 1632, 1605, 1528, 1504,
1367, 1254, 1136, 1117, 1086 cm-1. HRMS (ESI+) m/z 597.2434 (M
+ H+, C31H37N2O10 requires m/z 297.2448).
1
foam: [R]22 ) -19.7° (c ) 1.54, 20% MeOH in DCM). H NMR
D
(CDCl3, 400 MHz): δ 8.27 (s, 1H), 7.85 (s, 1H), 7.55-7.35 (m, 5H),
7.29 (d, J ) 2.9 Hz, 1H), 7.11 (d, J ) 8.7 Hz, 1H), 5.77 (d, J ) 1.9
Hz, 1H), 5.23 (s, 2H), 5.05 (d, J ) 1.9 Hz, 1H), 4.95 (t, J ) 7.7 Hz,
1H), 3.59 (s, 3H), 3.30 (d, J ) 7.6 Hz, 1H), 2.27 (s, 3H), 1.34 (s, 3H),
1.19 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 159.0, 155.2, 153.6,
153.6, 149.2, 136.0, 129.1 (2C), 129.0, 128.7 (2C), 125.8, 122.6, 122.1,
115.2, 115.1, 111.6, 94.8, 83.3, 78.4, 77.6, 77.0, 67.9, 61.0, 27.9, 22.6,
8.8. IR (film): νmax 3402, 3319, 3063, 3034, 2984, 2939, 2839, 1817,
1709, 1634, 1609, 1587, 1522, 1456, 1383, 1366, 1331, 1296, 1263,
1229, 1205, 1175 cm-1. HRMS (ESI+) m/z 526.1688 (M + H+, C27H28-
NO10 requires m/z 526.1713).
N-(7-((2R,3R,4S,5R)-3,4-Dihydroxy-5-methoxy-6,6-dimethyltet-
rahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-yl)-4-hy-
droxy-3-(3-methylbut-2-enyl)benzylamide (DHN2). A solution of
carbonate 17 (12 mg, 19.3 µmol) in 10/1 methanol/Et3N (220 µL) was
stirred for 14 h. The solvent was removed and the residue purified by
preparative TLC (SiO2, 10:1, CH2Cl2:methanol) to afford DHN2 (8
mg, 75%) as a colorless solid: [R]31D ) -12.9° (c ) 0.310, 10% MeOH
3-Amino-7-((3aR,4R,7R,7aR)-7-methoxy-6,6-dimethyl-2-oxotet-
rahydro-3aH-[1.3]dioxolo[4,5-c]pyran-4-yloxy)-8-methyl-2H-chromen-
2-one (16). Palladium on carbon (10%, 67 mg) was added to a solution
of carbamate 15 (670 mg, 1.31 mmol) in THF (13 mL). The suspension
was stirred for 6 h under a hydrogen atmosphere and was filtered
through a plug of silica gel. The solvent was removed and the residue
purified by chromatography (100:1 f 50:1, CH2Cl2:acetone) to afford
16 (425 mg, 83%) as a pale yellow foam: [R]23D ) -26.4° (c ) 0.780,
1
in DCM). H NMR (CDCl3, 400 MHz): δ 8.78 (s, 1H), 8.66 (s, 1H),
7.71 (d, J ) 2.2 Hz, 1H), 7.68 (dd, J ) 2.2, 8.3 Hz, 1H), 7.33 (d, J )
8.8 Hz, 1H), 7.19 (d, J ) 8.8 Hz, 1H), 6.90 (d, J ) 8.3 Hz, 1H), 6.05
(s, 1H), 5.61 (d, J ) 1.6 Hz, 1H), 5.33 (t, J ) 7.1 Hz, 1H), 4.27-4.23
(m, 2H), 3.61 (s, 3H), 3.45-3.35 (m, 3H), 2.77 (s, 1H), 2.67 (s, 1H),
1
20% MeOH in DCM). H NMR (CDCl3, 400 MHz): δ 7.10 (d, J )
8.6 Hz, 1H), 7.05 (d, J ) 8.6 Hz, 1H), 6.68 (s, 1H), 5.73 (d, J ) 2.0
Hz, 1H), 5.04 (dd, J ) 2.0, 7.9 Hz, 1H), 4.95 (t, J ) 7.7 Hz, 1H), 4.11
(s, 2H), 3.54 (s, 3H), 3.29 (d, J ) 7.6 Hz, 1H), 2.28 (s, 3H), 1.34 (s,
3H), 1.21 (s, 3H). 13C NMR (CDCl3, 200 MHz): δ 159.6, 153.3, 153.0,
148.1, 130.2, 122.7, 116.1, 114.8, 111.9, 111.0, 94.5, 83.0, 78.0, 77.3,
76.4, 60.6, 27.5, 22.2, 8.6. IR (film): νmax 3462, 3362, 2984, 2937,
2839, 1807, 1707, 1636, 1595, 1497, 1387, 1371, 1331, 1263, 1169,
1109, 1078, 1036 cm-1. HRMS (ESI+) m/z 392.1357 (M + H+, C19H22-
NO8 requires m/z 392.1346).
2.05 (s, 3H), 1.80 (s, 3H), 1.79 (s, 3H), 1.38 (s, 3H), 1.14 (s, 3H). 13
C
NMR (CDCl3, 125 MHz): δ 165.9, 159.5, 158.3, 155.9, 149.0, 135.8,
129.5, 127.5, 126.9, 125.9, 125.8, 124.2, 122.0, 120.9, 115.9, 114.2,
114.1, 111.2, 97.7, 84.3, 78.6, 71.2, 68.6, 62.0, 29.6, 29.3, 25.8, 22.5,
18.0, 8.2. IR (film): νmax 3402, 2974, 2928, 2854, 1717, 1701, 1645,
1605, 1526, 1506, 1367, 1254, 1088 cm-1. HRMS (ESI+) m/z 554.2363
(M + H+, C30H36NO9 requires m/z 554.2390).
4-((7-((3aR,4R,7R,7aR)-7-Methoxy-6,6-dimethyl-2-oxotetrahydro-
3aH-[1.3]dioxolo[4,5-c]pyran-4-yloxy)-8-methyl-2-oxo-2H-chromen-
3-ylcarbamoyl)-2-(3-methylbut-2-enyl) Phenyl Acetate (17). Oxalyl
chloride (15 mg, 119 µmol) was added to a solution of benzoic acid 9
(28 mg, 113 µmol) in CH2Cl2 (0.5 mL), followed by the addition of
catalytic DMF. After the mixture was stirred for 2.5 h, the acid
chloride (10) was concentrated. The yellow solid was redissolved in
CH2Cl2 (0.5 mL) and added dropwise over 3 min to a stirred solution
of aniline 16 (34 mg, 87 µmol) in pyridine (0.5 mL) at 0 °C. The
resulting solution was stirred at room temperature for 3.5 h and
concentrated. The residue was purified by preparative TLC (SiO2, 40:
1, CH2Cl2:acetone) to afford 17 (31 mg, 57%) as a colorless solid:
[R]22D ) -21.7° (c ) 0.840, 20% MeOH in CH2Cl2). 1H NMR (CDCl3,
500 MHz): δ 8.72 (s, 1H), 8.64 (s, 1H), 7.73 (d, J ) 2.5 Hz, 1H),
7.69 (dd, J ) 2.5, 8.0 Hz, 1H), 7.29 (d, J ) 6.8 Hz, 1H) 7.11 (d, J )
8.0 Hz, 1H), 7.07 (d, J ) 9.0 Hz, 1H), 5.72 (d, J ) 2.0 Hz, 1H), 5.18-
5.14 (m, 1H), 4.99 (dd, J ) 1.5, 7.5 Hz, 1H), 4.89 (t, J ) 8.0 Hz, 1H),
3.53 (s, 3H), 3.26-3.22 (m, 3H), 2.27 (s, 3H), 2.23 (s, 3H), 1.70 (s,
3H), 1.66 (s, 3H), 1.29 (s, 3H), 1.13 (s, 3H). 13C NMR (CDCl3, 125
MHz): δ 167.9, 164.4, 158.1, 154.1, 152.2, 151.1, 148.1, 133.6, 133.3,
131.0, 128.6, 124.9, 123.1, 122.0 (2C), 121.2, 119.6, 113.8, 113.7,
110.2, 93.3, 81.9, 76.9, 76.3, 75.6, 59.5, 27.8, 26.5, 24.7, 21.1, 19.9,
16.9, 7.4. IR (film): νmax 3400, 2982, 2935, 2856, 1811, 1763, 1715,
1674, 1634, 1607, 1526, 1489, 1437, 1369, 1250, 1202, 1175, 1111,
1090 cm-1. HRMS (ESI+) m/z 622.2277 (M + H+, C33H36NO11
requires m/z 622.2289).
Cells and Reagents. SKBr3 and MCF7 human breast cancer cells
were purchased from the American Type Culture Collection and grown
as previously described.64 Antibodies for R-tubulin and ErbB2 (Ab-2
and Ab-5) were from Calbiochem (La Jolla, CA).
Western Blotting. Cells were washed once with cold phosphate-
buffered saline (pH 7.0) and lysed by scraping in TMNS (50 mM Tris-
HCl, pH 7.5, 20 mM Na2MoO4, 0.1% NP-40, 150 mM NaCl)
supplemented with 20 µg/mL aprotinin, 20 µg/mL leupeptin, and 1
mM phenylmethanesulfonyl fluoride. Cell lysate was clarified by
centrifugation at 14 000 rpm at 4 °C for 15 min, and protein
concentration was determined by using the BCA method (Pierce,
Rockford, IL). Twenty micrograms of total protein from cell lysates
was separated by 4-20% gradient SDS-PAGE (Bio-Rad, Hercules,
CA). Western blotting for ErbB2 was performed as described
previously.64 Blotting for R-tubulin was used to verify equal loading
of lanes.
MTT Assay. Cell growth was monitored using methylthiazol-
tetrazolium (MTT). Briefly, cells (5 × 103) were plated in 96-well
microtiter plates (Costar) in a volume of 0.1 mL of DMEM
containing 0.1% FBS. After 12 h, cells were exposed to drugs (final
volume 0.2 mL/well). At various times after drug addition, 20 µL of 5
mg/mL MTT solution in PBS was added to each well for 4 h. After
removal of medium, 0.1 mL of DMSO was added to each well to
dissolve the formazan crystals. Absorbance at 562 nm was determined
using an ELx 808 microplate reader (Bio-Tek, Winooski, VT). Six wells
were assayed at each concentration, and the mean absorbance was
determined. Absorbance at 562 nm is directly proportional to viable
cell number.
(3R,4S,5R,6R)-5-Hydroxy-6-(3-(4-hydroxy-3-(3-methylbut-2-
enyl) benzylamino)-8-methyl-2-oxo-2H-chromen-7-yloxy)-3-meth-
oxy-2,2-dimethyltetrahydro-2H-pyran-4-yl Carbamate (DHN1). A
solution of carbonate 17 (32 mg, 52 µmol) in 7 M methanolic ammonia
9
J. AM. CHEM. SOC. VOL. 128, NO. 48, 2006 15535