1308 J. Am. Chem. Soc., Vol. 118, No. 6, 1996
NoVak and Lin
water saturated 1-butanol. The 1-butanol extracts were combined and
evaporated on a rotary evaporator at 40 °C. The residue which
remained after evaporation was dissolved in a minimum volume of
0.2 M aqueous ammonium formate (pH 7.4). This solution was applied
to a 1.5 cm × 25 cm DEAE sephadex A-25 column which had been
equilibrated with 0.2 M aqueous ammonium formate. The column was
eluted with a stepwise gradient of 0.2 M (50 mL), 0.6 M (350 mL),
and 1.0 M (100 mL) aqueous ammonium formate (pH 7.4). Fractions
(5 mL) were collected and monitored by UV absorbance at 254 nm.
Two main peaks (150-225 mL and 250-350 mL) were pooled. These
solutions were extracted with 1-butanol as described above, and the
residues which remained after evaporation were dissolved in a minimum
volume of 1/1 MeOH/H2O containing 0.02 M AcOH. These two
solutions were separately eluted through a 1 cm × 20 cm reverse phase
(C18) silica gel 100 column with the same solvent. In each case one
major product was observed by UV analysis of fractions. Fractions
containing these products were pooled and lyophilized. The two
products 9 (from the 250-350 mL fractions) and 10 (from the 150-
225 mL fraction) were identified by analysis of spectral data and (for
9) comparison of the alkaline cleavage product with an authentic sample.
4-(N-Acetylamino)-3-(glutathion-S-yl)biphenyl (9): 1H NMR (300
MHz, D2O) δ 1.94-2.01 (2H, m), 2.22 (3H, s), 2.28-2.44 (2H, m),
3.26 (1H, dd, J ) 7.9, 14.6 Hz), 3.43 (1H, dd, J ) 4.9, 14.6 Hz),
3.50-3.58 (2H, m), 3.61-3.65 (1H, m), 4.46 (1H, dd, J ) 4.9, 7.8
Hz), 7.41-7.46 (1H, m), 7.49-7.54 (3H, m), 7.59 (1H, dd, J ) 2.0,
8.3 Hz), 7.66-7.69 (2H, m), 7.79 (1H, d, J ) 2.0 Hz); 13C NMR (75.5
MHz, D2O) δ 25.1 (CH3), 28.8 (CH2), 34.1 (CH2), 45.9 (CH2), 56.1
(CH), 56.8 (CH), 129.4 (CH), 129.6 (CH), 129.8 (CH), 130.8 (CH),
131.9 (CH), 132.3 (C), 133.5 (CH), 138.5 (C), 141.8 (C), 142.4 (C),
174.0 (C), 176.3 (C), 176.5 (C), 177.3 (C), 178.7 (C); high resolution
aqueous solution containing these materials was lyophilized after
extraction with Et2O and 1-butanol. The residue was dissolved in a
minimum volume of 1/1 MeOH/H2O containing 0.02 M HOAc and
subjected to reverse phase chromatography as described above. The
two materials coeluted from the column and no effective method of
separation was found. COSY and NOESY cross correlation experi-
ments made it possible to assign most of the 1H NMR peaks to one of
the two isomers.
(E)-4-(Acetylamino)-2-(glutathion-S-yl)-1-phenyl-3,5-cyclohexa-
dien-1-ol (11): 1H NMR (300 MHz, D2O) δ 2.04 (3H, s), 2.06-2.16
(2H, m), 2.39-2.51 (2H, m), 2.81 (1H, dd, J ) 8.9, 14.2 Hz), 3.03
(1H, dd, J ) 4.8, 14.2 Hz), 3.68-3.77 (3H, m) 4.06 (1H, d, J ) 4.9
Hz), 4.36 (1H, dd, J ) 4.8, 8.9 Hz), 5.95 (1H, d, J ) 10.1 Hz), 5.99
(1H, m), 6.21 (1H, dd, J ) 1.9, 10.0 Hz), 7.35-7.44 (3H, m), 7.57
(2H, t, J ) 8.0 Hz).
(Z)-4-(Acetylamino)-2-(glutathion-S-yl)-1-phenyl-3,5-cyclohexa-
dien-1-ol (12): 1H NMR (300 MHz, D2O) δ 2.04 (3H, s), 2.06-2.16
(2H, m), 2.39-2.51 (2H, m) 2.80 (1H, dd, J ) 8.7, 14.1 Hz), 3.00
(1H, dd, J ) 5.3, 14.1 Hz), 3.68-3.77 (3H, m), 4.09 (1H, d, J ) 5.3
Hz), 4.52 (1H, dd, J ) 5.3, 8.7 Hz), 5.96 (1H, d, J ) 10.0 Hz), 5.98
(1H, m), 6.19 (1H, dd, J ) 1.9, 10.0 Hz), 7.35-7.44 (3H, m), 7.57
(2H, t, J ) 8.0 Hz).
All 44 expected 13C NMR peaks were located: 13C NMR (75.5 MHz,
D2O) δ 25.46 (CH3), 25.51 (CH3), 28.97 (CH2), 29.02 (CH2), 34.11
(CH2), 34.17 (CH2), 34.98 (CH2), 35.26 (CH2), 46.08 (CH2), 46.08
(CH2), 55.42 (CH), 55.46 (CH), 56.13 (CH), 56.24 (CH), 56.85 (CH),
56.85 (CH), 76.93, (C), 77.22, (C), 115.17 (CH), 115.77 (CH), 126.99
(CH), 127.04 (CH), 128.47 (CH), 128.49 (CH), 130.84 (CH), 130.96
(CH), 131.15 (CH), 131.23 (CH), 134.53 (C), 134.85 (C), 136.31 (CH),
136.58 (CH), 146.13 (C), 146.83 (C), 174.43 (C), 174.56 (C), 174.60
(C), 175.94 (C), 175.98 (C), 176.73 (C), 177.42 (C), 177.54 (C), 177.93
(C), 178.88 (C); high resolution MS (FAB) m/e 601.141, C24H28N4O8-
+
MS (FAB) m/e 631.047, C24H26N4O7SK3 requires 631.043.
4-(N-Acetylamino)-4′-(glutathion-S-yl)biphenyl (10): 1H NMR
(300 MHz, D2O) δ 2.01-2.08 (2H, m), 2.16 (3H, s), 2.35-2.41 (2H,
m) 3.32 (1H, dd, J ) 8.2, 14.6 Hz) 3.48 (1H, dd, J ) 4.7, 14.6 Hz),
3.50-3.57 (2H, m), 3.64-3.69 (1H, m) 4.55 (1H, dd, J ) 4.8, 8.1 Hz)
7.48-7.54 (4 H, m), 7.61-7.68 (4H, m); 1H NMR (300 MHz, DMSO-
d6) (aromatics only) δ 7.42 (2H, d, J ) 8.4 Hz), 7.56 (2H, d, J ) 8.5
Hz), 7.58 (2H, d, J ) 8.9 Hz), 7.65 (2H, d, J ) 8.8 Hz); 13C NMR
(75.5 MHz, D2O) δ 25.7 (CH3), 29.8 (CH2), 34.2 (CH2), 37.6 (CH2),
40.0 (CH2) 56.0 (CH) 57.1 (CH), 124.5 (CH), 129.9 (CH), 130.0 (CH),
133.7 (CH), 135.4 (C), 138.8 (C), 139.4 (C), 141.2 (C), 173.8 (C),
174.2 (C), 175.4 (C), 177.7 (C), 178.9 (C); high resolution MS (FAB)
+
SNa3 requires 601.132.
Kinetic and Product Studies. The decomposition of 1b and 19 or
the formation of GSH adduct 10 and reduction product 6b were
monitored by HPLC on a µ-Bondapak C-18 column with UV
absorbance monitored at 280 nm. The eluent was 7/3 or 8/2 MeOH/
H2O containing 0.02 M 1/1 HOAc/NaOAc. In each case 25 µL of a
0.02 M DMF solution of 1b or 19 was injected into 5 mL of the GSH
buffer (10 mM or 20 mM GSHT, pH 8.52) or Tris buffer (pH 8.5)
which had been incubating at 20 °C for at least 15 min. Aliquots of
20 µL were withdrawn periodically for HPLC analysis. Peak area vs
time data were fit to the first-order rate equation by nonlinear least
squares procedures.
For product analyses, initial concentrations of 1a of ca. 5.7 × 10-5
M and 1b of ca. 1.5 × 10-4 M were obtained in GSH buffers by
injection of 25 µL of an appropriate stock solution of 1a or 1b in DMF
into 5 mL of the buffer. All reactions were allowed to go to completion
at 20 °C before analyses were performed by HPLC (triplicate 20 µL
injections) on a µ-Bondapak C18 column or a Beckman C-8 column.
MeOH/H2O containing 0.02 M HOAc was used at the eluent and UV
absorbance was monitored at 280 nm. Products were identified by
HPLC comparison to isolated samples. Yields were determined from
average peak areas and extinction coefficients obtained from the isolated
samples.
+
m/e 583.129, C24H26N4O7SNa3 requires 583.121.
Bis[3-[4-(acetylamino)biphenylyl]] Disulfide (15). This material
was obtained from the thiazthionium hydrate 14 which was synthesized,
in turn, from 4-aminobiphenyl by a known procedure.30 A 100 mg
sample of 14 (0.47 mmol) was slurried with 1-2 mL of H2O under N2
at 0 °C while 160 µL of 10 M NaOH was added to the mixture. The
mixture was stirred for about 1 h as it was allowed to reach room
temperature. Then, a 152 µL (1.6 mmol) aliquot of Ac2O was added.
After a few minutes the reaction mixture was extracted with CH2Cl2
(3 × 5 mL). The extracts were combined, and the solvent was
evaporated. The residue was applied to a 1 cm × 20 cm silica gel
column and eluted with 1/9 EtOAc/CH2Cl2. The major band was
collected and evaporated to yield 15: mp 236-237 °C; 1R (KBr) 3200,
1664, 1522, 1378, 1296, 758, 696 cm-1; 1H NMR (300 MHz, DMSO-
d6) δ 2.05 (6H, s), 7.30-7.35 (2H, m), 7.37-7.42 (4H, m) 7.47-7.54
(6H, m), 7.58 (2H, dd, J ) 2.0, 8.2 Hz) 7.82 (2H, d, J ) 2.0 Hz), 9.77
(2H, s); 13C NMR (75.5 MHz, DMSO-d6) δ 23.1 (CH3), 126.0 (CH),
126.4 (CH), 126.8 (CH), 127.7 (CH), 127.9 (CH), 129.0 (CH), 131.5
(C), 136.0 (C), 137.9 (C), 138.8 (C), 168.9 (C); high resolution MS
m/e 484.129, C28H24N2O2S2 requires 484.128.
Acknowledgment. This work was supported by a grant from
the American Cancer Society (CN-23K). The 300 MHz 1H and
75.5 MHz 13C NMR spectra were obtained with equipment
funded by NSF Grant No. CHE9012532.
Supporting Information Available: Reaction product yields
for the decomposition of 1a and 1b in GSH buffers (2 pages).
This material is contained in many libraries on microfiche,
immediately follows this article in the microfilm version of the
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information and Internet access instructions.
Alkaline Cleavage of 9. A 10 mg sample of 9 was dissolved in 5
mL of 0.1 M NaOH. After stirring overnight the solution was
neutralized with 0.1 M HCl, and the precipitate was collected by
filtration. After purification by TLC on silica gel (2/3 EtOAc/CH2-
Cl2), the 1H and 13C NMR spectra of the cleavage product were found
to be identical to that of authentic 15.
The adducts 11 and 12 would not extract with 1-butanol. The
(30) Leaper, J. M. F. J. Am. Chem. Soc. 1931, 53, 1981-1894.
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