754 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Shen and Dryhurst
at 40 mV in phosphate buffer (pH 7.4) resulted in formation of
DHBT-6 (major product) and DHBT-2 (minor product) as a
result of intramolecular cyclization of the 2-S- and 5-S-cysteinyl
residues, respectively.
oxidation (40 mV, pH 7.4) of 5,6-bi-S-CyS-DA. Controlled
potential electro-oxidation (40 mV, pH 7.4) of DHBT-3 in the
presence of free CySH gave DHBT-4 as the major initial product.
Furthermore, controlled potential electro-oxidation (40 mV, pH
7.4) of DHBT-1 in the presence of free CySH gave initially a
mixture of DHBT-3 (minor product) and DHBT-2 (major prod-
uct) that were subsequently oxidized to DHBT-4. Together,
these observations confirmed that DHBT-3 is the 8-S-cysteinyl
conjugate of DHBT-1.
5,6-Bi-S-cystein yldopam in e (5,6-Bi-S-CyS-DA). This com-
pound was isolated as a white solid. In the HPLC mobile phase
(pH 2.15) 5,6-bi-S-CyS-DA exhibited UV bands at λmax ) 306
and 218 nm. FAB-MS (3-nitrobenzyl alcohol matrix) gave m/z
) 392.0958 (MH+, 100, C14H22N3O6S2; calcd m/z ) 392.0950).
1H NMR (D2O) gave δ: 6.94 (s, 1H, C(2)-H), 4.10 (dd, J ) 6.0,
4.8 Hz, 1H, C(b′)-H), 4.03 (dd, J ) 5.7, 5.1 Hz, 1H, C(b)-H), 3.56
(dd, J ) 15.0, 5.7 Hz, 1H, C(a)-H), 3.40-3.26 (m, 3H, C(a)-H,
C(a′)-H2), 3.23-3.10 (m, 4H, C(R)-H2, C(â)-H2). The structure
of 5,6-bi-S-CyS-DA was further confirmed by the fact that
controlled potential electro-oxidation at 40 mV in phosphate
buffer pH 7.4 resulted in formation of DHBT-3 as the initial
reaction product.
6,8-Bi-S-cyst ein yl-7-(2-a m in oet h yl)-3,4-d ih yd r o-5-h y-
d r oxy-2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (DHBT-4).
DHBT-4 was isolated as a very pale pink solid. In the HPLC
mobile phase (pH 2.15) DHBT-4 exhibited UV bands at λmax
330 and 256 nm. At pH 7.4, the UV spectrum showed bands at
max, nm (log ꢀmax, M-1 cm-1), 332 (3.59) and 264 (4.42),
)
λ
calculated as the 2.5TFA salt. FAB-MS (glycerol/TFA matrix)
gave m/z ) 493.0896 (MH+, 100, C17H25N4O7S3; calcd m/z )
493.0885). 1H NMR (D2O) gave δ: 4.58 (dd, J ) 4.2, 3.3 Hz,
1H, C(3)-H), 3.96 (dd, J ) 6.3, 5.1 Hz, 1H, C(b)-H), 3.94 (dd, J
) 6.0, 5.4 Hz, 1H, C(b′)-H), 3.48 (m, 2H, C(R)-H2), 3.37 (dd, J )
12.9, 4.2 Hz, 1H, C(2)-H), 3.31-3.13 (m, 5H, C(a)-H2, C(2)-H,
C(a′)-H2), 3.11 (m, 2H, C(â)-H2). DHBT-4 was formed as an
initial product of controlled potential electro-oxidation (40 mV,
pH 7.4) of 2,5,6-tri-S-CyS-DA and as a result of oxidation (40
mV, pH 7.4) of DHBT-1, DHBT-2, and DHBT-3 in the presence
of free CySH. Together, these observations confirmed that
DHBT-4 is the 6,8-bi-S-cysteinyl conjugate of DHBT-1.
2,5,6-Tr i-S-cystein yldopam in e (2,5,6-Tr i-S-CyS-DA). This
compound was isolated as a white solid. In the HPLC mobile
phase (pH 2.15) this compound exhibited UV bands at λmax
)
316 and 226 nm. At pH 7.4, the UV spectrum showed λmax, nm
(log ꢀmax, M-1 cm-1), at 332 (3.68), 260 (sh, 4.10), and 236 (4.33).
FAB-MS (glycerol/TFA matrix) gave m/z ) 511.1011 (MH+, 100,
C17H27N4O8S3; calcd m/z ) 511.0991). 1H NMR (D2O) gave δ:
4.12 (dd, J ) 6.0, 4.8 Hz, 1H, C(b′′)-H), 4.11 (t, J ) 5.1 Hz, 1H,
C(b′)-H), 4.08 (t, J ) 5.1 Hz, 1H, C(b)-H), 3.65 (dd, J ) 15.0, 5.1
Hz, 1H, C(a)-H), 3.60-3.50 (m, 3H, C(R)-H2, C(a′)-H), 3.39 (dd,
J ) 14.7, 6.0 Hz, 1H, C(a′′)-H), 3.32 (dd, J ) 14.7, 4.8 Hz, 1H,
C(a′′)-H), 3.31 (dd, J ) 15.0, 5.1 Hz, 1H, C(a)-H), 3.28 (dd, J )
14.7, 5.1 Hz, 1H, C(a′)-H), 3.08 (t, J ) 8.1 Hz, 2H, C(â)-H2).
Elemental analysis gave C 30.63, H 3.10, N 5.67, S 9.66, F 23.32,
corresponding to 2,5,6-tri-S-CyS-DA‚4TFA (C25H30N4O16S3F12);
theory C 31.06, H 3.11, N 5.80, S 9.94, F 23.60.
8-(2-Am in oeth yl)-3,4-dih ydr o-5-h ydr oxy-2H-1,4-ben zoth i-
a zin e-3-ca r boxylic Acid (DHBT-5). DHBT-5 was isolated as
a white solid. In the HPLC mobile phase, DHBT-5 exhibited
bands at λmax ) 302 and 226 nm. At pH 7.4, the UV spectrum
showed λmax, nm (log ꢀmax, M-1 cm-1), at 304 (3.36) and 230
(4.35), calculated as the 1.5TFA salt. FAB-MS (3-nitrobenzyl
alcohol matrix) gave m/z ) 255.0802 (MH+, 100, C11H15N2O3S;
7-(2-Am in oeth yl)-3,4-dih ydr o-5-h ydr oxy-2H-1,4-ben zoth i-
a zin e-3-ca r boxylic Acid (DHBT-1). DHBT-1 was isolated as
a white solid. The UV, mass, and 1H NMR spectra of DHBT-1
have been presented in detail elsewhere (23). Elemental
analysis gave C 39.48, H 3.86, N 6.63, S 6.95, F 19.16,
corresponding to DHBT-1‚1.5TFA (C14H15.5N2O6S1F4.5); theory
C 39.53, H 3.65, N 6.59, S 7.53, F 20.12. Controlled potential
electro-oxidation (20 mV, pH 7.4) of 5-S-CyS-DA gave DHBT-1
as the major initial product, confirming the structure of the
latter compound.
calcd m/z ) 255.0803). 1H NMR (D2O) gave δ: 6.63 (d, J 6,7
)
8.1 Hz, 1H, C(6)-H), 6.57 (d, J 6,7 ) 8.1 Hz, 1H, C(7)-H), 4.55
(dd, J ) 4.8, 3.6 Hz, 1H, C(3)-H), 3.33 (dd, J ) 13.2, 4.8 Hz, 1H,
C(2)-H), 3.19 (dd, J ) 13.2, 3.6 Hz, 1H, C(2)-H), 3.13 (m, 2H,
C(â)-H2), 2.84 (m, 2H, C(R)-H2). Long-range 2D COSY experi-
ments confirmed cross coupling between the resonances at δ
6.57 (d, C(7)-H) and 2.84 (m, C(R)-H2). Furthermore, controlled
potential electro-oxidation (20 mV, pH 7.4) of 2-S-CyS-DA gave
DHBT-5 as the major initial product, confirming the structure
of this compound.
6-S-Cystein yl-7-(2-a m in oeth yl)-3,4-d ih yd r o-5-h yd r oxy-
2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (DHBT-2). DH-
BT-2 was isolated as a very pale yellow solid. At pH 7.4, the
UV-visible spectrum exhibited bands at λmax, nm (log ꢀmax, M-1
cm-1), 322 (3.57), 278 (sh, 3.90), and 248 (4.37), calculated as
the 2TFA salt. FAB-MS (3-nitrobenzyl alcohol matrix) give m/z
) 374.0871 (MH+, 100, C14H20N3O5S2; calcd m/z ) 374.0844).
1H NMR (D2O) gave δ: 6.68 (s, 1H, C(8)-H), 4.57 (dd, J ) 4.2,
3.6 Hz, 1H, C(3)-H), 3.90 (t, J ) 5.7 Hz, 1H, C(b)-H), 3.31 (dd,
J ) 13.2, 4.2 Hz, 1H, C(2)-H), 3.23 (dd, J ) 13.2, 3.6 Hz, 1H,
C(2)-H), 3.20-3.02 (m, 6H, C(a)-H2, C(R)-H2, C(â)-H2). DHBT-2
was formed as an initial product of controlled potential electro-
oxidation of 2,5-bi-S-CyS-DA (40 mV, pH 7.4) along with DHBT-
6. Furthermore, DHBT-2 was the major initial product of
controlled potential electro-oxidation (40 mV, pH 7.4) of DHBT-1
in the presence of free CySH and was then further oxidized to
DHBT-4. Together, these observations confirm that DHBT-2
is the 6-S-cysteinyl conjugate of DHBT-1, not the 8-S-cysteinyl
conjugate as was proposed previously (23).
6-S-Cystein yl-8-(2-a m in oeth yl)-3,4-d ih yd r o-5-h yd r oxy-
2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (DHBT-6). DH-
BT-6 was isolated as a very pale yellow solid. In pH 7.4
phosphate buffer, the UV spectrum of DHBT-6 showed λmax, nm
(log ꢀmax, M-1 cm-1), at 320 (3.48), 278 (3.81), and 248 (4.35),
calculated as the 2TFA salt. FAB-MS (thioglycerol/glycerol
matrix) gave m/z ) 374.0813 (MH+, 100, C14H20N3O5S2; calcd
m/z ) 374.0844). 1H NMR (D2O) gave δ: 6.79 (s, 1H, C(7)-H),
4.59 (dd, J ) 4.2, 3.6 Hz, 1H, C(3)-H), 3.91 (t, J ) 5.7 Hz, 1H,
C(b)-H), 3.34 (dd, J ) 13.2, 4.2 Hz, 1H, C(2)-H), 3.28 (d, J ) 5.7
Hz, 2H, C(a)-H2), 3.19-3.11 (m, 3H, C(2)-H, C(â)-H2), 2.94-2.77
(m, 2H, C(R)-H2). DHBT-6 (with DHBT-2) was formed as the
major initial product of controlled potential electro-oxidation (40
mV, pH 7.4) of 2,5-bi-S-CyS-DA. DHBT-6 was also the major
initial product of controlled potential electro-oxidation (40 mV,
pH 7.4) of DHBT-5 in the presence of free CySH and was further
oxidized under these conditions to DHBT-7. Together, these
results confirmed that DHBT-6 was the 6-S-cysteinyl conjugate
of DHBT-5, not the 6-S-cysteinyl conjugate of DHBT-1 as was
proposed previously (23).
8-S-Cystein yl-7-(2-a m in oeth yl)-3,4-d ih yd r o-5-h yd r oxy-
2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (DHBT-3). DH-
BT-3 was isolated as a very pale pink solid. In the HPLC mobile
phase (pH 2.15) DHBT-3 exhibited UV bands at λmax ) 318 and
244 nm. FAB-MS (3-nitrobenzyl alcohol matrix) gave m/z )
374.0847 (MH+, 80, C14H20N3O5S2; calcd m/z ) 374.0844). 1H
NMR (D2O) gave δ: 6.66 (s, 1H, C(6)-H), 4.52 (dd, J ) 4.2, 3.6
Hz, 1H, C(3)-H), 3.84 (dd, J ) 7.2, 4.5 Hz, 1H, C(b)-H), 3.36
(dd, J ) 13.2, 4.2 Hz, 1H, C(2)-H), 3.26-3.08 (m, 5H, C(2)-H,
C(a)-H2, C(â)-H2), 3.02-2.92 (m, 2H, C(R)-H2). DHBT-3 was
formed as the initial product of controlled potential electro-
6,7-Bi-S-cysteinyl-8-(2-aminoethyl)-3,4-dihydro-5-hydroxy-
2H-1,4-ben zoth ia zin e-3-ca r boxylic Acid (DHBT-7). DH-
BT-7 was isolated as a very pale pink solid. In pH 7.4 phosphate
buffer, the UV spectrum of DHBT-7 showed λmax, nm (log ꢀmax
,
M-1 cm-1), at 334 (sh, 3.71), 298 (sh, 3.79), and 262 (4.42),
calculated as the 2.5TFA salt. FAB-MS (thioglycerol/glycerol
matrix) gave m/z ) 493.0836 (MH+, 61, C17H25N4O7S3; calcd m/z
) 493.0885). 1H NMR (D2O) gave δ: 4.65 (dd, J ) 4.2, 3.6 Hz,
1H, C(3)-H), 4.00 (t, J ) 5.4 Hz, 1H, C(b)-H), 3.91 (dd, J ) 6.0,