A. M. Jordan et al. / Bioorg. Med. Chem. 7 (1999) 1775±1780
1779
in vacuo. Column chromatography (silica, dichloro-
methane) gave the di-chloride (1.38 g, 59%) as a white
powder; mp 108±109ꢀC (lit.18 105±06ꢀC); Rf (silica,
d, J=7.7 Hz, Ar), 6.88 (2H, d, J=7.9 Hz, Ar); 13C
NMR (100 MHz, CDCl3) (35.3 (CH2), 40.7 (CH2), 42.7
(CH2), 53.9 (CH2), 112.8 (CH), 115.6 (CH), 116.8 (CH),
120.9 (CH), 123.0 (CH), 130.9 (C), 142.5 (C), 143.0 (C),
144.1 (C), 144.4 (C), 156.1(C); m/z (CI) 413 ([M+H]+,
10%), 233 (50), 184 (100), 123 (35); (CI: found:
[M+H]+, 413.1045. C19H22Cl2N2O4 requires [M+H]+,
413.1034).
1
dichloromethane) 0.7; H NMR (400 MHz, CDCl3) d
3.62 (8H, m, 4ÂCH2), 5.00 (2H, s, PhCH2), 6.73 (2H, d,
J=8.6 Hz, Ar), 6.92 (2H, d, J=8.6 Hz, Ar), 7.29±7.44
(5H, m, Ar).
p-(Bis-2-chloroethylamino)phenol hydrochloride (6).
Hydrogen chloride gas was bubbled through a solution
of the di-chloride 5 (3.3 g, 0.01 mol) in methanol (35
mL), until complete dissolution occurred. Filtration and
concentration in vacuo gave the hydrochloride salt as a
white powder (mp 140±141ꢀC (lit.8 135±136ꢀC)), which
was immediately re-suspended in ethanol (40 mL) con-
taining 10% palladium on carbon (0.17 g). The suspen-
sion was stirred under an atmosphere of hydrogen until
no starting material was detected by TLC. The suspen-
sion was then ®ltered over Celite1 and concentrated in
vacuo to give the hydrochloride as a white solid (1.37 g,
Scanning oximetry. Tyrosinase solution (3.65 mL)
(phosphate buer, pH 7.2, 300 units of mushroom
tyrosinase (Sigma) per mL) was placed in the scanning
oximetry apparatus and to this cell was added 100 mL
of a 10 mM solution of the prodrug under investigation.
With constant stirring, the U.V. spectrum and oxygen
uptake were then measured at 5 s intervals for a period
of 15 min. The overall change in the U.V. spectrum
could then be calculated by subtracting the spectrum
recorded at 5 s from the spectrum recorded at 15 min.
51%); mp 176±178ꢀC (lit.18 170±173ꢀC); H NMR (400
Tyrosinase degradation studies. To a solution of mush-
room tyrosinase (Sigma, 3000 units mg 1) in phosphate
buer (pH 7.2, 0.2 mL) was added a solution of the
prodrug under investigation (2 mg) in DMSO (0.2 mL).
The mixture was then stirred gently for 1 h in a water
bath maintained at 25ꢀC. Aliquots (0.5 L) were injected
into the GC±MS system with an oven temperature of
150ꢀC for 5 min, warming to 250ꢀC over a period of
5 min and maintaining this ®nal temperature for a
further 40 min. The liberated free mustard was detected
by scanning the eluted components from the GC,
observing m/z 233 ([M]+). The free mustard was found
to elute at ca. 10.2 min under these conditions.
1
MHz, CDCl3, free amine) d 3.52 (8H, m, 4ÂCH2), 6.57
(2H, d, J=9.0 Hz, Ar), 6.67 (2H, d, J=9.0 Hz, Ar).
Carbonic acid p-(bis-2-chloroethylamino)phenol ester-p-
nitrophenyl ester (PNMC) (1). The bis-chloroethylamine
hydrochloride salt 6 (1.35 g, 0.057 mol) and triethyla-
mine (1.17 g, 1.61 mL, 0.114 mol) in toluene (15 mL)
were slowly added, over 15 min, to a re¯uxing solution
of p-nitrophenyl chloroformate (1 g, 0.05 mol) in toluene
(15 mL) and the mixture re¯uxed for 1 h. The reaction
was then cooled, concentrated in vacuo and puri®ed by
column chromatography (silica, dichloromethane) to
give the diester as a yellow oil (1.27 g, 64%), which
solidi®ed upon standing; mp 97±99ꢀC; Rf 0.77 (silica,
dichloromethane); vmax (KBr disc) 1767, 1615, 1594,
;
1H NMR (400 MHz,
Acknowledgements
1
1512, 1347, 1180, 814 cm
CDCl3) d 3.61 (4H, m, 2ÂCH2), 3.71 (4H, m, 2ÂCH2),
6.70 (2H, d, J=9.2 Hz, Ar), 7.16 (2H, d, J=9.2 Hz, Ar),
7.47 (2H, d, J=9.1 Hz, Ar), 8.30 (2H, d, J=9.1 Hz, Ar);
13C NMR (100 MHz, CDCl3) d 40.2 (CH2), 53.6 (CH2),
112.4 (CH), 121.7 (CH), 121.8 (CH), 125.3 (CH), 140.5
(C), 141.3 (C), 142.7 (C), 143.1 (C), 153.6 (C); m/z (CI)
399 ([M+H]+, 75%), 348 (100), 120 (20), 63 (15); (CI:
found: [M+H]+, 399.0525. C17H16Cl2N2O5 requires
[M+H]+, 399.0514).
We gratefully acknowledge the BBSRC (Post-doctoral
fellowship to A.M.J., grant no. 45/SBDO7534) and the
Royal Society for their ®nancial support of this work.
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{20-(300,400-Di-hydroxyphenyl)-ethyl} carbamic acid p-
(bis-2-chloroethylamino)phenyl ester (2). A solution of
the carbonate 1 (0.1 g, 0.26 mmol), 3-hydroxytyramine
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!9:1, v/v) gave the carbamate (0.08 g, 73%) as a
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1
1507, 1218 cm 1; H NMR (400 MHz, CDCl3) d 2.60
(2H, br t, J=6.3 Hz, PhCH2), 3.32 (2H, br q, J=6.3 Hz,
CH2NH), 3.48 (4H, t, J=6.2 Hz, 2ÂNCH2), 3.55 (4H, t,
J=6.2 Hz, 2ÂCH2Cl), 5.17 (1H, br t, J=6.3 Hz, NH),
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