Bioactivation of Pyrrolopyrimidine (U-89843)
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1233
of water instead of 50% aqueous CH3CN in the second washing.
Changing the wash solvent ensured complete removal of re-
sidual GSH since GSH is more soluble in water than in organic
solvents.
3-Br om o-2-oxop r op yl Ben zoa te (7). A flame-dried, three-
neck 500 mL round bottom flask was charged with benzoate 6
(14.5 g, 56.0 mmol), dicyclohexylcarbodiimide (21.6 g, 10.5
mmol), DMSO (5 mL), pyridine (0.75 mL), and diethyl ether (150
mL). The stirred solution was cooled to -5 °C, and trifluoro-
acetic acid (0.75 mL) was added. The reaction mixture was
allowed to warm and was maintained at 18 °C by using an ice
bath to control a brief exotherm at 20 min into the reaction.
Then, the reaction mixture was stirred for 1 h and recooled to
0 °C. Oxalic acid (6 g) in CH3OH (12 mL) was added with
diethyl ether. The reaction mixture was filtered, and filtrate
was concentrated in vacuo to yield a yellow oil. Hexane (800
mL) was added to the oil, and the resulting mixture was heated
to reflux. The hot hexane solution was allowed to cool, and the
resulting white crystalline solid was removed by filtration,
washed with chilled hexane, and dried under high vacuum at
room temperature to afford the desired product (6.4 g, 44.5%):
1H NMR (CDCl3) δ 8.08-8.11 (m, 2H), 7.59-7.65 (m, 1H), 7.46-
7.50 (m, 2H), 5.15 (s, 2H), 4.03 (s, 2H); IR (mull) cm-1 2953,
1747, 1715, 1281; MS (EI) m/ z 163, 122, 105, 77.
In Vitr o Cova len t Bin d in g to DNA. The measurement of
drug related radioactive materials covalently bound to genomic
DNA was performed in a manner analogous to reported proce-
dures (16, 17). A stock solution of calf thymus DNA was
prepared by dissolution overnight at 65 °C in TE buffer (15 mM
Tris buffer, 1 mM EDTA, pH 8.0). [14C]U-89843 (specific activity
30.8 µCi/mg; 9.91 mCi/mmol, 50 µM) was incubated with 0.5
mg/mL untreated rat liver microsomal protein and 2 mg/mL calf
thymus DNA (Sigma) and 0, 1, or 5 mM reduced glutathione
(GSH) in 100 mM pH 7.4 potassium phosphate buffer. Incuba-
tions were carried out at 37 °C for 30 min. Metabolic reactions
were started with the addition of an NADPH generating system
(NADP+/ICDH). Control incubations were carried out in the
absence of the NADPH generating system. Reactions were
stopped by placing the samples on ice followed by the addition
of 5.0 mL of DNA ISOLATOR (Genosys Biotechnologies, Inc.,
The Woodlands, TX, Genomic DNA isolation reagent, Catalog
No. DNA-ISO-050). A volume of 1.0 mL of chloroform was
added, and the tubes were inverted repeatably to allow mixing
and then were placed on ice for 5 min. The samples were
centrifuged at 2200g at 4 °C for 5 min, and the aqueous (top)
layers were transferred to clean tubes (200-300 µL was left
above protein interface to avoid contamination). A volume of
2.5 mL of isopropyl alcohol was added to the tubes containing
the aqueous layers to precipitate DNA. The samples were
placed on ice for 10 min. The precipitated DNA was transferred
to clean tubes containing 1.5 mL of 70% aqueous ethanol. The
tubes were mixed gently by inversion to wash the DNA and then
placed on ice. The wash was decanted, and the DNA was
washed twice more with 1.5 mL portions of 70% aqueous
ethanol. Following the final wash, the DNA was air-dried for
30 min under a nitrogen stream. DNA in the samples was
redissolved in 0.5 mL portions of TE buffer (pH 8.0). Samples
were heated at 65 °C for 5 min to effect DNA dissolution. A260
and A260/A280 spectrophotometric measurements on aliquots of
the DNA solutions were made to determine the DNA concentra-
tions and assess purity, respectively. Aliquots (400 µL) of the
DNA solutions were added to scintillation vials along with 10
mL of ULTIMA GOLD scintillation cocktail (Packard), and the
solutions were counted on a liquid scintillation counter. The
radioactivity (dpm/mL), µg/mL DNA calculated form the A260
reading, and specific activity (9.91 mCi/mmol) were used to
calculate the pmol equivalents of U-89843 bound per mg of DNA
for each sample.
Bis[7-m eth yl-2,4-d i-1-p yr r olid in yl-7H-p yr r olo[2,3-d ]p y-
r im id in -6-yl]m eth a n e (5). A flame-dried 100 mL round
bottom flask was charged with pyrimidine intermediate 8 (0.96
g, 3.89 mmol) in acetonitrile (50 mL). The solution was cooled
to 0 °C, and diisopropylethylamine (0.87 mL, 4.98 mmol) was
added to the solution followed by the bromoketone 7 (1.0 g, 3.89
mmol). The solution was allowed to warm to room temperature
and stirred overnight. The reaction mixture was heated to
reflux for 2 h, cooled to room temperature, and filtered, and the
cake was washed with CH3CN. A 1H NMR spectrum of the solid
revealed the absence of phenyl proton signals. The solid was
combined with the mother liquor and chromatographed on a
silica Prep-Pak eluting with 5/2/3 EtOAc/hexane/CH2Cl2. After
8 column volumes, a small peak was collected and concentrated
in vacuo to give 50 mg of a tan solid which was identified as
dimer U-98140: 1H NMR (CDCl3) δ 5.98 (s, 2H), 3.98 (s, 2H),
3.67-3.72 (m, 8H), 3.57-3.61 (m, 8H); 13C NMR (CDCl3) δ 157.8,
155.1, 154.4, 128.4, 99.4, 95.5, 47.4, 46.5, 27.8, 25.6, 25.3; MS
(FAB) m/z 555 (MH+), 554 (M+), 284, 270.
4-(1-Me t h ylh yd r a zin o)-2,6-d i-1-p yr r olid in ylp yr im i-
d in e (19). A suspension of 51 g of chloro intermediate 18 (4)
in 320 mL of methylhydrazine (Aldrich) was heated at reflux
under nitrogen for 4 h. Reaction mixture become a homoge-
neous light yellow solution as it was heated. The mixture was
then cooled to 0 °C, diluted with 350 mL of ice water, and
filtered. The solid product was washed with cold water (3 ×
250 mL) and then dried, first by pulling dry nitrogen through
the funnel, then at 25 °C, 0.02 mm for 24 h. The product
weighed 52.5 g (99% yield): mp 128-130 °C; IR (mull) cm-1
2954, 2925, 2855, 1573, 1553, 1469, 1452, 1346; 1H NMR (CDCl3)
δ 5.30 (s, 1H, CH), 4.19 (s, 2H, NH2), 3.55-3.51 (t, 4H, NCH2),
3.49-3.33 (m, 4H, NCH2), 3.16 (s, 3H, CH3), 1.94-1.87 (m, 8H,
CH2); 13C NMR (CDCl3) δ 165.6, 162.1, 159.9, 71.6, 46.1, 45.9,
39.7, 25.5, 25.2; MS m/z 262 (M+), 246, 234, 218, 206, 189, 177,
165, 148, 131, 121, 110, 70, 55; exact mass calcd for C13H22N6
m/ z 262.1906, found m/ z 262.1915.
In h ibition of U-97924 Dim er iza tion by Meth a n ol, GSH,
or N-Acetylcystein e. Inhibition of U-97924 dimerization by
GSH or N-acetylcysteine was conducted in acidic aqueous
acetonitrile (apparent pH 4) at room temperature with addition
of either GSH or N-acetylcysteine. The corresponding products
were analyzed by FAB/MS. Inhibition by MeOH was carried
out in acidic aqueous methanol (pH 4) at room temperature,
and the corresponding methoxy adduct was analyzed by GC/
MSD.
7-Meth yl-6-(tr iflu or om eth yl)-2,4-d ip yr r olid in yl-7H-p yr -
r olo[2,3-d ]p yr im id in e (20). A 250 mL, three-neck round
bottom flask was charged with hydrazine intermediate 19 (5.007
g, 19.08 mmol) in 125 mL of diphenyl ether. The suspension
was treated with 1,1,1-trifluoroacetone (3.5 mL, 39.11 mol) at
room temperature. The reaction mixture was then placed under
nitrogen and heated to 245 °C. After 3 h at 245 °C, the reaction
mixture was cooled and chromatographed directly on 900 g of
230-400 mesh silica gel. The column was packed with hexane
and eluted with 3200 mL of hexane, 2000 mL of 10% EtOAc/
hexane, 2000 mL of 20% EtOAc/hexane, and 4000 mL of 50%
EtOAc/hexane. An initial fraction of 3200 mL was collected
followed by 40 mL fraction. Fractions 66-100 contained the
desired trifluoromethyl analog and were combined, thereby
affording 3.7 g (58%) of the product as a light tan solid: 1H NMR
(CDCl3) δ 6.76 (s, 1H), 3.74 (m, 7H), 3.61 (m, 4H), 2.00-1.92
(m, 8H); MS m/ z 339 (M+), 311, 284, 270, 70; TLC (silica gel
Syn th esis. 3-Br om o-2-h yd r oxyp r op yl Ben zoa te (6). A
flame-dried, three-neck 500 mL round bottom flask was charged
with commercially available 3-bromo-1,2-propanediol (11.4 g,
73.5 mmol) in pyridine (100 mL). The solution was cooled to
-15 °C, and benzoyl chloride (10.3g, 73.5 mmol) was added at
a rate of 2 mL/h. Following the addition, the reaction was
allowed to warm to room temperature. After 28 h the solution
was concentrated in vacuo, and a solution of the residue in CH2-
Cl2 was washed with 5% H2SO4 (3 × 50 mL), saturated NaHCO3
(2 × 50 mL), and brine (1 × 50 mL). The organic layer was
dried (MgSO4), filtered, and concentrated in vacuo to give the
desired ester as a colorless oil (14.6 g, 76.0%): 1H NMR (CDCl3)
δ 8.04-8.07 (m, 2H), 7.57-7.62 (m, 1H), 7.41-7.49 (m, 2H),
4.47-4.50 (m, 2H), 4.19-4.24 (m, 1H), 3.52-3.65 (m, 2H), 2.66
(d, 1H).