PAPER
Synthesis of N-Acetoxy-N-benzoyl-2-aminofluorene by Lead Tetraacetate Oxidation
2417
Compound 10 was debenzoylated23 to give product 11.
The H NMR peak at = 8.76 ppm was attributed to the
Anal. Calcd for C20H15NO: C, 84.21; H, 5.26; N, 4.91. Found: C,
84.23; H, 5.25; N, 5.04.
1
NH proton; and its disappearance upon addition of D2O,
confirmed the structure 11.
N-Acetoxy-N-benzoyl-2-fluorenylamine (9)
A solution of nitrone 8 (500 mg, 1.76 mmol) in anhyd benzene (10
mL) was kept at –7 °C to 0 °C. LTA (800 mg, 2.46 mmol) was add-
ed in portions (exothermic reaction took place immediately), and
the reaction mixture was stirred for 10 min at 0 °C. Filtration of
white lead diacetate, and evaporation of the solvent at reduced pres-
sure afforded the crude solid product 9. Crude product was succes-
sively washed with cold (–20 °C) Et2O and CH2Cl2. The white solid
obtained was recrystallised several times from Et2O–hexane at 0 °C
to give pure 9, as a white crystalline solid (700 mg, 87%).
In conclusion, we have developed a facile activation path-
way to N-benzoylated ultimate carcinogen 9 from the
“precarcinogen” polynuclear nitrone 8. The activation13
of 8 which is required to produce the reactive metabolite
9 was achieved using lead tetraacetate (LTA), an excellent
oxidant. Compound 9 belongs to a new type of reactive
metabolites like its N-deacetylated and N-acetylated ana-
logues 5 and 6,3–9 respectively.
Mp 143–145 °C.
IR (KBr): 1670 (s, C=O, PhCON), 1788 (s, C=O, OCOCH3), 1482,
1490 (s, C-N), 1218 (s, C-O, OCOCH3) cm–1.
1H NMR (400 MHz, CDCl3): = 2.28 (s, 3 H, OCOCH3), 3.86 (s, 2
H, -CH2-), 7.34–7.82 (m, 12 H, Ar-H).
Silica gel (E-Merck GF254, 0.2 mm) with fluorescent indication was
used for TLC. The mobile phases used for TLC: CHCl3, benzene,
CHCl3–hexane (9:2), Et2O–hexane (1:1), MeOH–H2O (7:3) and
(9:1), EtOAc–hexane (1:1), MeOH, MeCN–MeOH–H2O (4:3:1),
and benzene–Et2O (1:1). HPLC was performed with one of the fol-
lowing mobile phases: H2O–MeCN (5:3), (7:3) and (7:1). Reagents
were obtained from commercial sources as indicated: dG (Aldrich),
2-nitrofluorene (E-Merck), LTA (Aldrich), benzaldehyde (E-Mer-
ck), silica gel-923 (Aldrich), saphadex G-15 (Aldrich), sodium cit-
rate (Aldrich), ammonia (s.d. fine). Mps (uncorrected) were
Anal. Calcd for C22H17NO3: C, 76.96; H, 4.95; N, 4.08. Found: C,
76.84; H, 4.94; N, 4.11.
N-(Benzoyl)-N-(deoxyguanosin-8-yl)-2-aminofluorene (10)
Significant modifications were made to the procedure used by Kriek
et al.24 in the synthesis of Gu-adducts.
1
recorded on a SELACO 605 melting point apparatus. H NMR
Compound 9 (291 mg, 0.89 mmol) in 95% EtOH (15 mL) was add-
ed to dG (49 mg, 0.17 mmol) in sodium citrate buffer (pH 7.0, 2
mM, 30 mL) at 55 °C over 2 h, and the mixture was stirred further
for 12 h at 60 °C. The reaction mixture was diluted with H2O (60
mL) and the EtOH was evaporated. The aqueous phase was extract-
ed with Et2O (5 10 mL) and EtOAc (5 15 mL). The Et2O extract
was discarded; the EtOAc extract was dried (Na2SO4) and evaporat-
ed to give crude solid 10. The crude product was first purified over
a silica gel column (MeOH–CHCl3, 9:2); then chromatographed on
sephadex G-15 (EtOH–CHCl3, 7:4) to give the product 10 (107 mg,
22%), which was stable in neutral aqueous solution for several
weeks at 0 °C. HPLC analysis (H2O–MeCN, 7:1) of the aqueous so-
lution of 10 indicated 98.9% purity.
spectra were recorded on Bruker AMX-400 (400 MHz) spectrome-
ter using CDCl3 or DMSO-d6 as solvent with TMS as internal stan-
dard. IR spectra were recorded on a Bio-Rad Win-IR spectrometer.
Elemental analyses were obtained on Vario-EL instrument. Low
temperature reactions were carried out using cryostat model MRP
700. HPLC was performed with a Lachrom-2000 Merck-Hitachi
L7100 pump with RP18.250-4 mm column, and UV Detector-UV-
VIS L7400.
N-(2-Fluorenyl)hydroxylamine (3)
Significant modifications were made to the method of Patrick.15
Zn dust (1.31 g, 0.02 mol) was added in portions over 45 min to a
stirred, cooled (20–30 °C) mixture of 2-nitrofluorene (7, 2.11 g,
0.01 mol), NH4Cl (2.12 g, 0.04 mol), and catalytic amount of
histidine16 in H2O (50 mL). The pH was kept at 7.4–7.5 by adding
small portions of ammonia. The reaction temperature was kept at
20–30 °C. The reaction mixture was stirred for an additional 45 min
and the mixture was filtered by suction at r.t. The filtrate was satu-
rated with common salt, and cooled in an ice bath for 2 h to ensure
maximum crystallization of product 3. The yellow solid was filtered
and dried. Crude product was washed with cyclohexane and recrys-
tallised from benzene to give 3, as a colourless solid (1.8 g, 91%);
mp 168–170 °C (Lit.15 mp 170 °C).
IR (KBr): 3340, 3032, 2920, 1676, 1645, 1560, 1400, 1064, 1030,
1011, 891 cm–1.
1
1H NMR (400 MHz, DMSO-d6): = 2.04 (m, 2 H2 , sug), 3.74 (m,
1
1
1
2 H5 , sug), 3.92 (s, 2 H9, AF), 3.98 (m, 1 H4 , sug), 4.41 (m, 1 H3 ,
1
1
sug), 5.38 (s, 1 H3 , OH), 6.04 (s, 1 H5 , OH, sug-OH), 6.37 (m, 1
H1 , sug), 6.52 (s, 2 H, Gu-NH2, D2O exchangeable), 7.24 (t, 1 H7,
1
AF), 7.36 (t, 1 H6, AF), 7.52–7.69 (m, 1 H8, AF and 5 H, Ar-H), 7.71
(d, 1 H3, AF), 7.78 (d, 1 H4, AF), 7.81 (d, 1 H5, AF), 8.12 (s, 1 H1,
AF), 10.59 (s, 1 H, Gu-NH, D2O exchangeable).
Anal. Calcd for C30H26N6O5: C, 65.45; H, 4.72; N, 15.27. Found: C,
65.10; H, 4.71; N, 15.67.
-Phenyl-N-(2-aminofluorenyl)nitrone (8)
N-(2 -Deoxyguanosin-8-yl)-2-aminofluorene (11)
Compound 11 was prepared by the method of Underwood et al.23
using the heterogeneous system (Na2CO3–MeOH) for debenzoyla-
tion of 10. Spectral data are in good agreement with those of an au-
thentic sample.25
Equimolar solutions of N-(2-fluorenyl)hydroxylamine (3) and ben-
zaldehyde in a minimum volume of EtOH was kept at 10–15 °C and
set aside in the dark for 4 h. The reaction mixture was stored over-
night at 0 °C. The crude nitrone was separated and crystallised from
EtOH to give 8, as a white crystalline solid (95%).
Mp 117–118 °C (dec.).
IR (paraffin): 1552 (C=N), 1088 (NO) cm–1.
Acknowledgement
1H NMR (400 MHz, CDCl3): = 3.98 (s, 2 H, -CH2-), 7.32–7.99
(m, 8 H, Ar-H), 8.12 (s, 1 H, CH=N), 8.31 (d, 2 H, Ar-H), 8.44 (d,
2 H, Ar-H).
We are grateful to the Department of Science and Technology
(DST), Government of India, New Delhi, for financial support un-
der the project vide No. SP/S1/GO5/97.
Synthesis 2001, No. 16, 2415–2418 ISSN 0039-7881 © Thieme Stuttgart · New York