Synthesis and Characterization of the Food-Derived Carcinogens 2-(Hydroxylamino)-α-carboline and 2-(Hydroxylamino)-3-methyl-α-carboline

Full text of DOI:10.1021/jo971786v

J . Org. Chem. 1998, 63, 895-897
895
Syn th esis a n d Ch a r a cter iza tion of th e
Sch em e 1
F ood -Der ived Ca r cin ogen s
-(Hyd r oxyla m in o)-r-ca r bolin e a n d
-(Hyd r oxyla m in o)-3-m eth yl-r-ca r bolin e
2
2
Shahrokh Kazerani and Michael Novak*
Department of Chemistry and Biochemistry, Miami
University, Oxford, Ohio 45056
Received September 25, 1997
Sch em e 2
In tr od u ction
2
-Amino-R-carboline (ARC) and 2-amino-3-methyl-R-
carboline (MeARC) are representative of a large group
of heterocyclic amines formed by pyrolysis of proteins and
amino acid mixtures.1 These materials have also been
isolated in parts per billion concentrations from broiled
and fried meats, fish, and protein-rich plant material and
have been shown to be mutagenic to Salmonella in the
presence of rat liver homogenates.1 The compounds are
also carcinogenic in mice and rats, with cancers of the
liver and intestine being most prominent.2 These het-
erocyclic amines are now considered to be probable
human carcinogens.2 Average daily intake of hetero-
,3
cyclic amines is diet-dependent but appears to range from
4
0
.1 to 15 µg/person in most developed countries. At
these levels it is believed that heterocyclic amines
constitute a significant cancer risk to human popula-
tions.5
been isolated or are incompletely characterized in the
literature. In the case of the R-carbolines, the synthesis
of 2-(hydroxylamino)-R-carboline (NHOHRC) has recently
The heterocyclic amines are promutagens/procarcino-
gens that require oxidative metabolism for activation.
6
been reported from 2-nitro-R-carboline (NO
experimental details and incomplete product character-
ization.7 The nitro derivative of MeARC, MeNO
RC, has
2
RC) with few
The likely activation processes are shown in Scheme 1.
Although the heterocyclic hydroxylamines are central to
this process, in many cases these compounds have not
2
also been reported in the literature, but attempts to
synthesize the hydroxylamine, MeNHOHRC, from the
nitro compound were unsuccessful.8 Since we have
embarked on a study of the chemical basis of heterocyclic
amine carcinogenesis, we needed to develop reliable
procedures for the synthesis and purification of the
hydroxylamine derivatives of the heterocyclic amines.
Herein we report the synthesis and purification of
NHOHRC and MeNHOHRC and complete spectral char-
acterization of both compounds.
*
To whom correspondence should be addressed. Phone:
13-529-2813. Fax: 513-529-5715. E-mail: MINOVAK@
MIAMIU.ACS.MUOHIO.EDU.
1) Hatch, F. T.; Knize, M. G.; Felton, J . S. Environ. Mol. Mutagen.
991, 17, 4-19. Felton, J . S.; Knize, M. G.; Shen, N. H.; Andersen, B.
D.; Bjeldanes, L. F.; Hatch, F. T. Environ. Health Perspect. 1986, 67,
7-24. Sugimura, T. Environ. Health Perspect. 1986, 67, 5-10.
5
(
1
1
(
2) Sugimura, T. Mutat. Res. 1985, 150, 33-41. Ohgaki, H.;
Matsukura, N.; Morino, K.; Kanachi, T.; Sugimura, T.; Takayama, S.
Carcinogenesis 1984, 5, 815-819.
(
3) Eisenbrand, O.; Tang, W. Toxicology 1993, 84, 1-82.
(4) Murray, S.; Gooderham, N. J .; Boobis, A. R.; Davies. Carcino-
genesis 1989, 10, 763-765. Ushiyama, H.; Wakabayashi, K.; Hirose,
M.; Itoh, M.; Sugimura, T.; Nagao, M. Carcinogenesis 1991, 12, 1417-
Resu lts a n d Discu ssion
1
422. Wakabayashi, K.; Nagao, M.; Esumi, H.; Sugimura, T. Cancer
Res. 1992, 52 (Suppl.), 2092s-2098s.
The parent amines (ARC and MeARC) were synthe-
sized, with minor modifications, from published proce-
dures.9 The amines are also commercially available, but
expensive. Our synthetic procedures for the formation
of the hydroxylamines are outlined in Scheme 2.
Many different routes were attempted for the synthesis
(
5) Adamson, R. H.; Thorgeirsson, U. P.; Snyderwine, E. G.; Thor-
geirsson, S. S.; Reeves, J .; Dalgard, D. W.; Takayama, S.; Sugimura,
T. J pn. J . Cancer Res. 1990, 81, 10-14. Felton, J . S.; Knize, M. G.;
Roper, M.; Fultz, E.; Shen, N. H.; Turtetaub, K. W. Cancer Res. 1992,
10
5
2 (Suppl.), 2103s-2107s. Gaylord, P. W.; Kadlubar, F. F. In Mutagens
in Food. Detection and Prevention; Hayatsu, H., Ed.; CRC Press: Boca
Raton, FL, 1991; pp 229-236.
(6) Yamazoe, Y.; Shimada, M.; Kamataki, T.; Kato, R. Cancer Res.
of NO
2
RC and MeNO RC, but most failed, presumably
2
1
983, 43, 5768-5774. Yamazoe, Y.; Tada, M.; Kamataki, T.; Kato, R.
Biochem. Biophys. Res. Commun. 1981, 102, 432-439. Gilissen, R.
A. H. J .; Bamforth, K. J .; Stavenuiter, J . F. C.; Coughtrie, M. W. H.;
Meerman, J . H. N. Carcinogenesis 1994, 15, 39-45. Shinohara, A.;
Saito, K.; Yamazoe, Y.; Kamataki, T.; Kato, R. Cancer Res. 1986, 46,
(7) Raza, H.; King, R. S.; Squires, B.; Guengerich, F. P.; Miller, D.
W.; Freeman, J . P.; Lang, N. P.; Kadlubar, F. F. Drug Metab. Dispos.
1996, 24, 395-400.
4
362-4367. Yamazoe, Y.; Shimada, M.; Kamataki, T.; Kato, R.
(8) Pfau, W.; Brocksted, U.; Shulze, C.; Neurath, G.; Marquardet,
H. Carcinogenesis 1996, 17, 2727-2732.
Biochem. Biophys. Res. Commun. 1982, 107, 165-172. Snyderwine,
E. G.; Wirth, P.J .; Roller, P. P.; Adamson, R. H.; Sato, S.; Thorgeirsson,
S. S. Carcinogenesis 1988, 9, 411-418. Meerman, J . H. N.; Ringer,
D. P.; Coughtrie, M. W. H.; Bamforth, K. J .; Gilissen, R. A. H. J . Chem.-
Biol. Interact. 1994, 92, 321-328.
(9) Hibino, S.; Sugino, E.; Kuwada, T.; Ogura, N.; Shintani, Y.;
Satoh, K. Chem. Pharm. Bull. 1991, 39 (1), 79-80.
(10) Toronto Research Chemicals, Inc., North York, Ontario, Canada
(in U.S. dollar, MeARC ca. $430/100 mg, ARC ca. $300/100 mg).
S0022-3263(97)01786-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/14/1998

8
96 J . Org. Chem., Vol. 63, No. 3, 1998
Notes
due to the relatively strongly acidic conditions employed.
This oxidation step has proven to be a significant
NHOHRC and MeNHOHRC were fully characterized
1
13
by H, C, DEPT, NOESY, and COSY NMR, MS, and IR.
1
13
synthetic problem in the chemistry of these carcino-
Copies of H and C NMR spectra and tables of NOESY
and COSY NMR correlations are provided in the Sup-
porting Information.
gens.7
,8,11,12
We tried to take advantage of a procedure
1
1
using mildly acidic conditions published by Grivas. In
this procedure the amine is dissolved in a 50:50 mixture
of DMF/AcOH, which is then added to a stirred concen-
1
The H NMR spectra of NHOHRC and MeNHOHRC
show the appearance of two new exchangeable protons
at 8.3-8.8 ppm that correspond to the NH and OH
protons of the hydroxylamines. All aromatic protons are
shifted upfield as expected. In the case of MeNHOHRC,
there is also an upfield shift of the methyl group from
trated solution of NaNO
method provides NO RC and MeNO
less than 10%). Later, our attention was focused on a
2
at room temperature. This
2
2
RC in very low yields
(
procedure, reported by Saito et al., in which Trp-P-2 (a
heterocyclic amine carcinogen structurally similar to
ARC) was oxidized to its nitro derivative.12 With a few
1
13
2.58 to 2.17 ppm. H and C NMR spectra of NHOHRC
and MeNHOHRC are nearly identical with those of the
corresponding amines, ARC and MeARC, with the excep-
tion of the NHOH protons of the hydroxylamines and the
modifications, this method provided MeNO
in 20-37% yield. The yields are low but are superior to
the other methods in our hands.
2
RC and NO RC
2
2
NH protons of the amines. This indicates that the
Both MeNO
following manner: The amine (ARC or MeARC) was
dissolved in a mixture of CH OH and acetic acid (500:1,
v/v). To this solution were added 30% H and Na
WO ‚2H O. The mixture was stirred for 24 h at 50-55
C. Reaction progress was monitored by HPLC. After
2
RC and NO
2
RC were synthesized in the
hydroxylamine tautomer, not oxime tautomer (eq 1), is
the correct structural representation for these com-
pounds.
3
2
O
2
2
-
4
2
°
workup and column chromatography, a yellow solid
product was isolated. Complete spectral characterization
of these compounds is provided in the Experimental
We are attempting to improve the yields of the nitro
compounds and are testing the applicability of these
procedures to the synthesis of hydroxylamine derivatives
of the other carcinogenic heterocyclic amines. The results
of these studies and characterization of the aqueous
solution chemistry of the hydroxylamines and their
carboxylic or sulfuric acid esters will be presented
elsewhere.
Section and Supporting Information.
1
H NMR spectra of MeNO
2
RC and NO
2
RC show the
disappearance of the singlet for NH
2
protons observed
near 6.0 ppm for ARC and MeARC. All aromatic proton
peaks are shifted downfield due to the deshielding effect
of the nitro group. In the case of MeNO
a distinct shift of the methyl group from 2.15 to 2.58 ppm.
Reduction of MeNO RC and NO RC by N ‚H O using
a modification of a procedure reported by Westra
2
RC, there is also
2
2
2
H
4
2
1
3
Exp er im en ta l Section
generated NHOHRC and MeNHOHRC in good yield.
These hydroxylamines are not very stable neat or in
Ca u tion : All compounds reported in this section are known8
or likely mutagens and should be treated as probable human
carcinogens.
solution at room temperature but are stable enough in
DMSO that 1H and 13C NMR spectra can be obtained.
2
-Nitr o-r-ca r bolin e (2-Nitr o-9H-p yr id o[2,3-b]in d ole) a n d
-Meth yl-2-n itr o-r-ca r bolin e (3-Meth yl-2-n itr o-9H-p yr id o-
2,3-b]in d ole). Gen er a l P r oced u r e. In a 250-mL round-
No decomposition of these products was observed when
the hydroxylamines were kept under nitrogen at -80 °C
for a period of 60 days. Both compounds are subject to
air oxidation in solution and also undergo apparent acid-
3
[
bottom flask, 0.5 mmol of ARC (92 mg) or MeARC (99 mg) was
dissolved in a mixture of 50 mL of MeOH and 0.1 mL of
concentrated acetic acid. To this solution was added 50 mL of
2
catalyzed decomposition in H O.
3
0% H
2
O
2
. While stirring, 1.0 g (3 mmol) of Na
2
WO
4
2
‚2H O was
Generally, ca. 100 mg of the nitro compound was
dissolved in 100 mL of dry, peroxide-free (freshly dis-
tilled) THF in the presence of 135 mg of 5% Pd/C at -20
added to the solution, and the temperature was elevated and
kept at 50-55 °C for 24 h. To maintain solution homogeneity,
2
5 mL of MeOH was added to the reaction mixture ca. 5 h after
°
C. Then 200 µL of N
in four portions of 50 µL each. About 15 min after each
addition of N ‚H O, the temperature was allowed to
2
H
4
‚H
2
O was added to the mixture
addition of the Na
monitored by HPLC using a C18 reverse-phase column at λ )
255 nm; 60:40 MeOH/H O, 0.05 M 1:1 KOAc/AcOH buffer was
2
WO
4
‚2H
2
O. The reaction progress was
2
H
4
2
2
used as the mobile phase. After 24 h, the MeOH was removed
by rotary evaporation at room temperature and the solution was
increase from -20 to 0 °C and remain at 0 °C for ca. 1
min. A sample was removed for HPLC, the temperature
was reduced again to -20 °C, and the next portion of
neutralized with 10% NaHCO
times with 50-mL portions of CHCl
washed with 5% NaHCO and distilled water and dried over
3
. The product was extracted three
3
.
The organic layer was
N
2
H
4
2
‚H O was added to the mixture. After 1.5 h the
3
Pd/C was filtered off, and the solvent was removed by
rotary evaporation. The product was rapidly recrystal-
lized from EtOAc and hexanes at -20 °C to provide a
MgSO4. After filtration, the CHCl3 was removed by rotary
evaporation and the crude product was purified by column
chromatography (silica gel) using 85:15 CH
2 mg (20-37%) of products.
-Nitr o-9H-p yr id o[2,3-b]in d ole (NO
2 2
Cl /THF to yield 24-
4
7
5-85% yield of the hydroxylamine.
2
2
rC): mp >270 °C; IR
KBr) 3299, 1540, 1343, 1309 cm ; H NMR (300 MHz, DMSO-
), δ 12.49 (1H, s, exchangeable), 8.87 (1H, d, J ) 8.2 Hz), 8.32
(1H, d, J ) 7.9 Hz), 8.19 (1H, d, J ) 8.2 Hz), 7.60-7.58 (2H, m),
Further reduction of the hydroxylamine to the amine
-1 1
(
was observed when the reaction was performed at
uniform higher temperatures (-5 to 0 °C). Mixtures of
both NHOHRC and ARC were obtained, even with
d
6
1
3
7.35-7.30 (1H, m); C NMR (75.5 MHz, DMSO-d
49.5 (C), 141.4 (C), 131.3 (CH), 129.2 (CH), 122.8 (CH), 120.9
CH), 120.9 (C), 119.5 (C), 112.2 (CH), 109.2 (CH); high-
resolution MS m/e 213.0536, C11 requires 213.0539.
-Meth yl-2-n itr o-9H-p yr id o[2,3-b]in d ole (MeNO rC): mp
>270 °C; IR (KBr) 3244, 1528, 1319 cm ; H NMR (300 MHz,
DMSO-d ) δ 12.24 (1H, s, exchangeable), 8.73 (1H, s), 8.23 (1H,
6
) δ 153.3 (C),
1
shorter reaction times and reduced amounts of N
2
H
4
2
‚H O.
(
7 3 2
H N O
(
(
11) Grivas, S. J . Chem. Res. (S) 1988, 84.
12) Saito, K.; Yamazoe, Y.; Kamatachi, T.; Kato, R. Carcinogenesis
3
2
-
1 1
1
983, 4, 1547-1550.
(
13) Westra, J . G. Carcinogenesis 1981, 2, 355-357.
6

Notes
J . Org. Chem., Vol. 63, No. 3, 1998 897
d, J ) 7.9 Hz), 7.56-7.55 (2H, m), 7.33-7.27 (1H, m), 2.58 (3H,
able), 8.78 (1H, s, exchangeable), 8.63 (1H, d, J ) 1.5 Hz,
exchangeable), 8.24 (1H, d, J ) 8.4 Hz), 7.90 (1H, d, J ) 7.7
Hz), 7.35 (1H, d, J ) 8.0 Hz), 7.25 (1H, m), 7.10 (1H, m), 6.72
1
3
s); C NMR (75.5 MHz, DMSO-d
6
) δ 154.1 (C), 147.9 (C), 141.3
(C), 134.0 (CH), 128.8 (CH), 122.4 (CH), 120.7 (CH), 119.8 (C),
1
19.3 (C), 117.0 (C), 112.1 (CH), 18.3 (CH
requires 227.0696.
-(Hyd r oxyla m in o)-r-ca r bolin e (2-(Hyd r oxyla m in o)-9H-
3
); high-resolution MS
(1H, d, J ) 8.4 Hz); 13C NMR (75.5 MHz, DMSO-d
6
) δ 162.2 (C),
m/e 227.0695, C12
H
9
N
3
O
2
151.3 (C), 137.9 (C), 130.3 (CH), 124.2 (CH), 121.7 (C), 119.3
(CH), 119.2 (CH), 110.9 (CH), 108.2 (C), 99.7 (CH); high-
2
p yr id o[2,3-b]in d ole) a n d 2-(Hyd r oxyla m in o)-3-m et h yl-r-
ca r bolin e (2-(Hyd r oxyla m in o)-3-m eth yl-9H-p yr id o[2,3-b]-
in d ole). Gen er a l P r oced u r e. In a two-neck 125-mL round-
bottom flask, 100 mg of the nitro compound was dissolved in
resolution MS m/e 199.0749, C11
-(H yd r oxyla m in o)-3-m e t h yl-9H -p yr id o[2,3-b]in d ole
MeNHOHrC): mp 195-197 °C dec; IR (KBr) 3312, 1629, 1610,
9 3
H N O requires 199.0746.
2
(
1
-1
1
6
572, 1412, 1300 cm ; H NMR (300 MHz, DMSO-d ) δ 11.37
1
00 mL of dry, peroxide-free THF (freshly distilled). While
(1H, s, exchangeable), 8.70 (1H, s, exchangeable), 8.34 (1H, s,
stirring under nitrogen, 140 mg of 5% Pd/C was added to the
exchangeable), 7.97 (1H, s), 7.86 (1H, d, J ) 7.6 Hz), 7.36 (1H,
solution that was then cooled to -20 °C in a dry ice-ethylene
d, J ) 7.9 Hz), 7.22 (1H, t, J ) 7.5 Hz), 7.08 (1H, t, J ) 7.4 Hz),
glycol bath. After 10 min, 200 µL of N
2
H
4
‚H
2
O in four portions
13
2
(
1
6
.18 (3H, s); C NMR (75.5 MHz, DMSO-d ) δ 158.3 (C), 150.1
of 50 µL was added to the reaction mixture by syringe over a
period of 1 h. About 15 min after each addition the temperature
was allowed to increase from -20 to 0 °C and remain at 0 °C
for 1 min. While at 0 °C, an aliquot was taken for HPLC
analysis, the temperature was lowered to -20 °C, and the next
C), 137.5 (C), 130.0 (CH), 123.7 (CH), 121.6 (C), 119.2 (CH),
19.0 (CH), 110.9 (CH), 109.3 (C), 107.1 (C), 16.8 (CH ); high-
resolution MS m/e 213.0886, C12 0 requires 213.0903.
3
11 3
H N
Ack n ow led gm en t. This work was supported by a
grant from the American Cancer Society (CN-172).
portion of N
this time, the aliquot was analyzed by HPLC at λ ) 255 nm
using 60:40 MeOH/H O, 0.05 M 1:1 KOAc/AcOH buffer and C18
reverse-phase column. About 1.5 h after the first addition of
‚H O, the reaction was stopped by filtering the Pd/C. The
2
H
4
‚H
2
O was added to the reaction mixture. During
2
Su p p or tin g In for m a tion Ava ila ble: Tables of spectro-
N
2
H
4
2
1
13
scopic data and copies of H and C NMR spectra for all nitro
and hydroxylamine compounds (12 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
filtered solution was kept in an EtOH-dry ice bath, and then
the solvent was removed by rotary evaporation at 20 °C. The
product was recrystallized from EtOAc/hexanes at -20 °C to
provide 70-80 mg of white solid (75-85% yield).
2
-(Hyd r oxyla m in o)-9H-p yr id o[2,3-b]in d ole (NHOHrC):
mp 140-148 °C dec; IR (KBr) 3278, 3226, 1629, 1608, 1578, 1419
-
1 1
cm ; H NMR (300 MHz, DMSO-d
6
) δ 11.30 (1H, s, exchange-
J O971786V