Monoamine Oxidase-Catalyzed Oxidative Rearrangement of trans,trans-1-(Aminomethyl)-2-methoxy-3-phenylcyclopropane

Article abstract of DOI:10.1021/jo961540a

trans,trans-1-(Aminomethyl)-2-methoxy-3-phenylcyclopropane (3) was synthesized in three steps from (Z)-β-methoxystyrene and ethyl diazoacetate. Compound 3 was shown to be a substrate and inactivator of mitochondrial beef liver monoamine oxidase (MAO) with

Full text of DOI:10.1021/jo961540a

J . Org. Chem. 1996, 61, 8961-8966
8961
Mon oa m in e Oxid a se-Ca ta lyzed Oxid a tive Rea r r a n gem en t of
tr a n s,tr a n s-1-(Am in om eth yl)-2-m eth oxy-3-p h en ylcyclop r op a n e
Xingliang Lu, Shengtian Yang, and Richard B. Silverman*
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology,
Northwestern University, Evanston, Illinois 60208-3113
Received August 7, 1996X
trans,trans-1-(Aminomethyl)-2-methoxy-3-phenylcyclopropane (3) was synthesized in three steps
from (Z)-â-methoxystyrene and ethyl diazoacetate. Compound 3 was shown to be a substrate and
inactivator of mitochondrial beef liver monoamine oxidase (MAO) with a partition ratio of 1428.
MAO-catalyzed oxidation of 3 produces one major metabolite, isolated and identified by GCOSY,
GHMQC, and GHMBC NMR techniques to be trans,trans-2-methoxy-3-phenyl-1-N-[(3-phenyl-N-
pyrrolyl)methyl]cyclopropane (7). A mechanism, supported by a model reaction, is proposed for
the formation of this metabolite.
Monoamine oxidase (MAO, EC 1.4.3.4) catalyzes the
oxidation of a variety of amine neurotransmitters to the
corresponding imine, which becomes hydrolyzed to the
aldehyde upon release into the aqueous medium.1 There
is much evidence to support a radical mechanism for
MAO.2 Newcomb and Chestney3 have reported a clever
approach to the differentiation of a radical from a
carbocation intermediate in which the incipient high-
energy site is generated adjacent to a trans,trans-2-
methoxy-3-phenylcyclopropyl moiety (Scheme 1). It was
shown that when a radical was generated (route A), the
cyclopropyl C-C bond to which the phenyl group was
attached was the one that broke exclusively, leading to
the formation of the benzylic radical (1), whereas when
a carbocation was generated (route B), the cyclopropyl
C-C bond producing the methoxyl-stabilized cation (2)
broke. We attempted to utilize this approach to gain
further evidence for the formation of an R-radical during
MAO-catalyzed amine oxidations. Consequently, trans,
tra ns-1-(aminomethyl)-2-methoxy-3-phenylcyclopro-
pane (3) was synthesized, and the major product gener-
Resu lts
Syn th esis of tr a n s,tr a n s-1-(Am in om eth yl)-2-m eth -
oxy-3-p h en ylcyclop r op a n e. The route to the synthesis
of trans,trans-1-(aminomethyl)-2-methoxy-3-phenylcyclo-
propane (3) is shown in Scheme 2. The reaction of (Z)-
2-methoxystyrene with ethyl diazoacetate in the presence
of copper sulfate produced two diastereomers of ethyl
2-methoxy-3-phenylcyclopropanecarboxylate (4a and 4b).
The diastereomers were separated, but it was found that
treatment with base in the next step converted the cis,cis-
isomer 4b to the trans,trans-isomer 4a . Consequently,
4a was used for the remainder of the synthesis. Two
routes to 3 were found to be successful. The shortest
route was conversion of the ester to the amide 5 followed
by lithium aluminum hydride reduction. Alternatively,
the ester was saponified to the carboxylic acid 6, which
was converted to the amide 5 by dicyclohexylcarbodiimide
coupling to ammonia. Lithium aluminum hydride reduc-
tion of the amide gave 3.
Mon oa m in e Oxid a se B-Ca ta lyzed Oxid a tion of 3.
Compound 3 was a substrate and inactivator of MAO.
The Km and kcat values were determined to be 10.8 mM
and 1090 min-1, respectively. As determined by the
method of Kitz and Wilson4 the KI and kinact values for
inactivation are 48.5 mM and 1.31 min-1, respectively.
The partition ratio, the number of substrate molecules
converted to product per inactivation event, is kcat/kinact
) 1428. Because of the high partition ratio, it was
thought that it would not be necessary to synthesize 3
in a radiolabeled form; sufficient product could be ob-
tained for NMR structural studies with the unlabeled
compound. Incubation of 3 with MAO led to the forma-
tion of a precipitate, which was removed by centrifuga-
tion. HPLC and TLC analysis of the supernatant indi-
cated that the major component was 3. Centrifugation
and extraction of the precipitate with chloroform gave
an insoluble precipitate shown to be denatured protein
(NaDodSO4-PAGE electrophoresis showed that it had
a mass of 60-70 kDa, consistent with MAO subunits).
The chloroform extract was analyzed by reversed phase
HPLC and by TLC, and only one metabolite was detected.
ated by incubation with MAO was isolated and its
structure determined by heteronuclear NMR techniques
using gradient inverse detection methods. Evidence for
an intermediate radical was not obtained; however, the
product that formed appears to be the result of oxidation
of 3 to the corresponding imine, followed by ring cleavage,
leading to an unusual rearrangement following transimi-
nation with a second molecule of 3. This paper describes
the NMR structure determination of this product and
provides evidence for the mechanism by which it forms.
* Address correspondence to this author at the Department of
Chemistry. Phone: (847) 491-5653. FAX: (847) 491-7713. E-mail:
Agman@chem.nwu.edu.
X Abstract published in Advance ACS Abstracts, November 15, 1996.
(1) Woo, J . C. G.; Silverman, R. B. J . Am. Chem. Soc. 1995, 117,
1663-1664.
(2) Silverman, R. B. Acc. Chem. Res. 1995, 28, 335-342.
(3) Newcomb, M.; Chestney, D. L. J . Am. Chem. Soc. 1994, 116,
9753-9754.
(4) Kitz, R.; Wilson, I. B. J . Biol. Chem. 1962, 237, 3245-3249.
S0022-3263(96)01540-X CCC: $12.00 © 1996 American Chemical Society

8962 J . Org. Chem., Vol. 61, No. 25, 1996
Lu et al.
Sch em e 1
Sch em e 2
On the basis of proton and 13C NMR spectroscopy and
high-resolution mass spectrometry, it was apparent that
a dimer of some kind was formed.
NMR An a lysis of th e Meta bolite. Because of the
small amount of sample, it took about 20 h of acquisition
time to obtain a reasonable 13C NMR spectrum, which
indicated the presence of two phenyl groups and two
double-bond carbons. The low sensitivity of the spec-
trum, however, prevented conventional 2D C-H hetero-
nuclear measurements. Inverse detection experiments,
which measure the proton nuclear response, though,
could be used to improve sensitivity. The sensitivity
enhancement was further aided by the application of
gradient inverse detection methods in the C-H hetero-
nuclear experiments, which avoid long phase cycling
procedures and increase sensitivity.
F igu r e 1. Partial GCOSY spectrum of the metabolite from
MAO-catalyzed oxidation of 3.
Ta ble 1. GCOSY Da ta (H-H Cor r ela tion s) for th e
Meta bolite fr om MAO-Ca ta lyzed Oxid a tion of 3 (in p p m )a
7.48 T 7.32 T 7.14
7.29 T 7.23 T 7.19
7.07 T 6.78 T 6.44
4.10 T 3.95 T 1.89
3.47 T 2.05 T 1.89
GCOSY in the aromatic and double-bond region shows
three spin systems (Figure 1). The peaks at 7.48, 7.32,
and 7.14 ppm and 7.29, 7.23, and 7.19 ppm in the 1H
NMR are from two different phenyl groups; the peaks at
7.07, 6.78, and 6.44 ppm are from double bonds. The
peaks at 7.29, 7.23, and 7.19 ppm are very similar to the
phenyl group protons of 3. GCOSY in the 1-5 ppm
region shows only one spin system (Table 1); the spin-
spin connectivities of the peaks are very similar to those
of 3. The high-resolution mass spectrum suggests that
the product is a dimeric compound with the molecular
formula C21H21NO. Mass fragments indicate the pres-
ence of a cyclopropyl group with substituted phenyl and
methoxyl groups. The only large chemical shift differ-
ences in the metabolite and 3 are reflected in the change
in the 13C spectrum (Figure 2) from 42.7 ppm in com-
pound 3 to 51.5 ppm in the metabolite and in the 1H
spectrum (Figure 3) from 2.80 and 2.90 ppm in 3 to 3.95
and 4.10 ppm in the metabolite. Furthermore, irradia-
tion of the proton at 3.47 ppm (on the cyclopropyl group
at the carbon with the methoxyl group) gave an NOE at
2.05 ppm (cyclopropyl proton on the carbon with the
phenyl group), indicating that these protons are cis to
a
The symbol T denotes which protons correlate. See Figure
1.
one another (Table 2). Irradiation at 1.89 ppm produced
an NOE at 7.23 ppm, indicating that the cyclopropyl
proton on the carbon with the methylene group is cis to
the phenyl group on the cyclopropyl group. This suggests
a similarity of part of the metabolite structure to that of
3 and that the major difference is in the attachment of
another moiety at the nitrogen of 3 (Figure 4).
GHMQC experiments were performed to establish
C-H connections (Figure 5 and Table 3). Contrary to
the one-dimensional 13C NMR spectrum, the GHMQC
spectra exhibit an excellent signal-to-noise ratio with an
acquisition time of 30 h. To establish links between the
separate spin systems, GHMBC experiments were per-
formed (Figure 6 and Table 4). From these studies it is
apparent that the proton at 2.05 ppm correlates with the
carbon of the phenyl at 128.3 ppm, with the carbon of
the aminomethyl group at 51.5 ppm, and with the carbon
of the cyclopropyl group at 27.3 ppm. The methoxyl
protons at 3.12 ppm correlate with the cyclopropyl carbon

Monoamine Oxidase-Catalyzed Rearrangement
J . Org. Chem., Vol. 61, No. 25, 1996 8963
F igu r e 2. 13C NMR spectra of 3 (top) and the metabolite from MAO-catalyzed oxidation of 3 (bottom). The down arrows indicate
the shift in the spectra from 42.7 ppm in 3 to 51.5 ppm in the metabolite; the inverted triangles indicate impurities; the asterisk
marks the CD2Cl2 carbons.
F igu r e 3. 1H NMR spectra of 3 (top) and the metabolite from MAO-catalyzed oxidation of 3 (bottom). The down arrows indicate
the shift in the spectra from 2.80 and 2.90 ppm in 3 to 3.95 and 4.10 ppm in the metabolite; the inverted triangles indicate
impurities; the asterisk in the top spectrum marks the water peak and in the bottom spectrum marks the CDCl3 proton.
Ta ble 2. NOE Da ta (H-H Sp a ce Cor r ela tion s) for th e
Meta bolite fr om MAO-Ca ta lyzed Oxid a tion of 3 (in p p m )
irradiate on 7.48, NOE on 7.32 (8.6%), 7.07 (4.3%), 6.44 (4.9%)
irradiate on 7.07, NOE on 7.48 (12.4%)
irradiate on 6.78, NOE on 6.44 (7.8%)
irradiate on 6.44, NOE on 7.48 (9.4%), 6.78 (8.3%)
irradiate on 3.95, NOE on 7.07 (3.4%), 6.78 (3.2%), 2.05(5.3%)
irradiate on 4.10, NOE on 7.07 (3.2%), 6.78 (4.1%), 3.47 (2.3%),
1.89 (3.4%)
irradiate on 3.47, NOE on 3.15 (3.4%), 2.05 (7.8%)
irradiate on 2.05, NOE on 7.23 (11.8%), 3.47 (10.5%)
irradiate on 1.89, NOE on 7.23 (14.6%)
irradiate on 7.19, NOE on 7.29 (4.9%)
to which the methoxyl group is attached (65.2 ppm). The
aminomethylene protons at 3.95 and 4.10 ppm both
correlate with the cyclopropyl carbon atoms at 65.2 and
F igu r e 4. A comparison of the general metabolite structure
with that of 3.
27.3 ppm, but only the 3.95 ppm proton correlates with
the 29.3 ppm cyclopropyl carbon. These results support
the existence of the starting material structure within
the full metabolite structure. Further examination of the
spectrum indicates that the protons at 3.95 and 4.10 ppm
also are long-range coupled to the two double-bond
carbons at 117.6 and 121.9 ppm, which suggests that the
aminomethyl nitrogen is directly bonded to the two
double-bond carbons. Since the GCOSY spectrum shows
that the three double-bond protons are spin correlated
with each other, it is apparent that a five-membered

8964 J . Org. Chem., Vol. 61, No. 25, 1996
Lu et al.
pyrrole structure is present. The C-H correlations
shown in Figures 5 and 6 and listed in Tables 3 and 4
are consistent with either structure 7 or 8. Further NOE
F igu r e 5. Partial GHMQC spectrum of the metabolite from
MAO-catalyzed oxidation of 3.
Ta ble 3. GHMQC Da ta (C-H Cor r ela tion s) for th e
difference experiments (Table 2) ruled out structure 8.
Irradiation of protons at 7.48 and 6.44 ppm indicates that
these protons are close in space, and irradiation of the
7.48 and 7.07 ppm protons shows that they are close.
These results are only consistent with structure 7.
Finally, the 1H and 13C chemical shifts of 3-phenylpyrrole
were very similar to the chemical shifts of the moiety
attached to the nitrogen of 3.
Meta bolite fr om MAO-Ca ta lyzed Oxid a tion of 3 (in p p m )a
7.48 T 125.1
7.32 T 128.9
7.29 T 128.4
7.23 T 128.3
7.19 T 126.2
7.14 T 125.5
7.07 T 117.6
6.78 T 121.9
6.44 T 106.6
4.10 T 51.5
3.95 T 51.5
3.47 T 65.2
3.15 T 58.3
2.05 T 29.3
1.89 T 27.3
Mod el Stu d y for th e F or m a tion of 7. To test the
hypothesized mechanism for the formation of 7 from the
MAO-catalyzed oxidation of 3, the reaction of 2-phenyl-
succinaldehyde with 3 was carried out at -78 °C.
Compound 7 was isolated as the major product.
a
The symbol T denotes which protons correlate with which
carbons. See Figure 5.
Discu ssion
trans,trans-1-(Aminomethyl)-2-methoxy-3-phenylcyclo-
propane (3) was synthesized as a potential substrate for
MAO to determine if radical character at the R-position
could be detected. On the basis of the work of Newcomb
and Chestney,3 if an R-radical was formed and was
relatively stable, then aldehyde 9 (Scheme 3) would be
expected to be produced; further reaction with 3 might
lead to 8. Whereas R-cation (imine) formation would lead
to 2-phenylsuccinaldehyde (10), reaction with 3 might
give 7 (Scheme 4). It should be noted that 7 is obtained
regardless of which aldehyde carbonyl is initially at-
tacked by 3. This product, however, does not provide
direct information regarding potential intermediates in
the reaction pathway because it is derived from the
expected iminium product.
The molecular weight obtained by high-resolution mass
spectrometry of the metabolite formed from MAO-
catalyzed oxidation of 3 indicated that a dimeric material
was produced. By a combination of GCOSY, GHMQC,
and GHMBC homonuclear and heteronuclear NMR
techniques (see Figures 1, 5, and 6), a phenyl-substituted
pyrrole attached to the 2-methoxy-3-phenylcyclopropane
(7) was identified. To test the hypothesized mechanism
for the formation of 7 (Scheme 4), 3 was allowed to react
with 2-phenylsuccinaldehyde (10), and 7 was isolated.
The results presented describe an interesting nonen-
zymatic rearrangement from the MAO-catalyzed oxida-
tion of 3. If the intermediate R-radical is formed, it does
F igu r e 6. Partial GHMBC spectrum of the metabolite from
MAO-catalyzed oxidation of 3.
not lead to cyclopropyl ring cleavage prior to second
electron oxidation to the product imine. A geometric
constraint, as a result of interactions with the active site
of the enzyme, theoretically could preclude ring cleavage,
although Newcomb and co-workers have found no ex-
perimental evidence to support a kinetic effect by an
enzyme-induced geometric constraint.5 Previous stud-
ies,6 however, strongly support the formation of an
R-radical in the MAO-catalyzed oxidations of amines.

Monoamine Oxidase-Catalyzed Rearrangement
J . Org. Chem., Vol. 61, No. 25, 1996 8965
Ta ble 4. GHMBC Da ta (C-H Lon g-Ra n ge Cor r ela tion s)
for th e Meta bolite fr om MAO-Ca ta lyzed Oxid a tion of 3
(in p p m )a
with lithium aluminum hydride (1.2 g, 37 mmol) in 25 mL of
anhydrous THF at reflux under a positive pressure of nitrogen
for 4 h followed by quenching with water in THF. Aqueous
20% sodium hydroxide (4 mL) was added to remove the
aluminate salts. The THF solution was extracted with 2 N
HCl (3 × 10 mL), the acid extracts were back-extracted with
ether (10 mL), the aqueous solution was evaporated to dryness,
and the residue was crystallized with ethanol-ethyl acetate
to give 1.9 g (85%) of 3: NMR (CDCl3) δ 8.26 (br, 2 H), 7.2-
7.3 (m, 5 H), 3.42 (dd, 1 H), 3.11 (s, 3 H), 2.6-2.8 (br, 2 H),
2.06 (t, 1 H), 1.90 (dd, 1 H). Anal. Calcd for C11H16ClNO: C,
61.82; H, 7.49; N, 6.56; Cl, 16.63. Found: C, 61.50; H, 7.49;
N, 6.50; Cl, 16.83.
7.48 T 125.0, 125.5, 128.9
7.32 T 136.3, 128.9, 125.1
7.29 T 126.2, 128.3, 137.0
7.23 T 126.2, 128.4
7.14 T 125.1
7.19 T 128.3
7.07 T 125.0, 121.9, 106.6
6.78 T 125.0, 117.6, 106.6
6.44 T 117.6, 121.9
a
The symbol T denotes which protons correlate with which
t r a n s,t r a n s-2-Me t h oxy-3-p h e n ylc yc lop r op a n e c a r -
boxylic Acid (6). Compound 4a (1.0 g, 4.5 mmol) was
saponified by the addition of 1.5 g of potassium hydroxide in
10 mL of ethanol at rt with stirring for 4 h. After acidification
with 15 mL of 3 N HCl, 6 (0.79 g, 90%) was obtained by
extraction into ether followed by evaporation: NMR (CDCl3)
δ 7.2-7.4 (m, 5 H), 3.89 (dd, 1 H), 3.35 (s, 3 H), 2.78 (t, 1 H),
2.20 (dd, 1 H); HRMS calcd 192.0786, found 192.0798, m/z 192
(192, 162, 147, 131, 115, 103, 91, 77, 51).
carbons. See Figure 6.
Sch em e 3
tr a n s,tr a n s-2-Meth oxy-3-p h en ylcyclop r op a n eca r boxa -
m id e (5). A second procedure for the synthesis of 5 is as
follows: To a stirred mixture of the compound 6 (192 mg, 1.0
mmol) and N-hydroxysuccinimide (138 mg, 1.2 mmol) in
methylene chloride (10 mL) at 0 °C was added N,N′-dicyclo-
hexylcarbodiimide (247 mg, 1.2 mmol). The reaction mixture
was stirred for 1.5 h under the same conditions, and 30%
aqueous ammonia (166 µL) was added to the stirred solution.
The reaction mixture was allowed to continue to stir for 1 h.
Dicyclohexylurea, which precipitated during the reaction, was
filtered off. The filtrate was evaporated under reduced pres-
sure to dryness, giving 5 (150 mg, 78%).
Dieth yl P h en ylsu ccin a te. To a 25-mL round bottom flask
fitted with a Dean-Stark trap were added phenylsuccinic acid
(1.0 g, 5.15 mmol), ethyl alcohol (5 mL), p-toluenesulfonic acid
(50 mg), and benzene (10 mL). Following overnight reflux,
the benzene solution was washed with saturated sodium
bicarbonate (2 × 5 mL) and water (2 × 5 mL) and dried over
sodium sulfate. Removal of the solvent resulted in a colorless
oil (1.1 g, 86%): 1H NMR (CDCl3) δ 7.30 (m, 5 H), 4.13 (m, 5
H), 3.18 (dd,1 H), 2.68 (dd, 1 H), 1.21 (m, 6 H), agrees with
the spectrum reported.8
Sch em e 4
Rea ction of 3 w ith 2-P h en ylsu ccin a ld eh yd e. To a
stirred solution of diethyl phenylsuccinate (1.0 g, 4.02 mmol)
in dry toluene (10 mL) at -78 °C was added a -78 °C solution
of 1.5 M diisobutylaluminum hydride (DIBAL) in toluene (10
mL) via cannula. The rate of addition was adjusted so as to
keep the internal temperature below -65 °C over the ca. 45
min addition time. The 2-phenylsuccinaldehyde produced was
too unstable to isolate. The reaction mixture was stirred for
an additional 3 h at -78 °C, and then the reaction was
quenched by slow addition of 200 mg (1 mmol) of compound 3
in 3 mL of anhydrous methanol at -78 °C, again so as to keep
the internal temperature below -65 °C. The resulting white
emulsion was allowed to warm to rt and was poured into 50
mL of 1 N HCl with swirling over 15 min. The aqueous
mixture was then extracted with methylene chloride (3 × 50
mL). The combined organic layers were washed with brine
(50 mL), dried over Na2SO4, filtered, and concentrated in vacuo
to give 50 mg of crude product as a yellow oil (4%). The
Exp er im en ta l Section
Rea gen ts. Compounds 4a and 4b were synthesized as
reported.7 All reagents were purchased from Aldrich Chemical
Co.
(5) (a) Newcomb, M.; Le Tadic-Biadatti, M.-H.; Chestney, D. L.;
Roberts, E. S.; Hollenberg, P. F. J . Am. Chem. Soc. 1995, 117, 12085-
12091. (b) Atkinson, J . K.; Hollenberg, P. F.; Ingold, K. U.; J ohnson,
C. C.; Le Tadic, M.-H.; Newcomb, M.; Putt, D. A. Biochemistry 1994,
33, 10630-10637. (c) Martin-Esker, A. A.; J ohnson, C. C.; Horner, J .
H.; Newcomb, M. J . Am. Chem. Soc. 1994, 116, 9174-9181.
(6) (a) Silverman, R. B.; Zhou, J . P.; Eaton, P. E. J . Am. Chem. Soc.
1993, 115, 8841-8842. (b) Silverman, R. B.; Lu, X.; Zhou, J . J . P.;
Swihart, A. J . Am. Chem. Soc. 1994, 116, 11590-11591. (c) Silverman,
R. B.; Zhou, J . J . P.; Ding, C. Z.; Lu, X. J . Am. Chem. Soc. 1995, 117,
12895-12896.
tr a n s,tr a n s-2-Meth oxy-3-p h en ylcyclop r op a n eca r boxa -
m id e (5). Ammonium hydroxide (20 mL) was added to 4a
(1.0 g, 4.5 mmol), and the mixture was stirred at rt overnight.
The precipitate that formed was washed with hexane to give
a pale yellow solid (0.78 g, 90%): 1H NMR (CDCl3) δ 7.2-7.4
(m, 5 H), 5.71 (br, 1 H), 5.59 (br, 1 H), 3.86 (dd, 1 H), 3.28 (s,
3 H), 2.71 (t, 1 H), 2.04 (dd, 1 H); HRMS calcd 191.0946, found
191.0930, m/z 191 (191, 159, 147, 131, 115, 103, 91, 77, 51).
tr a n s,tr a n s-1-(Am in om et h yl)-2-m et h oxy-3-p h en ylcy-
clop r op a n e (3). Compound 5 (2.0 g, 10.5 mmol) was reduced
(7) Newcomb, M.; Chestney, D. L. J . Chem. Soc., Perkin Trans. 2
1996, 1467-1473.
(8) Chadha, V. K.; Leidal, K. G.; Plapp, B. V. J . Med. Chem. 1985,
28, 36-40.

8966 J . Org. Chem., Vol. 61, No. 25, 1996
Lu et al.
compound was purified by flash chromatography, eluting with
10% ethyl acetate in hexane, and by TLC on silica gel, eluting
with methylene chloride: 1H NMR (CDCl3) δ 7.48 (d, 2 H),
7.14-7.32 (m, 8 H), 7.07 (m, 1 H), 6.78 (m, 1 H), 6.44 (m, 1
H), 4.10 (m, 1 H), 3.95 (m, 1 H), 3.47 (m, 1 H), 3.12 (s, 3 H),
2.05 (m, 1 H), 1.89 (m, 1 H); 13C NMR (CH2Cl2) δ 129.0, 128.5,
128.4, 126.1, 125.6, 125.2, 122.0, 117.5, 106.7, 65.3, 58.2, 51.5,
29.3, 27.4; HRMS calcd 303.1623, found 303.1661.
the CHCl3-insoluble material was determined by NaDodSO4
gel electrophoresis to be 60-70 kDa, which is denatured
protein. The CHCl3 extract was analyzed by reversed phase
HPLC on a C-18 column as above, which showed that only
one compound was formed. The extract also was analyzed by
silica gel TLC, eluting with ethyl acetate/hexane (20/80) or
1-butanol/H2O/HOAC (12/5/3), which showed that only one
metabolite was present. The extract was washed with 2% HCl
to remove some 3 and, after evaporation of the solvent, was
En zym e a n d Assa y. Mitochondrial beef liver MAO was
isolated and assayed as previously reported.9
1
analyzed by H NMR and 13C NMR: 1H NMR (CD2Cl2) δ 7.48
MAO-Ca ta lyzed Oxid a tion of 3. Compound 3 (15 mg) in
1.00 mL of 100 mM sodium phosphate buffer (pH 7.2) was
incubated with MAO B (100 µL, 200 µM) at rt for 2 days. The
activity of the MAO B, compared with a control experiment
without compound 3, was less than 10%. During incubation
the solution gradually turned cloudy; the cloudy mixture was
centrifuged in a microcentrifuge to isolate the precipitate. The
supernatant was analyzed by reversed phase HPLC using a 5
µm, 4.6 × 250 mm C-18 column, eluting with 40% water in
acetonitrile and monitoring at 254 nm. The main component
was the substrate. The same result was shown in the TLC
analysis, eluting with 1-butanol/H2O/HOAC (12/5/3) on a silica
gel plate with ninhydrin detection.
(d, 2 H), 7.32 (m, 2 H), 7.29 (m, 2 H), 7.23 (m, 2 H), 7.19 (m,
1 H), 7.14 (m, 1 H), 7.07 (m, 1 H), 6.78 (m, 1 H), 6.44 (m, 1 H),
4.10 (m, 1 H), 3.95 (m, 1 H), 3.47 (m, 1 H), 3.12 (s, 3 H), 2.10
(m, 1 H), 1.89 (m, 1 H); 13C NMR (CD2Cl2) δ 137.0, 136.3, 128.9
(2 C), 128.4 (2 C), 128.3 (2 C), 126.2, 125.5, 125.1 (2 C), 121.9,
117.6, 106.6, 65.2, 58.3, 51.5, 29.3, 27.3. In addition, GCOSY
and GHMQC were used for determination of the H-H cor-
relations and C-H correlations, respectively. The molecular
ion of the metabolite was determined by HRMS (calcd 303.1623,
found 303.1634) or as M + 1 (calcd 304.1681, found 304.1653);
the main fragments are 272, 156, 147, 117, 91, 43, and 41.
The precipitate was dried with a speed vac concentrator
followed by extraction with CHCl3. The molecular weight of
Ack n ow led gm en t. Financial support of this re-
search from the National Institutes of Health (GM
32634) is gratefully acknowledged.
(9) Banik, G. M.; Silverman, R. B. J . Am. Chem. Soc. 1990, 112,
4499-4507.
J O961540A