Monoamine oxidase-catalyzed oxidation of endo,endo-2-amino-6-[(Z)-2'-phenyl]ethenylbicyclo[2.2.1]heptane, a potential probe for a radical cation intermediate

Article abstract of DOI:10.1016/S0968-0896(00)00094-8

An 11-step synthesis of endo,endo-2-amino-6-[(E)-2'-phenyl]ethenylbicyclo[2.2.1]heptane (6) and the corresponding (Z)-isomer (7) was carried out in an attempt to make a compound that could trap the purported amine radical cation intermediate during monoamine oxidase (MAO)-catalyzed oxidation of amines. The E-isomer was not a substrate for MAO, and the Z-isomer was a very poor substrate. No trapping product was observed. Possible explanations for the inability of these compounds to trap a potential radical cation intermediate are discussed. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00094-8

Bioorganic & Medicinal Chemistry 8 (2000) 1645±1651
Monoamine Oxidase-Catalyzed Oxidation of
endo,endo-2-Amino-6-[(Z)-20-phenyl]ethenylbicyclo[2.2.1]heptane,
a Potential Probe for a Radical Cation Intermediate
Xueqing Wang and Richard B. Silverman*
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University,
Evanston, IL 60208-3113, USA
Received 4 February 2000; accepted 8 March 2000
AbstractÐAn 11-step synthesis of endo,endo-2-amino-6-[(E)-20-phenyl]ethenylbicyclo[2.2.1]heptane (6) and the corresponding (Z)-
isomer (7) was carried out in an attempt to make a compound that could trap the purported amine radical cation intermediate
during monoamine oxidase (MAO)-catalyzed oxidation of amines. The E-isomer was not a substrate for MAO, and the Z-isomer
was a very poor substrate. No trapping product was observed. Possible explanations for the inability of these compounds to trap a
potential radical cation intermediate are discussed. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Earlier we had synthesized (E)- and (Z)-2-(iodoeth-
enyl)benzyl amine (5) as a potential probe for the for-
mation of a radical cation;7 it was hoped that the rate of
cyclization of the double bond to the purported radical
cation intermediate (Scheme 4) would be sucient to
trap it. Unfortunately, only amine oxidation of the Z-
isomer was observed without cyclization. To enhance
the chance for intramolecular cyclization, we synthe-
sized (E)- and (Z)-endo,endo-2-amino-6-[20-phenyl]eth-
enylbicyclo[2.2.1]heptane (6 and 7, respectively).
Unfortunately, we again were unable to detect the for-
mation of any cyclized products.
Monoamine oxidase (MAO, EC 1.4.3.4), a ¯avoenzyme
in the outer mitochondrial membrane, catalyzes the
oxidative deamination of a wide variety of biogenic and
xenobiotic amines1 to the corresponding imines, which
are nonenzymatically hydrolyzed to aldehydes.2 The
mechanism of this enzyme-catalyzed reaction has long
been debated; there are three principal pathways that
have been proposed. Scheme 1 depicts a single-electron
transfer mechanism, via the amine radical cation (1) to
the a-radical (2), followed by either second electron
transfer to the imine or radical combination with a
group on the enzyme to 3 and then to the imine. The
amine radical cation intermediate is supported by a
variety of substrate analogue and inactivator studies.3
The second mechanism (Scheme 2) bypasses the initial
electron transfer in favor of direct hydrogen atom
abstraction.4 The support for this appears to come
mainly from the inability to detect an initial amine
radical cation intermediate spectroscopically. Scheme 3
shows the group transfer mechanism, in which the
amine undergoes nucleophilic attack of the ¯avin to give
a covalent adduct (4), which transfers electrons by
elimination. This mechanism is supported by chemical
model studies5 and Hammett studies.6
Results and Discussion
Syntheses of endo,endo-2-amino-6-[(E)-20-phenyl]-
ethenylbicyclo[2.2.1]heptane (6) and endo,endo-2-amino-
6-[(Z)-20phenyl]ethenylbicyclo[2.2.1]heptane (7)
Amines 6 and 7 were synthesized as shown in Scheme 5.
Ketal ester 12 was prepared from cyclopentadiene in 5
steps.8 Reduction of 12 with DIBAL did not stop at the
*Corresponding author. Tel.: +1-847-491-5653; fax: +1-847-491-
7713; e-mail: agman@chem.nwu.edu
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00094-8

1646
X. Wang, R. B. Silverman / Bioorg. Med. Chem. 8 (2000) 1645±1651
Scheme 1.
Enzymatic studies of 6 and 7
Both 6 and 7 were found to be competitive inhibitors of
beef liver MAO B as determined by Dixon10 and Cor-
nish-Bowden plots,11 although 6 is slightly more potent
than 7. Compound 6 has a Ki of 70 mM, and the Ki
for 7 is 180 mM. No time-dependent inactivation was
observed. Prolonged incubation of both did not reveal
any cyclized products by GC±MS detection. However,
after ®ve days of incubation, 7 was found to be con-
verted to the normal turnover product, ketone 17, as
con®rmed by comparison with an authentic sample.
Scheme 2.
desired aldehyde but was reduced to alcohol 13, whose
structure was con®rmed by X-ray di€raction analysis of
the corresponding carboxylic acid (data not shown). A
Wittig reaction with 14 a€orded a mixture of both E-
and Z-isomers (15) in a 1:1.3 ratio, which were insepar-
able by column chromatography. After deprotection,
however, the corresponding (E)-16 and (Z)-17 ketones
were isolated cleanly by preparative thin layer chro-
matography. Direct reductive amination of 16 and 17
with NH4OAc/NaCNBH3 did not yield any of the
desired product. The corresponding oximes were syn-
thesized, and various reducing reagents were tried to
reduce the oximes. Among them, TiCl3/NaCNBH3 was
the most successful.9 Both the amino group and the
phenylethenyl group were shown to be in the endo
positions as con®rmed by X-ray di€raction analysis of 7
(Fig. 1).
It is not clear why cyclization did not occur, but there
are several possible explanations. The fact that both 6
and 7 are competitive inhibitors (with Ki values lower
than the Km value for good substrates) indicates that
both compounds bind to the enzyme active site well.
However, the fact that both are poor substrates (no
turnover for 6 and very slow turnover for 7) may
suggest misorientation of the amino group or a-hydro-
gen with the ¯avin at the enzyme active site. This may
result in a slow enzyme-catalyzed process, or possibly a
slower than usual initial electron transfer, giving a
shorter lived amine radical cation. The misorientation
may be responsible for the slower rate of cyclization.
Scheme 3.
Scheme 4.

X. Wang, R. B. Silverman / Bioorg. Med. Chem. 8 (2000) 1645±1651
1647
Scheme 5.
Alternatively, it is estimated that the amine radical
cation intermediate 1 in Scheme 1 has a lifetime no
for cyclization is 120ꢀ, as predicted from the chair-like
transition state of the cyclization in hex-5-enyl sys-
tems.16 Therefore, in the solid state, the amino group
and the double bond are not in a favorable orientation
for cyclization. If this occurs in the active site of the
enzyme, the orientation of the amine radical orbital and
the p-orbital of the double bond will be less than opti-
mal, and the rate of cyclization will be slow relative to
deprotonation and second electron transfer (Scheme 6).
6
longer than 10 s.4c Although the ring cyclizations for
aminium radical cations have estimated rate constants
1 12
of the order of 107±109 s
,
the corresponding aminyl
radicals (the neutral form) react at rates 103±105 times
slower than the corresponding aminium radical
cations.13 Given that the pH optimum for MAO is 9
and the pKa of an aminium radical cation is about 7,13b
it is likely that the intermediate in the MAO-catalyzed
reaction is the neutral, less reactive, amine radical form,
which would have a much slower cyclization rate rela-
tive to a-deprotonation. Another possible explanation
comes from the work of Zelechonok and Silverman,14
who found that, in a chemical model study, formation
of an a-radical cyclopropylamine led to cyclopropyl ring
opening, but MAO-catalyzed oxidation of the corre-
sponding parent cyclopropylamine gave the imine (iso-
lated as the aldehyde) without cyclopropyl ring opening.
It was suggested that when the intermediate cyclopro-
pylamine radical is enzyme bound, it does not have the
appropriate geometry for the cyclopropane orbitals to
overlap with the a-radical, and ring cleavage becomes
slow relative to a-proton removal. Support for that
hypothesis came from the reaction of MAO with (amino-
methyl)cubane,15 which gives fragmentation of the
cubane, indicating the formation of the a-radical; in this
case there are more orientations of orbitals to overlap
with the a-radical, so ring cleavage is favored. If the
alkene p-orbitals of 7 are not aligned for addition by the
amine radical (cation), cyclization will not occur or will
occur at a much reduced rate.
Conclusion
endo,endo-2-Amino-6-[(E)-20-phenyl]ethenylbicyclo[2.2.1]-
heptane (6) and the corresponding (Z)-isomer (7) were
synthesized in an attempt to make a compound that
could trap the purported amine radical cation inter-
mediate during monoamine oxidase (MAO)-catalyzed
oxidation of amines. The E-isomer was not a substrate
for MAO, and the Z-isomer was a very poor substrate.
No trapping product was observed. Although these
speci®c compounds were not successful for the study of
monoamine oxidase, this general approach has the
potential to be applied to trapping of radical inter-
mediates in other enzyme-catalyzed reactions.
Experimental
General methods
Optical spectra and MAO assays were recorded on a
Perkin±Elmer Lambda 1, a Perkin±Elmer Lambda 10,
or a Beckman DU-40 DU-40 UV/Vis spectro-
photometer. NMR spectra were recorded on a Varian
Gemini 300 MHz, a Varian VXR 300 MHz, or a Varian
Consistent with the last explanation, the X-ray crystal
structure of 7 (Fig. 1) shows that the torsional angle of
C6±C5±C8±C9 is 99.7ꢀ, but an optimal torsional angle

1648
X. Wang, R. B. Silverman / Bioorg. Med. Chem. 8 (2000) 1645±1651
Figure 1. ORTEP drawing of the X-ray crystal structure of 7.
Unity plus 400 spectrometer. Chemical shifts are repor-
ted as d values in parts per million down®eld from
Me4Si as the internal standard in CDCl3. An Orion
Research Model 701 pH meter with a general combina-
tion electrode was used for pH measurements. Mass
spectra were obtained on a VG Instrument VG70-
250SE high resolution spectrometer with Maspec Data
System. Elemental analyses were performed by either
Oneida Research Service Inc. (Whiteboro, NY) or the
Department of Geological Sciences at Northwestern
University. Melting points were taken on a Fisher±
Johns melting point apparatus. Small amounts of sam-
ples were centrifuged in a Beckman Microfuge B. The
¯ash column chromatography was carried out with
Merck silica gel 60 (230±400 mesh ASTM). TLC was
run with EM Science silica gel 60 F254 precoated glass
plates. Lyophilization was carried out on a Heto CT60e
lyophilizer. X-ray structure determinations were carried
out on a Bruker SMART-1000 di€ractometer. GC±MS
studies were performed on a Hewlett Packard (HP) 6890
GC±MSD with an HP-5 MS phenyl methyl siloxane
column (30 m 250 mmÂ0.25 mm ®lm thickness).
Cambridge Isotope Laboratories. Other reagents and
solvents were purchased from Aldrich or Fisher and
were used without further puri®cation unless otherwise
stated. Dichloromethane, triethylamine, and toluene
were freshly distilled from CaH2. THF and ether were
freshly distilled from sodium metal. Glassware was oven
dried. Acetone was dried over 3 A molecular sieves. All
of the reactions were carried out in an atmosphere of
inert gas.
Crystal structure data for Figure 1
The nitrogen and chlorine atoms were re®ned aniso-
tropically and the rest isotropically.
Cell parameters
a=33.157 (6) A
b=11.214 (2) A
c=16.633 (3) A
ꢀ=104.557 (3)ꢀ
V=5985.8 (15) A3
Space group
C2/c (#15)
Reagents
Deuterium oxide, chloroform-d, dimethyl sulfoxide-d6,
and methanol-d4 were purchased either from Aldrich or
Z value
16 (2 independent molecules in an asymmetric unit)
No. of re¯ections
54,180 (14,833 unique)
No. of observations
1573
No. of variables
175
Scheme 6.

X. Wang, R. B. Silverman / Bioorg. Med. Chem. 8 (2000) 1645±1651
1649
Residuals: R1; wR2
0.142; 0.292
1.98±2.13 (m, 3H), 1.70±1.90 (m, 3H); MS (EI) m/z 168
(M+), 140, 108, 81; HRMS (EI) m/z calcd for C9H12O3
168.0786 (M+), found 168.0774.
Goodness of ®t
4.62
endo-6-Carbomethoxyspiro[1,3-dioxolane-2,20 -bicyclo-
[2.2.1]heptane] (12). A solution containing keto ester 11
(7.0 g, 4.16 mmol), ethane-1,2-diol (6.0 mL, 107 mmol),
and p-toluenesulfonic acid (100 mg) in benzene (40 mL)
was heated to re¯ux for 6 h in a ¯ask equipped with a
Dean±Stark trap. After removal of the solvent under
reduced pressure, saturated NaHCO3 (30 mL) was
added, and the mixture was extracted with ether (3Â30
mL). The organic extracts were combined, washed with
brine, dried over Na2SO4, concentrated, and chromato-
graphed (15% EtOAc:85% hexanes) to a€ord 12 (7.4 g,
85%) as a colorless liquid; 1H NMR (CDCl3, 300 MHz)
d 3.88±3.98 (m, 3H) 3.74±3.79 (m, 1H), 3.71 (s, 3H),
2.66±2.77 (m, 2H), 2.31 (m, 1H), 1.80±1.97 (m, 3H),
1.65±1.74 (m, 2H), 1.42±1.46 (m, 1H); HRMS (EI) m/z
calcd for C11H16O4 212.1048 (M+), found 212.1054.
endo-6-Carboxybicyclo[2.2.1]hept-2-ene (8). The proce-
dure of Alder and Stein17 was followed. To acrylic acid
(6.05 g, 84 mmol) was added freshly cracked cyclo-
pentadiene (7.2 mL, 90 mmol) at 0 ꢀC. The reaction was
stirred for two days and distilled at 75±80 ꢀC/0.1 mm
Hg. Compound 8 was obtained as a colorless liquid
(10.0 g, 85%). As reported,18 it contained 10% of the
exo isomer.
endo-2-Carboxy-exo-5-bromo-endo-6-hydroxynorbornane
lactone (9). Compound 9 was prepared according to a
procedure reported by Roberts et al.18 To a solution of
8 (4.0 g, 29 mmol) in aqueous sodium bicarbonate (6%,
80 mL) at 0 ꢀC was added bromine (1.5 mL, 29 mmol)
dropwise. After 0.5 h the yellow organic layer was
extracted with ether (3Â20 mL). The organic layers were
combined, washed with 10% Na2S2O3 (15 mL), brine
and dried over Na2SO4. The solvent was evaporated in
vacuo, recrystallized from EtOAc and hexane, and 3.6 g
endo-6-(Hydroxymethyl)spiro[1,3-dioxolane-2,20-bicyclo-
[2.2.1]heptane] (13). To a solution of 12 (5.5 g, 2.6
mmol) in toluene at 78 ꢀC was added DIBAL (1.5 M
in toluene, 45 mL) dropwise. After being stirred for 1 h
the reaction was quenched with KOH solution (30%, 10
mL), and the temperature was slowly brought up to
room temperature. More KOH solution was added
(30%, 40 mL), and the reaction mixture was extracted
with ether (3Â50 mL). The organic extracts were com-
bined, washed with brine, dried over MgSO4, and eva-
porated in vacuo to give 13 (4.6 g, 90%) as a colorless
of solid was obtained (57%); mp 62±65 ꢀC; H NMR
1
(CDCl3, 300 MHz) d 4.84±4.86 (d, 1H), 3.79±3.80 (d,
1H), 3.17±3.20 (t, 1H), 2.60±2.61 (m, 1H), 2.47±2.52
(dd, 1H), 2.22±2.26 (m, 1H), 2.05±2.12 (m, 1H), 1.66±
1.74 (m, 2H); MS (EI) m/z 216 (M+), 109, 93, 79;
HRMS (EI) m/z calcd for C8H779BrO2 (M H+):
215.9786, found 215.9794.
1
liquid; H NMR (CDCl3, 300 MHz) d 3.82±3.98 (m,
4H), 3.66±3.80 (m, 2H), 2.91 (m, 1H), 2.16±2.30 (m,
3H), 1.70±1.90 (m, 3H), 1.35±1.45 (m, 2H), 1.05±1.10
(m, 1H); MS (EI) m/z 184 (M+), 153, 99, 73; HRMS
(EI) m/z calcd for C10H16O3 184.1099 (M+), found
184.1101.
endo-6-Carboxybicyclo[2.2.1]heptan-2-one (10). Prepara-
tion of 10 was carried out following the procedure of
Iles and Worrall.8 Lactone 9 (3 g, 14 mmol) was added
to a solution of sodium hydroxide (2.24 g, 56 mmol) in
water (40 mL) at 25 ꢀC for 1 h. Concentrated HCl (3.52
mL) was added slowly. A trace of coagulated insoluble
gum was removed by ®ltration. The ®ltrate was con-
centrated in vacuo without external heating and extrac-
ted with CHCl3 (3Â30 mL). The organic layers were
combined, washed with brine, and dried over Na2SO4.
The solvent was removed in vacuo, and the crude acid
was puri®ed by column chromatography (2.5%
MeOH:97.5% CH2Cl2) to a€ord 10 as a white solid (1.4
endo-6-Formylspiro[1,3-dioxolane-2,20-bicyclo[2.2.1]hep-
tane] (14). To a vigorously stirred suspension of PCC
(6.4 g, 3.0 mmol) and powdered molecular sieves in
anhydrous dichloromethane (40 mL) was added alcohol
13 (3.0 g, 1.65 mmol). The reaction mixture was stirred
for 2 h before ether (60 mL) was added. The solution
was ®ltered through a pad of silica gel, concentrated
and chromatographed (17% EtOAc:83% hexanes) to
give 14 as a white solid; mp 92±94 ꢀC; 1H NMR (CDCl3,
300 MHz) d 9.76 (s, 1H), 3.76±3.99 (m, 4H), 2.75±2.76
(m, 1H), 2.55±2.61 (m, 1H), 2.38 (m, 1H), 1.70±1.95 (m,
4H), 1.56±1.61 (m, 1H), 1.44±1.48 (m, 1H); 13C NMR
(CDCl3, 75 MHz) d 204.3, 116.4, 65.4, 65.3, 51.5, 49.2,
44.8, 40.1, 36.3, 29.3; MS (EI) m/z 182 (M+), 153, 73;
HRMS (EI) m/z calcd for C10H14O3 182.0942 (M+),
g, 64%); mp 99±103 ꢀC; H NMR (CDCl3, 300 MHz) d
1
3.00±3.02 (m, 1H), 2.96±2.97 (m, 1H), 2.64±2.65 (m,
1H), 2.06±2.14 (m, 3H), 1.68±1.79 (m, 3H); MS (EI) m/z
154 (M+), 126, 108, 81; HRMS (EI) m/z calcd for
C8H10O3 154.0630 (M+), found 154.0629.
endo-6-Carbomethoxybicyclo[2.2.1]heptan-2-one (11). To
a solution of 10 (9.2 g, 6.0 mmol) in methanol (100 mL)
was added a solution of diazomethane in ether (100 mL,
ca. 10 mmol) until the yellow color persisted. The reac-
tion mixture was stirred for an additional 2 h. The sol-
vent was removed in vacuo, and the crude ester was
puri®ed by column chromatography (17% EtOAc:83%
hexanes) to a€ord 11 (9.4 g, 94%) as a white solid; mp
.
found 182.0952; anal. calcd for C10H14O3 0.6H2O: C,
62.22; H, 7.72; found: C, 62.19; H, 7.32.
endo-6-(20-Phenyl)ethenylspiro[1,3-dioxolane-2,20-bicyclo-
[2.2.1]heptane] (15). To a stirred suspension of benzyl
triphenylphosphonium chloride (1.56 g, 4.0 mmol) in
anhydrous THF (20 mL) was added n-butyl lithium (2.5
M in ether, 1.6 mL) dropwise. The above orange solu-
tion was transferred slowly via cannula to a solution of
ꢀ
1
46±48 C; H NMR (CDCl3, 300 MHz) d 3.70 (s, 1H),
3.02±3.10 (m, 1H), 2.86±2.87 (m, 1H), 2.73 (m, 1H),

1650
X. Wang, R. B. Silverman / Bioorg. Med. Chem. 8 (2000) 1645±1651
aldehyde 14 (720 mg, 4.0 mmol) in THF (10 mL). The
solution was stirred for another 30 min before saturated
NH4Cl solution was added (20 mL) and extracted with
ether (3Â30 mL). The organic extracts were combined,
washed with brine, evaporated in vacuo, and puri®ed by
chromatography (8% EtOAc/92% hexanes) to a€ord 15
(900 mg, 90%) as a mixture of the E- and Z-isomers in a
1.6:1 ratio.
MeOH:83% CH2Cl2). To a solution of the product in
ether (5 mL) at 0 ꢀC a solution of HCl gas in ether (3 N,
10 mL) was added dropwise. The solvent was removed
and the solid was recrystallized from CHCl3 and hexane
(1:1) to obtain white needles (50 mg, 40%); mp 224±
ꢀ
1
226 C; H NMR (CDCl3, 300 MHz) d 7.98 (b, 3H),
7.17±7.49 (m, 5H), 6.78±6.83 (d, 1H), 6.49±6.54 (d, 1H),
3.62 (m, 1H), 3.00 (m, 1H), 2.86 (m, 1H), 2.38 (m, 1H),
1.87±2.16 (m, 3H), 1.51±1.58 (m, 1H), 1.38±1.42 (m,
1H), 1.20±1.24 (m, 1H); 13C NMR (CDCl3, 75 MHz) d
136.6, 131.9, 131.5, 128.9, 127.9, 127.0, 53.9, 44.2, 42.2,
39.7, 37.2, 36.3, 33.3; MS (EI) m/z 213 (M+), 170, 115,
91; HRMS (EI) calcd for C15H19N (M+) 213.1517,
endo-6-[(E)-20 -Phenyl]ethenylbicyclo[2.2.1]heptan-2-one
(16) and endo-6-[(Z)-20-phenyl]ethenylbicyclo[2.2.1]hep-
tan-2-one (17). A solution of 15 (660 mg, 2.6 mmol) in
wet acetone (20 mL) containing pyridinium tosylate (50
mg, 0.2 mmol) was heated to re¯ux for 4 h. The solvent
was removed in vacuo, ether (30 mL) was added, and
the mixture was washed with NaHCO3 solution and
brine. The organic phase was dried over Na2SO4 and
evaporated in vacuo to give a mixture of ketone 16 and
17. The mixture was puri®ed as follows. Flash chroma-
tography gave 17 (180 mg) as a colorless oil. Preparative
TLC of the concentrated uncollected fractions a€orded
additional pure 17 (120 mg) and 16 (180 mg) as a col-
orless oil with an overall yield of 90%.
.
found 213.1517; anal. calcd for C15H20NCl 0.2CHCl3:
C, 66.22; H, 7.38; N, 5.12; found: C, 66.11; H, 7.50; N,
5.08.
endo,endo-2-Amino-6-[(Z)-20-phenyl]ethenylbicyclo[2.2.1]-
heptane (7). Compound 7 was prepared starting from 17
as described previously for 6 (55 mg, 45%); mp 217±
219 ꢀC; H NMR (CDCl3, 300 MHz) d 8.419 (b, 3H),
1
7.22±7.34 (m, 5H), 6.61±6.65 (d, 1H), 6.09±6.16 (t, 1H),
3.59 (m, 1H), 3.09 (m, 1H), 2.71 (m, 1H), 2.23±2.24 (m,
1H), 2.17 (m, 1H), 1.91±1.98 (m, 1H), 1.83 (m, 1H),
1.30±1.43 (m, 2H), 1.14±1.22 (m, 1H); 13C NMR
(CDCl3, 75 MHz) d 137.4, 134.7, 133.0, 129.0, 128.4,
127.1, 53.6, 46.5, 40.3, 38.9, 38.3, 37.6, 35.1; MS (EI)
m/z 213 (M+), 170, 115, 91; HRMS (EI) calcd for
C15H19N (M+) 213.1517, found 213.1517; anal. calcd
1
Compound 16: H NMR (CDCl3, 300 MHz) d 7.18±
7.34 (m, 5H), 6.40±6.45 (d, 1H), 5.92±6.00 (dd, 1H),
2.95±3.05 (m, 1H), 2.66±2.74 (m, 1H), 2.12±2.26 (m,
2H), 1.78±1.91 (m, 3H), 1.28±1.36 (m, 1H); 13C NMR
(CDCl3, 75 MHz) d 217.3, 138.1, 132.3, 132.0, 129.5,
128.3, 127.3, 57.2, 47.2, 42.5, 39.7, 36.6, 35.7; MS (EI)
m/z 212 (M+), 168, 128, 115, 91; HRMS (EI) m/z calcd
for C15H17O (M+H+) 213.1279, found 213.1270.
.
for C15H20NCl 0.1CHCl3: C, 69.29; H, 7.70; N, 5.35;
found: C, 68.90; H, 7.87; N, 5.58.
Enzyme and assay. Beef liver MAO B was isolated as
described previously19 and stored as a concentrated
solution (15±25 mg/mL) in sodium phosphate bu€er (50
mM, pH 7.2) at 4 ꢀC. The speci®c activity varied among
preparations, ranging from 3.5 to 7 units per mg, where
a unit of activity is the conversion of 1 mmol of benzyl-
amine to benzaldehyde per min at pH 9.0 and 30 ꢀC.
1
Compound 17: H NMR (CDCl3, 300 MHz) d 7.19±
7.34 (m, 5H), 6.44±6.47 (d, 1H), 5.34±5.39 (t, 1H), 3.19±
3.28 (m, 1H), 2.65 (m, 1H), 2.56 (m, 1H), 2.10±2.21 (m,
2H), 1.64±1.89 (m, 3H), 1.17±1.23 (m, 1H); 13C NMR
(CDCl3, 75 MHz) d 217.6, 138.1, 134.2, 131.3, 129.6,
129.3, 127.9, 57.1, 47.2, 39.8, 37.4, 36.7, 32.0; MS (EI)
m/z 212 (M+), 168, 128, 115, 91; HRMS (EI) m/z calcd
for C15H17O (M+H+) 213.1279, found 213.1280.
General procedure for inhibition of the MAO-catalyzed
oxidation of cinnamylamine. An MAO B solution was
prepared by diluting 15 mL of the stock MAO B solu-
tion with 285 mL of Tris±HCl bu€er (100 mM, pH 9.0).
The amount of inhibition of the oxidation of various
concentrations of cinnamylamine (0.2, 0.5, 0.67 and 0.8
mM in Tris±HCl bu€er, 100 mM, pH 9.0) by various
concentrations of inhibitor (0, 0.2, 0.4, 0.6, 0.8 mM in
Tris±HCl bu€er, 100 mM, pH 9.0) was determined by
adding 10 mL of the above MAO solution to 490 mL of
an inhibitor/substrate solution at 25 ꢀC, followed by
monitoring the increase in UV absorbance at 290 nm.
The Ki value was determined using a Dixon plot.10
Cornish-Bowden plots11 were used to determine the
type of inhibition.
endo,endo-2-Amino-6-[(E)-20-phenyl]ethenylbicyclo[2.2.1]-
heptane (6). To a solution of hydroxylamine hydro-
chloride (71 mg, 1.0 mmol) and sodium acetate (115 mg,
1.25 mmol) in water were added ketone 16 (105 mg, 0.5
mmol) and sucient ethanol to e€ect dissolution. This
mixture was heated in an oil bath (50 ꢀC) for 2 h, and
the solution was concentrated. The remaining solution
was extracted with CHCl3 (3Â10 mL). The combined
organic extracts were washed with brine, dried over
Na2SO4, and evaporated in vacuo to give the oxime
(115 mg, 98%) as a colorless oil. This was used without
further puri®cation. Titanium trichloride (20% aqueous
solution, 1.5 mL, 2.0 mmol) was added to a solution of
the above oxime (115 mg, 0.5 mmol), sodium cyanoboro-
hydride (135 mg, 2.2 mmol), and ammonium acetate (385
mg, 5 mmol) in methanol (10 mL) over a 14 h period. The
mixture was diluted with water, made basic (pH>10) with
dilute 1 N NaOH solution (2Â20 mL), and extracted with
CH2Cl2. After evaporation of the solvent in vacuo, the
product was puri®ed by chromatography (17%
General procedure for the isolation and detection of met-
abolites. To a solution of 6 (10.0 mM) in Tris±HCl buf-
fer (270 mL) was added MAO B (30 mL). Aliquots of 20
mL were periodically withdrawn, extracted with 40 mL of
CH2Cl2 by spinning in a centrifuge, and analyzed by gas
chromatography.

X. Wang, R. B. Silverman / Bioorg. Med. Chem. 8 (2000) 1645±1651
1651
Acknowledgement
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10. Dixon, M. Biochem. J. 1953, 55, 170.
We are grateful to the National Institutes of Health
(Grant GM32634) for ®nancial support of this research.
We thank Charlotte Stern for the crystal structures.
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