A novel and selective monoamine oxidase B substrate

Article abstract of DOI:10.1016/j.bmc.2005.05.059

Cyclic five- and six-membered tertiary allylamines constitute a unique class of monoamine oxidase substrates that undergo a net two-electron α-carbon oxidation to form the cyclic, conjugated eniminium metabolites. The corresponding saturated pyrrolidinyl

Full text of DOI:10.1016/j.bmc.2005.05.059

Bioorganic & Medicinal Chemistry 13 (2005) 5808–5813
A novel and selective monoamine oxidase B substrate
John M. Rimoldi,a,* Satish G. Puppali,a Emre Isin,b Philippe Bissel,b
Ashraf Khalilb and Neal Castagnoli, Jr.b,*
aDepartment of Medicinal Chemistry and Laboratory for Applied Drug Design and Synthesis, The University of Mississippi,
University, MS 38677, USA
bDepartment of Chemistry, Virginia Tech, Blacksburg, VA 24061-0212, USA
Received 23 April 2005; revised 25 May 2005; accepted 25 May 2005
Available online 29 June 2005
Abstract—Cyclic five- and six-membered tertiary allylamines constitute a unique class of monoamine oxidase substrates that under-
go a net two-electron a-carbon oxidation to form the cyclic, conjugated eniminium metabolites. The corresponding saturated pyr-
rolidinyl and piperidinyl systems are not substrates for this flavoenzyme system. In an attempt to evaluate possible contributions
that p-orbital stabilization of the putative a-carbon radical intermediates may play in the catalytic pathway, we have examined
the substrate properties of 3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane, the 3,4-cyclopropyl analog of the selective monoamine
oxidase B substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results, which document the first reported example
of a saturated, cyclic tertiary amine with monoamine oxidase substrate properties, are consistent with a-carbon radical stabilization
as a contributing factor in the catalytic pathway.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
a-carbon deprotonation of the resulting aminyl radical
cation 1Å+. A second 1-electron transfer then generates
the metabolite and the reduced flavin FADH2. In the
case of 1, conversion to the homoallylic radical 4Å does
not take place to any significant extent since the isomeric
dihydropyridinium species 5H+ is not observed as a pri-
mary metabolite although it is formed by rearrangement
of 3H+.8
The outer mitochondrial membrane flavoenzymes
monoamine oxidase A and B (MAO-A and MAO-B)
catalyze the two-electron a-carbon oxidation of a varie-
ty of primary and secondary amines including the bio-
genic amine neurotransmitters.1 Acyclic tertiary amine
substrates are rare2 and, with the exception of the class
of compounds under consideration in this study, no cyc-
lic tertiary amine has been unambiguously described as
an MAO substrate.3 This exceptional class of com-
pounds is a group of cyclic tertiary allylamines that
undergo MAO-catalyzed oxidation to give the corre-
sponding cyclic, conjugated eniminium metabolites.4
This pathway is illustrated in Scheme 1 by the MAO-
B-catalyzed oxidation of the parkinsonian inducing neu-
rotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
[MPTP (1)] that yields the corresponding dihydropyrid-
inium metabolite 3H+.5 The MAO catalytic pathway is
thought to proceed via a-carbon radical intermediates,
such as 2Å, that may be generated either directly via a
hydrogen atom transfer (HAT)6 step or via an initial
single electron transfer (SET)7 step and subsequent
The unique MAO substrate properties of this class of
compounds are dependent on the presence of the 4,5
p-bond of the heterocyclic moiety. Contrary to an earli-
er report,9 the corresponding piperidinyl analog 6 of
MPTP is not an MAO-B substrate.10 Similarly, 3-pyrr-
olinyl derivatives,11,12 such as 7, are good MAO-B sub-
strates; the corresponding pyrrolidinyl analog 8,
however, is stable in the presence of the enzyme.12 Also
noteworthy is the C(6)-regioselectivity of the a-carbon
oxidation of MPTP mentioned in Scheme 1.8 These
observations suggest that stabilization of the putative
a-carbon radical intermediates, such as 2Å, may contrib-
ute to the unique substrate properties of this class of
compounds and have prompted efforts to examine the
effects on substrate properties of molecules bearing
other a-carbon radical stabilizing groups.
Keywords: Monoamine oxidase B selective substrate; Cyclopropyl-
tetrahydropyridine; Regioselectivity; Cyclic iminium metabolite.
*
The cyclopropyl group shares some of the electron-
donating properties of the p-bond.13 For example, the
Corresponding authors. Tel.: +1 540 231 8200; fax: +1 540 231 8890
(N.C); e-mail addresses: jrimoldi@olemiss.edu; ncastagn@vt.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.059

J. M. Rimoldi et al. / Bioorg. Med. Chem. 13 (2005) 5808–5813
5809
Scheme 1.
kmax of the cyclopropyltropylium system is shifted to
longer wavelengths [247 nm (e 29,800) and 327 nm (e
12,300)] compared to the isopropyl analog [kmax of
225 nm (e 32,000) and 274 nm (e 14,400)].14 The calculat-
ed carbon–heteroatom bond rotational energy barriers
for vinylborane versus cyclopropylborane versus isopro-
pylborane are 7.1 versus 7.2 versus 0.7 kcal/mol, respec-
tively; the corresponding values for vinylamine versus
cyclopropylamine versus isopropylamine are 4.9 versus
4.9 versus 3.1 kcal/mol.15 Additionally, the optimized
Scheme 2.
The ESI/MS product ion spectrum of 9H+ (MH+ 188)
displayed a single fragment ion at m/z 44 that was
assigned structure i+. The formation of i+ is likely to
proceed via a reverse [2pi + 2pi + 2pi] cycloaddition
pathway with 2-phenyl-1,4-pentadiene (A) as the neutral
loss species (Scheme 3).
16
˚
length for the vinyl–cyclopropyl C–C bond, 1.475 A,
is considerably shorter than that of the vinyl–methyl
C–C bond, 1.503 A.17 In view of these p-bond character-
˚
istics of the cyclopropyl group, studies have been initiat-
ed to examine the MAO enzyme substrate properties of
3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane (9),
a
structurally rigid, chiral aminomethylcyclopropyl ana-
log of the aminomethylvinyl derivative MPTP. A litera-
ture search identified a report describing the MAO-B
substrate properties of trans, trans-1-(aminomethyl)-2-
methoxy-3-phenylcyclopropane (10), a primary amine
and somewhat distant structural analog of 9. Beef liver
MAO-B catalyzed the conversion of this compound
(Km = 10.8 mM) to a pyrrolyl metabolite via a complex
reaction sequence.18
The substrate properties of 9 were examined with
baboon liver mitochondrial MAO-B and human placen-
tal mitochondrial MAO-A.21 Spectrophotometric analy-
sis of the incubation mixtures showed the time-
dependent formation of a new chromophore (kmax
253 nm) with the baboon liver preparations but not with
the human placental preparations demonstrating that 9,
like MPTP,22 is an MAO-B selective substrate. (R)-de-
prenyl, a potent and selective mechanism-based inacti-
vator of MAO-B,23 completely inhibited the reaction.
2. Results and discussion
The synthesis of 9 via a direct Simmons–Smith cyclopro-
pylation reaction (Et2Zn, CH2I2) of MPTP (1) in
19
CH2Cl2 proceeded in 57% yield (Scheme 2). Strictly
anhydrous conditions and at least 6 equiv. of both dieth-
ylzinc and diiodomethane were necessary for the reac-
tion to proceed effectively.20 The proton 1H NMR
spectrum documented the replacement of the signal for
the C(5)-olefinic proton (d 5.86 ppm) of the starting
material (1H+ Æ ClÀ) with three signals [d 1.60 (m, 1H),
1.24 (m, 1H), and 1.14 (m, 1H)] corresponding to the
methide proton at C(3) and the methylene protons at
C(7) of the newly introduced cyclopropyl group.
Scheme 3.

5810
J. M. Rimoldi et al. / Bioorg. Med. Chem. 13 (2005) 5808–5813
A plot of the absorbance at 253 nm versus time was
linear for the first 15 min (0.048 OD units/min).
conjugation between the iminium and cyclopropyl
groups. Therefore, the observed kmax 253 nm of the
metabolite argues against the ꢀpseudohomoallylicꢁ struc-
ture 18H+ since this compound should have a chromo-
phore similar to that of the starting material (kmax
211 nm, e 5945). This chromophore of the metabolite,
however, does not rule out the dihydroazepinium struc-
ture 24H+.
The LC-ESI/MS total ion current tracing of the post-in-
cubation mixture showed two major peaks, one contain-
ing the starting material 9 and the second containing a
compound with MH+ 186 Da, consistent with a metab-
olite structure at an oxidation state two-electrons higher
than that of the substrate (MH+ 188). The two possible
a-carbon radical intermediates anticipated in this bio-
transformation, 15Å and 16Å, will lead to the correspond-
ing iminium metabolites 17H+ and 18H+ following the
second one-electron oxidation (Scheme 4). According
to the proposed a-carbon radical stabilization by the
cyclopropyl group, the cyclopropylcarbinyl radical 15Å
would be the preferred structure. Carbocyclic radicals
analogous to 15Å are reported to undergo spontaneous
rearrangements to ring-opened products, a reaction that
is promoted by release of ring strain. For example, the
bicyclo[4.1.0]heptyl radical 19Å is reported to rearrange
to the cyclohexylmethyl radical 20Å at a rate of 2 · 108/
s24 and the trans-2-phenylcyclopropylmethyl radical 21Å
to the open-chain benzylic radical 22Å at a rate of
1.9 · 1011/s.25 Consequently, 15Å also could undergo a
rearrangement reaction that most likely would lead to
the benzylic radical 23Å. Subsequent oxidation of this
intermediate would lead to a dihydroazepinium system
with the 2,3-dihydro-1H-azepinium species 24H+ as
the thermodynamically preferred product.26
This polar metabolite proved to be quite stable and
could be isolated in small quantities by preparative
TLC. GC-EI/MS analysis of the purified metabolite dis-
played two isomeric peaks with qualitatively similar
fragmentation patterns: Peak 1 (tR 10.0 min, major
product) 185 (MÅ+, 100%), 184 (20%), 157 (20%), 142
(80%), 141 (60%), 128 (15%), 115 (25%), and 82 (90%);
Peak 2 (tR 9.2 min, minor product) 185 (Mk+ 40%),
184 (100%) 157 (10%), 142 (10%), 141 (15%), 128
(10%), 115 (15%), and 82 (5%). These compounds could
be a pair of isomeric amines derived following thermal
deprotonation of a cationic (iminium) metabolite. The
complex fragmentation patterns did not help to differen-
tiate amongst the proposed structures. The LC-ESI/MS
total ion current tracing of this isolate showed a single
peak at tR 11 min. The product ion spectrum of the
MH+ ion also was complicated: [m/z 186 (100%), 171
(20%), 157 (40%), 155 (50%), 143 (50%), 129 (50%),
128 (50%), 105 (50%), 94 (80%), 91 (100%)] and again
did not help to distinguish amongst the proposed
structures.
By analogy with the shift observed with the cyclopropyl-
tropylium system,14 the UV spectrum of ꢀpseudoallylicꢁ
structure 17H+ would be expected to display a batho-
chromic shift relative to the starting amine due to the
The partially purified metabolite was treated with NaC-
NBD3 and the crude product was analyzed by mass
spectrometry. The GC-EI/MS of the product (MÅ+
Scheme 4.

J. M. Rimoldi et al. / Bioorg. Med. Chem. 13 (2005) 5808–5813
5811
Scheme 5.
188) had the same retention time (9.0 min) and fragmen-
tation pattern [188 (MÅ+, 40%), 159 (60%), 144 (65%),
130 (70%), 129 (70%), 115 (20%), 103 (30%), and 58
(100%)] as the starting amine 9 [187 (MÅ+, 40%), 159
(60%), 143 (65%), 130 (70%), 129 (100%), 115 (20%),
103 (30%), and 57 (100%)] except for mass shifts due
to the presence of a deuterium atom. The LC-ESI/MS
(MH+ 189) also displayed a single species with the same
retention time (12 min) as the starting amine. These data
eliminate the dihydroazepinium 24H+ as a possible
structure but do not distinguish between the two possi-
ble iminium species 17H+ and 18H+. The product ion
spectrum of MH+ (189 Da), however, led to a more
definitive outcome. The spectrum showed a single frag-
ment ion at m/z 44. A consideration of the reverse cyclo-
addition pathway proposed to account for the
fragmentation observed with the starting amine 9H+
provides convincing evidence that the MAO-B-catalyzed
oxidation of 9 proceeds exclusively to give the 3-methyl-
6-phenyl-3-azabicyclo[4.1.0]hept-2-enyl species 17H+.
Reduction of 17H+ with NaCNBD3 gives 9-2-d1that,
following collision-induced dissociation, will yield frag-
ment ion i+-d0 with a mass of 44 Da. The corresponding
reduction of the isomeric 3-methyl-6-phenyl-3-azabicy-
clo[4.1.0]hept-3-enyl species 18H+ will yield 9-6-d1. The
reverse cycloaddition fragment ion (i+-d1) that would
be obtained in the product ion spectrum of 9-6-d1 would
have a mass of 45 Da rather than the observed mass of
44 Da (Scheme 5). Based on these data, a tentative
assignment of 17H+ was made for the structure of the
MAO-B generated metabolite of 9.
subjected to Polonovskii reaction conditions (trifluoro-
acetic anhydride in CHCl3), generated a product that
was obtained in pure form as a stable perchlorate salt.
The 1H NMR spectrum of this product in DMSO-d6 dis-
played a complex signal centered at d 8.96 ppm that was
assigned to the C(2) proton of 17H+. The other signals
also were consistent with this structure (see Section 3).
The product ion spectrum of m/z 186 (MH+) was iden-
tical to that observed with the metabolite generated in
the MAO-B-catalyzed oxidation of 9. Finally, reduction
of this synthetic product with NaCNBD3 led to the same
monodeuterated reduction product, 9-2-d1, that was
obtained with the metabolite.
The results of these studies provide the first example of a
saturated, cyclic tertiary amine with MAO substrate
properties. Furthermore, the substrate properties of this
bicyclic compound parallel those of the corresponding
cyclic allylamine analog MPTP in terms of the MAO-
B selectivity and regiospecificity. We speculate that the
regiospecificity observed with these two compounds
may reflect stabilization of the corresponding a-carbon
radicals that are thought to be intermediates in the cat-
alytic pathways. Kinetic studies are underway to evalu-
ate this proposal and also to characterize the potential
enantioselectivity and the C(2)-diastereoselectivity of
this biotransformation.
3. Experimental
Note: Some 1, 4-disubstituted-1,2,3,6-tetrahydropyri-
dines are known nigrostriatal neurotoxins and should
be handled using disposable gloves in a properly venti-
lated hood following good laboratory practices.
Detailed procedures for the safe handling of MPTP have
been reported.28
The synthesis of 17H+ (Scheme 6) was achieved via a
reaction sequence used in the past to prepare 2,3-dihyd-
ropyridinium derivatives such as 3H+.27 The diastereo-
meric mixture of N-oxides 25/26 obtained by treatment
of 9 with m-chloroperoxybenzoic acid (m-CPBA), when
3.1. Chemistry
3.1.1. General methods. Unless otherwise noted, reagents
and starting materials were obtained from commercial
suppliers and were used without purification. Diethyl
ether was distilled over sodium and benzophenone.
˚
CH2Cl2 and CHCl3 were dried over Linde type 3 A
molecular sieves. All reactions were carried out using
flame-dried glassware under an atmosphere of argon.
Scheme 6.

5812
J. M. Rimoldi et al. / Bioorg. Med. Chem. 13 (2005) 5808–5813
Proton and carbon NMR spectra were recorded on a
JEOL 500 MHz spectrometer. Chemical shifts are
reported in ppm downfield from internal tetramethylsi-
lane. Spin multiplicities are given as s (singlet), bs (broad
singlet), or m (multiplet). All UV–vis spectra were
recorded on a Beckman DU 7400 spectrophotometer.
The high-resolution FAB mass spectra were obtained
on a JEOL HX-110 instrument.
25.5, 35.0, 49.1, 127.9, 128.0, 129.0, 140.9, 178.4; FAB-
HRMS: Calcd for C13H16N+: 186.1283. Found:
186.1286. Anal. Calcd for C13H16NClO4 Æ 0.14 H2O
(288.45): C, 54.13; H, 5.64; N, 4.86. Found: C, 54.13;
H, 5.25; N, 4.80.
3.2. Enzymology
3.2.1. General methods. Collections of human placenta
were approved by the Internal Review Boards of Mont-
gomery County Hospital and Virginia Tech and the col-
lections of baboon liver were approved by the Animal
Care Committee of Virginia Tech. Human placenta
and baboon liver mitochondrial homogenates were pre-
pared using the methodology reported earlier by Salach
and were stored at À70 ꢁC prior to use.29 The MAO-A
and MAO-B substrate properties of 9H+ Æ ClÀ were
examined on a Molecular Devices Spectra Max Plus
384 microplate spectrophotometer with the Softmax
Pro 4.7.1 software.
3.1.2. Hydrochloride salt of 3-methyl-6-phenyl-3-azabicy-
clo[4.1.0]heptane (9H+ Æ ClÀ). Diiodomethane (9.3 g,
35 mmol) was added dropwise to a solution of diethyl-
zinc (15% solution in toluene, 29 mL, 35 mmol) in
CH2Cl2 (10 mL) at 0 ꢁC under argon. After stirring for
0.5 h, a solution of amine 1 (1.0 g, 5.8 mmol) in CH2Cl2
(5.0 mL) was added dropwise at 0 ꢁC. The reaction mix-
ture was then stirred at room temperature for 18 h.
Aqueous saturated ammonium chloride (50 mL) was
added at 0 ꢁC. The inorganic salts were filtered and
washed with CH2Cl2(20 mL). The organic layer was
washed with water (3 · 30 mL), dried over Na2SO4,
and the solvent was removed under reduced pressure.
The crude product was purified by alumina chromatog-
raphy (Hexanes/EtOAc, 50:50) to give the free base 9
(0.62 g, 57%) as a colorless oil. To a solution of the free
base 9 (0.5 g, 2.7 mmol) in diethyl ether (15 mL), an
etheral solution of HCl (excess) was added at 0 ꢁC.
The precipitate was filtered to give an analytical pure
sample of 9H+ Æ ClÀ as a white solid (0.54 g, 92%): mp
286 ꢁC (dec); 1H NMR (500 MHz, CD3OD): d 1.14
(m, 1H), 1.24 (m, 1H), 1.60 (m, 1H), 2.43 (m, 2H),
2.81 (s, 3H), 2.90 (m, 1H), 3.11 (m, 1H), 3.35 (m, 1H),
4.00 (m, 1H), 7.21 (m, 1H), 7.31 (m, 2H), 7.40 (m,
2H); 13C NMR (125.8 MHz, CD3OD): d 15.0, 17.5,
22.9, 28.5, 42.0, 49.2, 53.9, 126.6, 127.6, 128.4, 145.0;
ESI/MS (MH+) 188 Da; UV (MeOH) kmax 211 nm (e
5945); FAB-HRMS: Calcd. for C13H18N+: 188.1439.
Found: 188.1441. Anal. Calcd for C13H18NCl Æ 0.24
H2O (228.08): C, 68.46; H, 8.10; N, 6.14. Found: C,
68.46; H, 8.08; N, 6.17.
3.2.2. MAO-B and MAO-A substrate properties of
9H+ Æ ClÀ. Mixtures of 9H+ Æ ClÀ (2 mM) and 0.06 mg
baboon liver mitochondrial protein (MAO-B) or human
placental mitochondrial protein (MAO-A) in phosphate
buffer (100 mM, pH 7.4) in a final volume of 200 lL
were incubated at 37 ꢁC in the microplate spectropho-
tometer. Scans of these mixtures (200–600 nm) estab-
lished the formation of a new chromophore (kmax
253 nm) in the baboon liver preparation only. A plot
of absorbance versus time established that the increase
in absorbance at 253 nm was linear for 15 min.
3.2.3. Studies on the regioselectivity of the MAO-B-
catalyzed oxidation of 9H+ Æ ClÀ. A total of 9.6 mg of
9H+ Æ ClÀ [eight flasks each containing substrate
(1.2 mg) and baboon liver mitochondria (0.3 mg pro-
tein) in phosphate buffer (10 mL, pH 7.4)] were incubat-
ed for 60 min at 37 ꢁC with gentle agitation in a water
bath. The pH of the combined incubation mixtures
was adjusted to 10 with saturated aqueous K2CO3 and
the resulting mixture was extracted with EtOAc
(3 · 50 mL). The organic layer was dried over anhy-
drous Na2SO4and the solvent removed under reduced
pressure. Preparative TLC on silica [EtOAc/
CHCl3(60:40) containing a few drops of NH4OH] sepa-
rated the starting material 9H+ (Rf 0.8) from the imini-
um metabolite 17H+ (Rf 0.2). The metabolite, which was
recovered from the silica gel with MeOH, was shown by
LC-ESI/MS to be free of starting material. A solution of
this isolate in MeOH (1 mL) was treated with excess of
NaCNBD3 at room temperature for 15 min. An equal
volume of water was added and the resulting mixture
was extracted with CH2Cl2 (3 · 1 mL), the combined
extracts dried over Na2SO4 and the solvent removed
under reduced pressure. GC-EI/MS, ESI/MS, and
ESI/MS/MS established the product to be the 9H+-2-d1.
3.1.3. 3-Methyl-6-phenyl-3-azabicyclo[4.1.0]hept-2-enyl
perchlorate (17H+ Æ HClO4À). A solution of m-CPBA
(1.0 g, 70%, 4.1 mmol) in CH2Cl2 (10 mL) was added
dropwise to a solution of 9 (0.55 g, 2.9 mmol) in
CH2Cl2(10 mL) at 0 ꢁC. After 4 h at room temperature,
the solvent was removed under reduced pressure to give
the crude diastereomeric N-oxides 25/26. The N-oxides
were used for the next step without purification. A solu-
tion of TFAA (4.2 mL) in CHCl3 (7.0 mL) was added
dropwise to a solution of 25/26 (0.70 g, 3.4 mmol) in
CHCl3(5.0 mL) at room temperature. After 1 h, the sol-
vent was removed under reduced pressure. The residue
was purified by alumina chromatography (CHCl3/
MeOH, 95:5). The oil was warmed in 3 mL of 10%
methanolic HClO4, cooled to 0 ꢁC and diethyl ether
was added. The precipitate was filtered and recrystal-
lized from CHCl3/diethyl ether to give 17H+ Æ HClO4
À
as white crystals (0.31 g, 32%): mp 142 ꢁC; UV (MeOH)
k
max 253 nm (e 4,700), 205 (4,080); 1H NMR (500 MHz,
Acknowledgments
DMSO-d6): d 2.00 (bs, 1H), 2.15 (m, 1H), 2.47 (bs, 3H),
3.56 (s, 3H), 3.74 (m, 2H), 7.29–7.42 (m, 5H), 8.96 (m,
1H); 13C NMR (125.8 MHz, DMSO-d6): d 23.1, 23.3,
This work was supported by a grant from the DHHS/
Centers for Disease Control and Prevention: U50/

J. M. Rimoldi et al. / Bioorg. Med. Chem. 13 (2005) 5808–5813
5813
confirmed the lack of substrate properties of 6 with a
sensitive GC-EI/MS assay.
11. Lee, Y.; Ling, K.-Q.; Lu, X.; Silverman, R. G.; Shepard,
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CCU423310-02 (University of Mississippi) and the
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Disease (Virginia Tech).
12. Wang, Y.-X.; Mabic, S.; Castagnoli, N., Jr. Bioorg. Med.
Chem. 1998, 6, 143.
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