2-(iodoethenyl)benzylamines as potential probes for radical intermediates formed during monoamine oxidase catalyzed oxidations

Article abstract of DOI:10.1021/jo980976i

An attempt to trap radical intermediates during the monoamine oxidase (MAO) catalyzed oxidation of amines by intramolecular cyclization with an activated alkene that is built into the substrate was unsuccessful. (E)-2- (Iodoethenyl)benzylamine (3a) was shown to be a reversible inhibitor of MAO B, but the corresponding Z isomer (3b) was a good substrate. By GC-MS analysis, the expected radical trapping product, isoquinoline (5), was observed as one of two products in 1:1 ratio. However, NMR analysis prior to GC-MS analysis showed no evidence of isoquinoline, suggesting that the isoquinoline was generated during gas chromatography. As a model for this GC- dependent reaction, the corresponding aldehyde, (Z)-2- (iodoethenyl)benzaldehyde (14), was treated with ammonia, and the product was analyzed by GC-MS; isoquinoline was detected. Likewise, the reaction of 14 with methylamine also produced isoquinoline by GC-MS analysis (but not by NMR analysis). These results are explained by electrocyclization of the corresponding imines at the elevated temperatures in the GC (Schemes 7 and 8). Substrate 3b and aldehyde 14 were recovered when the enzyme reaction products were chromatographed, although they were not detected by GC-MS. These products could arise via hydrolytic decomposition of the corresponding imine (17, Scheme 9). GC-MS analysis of 17 produced isoquinoline and (Z)-2- (iodoethenyl)benzyl iodide (18) in a 1:1 ratio; these are the two products observed in a 1:1 ratio after incubation of MAO with 3b and apparently arise from thermally induced electrocyclization and iodide ion cleavage (Scheme 9).

Full text of DOI:10.1021/jo980976i

J . Org. Chem. 1998, 63, 7357-7363
7357
2-(Iod oeth en yl)ben zyla m in es a s P oten tia l P r obes for Ra d ica l
In ter m ed ia tes F or m ed d u r in g Mon oa m in e Oxid a se Ca ta lyzed
Oxid a tion s
Xueqing Wang and Richard B. Silverman*
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology,
Northwestern University, Evanston, Illinois 60208-3113
Received May 21, 1998
An attempt to trap radical intermediates during the monoamine oxidase (MAO) catalyzed oxidation
of amines by intramolecular cyclization with an activated alkene that is built into the substrate
was unsuccessful. (E)-2-(Iodoethenyl)benzylamine (3a ) was shown to be a reversible inhibitor of
MAO B, but the corresponding Z isomer (3b) was a good substrate. By GC-MS analysis, the expected
radical trapping product, isoquinoline (5), was observed as one of two products in a 1:1 ratio.
However, NMR analysis prior to GC-MS analysis showed no evidence of isoquinoline, suggesting
that the isoquinoline was generated during gas chromatography. As a model for this GC-dependent
reaction, the corresponding aldehyde, (Z)-2-(iodoethenyl)benzaldehyde (14), was treated with
ammonia, and the product was analyzed by GC-MS; isoquinoline was detected. Likewise, the
reaction of 14 with methylamine also produced isoquinoline by GC-MS analysis (but not by NMR
analysis). These results are explained by electrocyclization of the corresponding imines at the
elevated temperatures in the GC (Schemes 7 and 8). Substrate 3b and aldehyde 14 were recovered
when the enzyme reaction products were chromatographed, although they were not detected by
GC-MS. These products could arise via hydrolytic decomposition of the corresponding imine (17;
Scheme 9). GC-MS analysis of 17 produced isoquinoline and (Z)-2-(iodoethenyl)benzyl iodide (18)
in a 1:1 ratio; these are the two products observed in a 1:1 ratio after incubation of MAO with 3b
and apparently arise from thermally induced electrocyclization and iodide ion cleavage (Scheme
9).
In tr od u ction
transfer mechanism by observing ring-cleavage reactions
that would be expected to accompany cycloalkylcarbinyl
or -aminyl intermediates.
Monoamine oxidase (MAO, EC 1.4.3.4), an integral
membrane protein, is one of the enzymes responsible for
the degradation of various biogenic and xenobiotic amines.1
It catalyzes the oxygen-dependent conversion of amine
substrates to the corresponding imines, which are non-
enzymatically hydrolyzed to aldehydes.2 MAO exists in
two principal isozymic forms known as MAO A and MAO
B.3 Compounds that inhibit MAO A exhibit antidepres-
sant effects,4 and those that inhibit MAO B are useful
adjuncts to L-dopa treatment for Parkinson’s disease.5
The catalytic mechanism for MAO has long been pro-
posed to involve radical intermediates 1 and/or 2 (Scheme
1); the electron-transfer mechanism shown in Scheme 16
and a hydrogen atom abstraction mechanism to go
directly to 27 are favored pathways. Previous studies
using cyclopropylamines, cyclobutylamines, and cubyl-
carbinylamines have provided evidence for an electron-
Related nonenzymatic amine oxidations via amine
radical cations have been demonstrated under electro-
chemical,8 photochemical,9 and chemical oxidizing10 con-
ditions. It has been shown that amine radical cations9c
can undergo ring cyclization to alkenes with rate con-
stants on the order of 108 s-1, and carbon radicals
undergo cyclization to an alkene with rate constants on
the order of 105-107 s-1 11
.
We envisioned incorporating a rotation-constraining
factor into an analogue of a substrate for MAO and
placing a double bond nearby to trap either the potential
amine radical cation or carbon radical intermediate;
formation of this intermediate would be detected by
(7) (a) Ottoboni, S.; Caldera, P.; Trevor, A.; Castagnoli, N., J r. J .
Biol. Chem. 1989, 264, 13684-13688. (b) Walker, M. C.; Edmondson,
D. E. Biochemistry 1994, 33, 7088-7098. (c) Miller, J . R.; Edmondson,
D. E.; Grissom, C. B. J . Am. Chem. Soc. 1995, 117, 7830-7831.
(8) (a) Mann, C. K.; Barnes, K. K. Electrochemical Reactions in Non-
Aqueous Systems; Marcel Dekker: New York, 1970; Chapter 9. (b)
Lewis, F. D.; Bassani, D. M.; Reddy, G. D. J . Org. Chem. 1993, 58,
6390-6393.
(9) (a) Cohen, S. G.; Parola, A.; Parsons, G. H. Chem. Rev. 1973,
73, 141. (b) Lewis, F. D.; Ho, T. J . Am. Chem. Soc. 1980, 102, 1751-
1752. (c) Newcomb, M.; Tanaka, N.; Bouvier, A.; Tronche, C.; Horner,
J . H.; Musa, O. M.; Martinez, F. N. J . Am. Chem. Soc. 1996, 118, 8505-
8506.
* To whom correspondence should be addressed at the Department
of Chemistry. Phone: (847) 491-5653. Fax: (847) 491-7713. E-mail:
Agman@chem.nwu.edu.
(1) Strolin Benedetti, M.; Dostert, P.; Tipton, K. F. Prog. Drug Metab.
1988, 11, 149-174.
(2) Woo, J . C. G.; Silverman, R. B. J . Am. Chem. Soc. 1995, 117,
1663-1664 and references therein.
(3) J ohnston, J . P. Biochem. Pharmacol. 1968, 17, 1285-1297.
(4) Knoll, J . Med. Res. Rev. 1989, 12, 505-524.
(5) Tetrud, V. W.; Langston, J . W. Science 1989, 245, 519-532.
(6) (a) Silverman, R. B. Acc. Chem. Res. 1995, 28, 335-342. (b)
Silverman, R. B.; Zhou, J . J . P.; Ding, C. Z.; Lu, X. J . Am. Chem. Soc.
1995, 117, 12895-12896.
(10) (a) Hull, L. A.; Davis, G. T.; Rosenblatt, D. H. J . Am. Chem.
Soc. 1969, 91, 6247. (b) Lindsay Smith, J . R.; Mead, L. A. V. J . Chem.
Soc., Perkin Trans. 2 1973, 206-210.
(11) Newcomb, M. Tetrahedron 1993, 49, 1151-1176.
S0022-3263(98)00976-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/23/1998

7358 J . Org. Chem., Vol. 63, No. 21, 1998
Wang and Silverman
Sch em e 1
Sch em e 2
isolation of the cyclized product. Compound 3 was
designed to lead to one of three possible cyclized products,
depending upon whether the amine radical cation or the
R-radical were trapped by either a 5-exo-trig or 6-endo-
trig cyclization process; the 5-endo-trig cyclization is
disfavored, according to the Baldwin rules12 (Scheme 2).
The 6-endo-trig cyclization would lead to a dihydroiso-
quinoline (4), which, upon further oxidation would give
isoquinoline (5). Isoquinoline was, in fact, detected by
monitoring the reaction by GC. However, it was subse-
quently determined that the isoquinoline was generated
only by the cis isomer of 3 and only during the gas
chromatography monitoring, not by the enzyme-catalyzed
reaction. Although this approach was not able to detect
the proposed enzyme-catalyzed intermediate, unexpected
oxidized products were identified.
yield from o-bromobenzaldehyde by palladium coupling.13
Iodination of alkyne 9 was readily realized by radical
addition of tributyltin hydride followed by iodine addi-
tion.14 The alcohol was converted to 3a by standard
functional group transformations.
Syn th esis of o-[(Z)-Iod oeth en yl]ben zyla m in e Hy-
d r och lor id e (3b). The key intermediate 14 was pre-
pared by a novel Wittig reaction (Scheme 4).15 To avoid
reaction with both aldehyde functionalities of o-phthal-
aldehyde, the ylide was generated and was added slowly
to an excess amount of o-phthalaldehyde. Conversion of
15 to 3b followed the same route that was used to prepare
3a .
Syn th esis of Con d en sa tion P r od u ct 17 a n d Me-
ta bolite 18. The condensation product 17 was prepared
according to a procedure by Enholm et al.16 (Scheme 5);
compound 18 was prepared from the corresponding
alcohol (Scheme 6).17
Resu lts a n d Discu ssion
(13) Austin, B. W.; Bilow, N.; Kelleghan, W. J .; Lau, K. S. J . Org.
Chem. 1981, 46, 2280-2286.
Syn th esis of o-[(E)-Iod oeth en yl]ben zyla m in e p-
Tolu en esu lfon a te (3a ). Amine 3a was synthesized as
shown in Scheme 3. Aldehyde 7 was synthesized in high
(14) Taber, D. F.; Wang, Y. J . Am. Chem. Soc. 1997, 119, 22-26.
(15) Stock, G.; Zhao, K. Tetrahedron Lett. 1989, 30, 2173-2174.
(16) Enholm, E. J .; Forbes, D. C.; Holub, D. P. Synth. Commun.
1990, 20, 981-987.
(12) Baldwin, J . E.; Cutting, J .; Dupont, W.; Kurse, L.; Silberman,
L.; Thomas, R. C. J . Chem. Soc., Chem. Commun. 1976, 736-738.
(17) Holton, R. A.; Zoeller, J . R. J . Am. Chem. Soc. 1985, 107, 2124-
2131.

Intermediates in MAO-Catalyzed Oxidations
J . Org. Chem., Vol. 63, No. 21, 1998 7359
Sch em e 3
Sch em e 4
Sch em e 5
tion of isoquinoline was not found to be strictly time-
dependent, and after short time periods, the formation
of isoquinoline ceased. A small contaminant appeared
to be consumed during enzyme-catalyzed oxidation of 3a .
Mass spectrometric analysis revealed that the contami-
nant was an isomer of 3a , suggesting the Z isomer 3b.
After 3b was synthesized, it was subjected to MAO B
oxidation and was found to be a substrate (Km ) 0.24
mM, kcat/Km ) 1490 mM-1 min-1). Two products were
observed, one of them being isoquinoline (5), as identified
by GC-MS analysis, and the other one was an unknown
compound.
En zym a tic Stu d ies. Compound 3a was synthesized
and found to be a competitive inhibitor of MAO B with
Ki ) 0.18 mM (Figure 1). Trace amounts of isoquinoline
(5) were detected by GC-MS when 3a was incubated with
MAO B. The 6-endo-trig mechanism in Scheme 2 could
explain the generation of isoquinoline. However, forma-
However, when the experiment was carried out on a
large enzyme scale (36 µmol), an NMR spectrum of the

7360 J . Org. Chem., Vol. 63, No. 21, 1998
Wang and Silverman
Sch em e 8
F igu r e 1. Dixon plot of the inhibition of MAO B by 3a in 100
mM Tris buffer, pH 9.0 at 25 °C; (b) 0.2 mM; (O) 0.35 mM;
(9) 0.5 mM; (0) 0.67 mM.
indicates that these two products might have come from
the same precursor by decomposition. The substrate 3b
and the aldehyde 14 were recovered when the enzyme
reaction products were chromatographed, although they
were not detected by GC. These products (3b and 14)
could arise via hydrolytic decomposition of the corre-
sponding imine (17; Scheme 9). The unknown second GC
product, then, is proposed to be 18. The structure of 17
was verified by GC, GC-MS, and NMR comparison with
the independently synthesized compound, prepared as
shown in Scheme 5. The structure of 18 was confirmed
by GC and GC-MS using synthetic 18 (Scheme 6).
Sch em e 6
Sch em e 7
Con clu sion s
Compounds 3a and 3b were designed to trap potential
radical intermediates of the MAO-catalyzed reaction
intramolecularly. However, the rate of cyclization must
be too slow relative to the rate of electron transfer,
possibly because of constraints within the active site, and
only “normal” oxidation to the corresponding imine
results. An important finding from this study, however,
is the artifactual formation of isoquinoline (5) during
product analysis by gas chromatography. GC is an easy
and fast technique for product study; however, it can
produce secondary products unrelated to the system that
it is being used to analyze, and therefore this may be a
drawback for monitoring sensitive enzyme-catalyzed
reactions. Although this general approach was not
successful in the case of monoamine oxidase, it has the
potential to be applied to the trapping of radical inter-
mediates in other enzyme-catalyzed reactions.
total products indicated that no isoquinoline was present.
Instead, it was shown that the products had the same
NMR vinyl proton chemical shifts and coupling constants
as those of the vinyl protons of the starting material. This
suggests that the formation of isoquinoline occurred
during the gas chromatography. As a test of this
hypothesis, aldehydes 12 and 14 were treated with
ammonia, and the incubation mixture was injected into
the GC; isoquinoline was detected. Electrocyclization in
the GC (Scheme 7) would rationalize that observation.
Isoquinoline also was observed by GC-MS when both 12
and 14 were treated with methylamine; the isolated
products (prior to GC analysis) were determined by NMR
and MS to be the corresponding imines 19 and 20
(Scheme 8). This result also can be explained by a GC-
induced electrocyclization, followed by demethylation.
The second product that was detected by GC was
always found to be in the ratio of approximately 1:1 with
5 by GC-MS analysis throughout the reaction. This
Exp er im en ta l Section
Gen er a l P r oced u r es. All reagents and solvents were
purchased from Aldrich or Fisher and were used without
further purification 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 Å
molecular sieves. All of the reactions were carried out under
an atmosphere of inert gas.

Intermediates in MAO-Catalyzed Oxidations
J . Org. Chem., Vol. 63, No. 21, 1998 7361
Sch em e 9
2-[(Tr im eth ylsilyl)eth yn yl]ben zaldeh yde (7) an d 2-Eth -
yn ylben za ld eh yd e (8). The procedure of Austin et al.13 was
utilized. A deaerated solution of 15.66 g (84.6 mmol) of
2-bromobenzaldehyde, 338 mg (1.29 mmol) of triphenylphos-
phine, and 167 mg (0.74 mmol) of palladium (II) acetate in
200 mL of anhydrous triethylamine was treated with 12.47 g
(12.70 mmol) of ethynyltrimethylsilane under argon. The
reaction mixture was rapidly heated to 80 °C for 6 h. After it
was cooled, the mixture was filtered, and the filtrate was
concentrated, mixed with water (100 mL), and extracted with
dichloromethane (3 × 50 mL). The combined organic phases
were dried over Na2SO4, concentrated, and distilled at 80-84
°C (0.1 Torr) to give 2-[(trimethylsilyl)ethynyl]benzaldehyde
0.53 mmol) in anhydrous toluene (30 mL) was added Bu3SnH
(2.91 g, 10 mmol) at room temperature under argon. The
reaction mixture was heated at 100-105 °C for 3 h and stirred
at room temperature overnight. The reaction mixture was
concentrated to afford a light yellowish liquid.
To the crude vinyl stannane (4.8 g) in ether (20 mL) was
added iodine (2.54 g, 10 mmol) dissolved in ether (10 mL). After
30 min, the reaction mixture was washed with 10% Na2S2O3
(15 mL). The aqueous layer was washed with ether (15 mL).
Organic layers were combined, washed with brine, dried over
Na2SO4, concentrated, and chromatographed (17% EtOAc/83%
hexane) to give 10 (1.04 g, 40%) as a white solid: mp 129-
131 °C; 1H NMR (CDCl3, 300 MHz) δ 7.75-7.80 (d, 1H), 7.31-
7.35 (m, 4H), 6.80-6.85 (d, 1H), 4.73 (s, 2H); 13C NMR (CDCl3,
300 MHz) 142.30, 136.89, 136.85, 128.65, 128.58, 128.44,
126.40, 118.94, 79.25, 63.40; MS (EI) m/z 260 (M+), 133, 115,
105, 77, 51; HRMS (EI) calcd for C9H9IO (M+) 259.9700, found
259.9699. Anal. Calcd for C9H9OI: C, 41.56; H; 3.49.
Found: C, 41.68: H, 3.79.
2-[(E)-2-Iod oeth en yl]ben zyla m in e (3a ). To a stirred
solution of 10 (743 mg, 2.86 mmol) and triethylamine (438 µL,
3.14 mmol) in anhydrous ether (30 mL) at 0 °C under nitrogen
was slowly added methanesulfonyl chloride (342 mg, 3 mmol).
After 2 h, water (10 mL) was added to the reaction mixture.
The organic layer was washed with brine, dried with Na2SO4,
and concentrated to give 11 as a yellow syrup (940 mg, 97%):
1H NMR (CDCl3, 300 MHz) δ 7.70-7.75 (d, 1H), 7.37-7.44
(m, 4H), 6.86-6.91 (d, 1H), 5.30 (s, 1H), 2.93 (s, 1H).
1
(7) as a yellow-brown solid (15.30 g, 90%): mp 47-48 °C; H
NMR (CDCl3, 400 MHz) δ 10.57 (s, 1H), 7.91-7.933 (d, 1H),
7.43-7.58 (m, 3H), 0.30 (s, 9H); 13C NMR (CDCl3, 400 MHz) δ
193.00, 137.24, 134.80, 134.60, 129.93, 127.96, 127.90, 103.53,
101.14, 0.88; MS (EI) m/z 201 (M - H+), 187, 161, 128; HRMS
(EI) m/z calcd for C12H13OSi (M - H+) 201.0736, found
201.0733.
The aldehyde 7 (8.0 g, 39.6 mmol) was treated with
potassium fluoride dihydrate (8.3 g, 88.2 mmol) in DMF (30
mL) under nitrogen at room temperature for 3 h. It was then
poured into water (60 mL) and extracted with dichloromethane
(3 × 30 mL), dried over Na2SO4, and concentrated to yield 8
(5.0 g, 97%) as a yellow solid. An analytical sample was
1
prepared by sublimation at 15 Torr: mp 61-62 °C; H NMR
(CDCl3, 400 MHz) δ 10.53 (s, 1H), 7.92-7.94 (d, 1H), 7.48-
7.60 (m, 3H), 3.46 (s, 1H); 13C NMR (CDCl3, 400 MHz) δ 192.57,
137.60, 134.99, 134.82, 130.30, 128.33, 126.57, 102.43, 85.36;
MS (EI) m/z 130 (M+), 102, 76, 51; HRMS (EI) m/z calcd for
C9H6O 130.0419 (M+), found 130.0415.
A stirred suspension of the sulfonate ester 11 (300 mg, 0.9
mmol) in anhydrous DMF (15 mL) containing sodium azide
(455 mg, 7 mmol) under nitrogen was heated to no greater
than 50 °C for 3 h. The reaction mixture was cooled to room
temperature, and water (30 mL) was added to bring excess
sodium azide into solution. The mixture was extracted with
ether (2 × 25 mL). The organic extracts were combined,
washed with water (20 mL) and brine (20 mL), and dried over
Na2SO4. Part of the solvent was removed, and to this ether
solution at 0 °C was added triphenylphosphine (264 mg, 1
mmol). After 2 h, 100 µL of water was added to the reaction
mixture. After 12 h, this ether solution was extracted with 1
N HCl (2 × 20 mL). The water layer was washed with ether
(3 × 10 mL) followed by addition of potassium hydroxide
pellets until strongly basic. The solution was then extracted
with ether (3 × 20 mL). The organic phases were combined,
washed with brine, dried over Na2SO4, and acidified with
p-toluenesulfonic acid and then recrystallized from ether-
ethanol to give 3a as white crystals (200 mg, 51%): mp 185-
2-Eth yn ylben zyl Alcoh ol (9). To a solution of 8 (2.56 g,
19.7 mmol) in anhydrous ethanol (30 mL) was added portion-
wise sodium borohydride (370 mg, 10 mmol) at 0 °C under
nitrogen. The reaction mixture was stirred at 0 °C for 1 h
before it was warmed to room temperature. The mixture was
concentrated, diluted with 20 mL of water, and extracted with
ether (3 × 15 mL). The combined organic layers were washed
with brine (20 mL), dried over Na2SO4, concentrated, and
purified by column chromatography (15% EtOAc/85% hexanes)
to give 9 (2.55 g, 97%) as a white solid: mp 61-62 °C; 1H NMR
(CDCl3, 300 MHz) δ 7.46-7.55 (m, 2H), 7.30-7.38 (m, 2H),
4.79 (s, 2H), 3.38 (s, 1H); 13C NMR (CDCl3, 300 MHz) δ 143.22,
132.92, 129.30, 127.53, 127.40, 120.26, 82.03, 81.30, 63.90; MS
(EI) m/z 132 (M+), 103, 77, 51; HRMS (EI) m/z calcd for C9H8O
132.0575 (M+), found 132.0570.
1
2-[(E)-2-Iod oeth en yl]ben zyl Alcoh ol (10). To a solution
of hydroxy alkyne 9 (1.32 g, 10 mmol) and AIBN (87.2 mg,
190 °C; H NMR (CDCl3, 300 MHz) 7.73-7.78 (d, 1H), 7.29-
7.37 (m, 4H), 6.75-6.80 (d, 1H), 3.90 (s, 1H); 13C NMR (CDCl3,

7362 J . Org. Chem., Vol. 63, No. 21, 1998
Wang and Silverman
300 MHz) 142.56, 139.49, 136.50, 128.72, 128.26, 127.49,
126.39; MS (EI) m/z 258 (M - H+), 132, 115, 77; HRMS (EI)
calcd for C9H10NI (M - H+) 259.9781, found 257.9785. Anal.
Calcd for C16H18NO3SI (tosylate salt): C, 44.56; H, 3.91; N,
3.25. Found: C, 44.34; H, 3.93; N, 3.17.
2-[(E)-2-Iod oeth en yl]ben za ld eh yd e (12). To a vigorously
stirred suspension of PCC (1.20 g, 5.57 mmol) in anhydrous
dichloromethane (20 mL) was added vinyl iodide 10 (1.00 g,
3.9 mmol). The reaction mixture was stirred for 2 h before 20
mL of ether was added. The solution was filtered through a
pad of silica gel and concentrated to give 12 as a colorless oil:
1H NMR (CDCl3, 300 MHz) δ 10.21 (s, 1H), 8.28-8.33 (d, 1H),
7.48-7.80 (m, 4H), 6.87-6.92 (d, 1H); 13C NMR (CDCl3, 300
MHz) 192.20, 141.85, 139.59, 134.00, 132.43, 132.05, 128.59,
127.52, 81.83; MS (EI) m./z 131, 103, 77, 51; HRMS (EI) calcd
for C9H7OI (M+) 257.9543, found 257.9538. The compound
slowly decomposed, prohibiting elemental analysis.
128.64, 128.58, 128.49, 128.40, 127.67, 126.97; MS (EI) m/z
497 (M - H+), 371, 245, 130, 116; HRMS (EI) calcd for C18H15
-
NI2 (M - H+) 497.9216, found 497.9218.
2-[(Z)-2-Iod oeth en yl]ben zyl Iod id e (18). To a solution
of the sulfonate ester 16 (62 mg, 0.19 mmol; prepared by the
same procedure as 11) in anhydrous acetone (5 mL) was added
a solution of sodium iodide (67.5 mg, 0.45 mmol) in acetone
(10 mL). The solution was allowed to reflux overnight. The
solvent was removed, water (10 mL) was added, and the
solution was extracted with ether (3 × 10 mL). The organic
layers were combined, dried, concentrated, and chromato-
graphed to afford 18 as a white solid: mp 40-42 °C; the color
changes rapidly upon exposure to light; 1H NMR (CDCl3, 300
MHz) δ 7.36-7.42 (m, 3H), 7.28-7.30 (m, 2H), 6.83-6.86 (d,
1H), 4.38 (s, 2H); 13C NMR (CDCl3, 300 MHz) δ 138.72, 138.29,
137.55, 130.59, 130.43, 129.87, 129.17, 86.25, 5.33; MS (EI)
m/z 370 (M+), 254, 243, 116, 63; HRMS (EI) calcd for C9H8I2
(M+) 369.8716, found 369.8713.
2-[(Z)-2-Iod oeth en yl]ben za ld eh yd e (14). To a stirred
suspension of (iodomethyl)triphenylphosphonium iodide18 (1.80
g, 3.2 mmol) in anhydrous THF (15 mL) was added sodium
hexamethyldisilazine (1 M, 3.2 mL) dropwise. After it was
stirred for 1 min, the solution was cooled in a dry ice-acetone
bath and HMPA (1 mL) was added. The above orange solution
was transferred via cannula slowly to a solution of phthalic
dicarboxaldehyde (1.72 g, 12.8 mmol) in THF (20 mL) at -78
°C. The cold bath was then removed, and stirring was
continued for 30 min. Hexane (40 mL) was added to the
reaction mixture. After filtration, the filtrate was washed with
water (3 × 15 mL) and brine (20 mL), dried over Na2SO4,
concentrated and chromatographed (11% EtOAc/89% hexane)
to give 14 (350 mg, 42%), which was inseparable from its trans
isomer. An analytical sample was obtained by column chro-
matographic separation of the corresponding alcohol 15 (see
below) followed by PCC oxidation: 1H NMR (CDCl3, 300 MHz)
δ 10.20 (s, 1H), 7.94-7.96 (d, 1H), 7.78-7.80 (d, 1H), 7.53-
7.68 (m, 3H), 6.89-6.92 (d, 1H); 13C NMR (CDCl3, 300 MHz)
δ 192.33, 141.02, 138.24, 134.32, 133.71, 131.21, 130.34,
129.19, 86.80; HRMS (EI) calcd for C9H7OI (M+) 257.9543,
found 257.9534. The compound slowly decomposed, prohibit-
ing elemental analysis.
2-[(Z)-2-Iod oeth en yl]ben zyl Alcoh ol (15). Compound 15
was prepared by the same method as 10 by NaBH4 reduction
of 14: mp 54-55 °C; 1H NMR (CDCl3, 300 MHz) δ 7.27-7.49
(m, 5H), 6.72-6.75 (d, 1H), 4.64 (s, 2H); 13C NMR (CDCl3, 300
MHz) δ 138.25, 138.12, 136.78, 128.70, 128.54, 128.02, 127.71,
84.70, 63.44; MS (EI) m/z 260 (M+), 133, 105, 77, 51; HRMS
(EI) calcd for C9H9IO (M+) 258.9699, found 259.9703. Anal.
Calcd for C9H9OI: C, 41.56; H, 3.49; Found: C, 41.60; H, 3.48.
2-[(Z)-2-Iod oeth en yl]ben zyla m in e (3b). Compound 3b
was prepared from 15 by the same method used to make 3a
from 10. It was analyzed as the HCl salt. The sulfonate ester
16 was first obtained: 1H NMR (CDCl3, 400 MHz) δ 7.43-
7.50 (m, 5H), 6.82-6.84 (d, 1H), 5.22 (s, 2H), 2.90 (s, 3H).
Compound 3b (HCl salt): mp 202-204 °C dec; 1H NMR
(D2O, 300 MHz) δ 7.48-7.54 (m, 5H), 6.96-6.99 (d, 1H), 4.16
(s, 2H); 13C NMR (D2O, 300 MHz) δ 138.64, 137.76, 129.82,
129.50, 129.32, 129.21, 129.00, 88.10, 40.59; MS (EI) m/z 258
(M+), 132, 115, 77, 51; HRMS (EI) calcd for C9H10IN (M+)
257.9781, found 257.9788. Anal. Calcd for C9H11NClI (HCl
salt): C, 36.58; H, 3.75; N, 4.74. Found: C, 36.28; H, 3.76; N,
4.86.
N-{2-[(Z)-2-Iodoeth en yl]ben zyliden e}m eth ylam in e (19).
A solution of 14 (10 mg, 0.04 mmol) and p-toluenesulfonic acid
hydrate (1.5 mg, 0.008 mmol) in benzene (5 mL) was bubbled
with methylamine for 3 min. The reaction mixture was stirred
for 1 h, washed with saturated Na2CO3 (3 mL), dried over
Na2SO4, and concentrated to afford 19 as a yellow oil (10 mg,
90%): 1H NMR (CDCl3, 300 MHz) δ 8.42 (d, 1H), 7.94-7.96
(m, 1H), 7.59-7.62 (d, 1H), 7.38-7.45 (m, 3H), 6.79-6.82 (d,
1H), 3.53 (d, 3H); MS (EI) m/z 272 (MH+), 144, 103, 55; HRMS
(EI) calcd for C10H11IN (MH+) 271.9936, found 271.9949.
N-{2-[(E)-2-Iodoeth en yl]ben zyliden e}m eth ylam in e (20).
Compound 20 was prepared in a manner identical with that
for 19, starting from 12: 1H NMR (CDCl3, 300 MHz) δ 8.56
(d, 1H), 7.97-8.02 (d, 1H), 7.79-7.80 (d, 1H), 7.35-7.38 (m,
3H), 6.72-6.77 (d, 1H), 3.56 (d, 3H); MS (EI) m/z 270 (M -
H+), 229, 144, 131, 115, 103, 77; HRMS (EI) calcd for C10H9NI
(M - H+) 269.9780, found 269.9766.
En zym e a n d Assa y. Beef liver MAO B was isolated as
described previously19 and stored as a concentrated solution
(15-25 mg/mL) in sodium phosphate buffer (50 mM, pH 7.2)
at 4 °C. The specific activity varied among preparations,
ranging from 3.5 to 7 units/mg, where a unit of activity is the
conversion of 1 µmol of benzylamine to benzaldehyde per
minute at pH 9.0 and 30 °C.
Gen er a l P r oced u r e in Or ga n ic Solven t. Stock MAO (40
µL) was pipetted into a vial, frozen in a -78 °C dry ice-acetone
bath, and lyophilized. The substrate solution (6 mM, 1000 µL)
was syringed into the vial containing the dried enzyme; water
was pipetted into the mixture to give a final water concentra-
tion of 0.5% (v/v). The mixture was sonicated for 10 s in an
ultrasonic cleaning bath. For analysis, 1 µL aliquots were
periodically removed and analyzed by capillary gas chroma-
tography.
Gen er a l P r oced u r e for In h ibition of th e MAO-Ca ta -
lyzed Oxid a tion of Cin n a m yla m in e. An MAO solution was
prepared by diluting 15 µL of the stock MAO solution with
285 µL of Tris-HCl buffer (100 mM, pH 9.0). The amount of
inhibition of the oxidation of various concentrations of cin-
namylamine (0.2, 0.35, 0.50, and 0.67 mM in Tris-HCl buffer,
100 mM, pH 9.0) by various concentrations of inhibitor 3a (0,
0.2, 0.4, 0.6, 0.8 mM in Tris-HCl buffer, 100 mM, pH 9.0) was
determined by adding 10 µL of the above MAO solution to 490
µL of an inhibitor/substrate solution at 25 °C, followed by
monitoring the increase in absorbance at 290 nm. Up to 30%
anhydrous DMSO was used as the cosolvent to dissolve 3a .
An enzyme control was incubated in DMSO-containing buffer
to factor out any effects of the cosolvent on the enzyme activity.
The Ki value was determined by a Dixon plot.20
Con d en sed Im in e of 3b a n d 14 (17). To a solution of the
free amine form of 3b (16.62 mg, 0.064 mmol) in benzene (5
mL) was added aldehyde 14 (16.5 mg, 0.064 mmol) in 5 mL of
benzene. The solvent was removed in vacuo, and benzene was
added and evaporated a couple of times to get rid of the water.
Compound 17 was obtained as a yellowish oil: 1H NMR
(CDCl3, 300 MHz) δ 8.48 (s, 1H), 8.02 (d, 1H), 7.60-7.62 (d,
1H), 7.52-7.55 (d, 1H), 7.32-7.49 (m, 7H), 6.78-6.81 (d, 1H),
6.71-6.74 (d, 1H), 4.67 (s, 2H); 13C NMR (CDCl3, 300 MHz)
160.36, 138.86, 138.65, 138.48, 136.91, 133.17, 130.33, 128.80,
Meta bolite Stu d ies. To a solution of 3b (12.4 mM) in Tris-
HCl buffer (3.0 mL, 100 mM, pH 9.0) was added MAO B (220
µL, 17.75 mg/mL). Aliquots of 20 µL were periodically
withdrawn, extracted with 40 µL of methylene chloride by
(19) Weyler, W.; Salach, J . I. Arch. Biochem. Biophys. 1981, 212,
147-153.
(20) Price, N. C.; Stevens, L. Fundamentals of Enzymology; Oxford
University Press: Oxford, U.K., 1989, pp 178-179.
(18) Seyferth, D.; Heeren, J . K.; Singh, G.; Grim, S. O.; Hughes, W.
B. J . Organomet. Chem. 1966, 5, 267-274.

Intermediates in MAO-Catalyzed Oxidations
J . Org. Chem., Vol. 63, No. 21, 1998 7363
spinning, concentrated by gently blowing nitrogen over the
surface of the liquid, and analyzed by gas chromatography.
When the starting material was consumed, the reaction
mixture was extracted with dichloromethane (2 × 5 mL). The
organic layers were combined, washed with brine, dried over
Na2SO4, and concentrated for NMR analysis: 1H NMR (CDCl3,
300 MHz) 8.47 (s, 1H), 8.03-8.05 (d, 1H), 7.59-7.62 (d, 1H),
7.51-7.54 (d, 1H), 7.32-7.48 (m, 7H), 6.78-6.81 (d, 1H), 6.70-
6.73 (d, 1H), 4.76 (s, 1H).
Ack n ow led gm en t. We are very grateful to the
National Institutes of Health (Grant No. GM32634) for
financial support of this research.
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