4-Substituted cubylcarbinylamines: A new class of mechanism-based monoamine oxidase B inactivators

Article abstract of DOI:10.1021/jm9606249

Cubylcarbinylamine (1a), (4-cyclopropylcubyl)carbinylamine (1b), and (4- phenylcubyl)carbinylamine (1c) were synthesized and shown to be time- dependent, irreversible inactivators of monoamine oxidase B (MAO B). Substrate protects the enzyme from inactiva

Full text of DOI:10.1021/jm9606249

J . Med. Chem. 1997, 40, 1165-1168
1165
Notes
4-Su bstitu ted Cu bylca r bin yla m in es: A New Cla ss of Mech a n ism -Ba sed
Mon oa m in e Oxid a se B In a ctiva tor s
J oseph J . P. Zhou,†,‡ J ianchang Li,§ Suhbash Upadhyaya,§ Philip E. Eaton,§ and Richard B. Silverman*,†
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University,
Evanston, Illinois 60208-3113, and Department of Chemistry, The University of Chicago, 5735 South Ellis Avenue,
Chicago, Illinois 60637
Received August 30, 1996X
Cubylcarbinylamine (1a ), (4-cyclopropylcubyl)carbinylamine (1b), and (4-phenylcubyl)carbi-
nylamine (1c) were synthesized and shown to be time-dependent, irreversible inactivators of
monoamine oxidase B (MAO B). Substrate protects the enzyme from inactivation, but
-mercaptoethanol does not, suggesting that these compounds are mechanism-based inacti-
vators. All three compounds were also substrates for MAO B with partition ratios ranging
from 152 to 536. The 4-substituted analogues were more potent inactivators than the
unsubstituted analogue, indicating a benefit to 4-substitution in this class of inactivators.
Sch em e 1
In tr od u ction
Monoamine oxidase (EC 1.4.3.4; MAO), a flavin-
dependent enzyme located in the outer membrane of the
mitochondria, is responsible for the degradation of
various neurotransmitters and xenobiotic amines.1 Two
major isozymes of this enzyme are known:2 MAO A
selectively degrades serotonin and norepinephrine and
MAO B is selective for 2-phenylethylamine and benzyl-
amine. Compounds that selectively inhibit MAO A
exhibit antidepressant activity; selective MAO B inhibi-
tors are used clinically as adjuncts in the treatment of
Parkinson’s disease.3
Sch em e 2
We have reported a variety of structures based on the
hypothesis that MAO proceeds by a single-electron
transfer mechanism (Scheme 1).4 In a mechanism-
based approach, new structures are designed to test the
mechanism, and this often provides unusual molecules
that can be used for further design of new selective
inhibitors. Our goal has not been to prepare selective
MAO inhibitors but rather to design new classes of
inhibitors as lead compounds for future selective inhibi-
tor design.
Resu lts a n d Discu ssion
Syn th esis. The syntheses of compounds 1a -c are
summarized in Scheme 2. 4-Substituted cubane 1-io-
dides (3) were made from 1,4-diiodocubane (2) via the
1,4-cubanediyl intermediate.6-9 Unlike many known
1,4-diradicals,10 cubane-1,4-diyl is singlet because there
is a strong through-bond interaction between the cubyl
1- and 4-positions. The cubane iodides were trans-
formed into the cubylcarbinylamines (1) by standard
procedures via the carboxylic acid (4) and the amide (5).
All reactions proceeded in good yields.
En zym ology. All of the cubylcarbinylamines (1a -
c) are time-dependent irreversible MAO B inactivators
and substrates (Table 1; see Figure 1 for representative
kinetic plots for 1b). The substrate activity of the
compounds is consistent with the increased activity of
arylalkylamine substrates. The kinetic constants were
determined by the method of Kitz and Wilson.11 Inac-
tivation of MAO B by cubylcarbinylamines 1a -c was
protected by a 10-fold excess of the substrate 2-phenyl-
ethylamine, indicating that inactivation occurred at the
active site. The addition of -mercaptoethanol to the
One of the unusual compounds that we reported a a
probe for the mechanism of MAO is (aminomethyl)-
cubane (1a ).5 This compound is a mechanism-based
inactivator and substrate of MAO B, the first cubane-
containing molecule demonstrated to be an inactivator
for any enzyme. Here we show that 4-substituted
cubane-containing analogues (1b,c) also are substrates
and inactivators of MAO B.
† Northwestern University.
‡ Current address: Versicor, Inc., 270 E. Grand Ave., South San
Francisco, CA 94080.
§ The University of Chicago.
X Abstract published in Advance ACS Abstracts, February 1, 1997.
S0022-2623(96)00624-3 CCC: $14.00 © 1997 American Chemical Society

1166 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Notes
Ta ble 1. Kinetic Constants for the Inactivation of Monoamine Oxidase B by Cubylcarbinylamines
compd
KI (mM)
kinact (min-1
)
kinact/KI (min-1 mM-1
)
Km (mM)
kcat (min-1
5.0 ( 0.2
150 ( 9
208 ( 10
)
kcat/Km (min-1 mM-1
)
partition ratio
1a a
1b
1c
0.17 ( 0.05
0.26 ( 0.04
0.12 ( 0.02
0.033 ( 0.004
0.28 ( 0.01
0.75 ( 0.06
0.19 ( 0.05
1.1 ( 0.2
6.3 ( 1.0
0.14 ( 0.01
1.54 ( 0.10
1.01 ( 0.07
36 ( 3
100 ( 6
210 ( 15
152
536
277
a
From ref 5.
Sch em e 3
(1a ). This appears to be the case (Table 1). Whereas
the KI for the unsubstituted analogue (1a ) and for the
4-phenyl-substituted analogue (1c) are essentially the
same, the kinact value of the 4-phenyl analogue is 23
times greater than that of the unsubstituted compound;
the 4-cyclopropyl analogue has an intermediate value.
This order of increased kinact values, however, also
follows the order of substrate activities (kcat), so it is not
yet clear if there is something intrinsic to the structures
or if the radical stabilization effect is more important.
The fact that the KI values for 1b and 1c differ from
their Km values suggests that either the inactivation
pathway diverges from the turnover pathway after E‚I
complex formation, as is shown in Scheme 3, or two
different E‚I complexes form, one that leads to product
and the other that leads to inactivation. More ana-
logues need to be synthesized to determine if the
proposed increased stabilization at the 4-position is
responsible for the increased inactivator efficiency.
F igu r e 1. (A) Time-dependent inactivation of monoamine
oxidase by 0.10 (0), 0.20 (9), 0.80 (O), 6.36 (b), and 12.73 (2)
mM (4-cyclopropylcubyl)carbinylamine (1b). See the Experi-
mental Section for details. (B) Kitz and Wilson replot11 of the
data in part A.
inactivation buffer had no effect on the rate of inactiva-
tion, suggesting that the reactive species does not escape
the active site prior to inactivation. Enzyme activity
did not return, even after exhaustive dialysis, indicating
an irreversible inactivation process. The efficiency of
the inactivators, as measured by the kinact/KI values,
indicates that it is favorable to have substituents at the
4-position. The inactivation mechanism is not com-
pletely understood; however, on the basis of metabolite
studies and model reaction studies,5,12,13 it is believed
that cubane ring opening reactions are responsible for
the enzyme inactivations (Scheme 3). Opening of the
cubane ring to a cyclooctatrienyl radical instead of to 6
is not predicted from orbital symmetry arguments and
was not observed in model reactions.13 Since the
cubylcarbinyl radical ring opening is expected to gener-
ate a radical center at the C-4 carbon atom, substitution
at the C-4 position of cubanes would eventually form
substituted radicals such as 6. These substituted
radicals would be stabilized, and the driving force for
the third ring-opening reaction would be larger. Since
the third bond cleavage is the slowest of the three,13 this
would suggest that the rates of ring-opening reactions
of 4-substituted cubylcarbinyl radicals should be faster
than those of the unsubstituted cubylcarbinyl radical,
and 4-substituted analogues (1b,c) would be more
efficient inactivators than the unsubstituted analogue
Exp er im en ta l Section
Ch em istr y. All reagents are from Aldrich and were used
without further purification. Cubanecarboxylic acid (4a ) was
prepared as previously reported.14 NMR spectra were run in
chloroform-d at ambient probe temperature. 1H NMR spectra
are at 400 MHz and were referenced to an internal standard
of tetramethylsilane; 13C NMR spectra are at 100.6 MHz and
were referenced to the main peak of the solvent.
4-P h en ylcu byl Iod id e (3c). Phenyllithium (12 mL, 0.022
mol, 1.8 M in cyclohexane/diethyl ether, 7:3) was added
dropwise to a cold and magnetically stirred suspension of 1,4-
diiodocubane6 (3.56 g, 0.01 mol) in ether (80 mL). The mixture
was then stirred at room temperature for 1 h during which
time it turned to a clear light yellow-brown solution. More
phenyllithium (6 mL, 0.011 mol) was added, and the solution
was stirred for 10 min. The solution was cooled (0 °C) and
the reaction quenched with methanol (20 mL). The organic

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1167
1
layer was washed with water and brine, dried (Na2SO4), and
concentrated to dryness to give 4-phenylcubyl iodide (3.06 g,
100%) as a light yellow solid which was used in the next step
°C dec; H-NMR (D2O) 3.10 (s, 2 H) 3.75 (m, 3 H), 3.88 (m,
3 H) ppm; 13C-NMR (D2O) 41.1, 43.3, 47.4, 55.2, 60.1, 124.6,
126.2, 128.8, 142.9 ppm. Anal. (C15H16ClN) C, H, Cl, N.
1
without further purification: mp 77-79 °C; H-NMR 4.32
(4-Cyclopr opylcu byl)car bin ylam in e Hydr och lor ide Salt
(1b‚HCl). This compound was synthesized in a 78% yield in
the same way as was 1c‚HCl described above: mp 230 °C dec;
1H NMR (CD3OD) 0.18-0.43 (m, 4 H), 1.02 (m, 1 H), 3.13 (s,
2 H), 3.58 (m, 3 H), 3.77 (m, 3 H); 13C-NMR (CD3OD) 0.3,
12.8, 42.6, 44.6, 45.1, 57.1, 62.2 ppm. Anal. (C12H16NCl) C,
H, Cl, N.
(s, 6 H), 7.09-7.19 (m, 5 H) ppm; 13C-NMR 39.0, 52.0, 54.3,
60.5, 124.7, 126.3, 128.5, 141.5 ppm.
4-P h en ylcu ba n eca r boxylic Acid (4c). A solution of
4-phenylcubyl iodide (3.06 g, 0.01 mol) in dry THF (160 mL)
was cooled to -78 °C. tert-Butyllithium in pentane (1.7 M,
11.8 mL, 20 mmol) was added dropwise, and the resulting pale
yellow solution was stirred at -78 °C for 30 min. Dry CO2
was bubbled through for 10 min, and the reaction mixture was
allowed to warm up to room temperature while CO2 passed
through for 1 h. The reaction mixture was mixed with 50 mL
of aqueous HCl (2 M) and was extracted with chloroform. The
combined organic solution was washed with aqueous HCl (2
M) and dried over Na2SO4. Removal of the solvents gave
4-phenylcubanecarboxylic acid as a colorless solid in 71%
Cu bylca r bin yla m in e Hyd r och lor id e (1a ‚HCl). This
was synthesized in an 81% yield from the corresponding amide
in the same way as was 1c‚HCl: mp 275 °C dec; 1H-NMR (D2O)
3.00 (s, 2 H) 3.73 (m, 6 H), 3.82 (m, 1 H) ppm; 13C-NMR
(D2O) 41.1, 43.8, 46.6, 48.2, 54.0 ppm. Anal. (C9H12ClN) C,
H, Cl, N.
En zym e a n d Assa ys. Mitochondrial MAO (EC 1.4.3.4) was
isolated15 as previously reported. MAO was assayed by a
modification of the procedure of Tabor et al.16 A typical assay
would be the addition of a solution of MAO (5 L) to 0.495 mL
of a 2 mM benzylamine solution in 20 mM Tris‚HCl buffer,
pH 9.0 at 25 °C. The change in absorbance at 250 nm was
observed with time.
1
yield: mp 178-179 °C; H-NMR 4.19 (m, 3 H), 4.30 (m, 3
H), two sets of multiple peaks centered at 7.22 and 7.37 (5 H)
ppm; 13C-NMR 46.1, 48.7, 56.1, 60.2, 124.7, 126.2, 128.5,
141.8, 178.3 ppm.
4-Cyclop r op ylcu ba n eca r boxylic Acid (4b). The same
procedure was used as described for 4c except substituting
4-cyclopropylcubyl iodide.9 Recrystallization of the crude
product in hexane gave pure 4-cyclopropylcubanecarboxylic
acid as colorless plates in 62% yield: mp 155-156 °C; 1H-NMR
(CDCl3) 0.18 and 0.43 (m, 4 H), 1.00 (m, 1 H), 3.65 (m, 3 H),
4.08 (m, 3 H); 13C-NMR (CDCl3) 0.1, 11.7, 45.0, 45.8, 56.3,
60.4, 178.1 ppm. Anal. (C12H12O2) H; C: calcd, 76.57; found,
76.12.
Su bstr a te Activity of th e Cu bylca r bin yla m in e Hyd r o-
ch lor id es. Kinetic constants (Km and kcat) were determined
by measuring the amount of hydrogen peroxide that was
formed with time as described previously.17
Tim e-Dep en d en t In a ctiva tion Exp er im en ts (Gen er a l
Meth od s). Solutions (200 L each) of cubylcarbinylamine
hydrochlorides of various concentrations in 100 mM Tris
buffer, pH 9.0 containing 4% v/v DMSO (freshly distilled), were
preincubated at 25 °C. To these solutions was added MAO B
(20 L of 2.6 mg/mL). After being mixed, the samples were
incubated at 25 °C, periodically agitated, and assayed for MAO
activity by removing 10 L of the mixture and adding it to
490 L of a 1.0 mM benzylamine solution in 100 mM Tris
buffer, pH 9.0. The enzyme activity thus determined was
corrected against a control containing no inactivator (control
activity set to 100%). Kinetic constants (KI and kinact) were
determined as described by Kitz and Wilson.11 The control
enzyme also contained 4% DMSO (it was shown that there
are no adverse effects on activity up to 10% DMSO). After
inactivation each enzyme solution was dialyzed against 100
mM Tris-HCl buffer, pH 9.0, for 48 h to determine reversibility.
Gen er a l P r oced u r e for th e Syn th esis of Cu ba n eca r -
boxa m id es (5a -c). The cubanecarboxylic acid was dissolved
in oxalyl chloride (ca. 10 mL/g) and was stirred at room
temperature for 20 min. The excess oxalyl chloride was
removed under vacuum. The residue was dissolved in dry CH2-
Cl2 (ca. 40 mL/g) in a round bottom flask equipped with a cold
finger filled with dry ice/acetone, and NH3 gas was condensed
into the flask. After being stirred for 20 min, the resulting
suspension was allowed to warm to room temperature, and
the excess NH3 was slowly evaporated. The reaction mixture
was mixed with water and extracted with CHCl3, and the
combined organic solution was dried over Na2SO4. Removal
of solvents gave the cubanecarboxamides as white solids.
1
5a (89% yield): mp 209-212 °C; H NMR 4.01 (m, 4 H),
4.22 (m, 3 H), 5.41 (br, 2 H) ppm; 13C-NMR 44.8, 47.9, 49.5,
57.1, 174.9 ppm.
Effects of â-Mer ca p toeth a n ol a n d 2-P h en yleth yla m in e
on th e Ra te of In a ctiva tion of MAO B by Cu bylca r bin yl-
5b (96% yield): mp 225 °C dec. The crude product was used
for the next step without purification. The sample for elemen-
tal analysis was obtained by recrystallization of the crude
product in EtOAc and CHCl3: 1H-NMR (CDCl3) 0.18 and
0.43 (m, 4 H), 1.02 (m, 1 H), 3.63 (m, 3 H), 4.00 (m, 3 H), 5.40
(br 2 H); 13C-NMR (DMSO-d6) 0.0, 11.7, 43.6, 44.8, 57.7, 59.2,
173.3 ppm. Anal. (C12H13ON) H, N; C: calcd, 76.98; found,
76.38.
a m in es Hyd r och lor id e. The following solutions (200
L
each) were prepared in 100 mM Tris buffer, pH 9.0, containing
4% v/v DMSO and were preincubated at 25 °C: (1) (4-
phenylcubyl)carbinylamine hydrochloride (0.16 mM); (2) (4-
phenylcubyl)carbinylamine hydrochloride (0.16 mM) and -mer-
captoethanol (2 mM); (3) (4-cyclopropylcubyl)carbinylamine
hydrochloride (0.22 mM); (4) (4-cyclopropylcubyl)carbinylamine
hydrochloride (0.22 mM) and -mercaptoethanol (2 mM); and
(4) a control with only the buffer. To these solutions was added
MAO B (20 L of 2.6 mg/mL). The mixtures were incubated
at 25 °C, and MAO activity was assayed as described above.
The same experiments were repeated except the 2 mM
-mercaptoethanol was replaced with 2.4 mM 2-phenylethyl-
amine.
1
5c (80% yield): mp 196-197 °C; H-NMR 4.17 (m, 3 H),
4.22 (m, 3 H), 7.21-7.36 (5 H), 5.45 (br, 2 H) ppm; 13C-NMR
46.1, 48.3, 57.9, 60.3, 124.7, 126.2, 128.5, 141.9, 174.4 ppm.
(4-P h en ylcu byl)car bin ylam in e Hydr och lor ide Salt (1c‚-
HCl). The procedure described below was used also for 1a
and 1b. LiAlH4 (0.64 g, 11 mmol) was added slowly to a
suspension of 4-phenylcubanecarboxamide (0.81 g, 3.6 mmol)
in THF (80 mL) at 0 °C. The mixture was heated to reflux for
15 h and then it was cooled to room temperature. The excess
LiAlH4 was destroyed at 0 °C by dropwise addition of aqueous
NaOH (saturated), and then the mixture was stirred at room
temperature for 1 h. The granular precipitate was filtered off
and was washed with CH2Cl2, and the filtrate was dried (Na2-
SO4). Evaporation of the solvents gave (4-phenylcubyl)-
carbinylamine as a pale yellow oil (0.62 g, 82%): 1H-NMR
2.93 (s, 2 H) 3.81 (m, 3 H), 4.02 (m, 3 H), 7.21-7.35 (m, 5 H)
ppm. The oil was dissolved in dry ether (50 mL), and HCl
gas was bubbled through the solution. The resulting precipi-
tate was collected by filtration and was recrystallized from
methanol to give 0.5 g (69%) of (4-phenylcubyl)carbinylamine
hydrochloride salt (1c‚HCl) as colorless crystals: mp 203-204
Ack n ow led gm en t. The authors are grateful to the
National Institutes of Health (GM32634 to R.B.S.) and
to the National Science Foundation (CHE-9313413 to
P.E.) for financial support of this research.
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