1-methyl-2-undecyl-4(1H)-quinolone as an irreversible and selective inhibitor of type B monoamine oxidase.

Article abstract of DOI:10.1248/cpb.51.409

The inhibitory compound of monoamine oxidase (MAO) activity was isolated from the CH(2)Cl(2) fraction of the fructus of Evodia rutaecarpa and identified as 1-methyl-2-undecyl-4(1H)-quinolone (1). Compound 1 showed a selective inhibition of type B MAO (MAO-B) activity with the IC(50) value of 15.3 microM using a substrate kynuramine, but did not inhibit type A MAO (MAO-A) activity. The kinetic analysis using Lineweaver-Burk plots indicated that compound 1 competitively inhibited MAO-B activity with the K(i) value of 9.91 microM. The inhibition of MAO-B by compound 1 was found to be irreversible by dialysis of the incubation mixture. These results suggest that compound 1 is a potent irreversible inhibitor of MAO-B, and may regulate catecholamine content in the neurons.

Full text of DOI:10.1248/cpb.51.409

April 2003
Notes
Chem. Pharm. Bull. 51(4) 409—411 (2003)
409
1-Methyl-2-undecyl-4(1H)-quinolone as an Irreversible and Selective
Inhibitor of Type B Monoamine Oxidase
a,b
c
a
d
a
Myung Koo LEE, Bang Yeon HWANG, Seon A LEE, Gab Jin OH, Woo Hoi CHOI,
a
a
,a
Seong Su HONG, Kyong Soon LEE, and Jai Seup RO*
a
b
College of Pharmacy, Chungbuk National University; Cheongju 361–763, Korea: Research Center for Bioresource and
c
Health, Chungbuk National University; Cheongju 361–763, Korea: Program for Collaborative Research in the
Pharmaceutical Science and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of
d
Illinois at Chicago; Chicago, IL 60612, U.S.A.: and Samjin Pharm. Co., LTD.; Hwasung 445–920, Korea.
Received August 26, 2002; accepted November 14, 2002
The inhibitory compound of monoamine oxidase (MAO) activity was isolated from the CH Cl fraction of
2
2
the fructus of Evodia rutaecarpa and identified as 1-methyl-2-undecyl-4(1H)-quinolone (1). Compound 1 showed
a selective inhibition of type B MAO (MAO-B) activity with the IC50 value of 15.3 mM using a substrate kynu-
ramine, but did not inhibit type A MAO (MAO-A) activity. The kinetic analysis using Lineweaver-Burk plots in-
dicated that compound 1 competitively inhibited MAO-B activity with the K value of 9.91 mM. The inhibition of
i
MAO-B by compound 1 was found to be irreversible by dialysis of the incubation mixture. These results suggest
that compound 1 is a potent irreversible inhibitor of MAO-B, and may regulate catecholamine content in the
neurons.
Key words 1-methyl-2-undecyl-4(1H)-quinolone; Evodia rutaecarpa; monoamine oxidase inhibitor
Monoamine oxidase (MAO; EC 1.4.3.4) is a flavin-con- was identified by comparison with the physical properties
1
13
taining enzyme that catalyzes the oxidation of a variety of and spectral data (UV, IR, MS, H- and C-NMR) with liter-
1
7,18)
amine-containing neurotransmitters such as catecholamines ature values.
and serotonin to yield the corresponding aldehyde.
1—3)
Compound 1 inhibited MAO activity in a concentration-
MAO exists in two isoforms, namely type A and type B dependent manner and the IC50 value was 35.2 mM: com-
MAO-A and MAO-B), which are characterized by their sen- pound 1 exhibited a less inhibitory effect than iproniazid
(
sitivity towards specific substrates and inhibitors. MAO-A (IC50 value: 12.5 mM) which was used as a positive control
preferentially deaminates serotonin and norepinephrine, and (data not shown).
is selectively inhibited by clorgyline. However, MAO-B pref-
The inhibitory activity of compound 1 against MAO-A
erentially deaminates b-phenylethylamine and benzylamine, and MAO-B activities in mouse brain were investigated. l-
4,5)
and is selectively inhibited by l-deprenyl and pargyline.
Deprenyl-treated MAO preparation was used for the mea-
The inhibitors of MAO-A are expected to be clinically surement of MAO-A activity, and a clorgyline-treated one
useful to treat depression and anxiety, while those of MAO-B was for MAO-B. MAO-A activity in our preparations was
appear to be helpful in the prevention and adjunct treatment about 32% of the total MAO activity and MAO-B activity
6,7)
of Parkinson’s disease. A number of inhibitors of MAO ac- was about 61% (Table 1).
8
,9)
10—12)
13,14)
tivity such as quinolines, isoquinolines,
and xanthones
coumarins,
Compound 1 preferentially inhibited the MAO-B activity
than MAO-A activity in a concentration-dependent manner
15,16)
have been reported.
As a part of our ongoing research for MAO inhibitors with the IC50 values of 15.3 and 338.2 mM, respectively
from natural resources, we found that a MeOH extract from (Table 1). These data indicate that compound 1 is a potent in-
the fruits of Evodia rutaecarpa BENTHAM (Rutaceae) strongly hibitor for MAO-B activity.
inhibited the MAO activity in mouse brain. The MeOH ex-
According to the kinetic properties of MAO-B from
tract was, therefore, subjected to the bioactivity-guided frac- mouse brain, the values of K and V by using kynuramine
m
max
tionations to isolate the active compound. The finally purified were 92.5ꢀ0.5 mM and 4.28ꢀ0.04 nmol/min/mg protein, re-
bioactive substance, compound 1 (Fig. 1), was identified by spectively (nꢁ5) (Fig. 2). Kinetic study was performed on
comparison of its spectral data as 1-methyl-2-undecyl-4(1H)- MAO-B at different concentrations of the substrate in the ab-
quinolone and was shown to be a selective inhibitor on sence or presence of compound 1. Lineweaver–Burk recipro-
MAO-B.
cal plot analyses clearly demonstrated that the inhibition of
MAO-B activity with compound 1 was competitive with the
Results and Discussion
In the search for MAO inhibitors from the medicinal
K value of 9.91ꢀ0.2 mM (nꢁ4) (Fig. 2).
i
MAO-B from mouse brain mitochondrial fraction (0.1 mg
plants, we found that CH Cl fraction of the dried unripe protein) was incubated with 15 mM compound 1 or 40 mM
2
2
fruits of E. rutaecarpa showed the potent inhibitory effects
on MAO in mouse brain. A CH Cl fraction of E. rutaecarpa
2
2
at a concentration of 200 mg/ml (89.1% inhibition) showed a
potent inhibitory activity against MAO in mouse brain. Ac-
tivity-guided separation and purification of the extract yielded
a known quinolone alkaloid, 1-methyl-2-undecyl-4(1H)-
Fig. 1. Chemical Structure of 1-Methyl-2-undecyl-4(1H)-quinolone
quinolone (1) as an active compound (Fig. 1). The structure (Compound 1) from Evodia rutaecarpa
*
To whom correspondence should be addressed. e-mail: jsroh@cbucc.chungbuk.ac.kr
© 2003 Pharmaceutical Society of Japan

410
Vol. 51, No. 4
Table 1. Inhibitory Effects of Compound 1 on MAO-A and MAO-B in Mouse Brain
MAO activity
(nmol/min/mg protein)
Concentration
Types
IC50 value
(mM)
(mM)
(% of control)
Control
2.305ꢀ0.11
MAO-A (l-deprenyl-treated)
338.2
15.3
(Controlꢂl-deprenyl)
0.738ꢀ0.08 (100)
Compound 1
50
0.595ꢀ0.03 (80.7)*
0.510ꢀ0.04 (69.1)**
0.416ꢀ0.05 (56.4)**
0.366ꢀ0.01 (49.6)**
1
00
00
00
2
4
MAO-B (clorgyline-treated)
(Controlꢂclorgyline)
1.406ꢀ0.10 (100)
Compound 1
3
6
1.102ꢀ0.07 (78.4)*
0.938ꢀ0.06 (66.7)**
0.687ꢀ0.03 (48.9)***
0.522ꢀ0.06 (37.1)***
1
5
3
0
The activities of MAO-A and MAO-B in mouse brain were measured in the presence of 1 mM l-deprenyl or clorgyline, respectively. Data represent the meansꢀS.E.M. of three
independent experiments performed in triplicate. Significantly different from the control value: * pꢃ0.05; ** pꢃ0.01; *** pꢃ0.001 (Student’s t-test).
Table 2. Effects of Dialysis on the Inhibition of MAO-B by Compound 1
MAO-B activity
(nmol/min/mg protein)
(% of control)
Before dialysis
MAO-B (clorgyline-treated)
Compound 1
Amitriptyline
0.694ꢀ0.06 (100)
0.398ꢀ0.04 (57.4)**
0.383ꢀ0.05 (55.7)**
15 mM
40 mM
After dialysis
Dialysate (compound 1)
Dialysate (amitriptyline)
Amitriptyline
0.363ꢀ0.03 (52.2)**
0.642ꢀ0.03 (92.5)
0.381ꢀ0.02 (59.3)**
40 mM
MAO-B (clorgyline-treated) were incubated with 15 mM compound 1 or 40 mM
amitriptyline for 30 min at 37 °C, and then dialyzed against 10 mM potassium phosphate
buffer, pH 7.4 at 4 °C over night. MAO-B activity in the dialysate was measured using
Fig. 2. Inhibition of Type B Monoamine Oxidase (MAO-B) by Compound
1
00 mM kynuramine in the absence of and in the presence of compound 1 or amitripty-
1
Added in the Enzyme Reaction Mixture
line. Amitriptyline, a reversible MAO-B inhibitor, was used for positive control. Results
represent the meansꢀS.E.M. (nꢁ3). Significantly different from the control value:
** pꢃ0.01 (Student’s t-test).
The reciprocal of MAO-B activities was plotted against the reciprocal of substrate
concentrations (nꢁ5). Concentrations of compound 1: (1) 0 mM; (2) 6 mM; (3) 15 mM.
amitriptyline for 30 min at 37 °C, and then the reaction mix-
In conclusion, this study described herein suggests that
ture was dialyzed against 10 mM potassium phosphate buffer compound 1, a quinolone alkaloid, from the fructus of E. ru-
pH 7.4 at 4 °C overnight. MAO-B activity in the dialysate taecarpa selectively inhibits the mouse brain MAO-B activ-
was examined using 100 mM kynuramine. MAO-B activity by ity and that the inhibitory pattern of compound 1 is irre-
compound 1 was not recovered, whereas the activity by versible and competitive. These results also suggest that
amitriptyline, a reversible inhibitor, was recovered (Table compound 1 may contribute to the treatment of Parkinson’s
19,20)
2).
These results indicated that compound 1 is an irre- disease or Alzheimer’s disease. The in vivo pharmacological
versible inhibitor.
investigations of compound 1 need to be studied further.
Irreversible MAO inhibitors such as phenelzine and tranyl-
cypromine have proven efficacy, not only in depression, such
as geriatric depression, but also in anxiety or stress-related
conditions associated with panic or phobias and in appetite
Experimental
General Experimental Procedures Melting points were measured
without correction on Electrothermal model 9100. IR spectra were taken on
a JASCO FT/IR 300E spectrometer. UV spectra were obtained on a Milton
21—23)
1
13
disorders.
Indeed, an increase of MAO-B activity has Roy 3000 spectrometer. H-NMR (500 MHz) and C-NMR (125 MHz) spec-
as a
been observed in some neurological disorders such as tra were obtained on a Varian Unity NMR spectrometer using CDCl
3
solvent. The low- and high-resolution FAB-MS were measured on a JEOL
JMS-HX110/110A mass spectrometer. Elemental analysis was obtained on a
flash EA 1112series/CE instruments. Sephadex LH-20 (Pharmacia Fine
Chemical Industries Co.) and silica gel (70—230 mesh) (Merck) were used
Parkinson’s disease, Alzheimer’s disease and Huntington’s
6,7)
chorea.
Compounds that selectively inhibit the MAO-B isozyme
are used in the treatment of Parkinson’s disease. Selegiline, a for column chromatography and silica gel 60 F254 (Merck) for TLC. Fluores-
MAO-B inactivator having a selectivity for inhibition of cence was measured on a Perkin Elmer Model LS50B luminescence
24)
(U.S.A.).
MAO-B/MAO-A of 39, is used to treat Parkinson’s disease.
Kynuramine, dihydrobromide, amitriptyline, 4-hydroxyquinoline, clorgy-
line, deprenyle and zinc sulfate were purchased from Sigma Chemical Co.
In this study, compound 1 as an irreversible inhibitor of
MAO-B has a selectivity for inhibition of MAO-B/MAO-A
of 22, and so it might be effective in Parkinson’s disease.
(St. Louis, MO, U.S.A.). All other chemicals were used of reagent grade.
Materials and Methods The dried unripe fruits of E. rutaecarpa

April 2003
411
BENTHAM (Rutaceae) were purchased at an herbal drug store in Cheongju,
Korea in April 2000 and identified by Prof. K. S. Lee, a plant taxonomist at
Chungbuk National University. A voucher specimen (No. CBNU00125) was
deposited at College of Pharmacy, Chungbuk National University.
References
1) Nagatsu T., Yamamoto T., Harada M., Enzymologia, 39, 15—25
(1970).
2) Tipton K. F., Cell Biochem. Funct., 4, 79—87 (1986).
3) Tipton K. F., O’Carrol A. M., McCrodden J. M., J. Neural Transm.
(Suppl.), 23, 25—35 (1987).
4) Bach A. W., Lan N. C., Johnson D. L., Abell C. W., Bembenek M. E.,
Kwan S. W., Seeburg P. H., Shin J. C., Proc. Natl. Acad. Sci. U.S.A.,
85, 4934—4938 (1988).
5) Kalgutkar A. S., Castagnoli N. J., Testa B., Med. Res. Rev., 15, 325—
388 (1995).
6) Fowler C. J., Ross S. B., Med. Res. Rev., 4, 323—358 (1984).
7) Thomas T., Neurobiol. Aging, 21, 343—348 (2000).
8) Naoi M., Nagatsu T., Life Sci., 40, 1075—1082 (1987).
9) Mitsui N., Noro T., Kuroyanagi M., Miyase T., Umehara K., Ueno A.,
Chem. Pharm. Bull., 37, 363—366 (1989).
10) Thull U., Kneubuhler S., Gaillard P., Carrupt P. A., Testa B., Altomare
C., Carotti A., Jenner P., McNaught K. St. P., Biochem. Pharmacol.,
50, 869—877 (1995).
Extraction and Activity-Guided Isolation The dried fruits of E. rutae-
carpa (3.0 kg) were extracted three times with MeOH at room temperature.
The MeOH extracts were combined and concentrated in vacuo at 40 °C. The
resulting extract (220 g) was suspended in 1% HCl and partitioned with n-
hexane. The water layer was made alkaline with NaOH and partitioned with
CH Cl . The CH Cl layer was concentrated to afford the alkaloid fraction.
2
2
2
2
This concentrated alkaloid fraction (20 g) was subjected to a silica gel col-
umn chromatography using a CH Cl –MeOH step gradient as eluents to give
2
2
seven fractions (A1—A7). The each fractions were tested for MAO activity,
the higher inhibitory activities on MAO were found in the Fr. A2 (82.6% in-
hibition at 150 mg/ml). Compound 1 (yield 1.1 g) was obtained from Fr. A2,
and identified as 1-methyl-2-undecyl-4(1H)-quinolone. The purity of com-
25)
pound 1 on HPLC was measured 97.6% (area percent).
1
-Methyl-2-undecyl-4(1H)-quinolone (1): White powder; mp 68—70 °C.
1
H-NMR (CDCl ) d: 8.44 (1H, d, Jꢁ8.0 Hz), 7.65 (1H, t, Jꢁ7.5 Hz), 7.50
1H, d, Jꢁ8.5 Hz), 7.36 (1H, t, Jꢁ7.5 Hz), 6.23 (1H, s), 3.73 (3H, s), 2.70 11) Bembenek M. E., Abell C. W., Chrisey L. A., Rozwadowska M. D.,
3
(
(
2H, t, Jꢁ8.0 Hz), 1.67 (2H, m), 1.42 (2H, m), 1.27 (14H, br s), 0.88 (3H, t,
Gessner W., Brossi A., J. Med. Chem., 33, 147—152 (1990).
12) Ro J. S., Lee S. S., Lee K. S., Lee M. K., Life Sci., 70, 639—645
(2001).
13) Gnerre C., Catto M., Leonetti F., Weber P., Carrupt P. A., Altomare C.,
Carotti A., Testa B., J. Med. Chem., 43, 4747—4758 (2000).
14) Huong D. T., Choi H. C., Rho T. C., Lee H. S., Lee M. K., Kim Y. H.,
Arch. Pharm. Res., 22, 324—326 (1999).
13
Jꢁ6.5 Hz). C-NMR (CDCl ) d: 154.8, 111.2, 177.8, 126.6, 126.7, 123.3,
1
3
32.0, 115.3, 142.0, 34.1, 34.8, 28.6, 29.3, 29.5, 29.6, 31.9, 22.7, 14.1. IR
ꢄ1
(KBr) cm : 2960, 2920, 2855, 1640, 1605, 1575, 1306, 1180, 775, 760. UV
lmax (MeOH) nm (log e): 215 (4.2), 240 (4.3), 320 (4.1), 335 (4.1). HR-
FAB-MS m/z: 314.2484 [MꢂH] (Calcd for C H NO: 314.2484). Anal.
Calcd for C H NO: C, 80.46; H, 9.97; N, 4.47; O, 5.10. Found: C, 79.59;
ꢂ
2
1
32
21
31
H, 10.84; N, 4.49; O, 5.34. Spectral data and physical constants were identi- 15) Suzuki O., Katsumata Y., Oya M., Chari V. M., Vermes B., Wagner H.,
17,18)
fied with the previous reports.
Hostettmann K., Planta Med., 42, 17—21 (1981).
MAO Preparation and Assay for MAO Activity The crude MAO was 16) Ohishi N., Suzuki T., Ogasawara T., Yagi K., J. Mol. Catal. B-Enzym.,
26)
12)
prepared by Naoi’s method with minor modification and kept at ꢄ70 °C
10, 291—294 (2000).
until use. The MAO activity with kynuramine as a substrate was assayed by 17) Tang Y. Q., Feng X. Z., Huang L., Phytochem., 43, 719—722 (1996).
27)
a modification of the fluorometric method of Kraml. The fluorescence in-
tensity of 4-hydroxyquinoline formed from kynuramine by MAO was mea-
sured at 380 nm (emission) with excitation at 315 nm in a fluorophotometer.
The activities of MAO-A and MAO-B in mouse brain were measured in the
presence of 1 M l-deprenyl (type B inhibitor) or clorgyline (type A inhibitor)
18) Sugimoto T., Miyase T., Kuroyanagi M., Ueno A., Chem. Pharm.
Bull., 36, 4453—4461 (1988).
19) Giller E., Jatlow P., Bialos D., Harkness L., Docherty J., Psychiatry
Res., 2, 259—265 (1980).
20) Roth J. A., J. Neurochem., 27, 1107—1112 (1976).
with a pre-incubation time of 15 min, respectively. The test solutions were 21) Sheehan D. V., J. Clin. Psychiat., 45, 29—36 (1984).
dissolved in dimethylsulfoxide, which was confirmed to have no effect on 22) Walsh B. T., Gladis M., Roose S. P., Stewart J. W., Stetner F., Glass-
MAO activity below 2.8% concentration.
Statistical Methods The values of the Michaelis constant (K ) and the
maximum velocity (Vmax) were obtained by Lineweaver–Burk’s plot using
man A. H., Arch. Gen. Psychiat., 45, 471—475 (1988).
23) Liebowitz M. R., Schneier F. R., Hollander E., Welkowitz L. A., Saoud
J. B., Feerick J., Campeas R., Fallon B. A., Street L., Gitow A., J. Clin.
Psychiat., 52, 10—15 (1991).
24) Tetrud J. W., Langston J. W., Science, 245, 519—522 (1989).
25) Sugimoto T., Miyase T., Kuroyanagi M., Ueno A., Chem. Pharm.
Bull., 36, 4453—4461 (1988).
m
various concentrations of kynuramine. The amount of protein was deter-
28)
mined by the method of Lowry et al. using bovine serum albumin as a
standard. The results were expressed as the meansꢀS.E.M. of three indepen-
dent experiments performed in triplicate. The significance of the difference
between the control and the drug treated groups was analyzed by Student’s t- 26) Naoi M., Nagatsu T., J. Neurochem., 46, 655—657 (1986).
test. 27) Kraml M., Biochem. Pharmacol., 14, 1684—1686 (1965).
28) Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J., J. Biol.
Acknowledgements This study was supported by a grant of the Korean
Chem., 193, 265—275 (1951).
Health 21 R & D Project, Ministry of Health and Welfare, Republic of
Korea (HMP-00-B-21600-00123).