Inhibition of monoamine oxidase B by selective adenosine A2A receptor antagonists

Bioorganic & Medicinal Chemistry 11 (2003) 1299–1310

Inhibition of Monoamine Oxidase B by Selective Adenosine

A_2AReceptor Antagonists

Jacobus P. Petzer,^a,{Salome Steyn,^aKay P. Castagnoli,^aJiang-Fan Chen,^b

Michael A. Schwarzschild,^bCornelis J. Van der Schyf^a,y

and Neal Castagnoli^a,*

^aDepartment of Chemistry, Virginia Tech, Blacksburg, VA 24061-0212, USA

^bDepartment of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129, USA

Received 4 September 2002; accepted 25 November 2002

Abstract—Adenosine receptor antagonists that are selective for the A_2Areceptor subtype (A_2Aantagonists) are under investigation

as possible therapeutic agents for the symptomatic treatment of the motor deﬁcits associated with Parkinson’s disease (PD). Results

ofrecent studies in the MPTP mouse model ofPD suggest that A

antagonists may possess neuroprotective properties. Since

2A

monoamine oxidase B (MAO-B) inhibitors also enhance motor function and reduce MPTP neurotoxicity, we have examined the

MAO-B inhibiting properties ofseveral A antagonists and structurally related compounds in an eﬀort to determine ifinhibition

2A

ofMAO-B may contribute to the observed neuroprotection. The results ofthese studies have established that all ofthe ( E)-8-styryl-

xanthinyl derived A_2Aantagonists examined display signiﬁcant MAO-B inhibitory properties in vitro with K_ivalues in the low

mM to nM range. Included in this series is (E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KW-6002), a potent A_2A

antagonist and neuroprotective agent that is in clinical trials. The results ofthese studies suggest that MAO-B inhibition may

contribute to the neuroprotective potential ofA _2Areceptor antagonists such as KW-6002 and open the possibility ofdesigning dual

targeting drugs that may have enhanced therapeutic potential in the treatment ofPD.

Introduction

the degenerative processes associated with exposure to

the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-

pyridine [(MPTP, (1)]. The MPTP-induced losses of

nigrostriatal neurons⁴in the human produce a syn-

drome that is neurochemically, behaviorally and patho-

logically similar to that observed in patients diagnosed

with PD. The toxic eﬀects ofMPTP are reported to be

mediated by the pyridinium species MPP⁺(3),⁵a mito-

chondrial toxin.^6,7MPP⁺is formed via the MAO-B

catalyzed oxidation ofthe parent tetrahydropyridinyl

protoxin which generates the unstable dihydropyr-

idinium intermediate MPDP⁺(2). A second 2-electron

oxidation yields MPP⁺(Fig. 1).^8,9

Idiopathic Parkinson’s disease (PD) is a neurodegen-

erative disorder characterized pathologically by a

marked loss ofdopaminergic nigrostriatal neurons and

clinically by disabling movement disorders. At present

the mainstay for the treatment of PD relies on dopa-

mine replacement therapy with the dopamine precursor

l-DOPA.¹Although this approach provides consider-

able symptomatic reliefin the early stages ofthis dis-

ease, advanced PD may be associated with poor quality

2,3

oflife and early death.

Drugs that target the mechan-

ism ofneuronal cell death and therefore delay or even halt

the progression ofthis disease may oﬀer improved ther-

apeutic approaches for the treatment of PD.

Early studies established that (R)-deprenyl (4), an irre-

versible MAO-B inhibitor and clinically useful anti-

parkinsonian agent, is neuroprotective in MPTP-treated

Attempts to develop neuroprotective agents have

focused on identifying compounds that protect against

animals.¹⁰Other inactivators and competitive inhibitors

11ꢀ14

ofMAO-B exhibit similar eﬀects.

Although this

neuroprotection may be linked to the blockade ofthe

metabolic bioactivation ofMPTP, the neuroprotective

properties of( R)-deprenyl in MPTP animal models,^10,14

*Corresponding author. Tel.: +1-540-231-8200; fax: +1-540-231-

8890; e-mail: ncastagn@vt.edu

^yCurrent address: Department ofPharmaceutical Sciences, Texas Tech

University Health Sciences Center School ofPharmacy, 1300 S. Coul-

ter Ave. Amarillo, TX 79106, USA.

also appear to involve unknown pathways that are

formation.^15ꢀ17

+

^{This research constitutes a part ofJ.P.P.’s PhD dissertation presented

to Potchefstroom University, South Africa.

independent ofthe inhibition ofMPP

doi:10.1016/S0968-0896(02)00648-X

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J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

Antagonists ofadenosine receptors (A

antagonists)

ryl moiety modiﬁed on the phenyl ring. We have exam-

ined the MAO-B inhibiting properties of16 known ( E)-

8-styrylxanthinyl A_2Aantagonists (5a–5h and 11a–11h)

including (E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-

xanthine [KW-6002 (5a)], a compound which is under-

going clinical trials as a novel, non-dopaminergic

agent for the treatment of PD.^19,20Furthermore, KW-6002

is reported to protect against the dopaminergic neuro-

toxicity ofMPTP in mice and of6-hydroxydopamine in

rats.^21,22We also have examined the photochemical

isomerization ofsome members ofthis series in order to

determine ifthe resulting cis isomers (speciﬁcally the

cis isomer 6a ofKW-6002) might mediate MAO-B

inhibition. As part ofa limited SAR analysis, we have

determined the MAO-B inhibiting properties ofthe two

xanthinyl analogues 7a and 7b in which the styryl double

bond has been reduced, and three (E)-2-styrylbenzimid-

azolyl analogues (8a–8c) (Fig. 2).

2A

are reported to ameliorate motor deﬁcits in MPTP-

treated animals^18ꢀ21and therefore may provide symp-

tomatic reliefin patients diagnosed with PD. Some A

2A

antagonists also display neuroprotective properties in

animal models ofPD. We previously demonstrated that

the nonselective A₁/A_2Aantagonist caﬀeine protects

against the MPTP induced nigrostriatal neurotoxicity in

22

the mouse model ofPD. Results ofmore recent stud-

ies have documented that the potent and selective A_2A

antagonist (E)-8-(3-chlorostyryl)caﬀeine [CSC (5b)]^23,24

also is neuroprotective in the MPTP mouse model.²⁵

This protection, however, may be dependent in part on

the inhibition ofthe MAO-B catalyzed bioactivation of

MPTP since CSC was also found to be a potent and

selective competitive MAO-B inhibitor with a K_ivalue of

100 nM in mouse brain mitochondrial preparations.²⁵

The combination ofthe neuroprotective and MAO-B

inhibiting properties ofCSC has prompted us to examine

the MAO-B inhibiting properties ofother A _2Aantagonists

and related compounds to determine ifsuch dual actions

are a common efature ofthese types ofheterocyclic

systems.

Results and Discussion

Chemistry

The majority ofthe reported A antagonists are 1,3-

disubstituted xanthinyl analogues bearing an (E)-8-sty-

The majority ofthe compounds examined here have

been reported previously as cited in the Experimental

2A

Figure 1. The MAO-B dependent bioactivation pathway for MPTP (1) via MPDP⁺(2) to MPP⁺(3) and the structure ofthe MAO-B inactivator

(R)-deprenyl (4).

Figure 2. Structures ofcompounds discussed in the text.

J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

1301

Figure 3. Synthetic pathway to (E)-8-styrylxanthinyl analogues 11a–11h and 5a–5h. Key: (i) EDAC, dioxane–H₂O; (ii) NaOH (aq), reﬂux; (iii)

CH₃I, K₂CO₃, DMF.

Procedures. The (E)-8-styrylxanthinyl analogues (Fig. 3)

were prepared by acylation of1,3-dimethyl- ( 9a) or 1,3-

diethyl- (9b) 5,6-diaminouracil²⁶with commercially

available trans-cinnamic acid (10a) or 3-chloro- (10b), 3-

ﬂuoro- (10c), 3-nitro- (10d), 3,4-dimethoxy- (10e) or 3,4-

methylenedioxy- (10f) trans-cinnamic acid in the pre-

sence ofa carbodiimide reagent (EDAC). The resulting

crude amides (most likely as mixtures of5- and 6- acy-

lated derivatives) underwent cyclization when heated

with base to give the corresponding 1,3-disubstituted

(E)-8-styrylxanthinyl derivatives 11a–11h.^23,27Treat-

ment of 11a–11h with iodomethane led to the desired 7-

methylated analogues 5a–5h. The structures and purity

ofthe compounds reported previously were veriﬁed by

In an eﬀort to determine the relative importance ofthe

xanthinyl moiety in the MAO-B inhibition properties of

(E)-8-styrylxanthinyl analogues, we synthesized a series

of( E)-2-styrylbenzimidazolyl derivatives (8a–8c). The

key intermediate, 2-methyl-1H-benzimidazole (13), was

prepared in high yield according to Phillips³¹by con-

densing o-phenylenediamine (12) with acetic acid. For

the preparation ofthe ( E)-2-styrylbenzimadazolyl ana-

logues we used the protocol as described by Dubey et

al.³²2-Methylbenzimidazole was treated with commer-

cially available benzaldehyde (14a) or 3-chloro- (14b) or

3-ﬂuoro- (14c) benzaldehyde at high temperatures to

give the corresponding substituted (E)-2-styryl-1H-ben-

zimidazoles 15a–15c which could be isolated from the

reaction mixtures as their oxalate salts. Treatment of

15a-15c with an equivalent ofiodomethane resulted in

the desired (E)-1-methyl-2-styrylbenzimidazoles 8a–8c.

In the presence oftwo equivalents ofiodomethane the

formation of the (E)-1,3-dimethylbenzimidazolium spe-

cies 16 as its iodide salt was observed (Fig. 4). The

structures were veriﬁed by comparing melting points,

1

comparing melting points, mass spectrometric and H

NMR properties with the corresponding literature

values. The trans geometry ofthe styryl derivatives was

conﬁrmed by proton-proton coupling constants (ꢁ15 Hz)

ofthe oleﬁnic proton signals. The same protocol was

followed for the synthesis of the previously unknown

8-(2-phenylethyl)xanthinyl derivatives 7a and 7b. In this

case, 1,3-dimethyl-5,6-diaminouracil (9a) was treated

with 3-phenylpropanoic acid rather than cinnamic acid.

1

mass spectrometric and H NMR properties with the

corresponding literature values where applicable. The

trans geometry ofthe styryl moiety was conﬁrmed by

proton-proton coupling constants (Jꢁ15 Hz) ofthe

oleﬁnic proton signals.

The cis isomer ofKW-6002 [( Z)-KW-6002 (6a)] was

prepared by exposing dilute solutions ofKW-6002 to

light followed by sequential recrystallizations to sepa-

rate the cis from the trans isomer (see Experimental

Procedures). An oleﬁnic proton-proton coupling con-

stant of12.4 Hz was consistent with the cis conﬁgur-

ation. This coupling constant corresponded with values

reported for related (Z)-8-styrylxanthinyl deriva-

tives.^28,29These studies conﬁrm speculation in the lit-

erature that the cis isomer is the principal

photochemical product formed when solutions of KW-

6002 are irradiated with light.³⁰

Photochemistry

We have examined the photochemical isomerization of

certain members ofthe ( E)-8-styrylxanthinyl series in

order to determine ifthe resulting cis isomers (speciﬁ-

cally the cis isomer 6a ofKW-6002) might mediate

MAO-B inhibition (Fig. 5). In all experiments ambient

laboratory light was used. Based on earlier reports

that certain (E)-8-styrylxanthines^28,29isomerize to the

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J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

corresponding cis isomers when exposed to light, it was

suggested that KW-6002 may undergo a similar photo-

chemical transformation.³⁰As determined by HPLC

analysis (see Experimental), when dilute solutions of

KW-6002 were exposed to light the concentration ofthe

trans isomer decreased as a function of exposure time.

Loss ofthe trans isomer was accompanied by the

appearance ofa second peak with a slightly shorter

retention time. In the case ofKW-6002, the compound

representing this second peak was identiﬁed as the cis

isomer 6a since it displayed identical absorption spectral

features and retention time as those observed with the

fully characterized sample of 6a described above. In

accordance with the literature,²⁸dilute solutions of 5a

isomerized more rapidly than did more concentrated

solutions. A 5 mM solution ofKW-6002 reached the

photostationary state (8.3% trans and 91.7% cis) after

approximately 150 min oflight exposure while a 50 mM

solution reached the same equilibrium composition only

after 360 min. A 500-mM solution ofKW-6002 did not

reach equilibrium even after 900 min of light exposure.

This isomerization reaction was stoichiometric — the

disappearance ofthe trans isomer was balanced by the

appearance ofthe cis isomer (Fig. 6). The eﬀect oflight

Figure 4. Synthetic pathway to substituted (E)-2-styrylbenzimidazolyl analogues 8a–8c and 16: (i) 4 N HCl, reﬂux 1 h; (ii) 180 ^ꢂC, 24 h; (iii) CH₃I

(1 equiv), K₂CO₃, DMF, rt; (iv) CH₃I (2 equiv), K₂CO₃, DMF, 100 ^ꢂC.

Figure 5. Photo-induced isomerization of( E)-8-styryl-7-methylxanthinyl derivatives.

J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

1303

on the cis isomer ofKW-6002 was also investigated.

When 6a (5 mM) was exposed to light the photosta-

tionary state (10.1% trans and 89.9% cis) was reached

after approximately 150 min.

cis isomer was also observed. In contrast, after shielding

these incubations by covering the incubation tubes with

aluminum foil, the amounts of 5a remaining in the

incubation mixtures were >99% ofthe initial concen-

tration. We conclude that, when appropriate measures

are taken to shield incubations from light, the photo-

chemical isomerization ofthe trans to the cis alkene is

negligible and therefore the MAO-B properties of KW-

6002 and other (E)-8-styrylxanthinyl derivatives are

independent ofthe formation ofthe cis isomers.

We also observed rapid photochemical isomerizations

with compounds 5d and 5g. Analogue 5d (4.5 mM) iso-

merized to the corresponding cis isomer with the pho-

tostationary state (12.7% trans) being achieved in

approximately 150 min. Similarly 5g (5 mM solution)

reached the photostationary state (13.8% trans) after

about 100 min. The rates of cis to trans isomerization of

(E)-8-styryl-7-methylxanthines bearing electron with-

drawing groups on the phenyl ring (5b, 5c, 5f and 5h)

were much slower than the other analogues (5a, 5d, 5e,

5g). For example, the photostationary state ofa 5 mM

solution of 5b had not been achieved after 4 h of

continuous light exposure.

Enzymology

The K_ivalues for the competitive inhibition of MAO-B

were determined in most instances by measuring the

extent by which various concentrations ofthe test

compounds slowed the rate ofthe a-carbon oxidation of

1-methyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine

[MMTP (17)] to the corresponding dihydropyridinium

metabolite, the 1-methyl-4-(1-methylpyrrol-2-yl)-2,3-

dihydropyridinium species MMDP⁺(18) (Fig. 7).³³

MMDP⁺was monitored spectrophotometrically at 420

nm, a wavelength that was distant from the chromo-

(E)-8-Styryl-7-methylxanthines bearing electron with-

drawing groups on the phenyl ring (5b, 5c, 5f and 5h)

have l_maxvalues of354 nm and molar extinction coe-f

ﬁcients ranging from 28,000 to 32,000 whereas the other

analogues have l_maxvalues near 362 nm and molar

extinction coeﬃcients greater than 41,000. This greater

eﬃciency oflight absorption ofthese analogues may

account for their more rapid rates of photo-induced

isomerization.

phores ofboth the substrate and the A

investigated in this study. The chemical stability of 18

antagonists

2A

obviated the need to monitor for the formation of the

1-methyl-4-(1-methylpyrrol-2-yl)pyridinium

species

MMP⁺(19).³⁴

Since all our in vitro MAO-B assays required that the

test compounds be in solution, we were concerned that

those compounds containing oleﬁnic bonds may iso-

merize under the assay conditions and in this way com-

promise the outcome ofthe experiments. We found that

signiﬁcant amounts ofthe trans isomer (5a) ofKW-6002

were converted to the cis isomer (6a) during a 45-min

incubation period. When appropriate measures were

taken to protect these samples from light, the amounts

of 6a detected in the samples signiﬁcantly decreased.

For example when 5, 10 and 20 mM solutions of 5a were

incubated for 45 min without shielding from light,

80.4ꢃ5.4, 87.6ꢃ1.7 and 90.7ꢃ0.6% ofthe initial con-

centration of 5a, respectively, were detected in the

samples. In each ofthese solutions the presence ofthe

In certain cases, the limited solubility ofthe antagonists

in the incubation mixture impacted negatively on our

ability to determine the K_ivalues accurately using this

spectrophotometric assay. For these compounds, con-

centrations that bracketed the K_ivalue and that were

within the solubility range did not result in enough

inhibition ofMAO-B to enable accurate measurements.

In these cases a reverse-phase HPLC assay was used to

measure the time dependent formation of MMDP⁺.

This approach allowed us to estimate K_ivalues for

inhibitors for which the limits of solubility and the K_i

values are spaced closely together. We also conﬁrmed

that MMDP⁺does not undergo any detectable oxida-

tion to the MMP⁺(19) in our in vitro assay because LC

tracings ofincubation mixtures failed to yield peaks that

corresponded in retention time to synthetic MMP⁺.³⁴

The MAO-B inhibitory properties ofKW-6002 ( 5a) and

CSC (5b) also were investigated using MPTP (1) instead

ofMMTP as enzyme substrate since MPTP has been

studied extensively as the proneurotoxin that undergoes

bioactivation by MAO-B. Since MPTP is the biologi-

cally more relevant substrate, it was included in this

study to compare with data generated with MMTP, our

substrate ofchoice ofr screening ofMAO activity. To

make quantitative estimations ofthe oxidation of

MPTP by MAO-B, the concentrations ofboth its

a-carbon oxidation product MPDP⁺(2) and the sub-

sequent 2-electron oxidation product MPP⁺(3) were

determined by reversed phase HPLC analysis as described

in the Experimental Procedures.

Figure 6. A graphical representation ofthe isomerization ofKW-6002

(circles) to its corresponding cis isomer (triangles) when a 5-mM solu-

tion was exposed to light for various periods of time.

For screening ofMAO-B inhibitory activity we used

baboon liver mitochondria as enzyme source because it

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J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

Figure 7. The MAO catalyzed oxidation ofMMTP ( 17) to the corresponding dihydropyridinium species (18). The further oxidation of 18 to the

pyridinium species 19 is not observed.

exhibits high MAO-B activity while MAO-A activity is

negligible.³⁴For select compounds C57BL/6 mouse

brain mitochondria also served as a source for MAO-B

since the C57BL/6 mouse is a well established model of

MPTP-induced parkinsonism.^35,36Finally, in some

experiments human liver mitochondria also served as

MAO-B source to determine ifthe baboon and human

derived inhibition values are comparable.

selective displacement ofthe pr

efrred A

ligand

2A

³H-CGS 21680 from the A_2Areceptor for CSC is 54

nM.²³Consequently, the aﬃnity ofCSC for the enzyme

and the receptor are comparable. Two other com-

pounds (5f and 5h) also proved to be potent inhibitors

ofMAO-B with K_ivalues in the nM range. Like CSC,

these compounds are (E)-8-styrylcaﬀeinyl analogues

bearing an electronegative group at the C-3 position of

the styryl ring.

(E)-8-Styrylxanthinyl derivatives

Structural modiﬁcations ofCSC led to diminished

MAO-B inhibitory activity. For example, replacement

ofthe 1,3-dimethyl groups ofthe xanthinyl moiety with

ethyl groups impacted negatively on the potency of

MAO-B inhibition (compare 5b vs 5c). Within this ser-

ies, the 7-N-methylxanthinyl analogues (Table 2) were

more potent inhibitors than the corresponding 7H-xan-

thinyl (Table 1) analogues (for example 5a vs 11a, 5b vs

11b and 5e vs 11e). As documented in Table 3, satura-

tion ofthe styryl double bond also led to reduced

activity (7a vs 11e and 7b vs 5e). The styryl moiety was

essential for inhibition since caﬀeine proved to be a very

weak inhibitor. Also, the trans geometry is required

The K_ivalues for the inhibition of MAO-B by the (E)-8-

styrylxanthinyl derivatives are presented in Tables 1 and

2. As illustrated by example with KW-6002 (Fig. 8), the

plots we obtained in all cases were classical for compe-

titive inhibition. While caﬀeine was found to be a very

weak inhibitor ofMAO-B, all ofthe other 8-substituted

xanthinyl analogues were moderate to potent competi-

tive inhibitors ofMAO-B. CSC ( 5b) was conﬁrmed to

be an exceptionally potent inhibitor with K_ivalues of70

and 235 nM in baboon and human liver mitochondria,

respectively. The reported K_ivalue in mouse brain

mitochondria is 100 nM.²⁵The reported K_ivalue for the

Table 1. The K_ivalues for the inhibition of MAO-B by various (E)-8-

styryl-7H-xanthinyl derivatives (the reported K_ivalues for antagonism

ofthe A _2Areceptor are also listed)

Table 2. The K_ivalues for the inhibition of MAO-B by various (E)-8-

styryl-7-methylxanthinyl derivatives (the reported K_ivalues for antag-

onism ofthe A _2Areceptor are also listed)

K_ivalue

Compd R¹/R³

X or X₂

MAO-B (mM)

A_2A(nM)

Compd R¹/R³

X or X₂

MAO-B (mM)

A_2A(nM)

2.2³⁰

11a

11b

11c

11d

11e

11f

11g

11h

Ethyl

Methyl

Ethyl

Methyl

3,4-Dimethoxy

3-Chloro

3,4-Dimethoxy

H

63^a

1.5^a; 8.0^b

23³⁷

Not reported

5a

5b

5c

5d

5e

5f

Ethyl

Methyl

Ethyl

Methyl 3,4-Dimethoxy

Methyl

3,4-Dimethoxy

3-Chloro

28^a; 21^b; 17^b,c

0.07^a; 0.1^b,c; 0.235^d54²³; 36²⁴

Not determined^cNot reported

3^a; 30^d

2.7^a; 11

3^a; 3

Not reported

18²⁴; 197²³

94²³

6^a

31^a

1100²³

291²³

516²³

15²³

H

3-Fluoro

3-ﬂuoro

1.9^a

0.4^a

83²³

Ethyl 3,4-Methylenedioxy

Methy 3-Nitro

2.5^a

5g

5h

Ethyl 3,4-Methylenedioxy

Methyl 3-Nitro

8^a

6.1³⁷

1.7^a; 9^b

438²³

0.16^a

195²³

^aBaboon liver mitochondria.

^bHuman liver mitochondria.

^cDue to limited solubility in the incubation mixture the K_i-value could

not be obtained for this compound.

^aBaboon liver mitochondria.

^bC57BL/6 mouse brain mitochondria.

^cMPTP was used as substrate instead ofMMTP.

^dHuman liver mitochondria.

J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

1305

Figure 8. Lineweaver–Burke plots ofthe rates ofthe mouse brain MAO-B catalyzed oxidation ofMPTP carried out in the absence (open circles)

and presence ofvarious concentrations ofKW-6002 (ﬁlled circles, 5 mM; open triangles, 10 mM; ﬁlled triangles, 20 mM). The secondary plot ofthe

slopes ofthe Lineweaver–Burke plots versus the concentrations ofKW-6002 (inset) gave a K_ivalue of17 mM.

because the cis isomer ofKW-6002 was inactive as an

MAO-B inhibitor.

bition ofMAO as a possible contributor to the neuro-

protective mechanism since the neuroprotective

properties ofthe potent MAO-B mechanism-based

inactivator (R)-deprenyl in the MPTP animal model

also appear to involve pathways that are independent of

the inhibition ofMPP ⁺formation.^15,16

KW-6002 (5a) was found to be a moderate MAO-B

inhibitor with K_ivalues of21 and 28 mM, respectively,

in mouse brain and baboon liver mitochondria. A com-

parable K_ivalue (17 mM) was obtained when MPTP

instead ofMMTP served as substrate of r mouse brain

(E)-2-Styrylbenzimidazolyl derivatives

30

MAO-B (Fig. 8). With a reported K_ivalue of2.2 nM

3

for the selective displacement of H-CGS 21680 from

In an eﬀort to determine the relative importance ofthe

xanthinyl moiety on the MAO-B inhibition properties

of( E)-8-styrylxanthinyl analogues, we synthesized a

series of( E)-2-styrylbenzimidazolyl derivatives (8a–8c).

Since the xanthinyl and benzimidazolyl moieties are both

planar structures, the latter could provide a useful system

to investigate the role ofthe xanthinyl moiety versus the

requirement for planarity in MAO-B inhibition. The

literature supports the idea that planarity is important

in MAO-B inhibition since a wide variety ofplanar,

heterocyclic systems have been found to be competitive

the A_2Areceptor, this compound binds much more

tightly to the A_2Areceptor compared to its binding to

the active site ofMAO-B. The extent to which MAO-B

inhibition may contribute to the neuroprotective prop-

erties ofKW-6002 is not clear at this time. Observations

by Ikeda et al.²¹argue against a role for MAO-B inhi-

bition since neuroprotective doses ofKW-6002 do not

alter MPP⁺levels in the striatum when measured 1–6 h

after MPTP administration. This does not exclude inhi-

38ꢀ40

inhibitors ofMAO-B.

Table 3. The K_ivalues for the inhibition of MAO-B by 8-(2-phenyl-

ethyl)xanthines 7a and 7b

The K_ivalues for the inhibition of MAO-B by the (E)-2-

styrylbenzimidazolyl derivatives are presented in Table 4.

All derivatives exhibited MAO-B inhibitory activity and

the Lineweaver–Burke plots obtained were classical for

competitive inhibition.⁴¹We observed that the (E)-1-

methyl-2-styrylbenzimidazolyl analogues (8a–8c) con-

sistently were more potent MAO-B inhibitors than their

corresponding 1H analogues (15a–15c). Ofall the

compounds tested, (E)-1-methyl-2-(3-chlorostyryl)-

benzimidazole (8b) proved to be the most potent

inhibitor with a K_ivalue of1.4 mM. The inhibitor

potency ofthis compound is 20 times less than what was

observed for the corresponding xanthinyl analogue CSC

Compd

R⁷

K_ivalue (mM)

7a

7b

H

Methyl

220^a; 183^b

30^a; 36^b

^aHuman liver mitochondria.

^bBaboon liver mitochondria.

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J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

(5b). Also the K_ivalue for the inhibition of MAO-B by

8c (2.6 mM) was signiﬁcantly lower than the corres-

ponding value for the (E)-8-(3-ﬂuorostyryl)xanthinyl

analogue 5f (0.4 mM). We conclude that, although the

planar character ofthe benzimidazolyl ring system

contributes signiﬁcantly to the competitive inhibition

ofMAO-B, the xanthinyl moiety is a better system

for the development of exceptionally potent MAO-B

inhibitors.

Experimental

Caution: Procedures for the safe handling of neurotoxic

compounds such as MPTP were followed as described

previously.⁴³

Chemistry

Materials and instrumentation. Chemicals and reagents

not described elsewhere were purchased from Aldrich

Chemical Co._ꢀ(Milwaukee, WI, USA). MPTP HCl (1),⁴⁴

MPDP⁺ClO₄(2),⁹MPP⁺I^ꢀ(3),⁴⁵MMTP HCl (17),³³

and 1-methyl-4-(1-methylpyrrol-2-yl)pyridinium iodide,

MMP⁺I^ꢀ(19)³³were synthesized according to litera-

ture procedures. Caﬀeine was purchased from Sigma

(St. Louis, MO, USA). Melting points (mp) were

obtained on a Thomas-Hoover melting point apparatus

and are uncorrected. NMR spectra were recorded at

360 MHz on a Bruker AM 360 (Aspect 3000) instru-

The 3-Cl (5b and 8b) and 3-ﬂuoro (5f and 8c) analogues

ofthe ( E)-1-methyl-2-styrylbenzimidazolyl series and

(E)-8-styryl-7-methylxanthinyl series, respectively, were

considerably more potent MAO-B inhibitors than were

the corresponding 3-H analogues 5e and 8a. This

observation reinforces the importance of an electron

withdrawing substituent on the styryl moiety for MAO-B

inhibition and further suggests that the (E)-8-styryl-

xanthinyl and (E)-2-styrylbenzimidazolyl derivatives

may interact in a similar manner with MAO-B.

The positively charged (E)-1,3-dimethyl-2-styryl-

benzimidazolium species 16 was found to be a weak

inhibitor (K_ivalue of85 mM with baboon liver mito-

chondria). This is in accordance with the reported

1

ment. Chemical shifts for H are expressed in parts per

million (d) relative to the signal from tetramethylsilane

added to an appropriate deuterated solvent (CDCl₃,

CD₃CN or DMSO-d₆). Direct insertion probe electron

ionization mass spectra (EIMS) were obtained on a VG

Quattro I mass spectrometer. HPLC analyses were per-

formed on a Hewlett Packard 1100HPLC system

equipped with a UV–Vis diode array (DA) detector, a

Rheodyne 7725I injector, a Zorbax SB-C8 analytical

column (4.6ꢄ250 mm, 5 mm) and an in-line pre-column

ﬁlter (2 mm; Upchurch Scientiﬁc Inc., Oak Harbor, WA,

USA). Milli-Q water was obtained from a Waters Milli-

Q UV Plus system (Millipore, Bedford, MA, USA).

Microanalyses were performed by Atlantic Microlab,

Inc., Norcross, GA, USA.

42

hydrophobic nature ofthe active site.

Conclusions

All ofthe ( E)-8-styrylxanthinyl derived A_2Aantagonists

examined in this study exhibited signiﬁcant inhibiting

activity towards MAO-B. This behavior suggests the

possibility ofdeveloping compounds that may provide

enhanced antiparkinsonian activity since both A_2A

antagonism and MAO-B inhibition may be linked to

neuroprotective mechanisms as well as symptomatic

enhancement ofmotor ufnction. ( E)-8-styrylcaﬀeine

derived structures with electron withdrawing groups on

C-3 ofthe styryl ring are worthy ofufrther investiga-

tions since they are particularly potent inhibitors of

MAO-B and antagonists ofthe A _2Areceptor.

Preparation of 1,3-dialkyl-5,6-diaminouracil (9a,b)

The syntheses of1,3-dimethyl-5,6-diaminouracil ( 9a)

and its corresponding hydrochloric acid salt were

achieved as described previously.²⁶The melting points

of the free base and hydrochloride salt were found to be

214 ^ꢂC (lit. mp 209 ^ꢂC) and >260 ^ꢂC (lit. mp >260 ^ꢂC),

respectively.²⁶Using the same procedure, 1,3-diethyl-

5,6-diaminouracil (9b) was prepared from 1,3-diethyl-

uracil. The hydrochloride salt was recrystallized from

ethanol to yield white crystals: mp >260 C; H NMR

(DMSO-d₆) d 9.47 (s, 3H), 7.78 (s, 2H), 3.92 (q, 2H,

J=6.8 Hz), 3.80 (q, 2H, J=6.8 Hz), 1.12 (t, 3H, J=7.2

Hz), 1.06 (t, 3H, J=7.2 Hz). Anal. calcd for

Table 4. The K_ivalues for the inhibition of MAO-B by (E)-2-styryl-

benzimidazolyl derivatives

ꢂ

1

.

C₈H₁₄N₄O₂HCl: C, 40.93; H, 6.44; N, 23.87. Found: C,

41.23; H, 6.58; N, 23.76.

Preparation of (E)-8-styrylxanthines (11a–11h). The

preparation of 11a–11g was accomplished using the

procedure described by Suzuki et al.²⁷or Jacobson et

al.²³The melting points ofcompounds reported pre-

viously were as follows: 11d mp >270 ^ꢂC (from DMF),

lit. mp >295 ^ꢂC;²³11e mp >270 ^ꢂC (from DMF), lit.

mp >280 ^ꢂC;²³11f mp >260 ^ꢂC (from DMF), lit. mp

>310 ^ꢂC.;²³11h mp >300 ^ꢂC (from DMF), lit. mp

>300 ^ꢂC.²³The characterizations of 11a, 11c and 11g,

which have been reported only in the patent literature,³⁷

and of 11b (previously unreported), are summarized below.

K_ivalues^a(mM)

Compd

R¹

X

MAO-B

15a

15b

15c

8a

8b

8c

H

Methyl

H

53

Chloro

Fluoro

H

Chloro

Fluoro

3.5

5.3

17

1.4

2.6

^aThe MAO-B source in all instances was baboon liver mitochondria

and the enzyme substrate MMTP.

J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

1307

(E)-1,3-Diethyl-8-(3,4-dimethoxystyryl)xanthine (11a).

This was obtained from 9b in 35% yield: mp 267 ^ꢂC

(from DMF), lit. mp 268.8–269.1;^{37 1}H NMR (DMSO-

d₆) d 13.45 (s, 1H, brs), 7.59 (d, 1H, J=16.2 Hz), 7.26

(d, 1H, J=1.4 Hz), 7.13 (dd, 1H, J=1.4, 8.3 Hz), 7.00

(1H, d, J=8.3 Hz), 6.95 (d, 1H J=16.2 Hz), 4.06 (q, 2H,

J=6.8 Hz), 3.94 (q, 2H, J=6.8 Hz), 3.83 (s, 3H), 3.79 (s,

3H), 1.26 (t, 3H, J=7.2 Hz), 1.14 (t, 3H, J=7.2 Hz); EIMS

m/z 370 (M^.+). Anal. calcd for C₁₉H₂₂N₄O₄: C, 61.61;

H, 5.99; N, 15.13. Found: C, 61.72; H, 6.04; N, 15.16.

(d, 1H, J=15.8 Hz), 4.17 (q, 2H, J=6.8 Hz), 4.05 (q,

2H, J=6.8 Hz), 4.04 (s, 3H), 1.35 (t, 3H, J=6.8 Hz),

1.23 (t, 3H, J=6.8 Hz); EIMS, m/z 358 (M^.+). Anal.

calcd for C₁₈H₁₉N₄O₂Cl: C, 60.25; H, 5.34; N, 15.61.

Found: C, 59.96; H, 5.36; N, 15.41. Anal. calcd for

.

C₁₈H₁₉N₄O₂Cl H₂O: C, 57.37; H, 5.62; N, 14.87.

Found: C, 57.40; H, 5.56; N, 14.82.

(E)-1,3-Diethyl-8-(3,4-methylenedioxystyryl)-7-methyl-

xanthine (5g). was obtained from 11g in 86% yield: mp

219–220 ^ꢂC (from toluene/n-hexane 3:1), lit. mp 219.4–

219.6 ^ꢂC;^{37 1}H NMR (CDCl₃) d 7.68 (d, 1H, J=15.9

Hz), 7.07 (d, 1H J=1.8 Hz), 7.03 (dd, 1H J=1.6, 8.1

Hz), 6.81 (d, 1H, J=7.9 Hz), 6.70 (d, 1H, J=15.7 Hz),

5.99 (s, 2H), 4.18 (q, 2H, J=7.2 Hz), 4.06 (q, 2H, J=7.2

Hz), 4.02 (s, 3H), 1.36 (t, 3H, J=7.2 Hz), 1.24 (t, 3H,

J=7.2 Hz); EIMS m/z 368 (M^.+). Anal. calcd for

C₁₉H₂₀N₄O₄: C, 61.95; H, 5.47; N, 15.21. Found: C,

62.00; H, 5.49; N, 15.22.

(E)-1,3-Dimethyl-8-(3-chlorostyryl)xanthine (11b). This

was obtained in 74.6% yield from 9a: mp >300 ^ꢂC

(from DMF); ¹H NMR (DMSO-d₆) d 7.69 (s, 1H),

7.63–7.55 (m, 2H), 7.47–7.38 (m, 2H), 7.08 (d, 1H, J=6.5

Hz), 4.11 (s, 1H,), 3.47 (s, 3H), 3.25 (s, 3H); EIMS m/z

316 (M^.+). Anal. calcd for C₁₅H₁₃N₄O₂Cl: C, 56.88; H,

4.14; N, 17.69. Found: C, 56.96; H, 4.19; N, 17.69.

(E)-1,3-Diethyl-8-(3-chlorostyryl)xanthine (11c). This

was obtained in 27% yield: mp >270 ^ꢂC (from DMF),

lit. mp >280 ^ꢂC;^{37 1}H NMR (DMSO-d₆) d 13.51 (s, 1H,

brs), 7.72 (s, 1H), 7.64–7.59 (m, 2H), 7.45–7.42 (m, 2H),

7.12 (d, 1H, J=16.4 Hz), 4.07 (q, 2H, J=6.8 Hz), 3.94

(q, 2H, J=6.8 Hz), 1.26 (t, 3H, J=6.8 Hz), 1.14 (t, 3H,

J=6.8 Hz); EIMS m/z 344 (M^.+). Anal. calcd for

C₁₇H₁₇N₄O₂Cl: C, 59.22; H, 4.97; N, 16.25. Found: C,

59.35; H, 4.98; N, 16.38.

1,3-Dimethyl-8-(2-phenylethyl)xanthine (7a). To a solu-

tion of 9a (7 mmol) and 1-ethyl-2-[3-(dimethylamino)-

propyl]carbodiimide hydrochloride (EDAC, 10.3 mmol)

in 80 mL dioxane/H₂O (1:1) was added 3-phenylpro-

pionic acid (7.6 mmol). The pH was adjusted to 5.0 with

2 N aqueous hydrochloric acid and stirring was con-

tinued for an additional 2 h by which time the reaction

was complete according to TLC analysis. The reaction

mixture was neutralized with 1 N aqueous sodium

hydroxide and cooled to 0 ^ꢂC. After the addition of 65

mL distilled water a precipitate formed which was col-

lected by ﬁltration. The crude product in 20 mL aqu-

eous sodium hydroxide (1 N)/dioxane (1:1) was heated

under reﬂux for 25 min. The solution was cooled to 0 ^ꢂC

and acidiﬁed to a pH of4 with 4 N aqueous hydrochlo-

ric acid. The resulting precipitate (7a) was collected by

ﬁltration and recrystallized from ethyl acetate to give

(E)-1,3-Diethyl-8-(3,4-methylenedioxystyryl)xanthine (11g).

This was synthesized from 9b in 30% yield: mp >260 ^ꢂC

(from DMF), lit. mp >275 ^ꢂC;^{37 1}H NMR (DMSO-d₆)

d 13.50 (s, 1H, brs), 7.57 (d, 1H, J=16.5 Hz), 7.31 (s,

1H), 7.08 (d, 1H, J=8.2 Hz), 6.95 (d, 1H, J=8.9 Hz),

6.90 (d, 1H, J=16.5 Hz), 6.08 (s, 2H), 4.06 (q, 2H,

J=7.2 Hz), 3.94 (q, 2H, J=7.2 Hz), 1.25 (t, 3H, J=6.8

Hz), 1.14 (t, 3H, J=6.8 Hz); EIMS m/z 354 (M^.+).

Anal. calcd for C₁₈H₁₈N₄O₄: C, 61.01; H, 5.12; N,

15.81. Found: C, 61.01; H, 5.25; N, 15.95.

the pure product in 41% yield: mp 259 ^ꢂC; H NMR

1

(CDCl₃) d 12.44 (s, 1H, brs), 7.23–7.10 (m, 5H), 3.64 (s,

3H), 3.42 (s, 3H), 3.14 (m, 4H); EIMS, m/z 284 (M^.+).

Anal. calcd for C₁₅H₁₆N₄O₂: C, 63.37; H, 5.67; N,

19.71. Found: C, 63.19; H, 5.55; N, 19.61.

Preparation of (E)-7-methyl-8-styrylxanthines (5a–5h).

The preparations of 5a–5g were accomplished using the

procedure described by Suzuki et al.²⁷For previously

reported compounds the melting points were as follows:

5a: mp 191–192 ^ꢂC (_ꢂfrom ethanol), lit. mp 190.4–

191.3 ^ꢂC;²⁷5b: mp 205 C (from methanol:ethyl acetate,

9:1), lit. mp 205 ^ꢂC;²³5d: mp 234 ^ꢂC (from chloro-

form:ethanol, 1:1), _ꢂlit. mp 230–232 ^ꢂC²³and 236–

238 ^ꢂC;²⁴5e: mp 219 C (from ethyl acetate), lit. mp 220–

222 ^ꢂC;²³; 5f: mp 208–209 ^ꢂC (from ethanol), lit. mp 208–

209 ^ꢂC.²³Analogue 5h was prepared accordin_ꢂg to the

procedure described by Jacobson et al.:²³mp 305 C (from

DMF), lit. mp 306–308 ^ꢂC.²³The characterizations of 5c

and 5g, which are reported only in the patent literature,³⁷

are as follows.

1,3-Dimethyl-8-(2-phenylethyl)-7-methylxanthine

(7b).

To a stirred solution of 7a (2.11 mmol) in 35 mL DMF

was added potassium carbonate (5.35 mmol) followed

by iodomethane (4.22 mmol). The reaction was com-

plete according to alumina TLC analysis (ethyl acetate/

chloroform, 8:3) after 2 h at which time the insoluble

materials were removed by ﬁltration. The addition of3

volumes ofwater led to a precipitate that was collected by

ﬁltration. Compound 7b (37% yield from methanol) mel-

ted at 154 ^ꢂC: ¹H NMR (CDCl₃) d 7.29–7.71 (m, 5H), 3.59

(s, 3H), 3.57 (s, 3H), 3.36 (s, 3H), 3.03 (m, 4H); EIMS, m/z

298 (M^.+). Anal. calcd for C₁₆H₁₈N₄O₂: C, 64.41; H, 6.08;

N, 18.78. Found: C, 64.43; H, 6.05; N, 18.84.

(E)-1,3-Diethyl-8-(3-chlorostyryl)-7-methylxanthine (5c).

was obtained from 11c in 87.6% yield. This compound

is reported in the literature as a dihydrate: mp 134.0–

134.4 ^ꢂC.³⁷We obtained an anhydrous form from ethanol

(mp 178–179 ^ꢂC) and _ꢂa monohydrate from ethanol:

water 4:1 (mp 154–155 C): ¹H NMR (CDCl₃) d 7.71 (d,

1H, J=15.5 Hz), 7.55 (s, 1H), 7.45–7.27 (m, 3H), 6.89

(Z)-1,3-Diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine

(6a). The isomerization of 5a (1.3 mM in acetone) to 6a

when exposed to light was monitored at 360 nm using

reversed-phase HPLC (75% acetonitrile/25% water;

ﬂow rate 1 mL/min) and UV–vis diode array detection

(see Materials and Instrumentation). The retention

1308

J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

times of 5a and 6a were found to be 3.8 and 4.2 min,

respectively. After 2 weeks, the peak height ratio of 5a

and 6a was found to be approximately 1:1. This ratio

corresponds to a concentration ratio ofapproximately

25% trans (5a) to 75% cis (6a). The acetone was

removed under reduced pressure and the residue was

crystallized from methanol to give the essentially pure

trans isomer 5a. The mother liquor was evaporated to

dryness and the residue was crystallized from hot aceto-

nitrile. The resulting light yellow crystals (16% yield)

were found to consist of at least 98.5% of the cis isomer

7.22 (m, 3H), 3.94 (s, 3H); EIMS m/z 252 (M^.+).

Although the spectral data were in accord with the

proposed structure 8c, the microanalytical values sug-

gested the presence ofthe solvent in the analytical sam-

ple. The unsatisfactory elemental analysis and broad

melting range may be due to the poor quality ofthe

crystals obtained with chloroform as solvent. Anal.

calcd for C₁₆H₁₃N₂F: C, 76.17; H, 5.19; N, 11.10. Calcd.

.

For C₁₆H₁₃N₂F 0.1CHCl₃: C,73.13; H, 4.96; N, 10.59.

Found: C, 73.27; H, 5.35; N, 10.66.

6a: mp 134–135 ^ꢂC; H NMR (CD₃CN) d 7.14–7.16 (m,

(E)-1,3-Dimethyl-2-styrylbenzimidazolium iodide (16).

Compound 16 was prepared as follows: 15a (3.41

mmol) was dissolved in 15 mL DMF containing potas-

sium carbonate (8.64 mmol). After the addition of

iodomethane (8.82 mmol) the reaction was stirred for 2

h at 100 ^ꢂC. The reaction mixture was cooled to room

temperature and the potassium carbonate was removed

by ﬁltration. After removal of the solvent under reduced

pressure, the residue was recrystallized from DMF to

give 16 in 56.6% yield: mp 282–286 ^ꢂC, lit. mp 235 ^ꢂC;⁴⁶

¹H NMR (DMSO-d₆) d 8.07 (m, 2H), 7.96 (m, 2H), 7.85

(d, 1H, J=6.9 Hz), 7.70 (m, 2H), 7.61–7.52 (m, 4H),

4.16 (s, 6H). Anal. calcd for C₁₇H₁₇N₂I: C, 54.27; H,

4.55; N, 7.44. Found: C, 54.17; H, 4.58; N, 7.32.

1

2H), 6.88–6.93 (m, 2H), 6.29 (d, 1H J=12.4 Hz), 4.04

(q, 2H, J=7.2 Hz), 4.98 (q, 2H, J=6.8 Hz), 3.80 (s,

3H), 3.75 (s, 3H), 3.66 (s, 3H), 1.23 (t, 3H, J=6.8 Hz),

1.17 (t, 3H, J=6.8 Hz); EIMS, m/z 384 (M^.+). Anal.

calcd for C₂₀H₂₄N₄O₄: C, 62.49; H, 6.29; N, 14.57.

Found: C, 62.58; H, 6.42; N, 14.69.

2-Methyl-1H-benzimidazole (13). This was prepared

from o-phenylenediamine (12) according to the method

ofPhilips: ³¹mp 176 ^ꢂC (from water), lit. mp 176 ^ꢂC.³¹

2-Styryl-1H-benzimidazole derivatives (15a–15c). These

were prepared from 13 according to a previously

described method:³²15a: mp 202 ^ꢂC (from toluene), lit.

mp 203–205 ^ꢂC³²and 201 ^ꢂC;⁴⁶15b: yield 68.2%; mp

226–228 ^ꢂC (from acetone); ¹H NMR (DMSO-d₆) d

12.73 (s, 1H, brs), 7.76 (s, 1H), 7.69–7.55 (m, 4H), 7.46–

7.30 (m, 3H), 7.195 (m, 2H); EIMS m/z 254 (M^.+).

Anal. calcd for C₁₅H₁₁N₂Cl: C, 70.73; H, 4.35; N, 10.99.

Found: C, 70.56; H, 4.34; N, 10.86; 15c: yield 59.8%;

Studies on the isomerization of (E)-8-styrylxanthinyl de-

rivatives. Solutions (5–500 mM) of( E)-8-styryl-7-

methylxanthinyl derivatives 5a–5h were prepared in

acetonitrile/water (50:50) in clear glass vials and

exposed to light. At various time intervals 200 mL of

these solutions were injected into the HPLC system as

described in Materials and Instrumentation. The mobile

phase consisted of75% acetonitrile and 25% Milli-Q

water at a ﬂow rate of1 mL/min. The eﬄuent was

monitored at 360 nm. Upon light exposure the concen-

tration ofeach trans isomer decreased with the con-

comitant appearance ofa second peak with a shorter

retention time from that of the trans isomer. For KW-

6002 (5a) this second peak was positively identiﬁed as

the cis isomer (6a) by comparing the absorption spec-

trum and retention time with those ofthe synthetically

prepared cis ifsomer (see above for preparation). The

l_maxvalue ofthe 5a (362 nm) was greater than that of

6a (345 nm); the retention times were 4.2 and 3.8 min,

respectively. This trend was observed for all of the (E)-

8-styryl-7-methylxanthinyl derivatives (5b–h) studied —

the newly formed peaks had shorter retention times and

lower l_maxvalues than those ofthe corresponding par-

ent compound. Based on these observations, we have

concluded that these peaks represent the cis isomers for

all compounds examined. Quantitative measurements

(performed in triplicate) of the trans isomers (5a–5h)

were made by comparing peak heights ofthe samples

with those oflinear calibration curves [0.5–7.0 mM in

acetonitrile/water (1:1)] prepared with the synthetic

standards.

mp 205–212 ^ꢂC (from chloroform); H NMR (DMSO-

1

d₆) d 12.67 (s, 1H, brs), 7.67 (d, 1H J=16.6 Hz), 7.59–

7.46 (m, 5H), 7.31 (d, 1H, J=16.6 Hz), 7.22–7.17 (m, 3H);

EIMS m/z 238 (M^.+). Anal. calcd for C₁₅H₁₁N₂F: C, 70.73;

H, 4.35; N, 10.99. Found: C, 70.56; H, 4.34; N, 10.86.

2-Styryl-1-methylbenzimidazole derivatives (8a–8c).

These were prepared in the following manner: The sty-

ryl derivative 15 (5.12 mmol) was dissolved in DMF (35

mL) containing potassium carbonate (12.95 mmol). The

mixture was cooled in a dry ice/acetone bath while

iodomethane (5.12 mmol) was added in a drop-wise

manner. The reaction was allowed to return to room

temperature and stirred for an additional 3 h at which

time the reaction mixture was ﬁltered to remove the

potassium carbonate. The DMF was removed under

reduced pressure and the residue in 40 mL chloroform

was washed with water (3ꢄ30 mL). The organic phase

was dried over anhydrous magnesium sulfate, ﬁltered

and removed under reduced pressure to give 8a: mp

119–121 ^ꢂC (from toluene), lit. mp 113 ^ꢂC.⁴⁶In a similar

way, analogue 8b was obtained in 84% yield: mp 101–

104 ^ꢂC (from methanol); ¹H NMR (DMSO-d₆) d 7.94 (s,

1H), 7.83 (d, 1H, J=16.1 Hz), 7.69–7.64 (m, 2H), 7.57

(d, 1H, J=15.7 Hz), 7.49–7.35 (m, 3H), 7.22 (m, 2H),

3.91 (s, 3H); EIMS m/z 268 (M^.+). Anal. calcd for

C₁₆H₁₃N₂Cl: C, 71.51; H, 4.88; N, 10.42. Found: C,

71.56; H, 4.90; N, 10.51; Analogue 8c was obtained in a

similar manner in 81.7% yield: mp 85–102 ^ꢂC (from

chloroform); ¹H NMR (DMSO-d₆) d 7.85 (d, 1H,

J=15.5 Hz), 7.76 (m, 1H), 7.60 (m, 4H), 7.47 (m, 1H),

Enzymology

In vitro inhibition studies with MAO-B. Intact mito-

chondria prepared from baboon liver, human liver and

C57BL/6 mouse (Harlan Sprague Dawley, Dublin, VA,

J. P. Petzer et al. / Bioorg. Med. Chem. 11 (2003) 1299–1310

1309

USA) brain served as MAO-B sources as discussed in

dation ofMPTP. The Lineweaver–Burke plots of1/[rate

+

the Results and Discussion section. Mitochondria were

prepared as described by Salach and Weyler⁴⁷and

stored in aliquots containing the equivalent of5–12 mg

protein at ꢀ70 ^ꢂC in Eppendorf. Before use, the original

mitochondrial isolate was suspended in 200 mL of

sodium phosphate buﬀer (100 mM, pH 7.4 containing

ofMPDP

plus MPP⁺formation (1/V)] versus 1/

[MPTP] with increasing concentrations ofthe A

2A

antagonist were constructed for KW-6002 (Fig. 8) and

CSC. The K_ivalue (ꢀx when y=0) was estimated from

a secondary plot in which the values ofthe slopes

obtained from the Lineweaver–Burke plots were

graphed as a function of the concentration of the test

compound.

50% glycerol, w/v) and the protein concentration was

48

determined by the method ofBradof rd.

For most of

the inhibition studies on MAO-B we utilized the MAO-

A/MAO-B mixed substrate MMTP.³³Human liver (0.3

mg protein/mL) and mouse brain (0.3 mg protein/mL)

mitochondrial preparations were preincubated for 15

min with 3.3ꢄ10^ꢀ8M clorgyline, to inactivate MAO-A.

Baboon liver mitochondria, which express no MAO-A

activity,³⁴were studied in the absence ofclorgyline. The

MAO-B inhibition characteristics ofKW-6002 ( 5a) and

CSC (5b) also were investigated using the MAO-B

selective substrate MPTP.⁴⁹All incubation mixtures

(500 mL ﬁnal volume in sodium phosphate buﬀer, pH

7.4) contained either MPTP (30–90 mM [K_mvalues

range from 40 mM (mouse brain)¹¹to 65 mM (human

liver)³⁴to 87 mM (baboon liver)³⁴]) or MMTP (30–120

mM [K_mvalues range from 32 mM (mouse brain)³⁴to 52

mM (human liver)³⁴to 61 mM (baboon liver)³⁴]), the

mitochondrial homogenate (ﬁnal concentration 0.15 mg

protein/mL) and the appropriate concentrations ofthe

test compounds which bracketed their K_ivalue. The test

compounds were dissolved in dimethyl sulfoxide

(DMSO) at concentrations which, following addition to

the incubation mixture, gave a ﬁnal concentration of

4% (v/v) DMSO. DMSO concentrations higher than

4% have been reported to inhibit MAO-B activity. The

samples were incubated at 37 ^ꢂC and the incubation

times were 15 min (MMTP) or 45 min (MPTP), time

periods for which dihydropyridinium metabolite for-

mation are reported to be linear.³⁴All samples were

protected from light by covering the incubation tubes

(1.5 mL microcentrifuge tubes) with aluminum foil. The

reactions were terminated by the addition of20 mL 70%

perchloric acid and the samples were centrifuged at

16,000g for 5 min. The supernatant fractions were

removed and assayed for the MAO-B generated dihy-

dropyridinium a-oxidation products ofMPTP or

MMTP. Diﬀerent analytical procedures were used to

determine these rates as described below.

MMTP (17) as substrate. The concentration of

MMDP⁺(18), the MAO-B generated oxidation pro-

duct ofMMTP, was determined either spectro-

photometrically or by HPLC analysis. In the former

+

case, the absorption ofMMDP

was monitored at a

wavelength of420 nm using a Beckman DU-7400 spec-

trophotometer (e=25,000 M^ꢀ1).³⁴The HPLC analyses

were performed using a mobile phase consisting of 70%

Milli-Q water [containing 0.6% (v/v) glacial acetic acid

and 1% (v/v) triethylamine], 20% methanol and 10%

acetonitrile. A volume of200 mL ofthe supernatant

fraction was injected into the HPLC system. The

MMDP⁺eluted at 4.57 min. The rates (peak heights at

420 nm/mg protein-min) ofthe MAO-B catalyzed oxi-

+

dation ofMMTP to MMDP were estimated by mea-

suring the peak height ofMMDP ⁺formed. From these

+

data Lineweaver–Burke plots of1/[rate ofMMDP

formation (1/V)] versus 1/[MMTP] were constructed

and the K_ivalue was calculated as described above.

Acknowledgements

This work was supported by the Harvey W. Peters

Center for the Study of Parkinson’s Disease, Depart-

ment ofChemistry, Virginia Tech and NIH grants

ES10804 and NS41083.

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