Structure - Activity studies leading to ( - )1-(Benzofuran-2-yl)-2-propylaminopentane, (( - )BPAP), a highly potent, selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain

Bioorganic & Medicinal Chemistry 9 (2001) 1197±1212

Structure±Activity Studies Leading to (À)1-(Benzofuran-2-yl)-2-

propylaminopentane, ((À)BPAP), a Highly Potent, Selective

Enhancer of the Impulse Propagation Mediated Release of

Catecholamines and Serotonin in the Brain

Fumio Yoneda,^a,* Toshiaki Moto,^aMasatoshi Sakae,^aHironori Ohde,^aBerta Knoll,^b

Ildiko Miklya^band Joseph Knoll^b,*

^aResearch Institute, Fujimoto Pharmaceutical Corporation, Matsubara, Osaka 580-8503, Japan

^bDepartment of Pharmacology, Semmelweis University of Medicine, POB 370, Budapest H-1445, Hungary

Received 7 September 2000; accepted 21 December 2000

AbstractÐThe catecholaminergic and serotoninergic neurons in the brain change their performance according to the physiological

need via a catecholaminergic/serotoninergic activity enhancer (CAE/SAE) mechanism. Phenylethylamine (PEA), tyramine and

tryptamine are the presently known endogenous CAE/SAE substances which enhance the impulse propagation mediated release of

catecholamines and serotonin in the brain. A PEA derivative, (À)deprenyl (selegiline), known as a selective inhibitor of MAO-B, is

for the time being the only CAE/SAE substance in clinical use. Aiming to develop a selective CAE/SAE substance much more

potent than (À)deprenyl, a series of new 1-aryl-2-alkylaminoalkanes, structurally unrelated to PEA and the amphetamines, was

designed and prepared. Among them, (À)1-(benzofuran-2-yl)-2-propylaminopentane ((À)BPAP) was selected as a promising can-

didate substance for further studies. (À)BPAP signi®cantly enhanced in rats the impulse propagation mediated release of catecho-

lamines and serotonin in the brain 30 min after acute injection of 0.36 nmol/kg sc. In the shuttle box, (À)BPAP was in rats about

130 times more potent than (À)deprenyl in antagonizing tetrabenazine induced inhibition of performance. (Æ)BPAP protected

cultured hippocampal neurons from the neurotoxic eect of b-amyloid in 10^À14À10^À15M concentration. # 2001 Elsevier Science

Introduction

disease and Alzheimer's disease, is for the time being the

only drug which acts primarily as a CAE substance.¹

Some neurons in the brain possess the previously

unknown ability to change their performance, according

to the physiological need, via endogenous activity

enhancer substances. The noradrenergic, dopaminergic

and serotoninergic neurons, the performance of which is

controlled via a catecholaminergic/serotoninergic activ-

ity enhancer (CAE/SAE) mechanism, are the most suit-

able neurons in the mammalian brain for studying this

peculiar regulation. Phenylethylamine (PEA), tyramine

and tryptamine are the presently known endogenous

CAE/SAE substances in the brain. A PEA derivative,

(À)deprenyl (selegiline), which is the peculiar neuropro-

tective agent used to slow the progress of Parkinson's

PEA and tyramine, the endogenous indirectly acting

sympathomimetic amines, are on the one hand, enhan-

cers of the impulse propagation mediated transmitter

release from the catecholaminergic neurons in the brain

(CAE eect), on the other hand, releasers of catechol-

amines from their storage sites. Amphetamine and

methamphetamine, PEA analogues with a long lasting

eect, act like their parent compound, the (À)enantio-

mers being the more potent CAE substances and the

(+)enantiomers being the more potent releasers of

catecholamines. All the unwanted eects of the amphet-

amines which seriously resticted their use in therapy are

due to the catecholamine releasing property of these

substances.²

(À)Deprenyl, the N-propargyl analogue of methamphet-

*Corresponding authors. Tel.: +81-723-32-5151; fax: +81-723-32-

8482; e-mail: soyaku@fujimoto-pharm.co.jp

amine, is at present the only clinically used amphet-

PII: S0968-0896(01)00002-5

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F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

amine derivative devoid of the catecholamine releasing

eect.³The attachment of a propargyl group to the

nitrogen of methamphetamine⁴resulted in the success to

separate for the ®rst time the catecholamine releasing

property from the CAE eect of the amphetamines. As

a CAE substance, (À)deprenyl is more potent than

(+)deprenyl.³The unique eects of (À)deprenyl such as

the prolongation of the life of rats,⁵the neuroprotective

eect,⁶the slowing of the functional decline of otherwise

untreated subjects with early Parkinson's disease,⁷and

the slowing of the progression of Alzheimer's disease,⁸

can reasonably be attributed to the CAE eect of this

drug⁹(Chart 1).

selective, potent enhancer of the impulse propagation

mediated release of dopamine, noradrenaline and sero-

tonin in the brain. The main aim of this paper is to

summarize the experiments leading to the selection of

(À)BPAP and to discuss the potential therapeutic

signi®cance of this new compound.

Synthesis and Primary Evaluation

The target compounds, 1-aryl-2-alkylaminopentanes,

were synthesized by the route outlined in Scheme 1. The

starting 1-aryl-2-nitro-1-pentenes (1) were prepared by

the condensation of appropriate arylaldehydes with

1-nitrobutane in the presence of ammonium acetate

(AcONH₄). Reduction of the compounds 1 with lithium

aluminum hydride (LiAlH₄) gave the corresponding

1-aryl-2-aminopentanes (2), which were treated with

propionyl choloride to aord the respective N-[2-(1-

aryl)pentyl]propionamides (3). The propionamides 3

thus obtained were reduced with aluminum hydride

(AlH₃) to give the racemic 1-aryl-2-propylaminpentanes

(4±7), which were converted into the corresponding

hydrochlorides. The newly synthesized compounds were

divided into the following four groups for the sake of

convenience (Tables 1±4) and were primarily evaluated

(À)Deprenyl was also the ®rst described highly selective,

potent inhibitor of B-type monoamine oxidase (MAO-

B).¹⁰Because of the great practical importance of this

property in exploring the nature of MAO-B, attention

was primarily focused on this eect of the drug and

consequently thousands of papers have been published

on (À)deprenyl, viewing the substance exclusively as a

selective MAO-B inhibitor. To furnish direct evidence

that the peculiar enhancement of catecholaminergic

activity in the brain in animals treated with a small dose

of (À)deprenyl is unrelated to MAO inhibition, we pre-

viously developed a family of (À)deprenyl analogues

devoid of a signi®cant MAO-B inhibitory eect.¹¹The

selected reference substance, (À)1-phenyl-2-propylami-

nopentane ((À)PPAP), enhanced the impulse propaga-

tion mediated release of catecholamines in the brain,

like (À)deprenyl, but it left MAO activity unchanged in

contrast to (À)deprenyl.

The discovery that also tryptamine, the indole analogue

of PEA, is a potent enhancer of the impulse propaga-

tion mediated release of catecholamines in the brain,

prompted us to design new potential CAE/SAE sub-

stances by changing the aromatic ring in PPAP. We

synthesized and investigated a series of new 1-aryl-2-

alkylaminoalkanes and selected, as a potential follower

of (À)deprenyl in the clinic and a promising experi-

mental tool for the analysis of the CAE/SAE mecha-

nism in the mammalian brain, (À)1-(benzofuran-2-yl)-2-

propylaminopentane ((À)BPAP), which was a highly

Chart 1.

Scheme 1. Preparation route to target compounds.

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

1199

Table 1. Group 1: substituted phenyl, furyl and thienyl derivatives of PPAP, and related compounds

Compound

Aer

R¹

R²

Formula (analysis)^a

mp (^ꢀC)

PPAP^b

4a

4b

4c

4d

4e

C₆H₅

4-Cl±C₆H₄

3-Cl±C₆H₄

2-Cl±C₆H₄

4-F±C₆H₄

n-C₃H₇

C₁₄H₂₃N^ꢁHCl (C,H,N)

C₁₄H₂₂ClN^ꢁHCl (C,H,N)

C₁₄H₂₂FN^ꢁHCl (C,H,N)

C₁₅H₂₅N^ꢁHCl (C,H,N)

C₁₅H₂₂F₃N^ꢁHCl (C,H,N)

C₁₇H₂₉N^ꢁHCl (C,H,N)

C₁₅H₂₅NO^ꢁHCl (C,H,N)

C₁₆H₂₇NO₂^ꢁHCl (C,H,N)

C₂₀H₂₇N^ꢁHCl (C,H,N)

C₁₂H₂₁NO^ꢁHCl (C,H,N)

C₁₂H₂₁NS^ꢁHCl (C,H,N)

123

130

128

115

134

107

119

125

118

122

100

107

158

111

93

3-F±C₆H₄

2-F±C₆H₄

4f

4g

4h

4i

4j

4k

4l

4m

4n

4o

4-CF₃±C₆H₄

4-CH₃±C₆H₄

i-C₃H₇±C₆H₄

4-CH₃O±C₆H₄

3,4-(CH₃O)₂±C₆H₃

4-Ph-C₆H₄

Furan-2-yl

Thiophen-2-yl

111

4p^c

C₁₃H₂₁N^ꢁHCl (C,H,N)

220

106

4q

C₁₅H₂₅N^ꢁHCl (C,H,N)

^aAnalytical results obtained for these elements were within Æ 0.4% of the theoretical values.

^bsee ref 23.

^csee ref 24.

Table 2. Group 2: methylendioxyphenyl derivatives of PPAP, and related compounds

Compound

Ar

R¹

R²

Formula (analysis)^a

mp (^ꢀC)

5a (MPAP)

3,4-(OCH₂O)±C₆H₃

2,3-(OCH₂O)±C₆H₃

3,4-(OCH₂O)±C₆H₃

3,4-(OCH₂CH₂O)±C₆H₃

3,4-(OCH₂CH₂)±C₆H₃

3,4-(CH₂)₃±C₆H₃

n-C₃H₇

n-C₄H₉

n-C₅H₁₁

sec-C₄H₉

(C₂H₅)₂CH

Ph±CH₂

CH₃±C₆H₄

n-C₃H₇

C₁₅H₂₃NO₂^ꢁHCl (C,H,N)

C₁₆H₂₅NO₂^ꢁHCl (C,H,N)

C₁₇H₂₇NO₂^ꢁHCl (C,H,N)

C₁₆H₂₅NO₂^ꢁHCl (C,H,N)

C₁₇H₂₇NO₂^ꢁHCl (C,H,N)

C₁₉H₂₃NO₂^ꢁHCl (C,H,N)

C₁₆H₂₅NO₂^ꢁHCl (C,H,N)

C₁₆H₂₅NO^ꢁHCl (C,H,N)

C₁₇H₂₇N^ꢁHCl (C,H,N)

142

105

115

88

167

177

137

136

148

140

155

5b

5c

5d

5e

5f

5g

5h

5i

5j

5k

n-C₃H₇

^aAnalytical results obtained for these elements were within Æ 0.4% of the theoretical values.

Table 3. Group 3: naphthyl derivatives of PPAP

Compound

Ar

R¹

R²

Formula (analysis)^a

mp (^ꢀC)

6a (NPAP)

2-Naphthyl

1-Naphthyl

2-Naphthyl

n-C₃H₇

C₂H₅

n-C₄H₉

n-C₅H₁₁

n-C₃H₇

C₂H₅

C₁₈H₂₅N^ꢁHCl (C,H,N)

C₁₇H₂₃N^ꢁHCl (C,H,N)

C₁₉H₂₇N^ꢁHCl (C,H,N)

C₂₀H₂₉N^ꢁHCl (C,H,N)

C₁₉H₂₇N^ꢁHCl (C,H,N)

C₂₀H₂₉N^ꢁHCl (C,H,N)

C₁₇H₂₃N^ꢁHCl (C,H,N)

C₁₉H₂₇N^ꢁHCl (C,H,N)

C₂₀H₂₉N^ꢁHCl (C,H,N)

C₁₉H₂₇NO^ꢁHCl (C,H,N)

183

109

166

143

108

158

146

161

182

127

118

187

190

135

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

n-C₄H₉

n-C₅H₁₁

iso-C₄H₉

iso-C₅H₁₁

neo-C₅H₁₁

n-C₃H₇

2-Naphthyl

2-(6-CH₃O-naphthyl)

1-(4-CH₃O-naphthyl)

1-(2-CH₃O-naphthyl)

^aAnalytical results obtained for these elements were within Æ 0.4% of the theoretical values.

1200

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

by the shuttle box experiment (vide infra) in comparison

with PPAP as a positive reference compound.

Group 3. Naphthyl derivatives of PPAP (Table 3)

The 2-naphthyl analogue of PPAP (NPAP, 6a) and the

corresponding (À) and (+)enantiomers ((À)NPAP and

(+)NPAP) (see Table 5) were used as reference sub-

stances of the group. In the shuttle box experiment

(À)NPAP, the most potent compound in this group,

was about twice as potent as PPAP.

Group 1. Substituted phenyl, furyl and thienyl derivatives

of PPAP (Table 1)

None of these substances (4a±l) was remarkably more

potent than PPAP. The furan-2-yl (4m), furan-3-yl (4n)

and thiophen-2-yl (4o) analogues of PPAP were less

potent than PPAP. The compounds in which the dis-

tance between the benzene ring and nitrogen was with

one carbon atom shorter (1-phenyl-1-propylaminobu-

tane (4p)) or longer (1-phenyl-3-propylaminohexane

(4q)) than in PPAP were less potent than PPAP in the

shuttle box experiment.

Group 4. Indolyl, benzothienyl and benzofuryl derivatives

of PPAP (Table 4)

The indol-3-yl analogue of PPAP (IPAP, 7a) and its (À)

and (+)enantiomers ((À)IPAP and (+)IPAP) (see

Table 5) were used as reference substances for the indol

analogues of PPAP. (À)IPAP was about 5 times more

potent than PPAP in the shuttle box experiment. The

compounds in which the distance between the indole

ring and nitrogen was with one carbon atom longer

(1-(indol-3-yl)-3-propylaminohexane (7j)) than in IPAP

was less potent than PPAP. The benzofuran-2-yl analo-

gue of PPAP (BPAP, 7i) was remarkably more potent

than PPAP and then was resolved into the (À) and (+)

enantiomers ((À)BPAP and (+)BPAP). Thus (À)BPAP,

the most potent member of the group, was about 100

times more potent than PPAP in the shuttle box

experiment.

Group 2. Methylenedioxy and related derivatives of

PPAP (Table 2)

The 3,4-methylenedioxyphenyl analogue of PPAP

(MPAP, 5a) was the most potent in the group and then

was resolved into the corresponding (À)enantiomer

((À)MPAP) and (+)enantiomer ((+)MPAP) (see Table

5). Ethylenedioxyphenyl (5i), dihydrobenzofuryl (5j)

and indanyl (5k) analogues of PPAP were less potent

than PPAP. (À)MPAP, the most potent member of the

group, was about 5 times more potent in the shuttle box

experiment than PPAP.

Table 4. Group 4: indolyl benzofuryl and benzothienyl derivatives of PPAP, and related compounds

Compound

Ar

R¹

R²

Formula (analysis)^a

mp (^ꢀC)

7a (IPAP)

7b

7c

7d

7e

7f

7g

7h

7i (BPAP)

7j

Indol-3-yl

Indol-2-yl

Indol-4-yl

Indol-6-yl

Indol-7-yl

n-C₃H₇

C₁₆H₂₄N₂^ꢁHCl (C,H,N)

C₁₆H₂₄N₂(C,H,N)

C₁₆H₂₃N₂Cl^ꢁHCl (C,H,N)

C₁₆H₂₃NS^ꢁHCl (C,H,N)

C₁₆H₂₃NO^ꢁHCl (C,H,N)

C₁₇H₂₆N₂^ꢁHCl (C,H,N)

151

62

152

173

Oil

67

159

119

136

162

5-Chloroindol-3-yl

Benzothiophen-2-yl

Benzothiophen-3-yl

Benzofuran-2-yl

Table 5. The respective enantiomers of the most representative new CAE/SAE substances

Racemate

Enantiomer

Formula (Analysis)^a

mp (^ꢀC)

[a]²_D⁰

5a (MPAP)

(À)MPAP

(+)MPAP

(À)NPAP

(+)NPAP

(À)IPAP

C₁₅H₂₃NO₂^ꢁHCl (C,H,N)

C₁₈H₂₅N^ꢁHCl (C,H,N)

C₁₆H₂₄N₂^ꢁHCl (C,H,N)

C₁₆H₂₃NO^ꢁHCl (C,H,N)

184

179

210

209

179

178

166

171

À4.29 (c=3.0, MeOH)

+3.90 (c=3.0, MeOH)

À10.62 (c=4.0, MeOH)

+9.95 (c=4.0, MeOH)

À14.03 (c=1.4, MeOH)

+13.47 (c=2.0, MeOH)

À4.08 (c=4.0, MeOH)

+4.01 (c=4.0, MeOH)

6a (NPAP)

7a (IPAP)

7i (BPAP)

(+)IPAP

(À)BPAP

(+)BPAP

^aAnalytical results obtained for these elements were within 0.4% of the theoretical values.

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

1201

The optical resolutions of the remarkably potent race-

mates into the corresponding enantiomers were carried

out by HPLC on a chiral stationary phase, the results of

which were summarized in Table 5.

to some drugs acting on the catecholaminergic and/or

serotoninergic neurons. (À)BPAP proved to be the most

potent CAE/SAE substance. Fig. 4 illustrates the high

eectiveness of (À)BPAP in the test.

Treatment of rats with single or repeated doses of

0.25 mg/kg (ꢂ1 mmol/kg) sc (À)deprenyl or (À)PPAP

signi®cantly enhanced the release of catecholamines

from selected distinct brain areas and decreased the

amount of serotonin released from the raphe.¹²The

newly synthesized CAE/SAE compounds acted simi-

larly. The two most eective substances, (À)IPAP and

(À)BPAP enhanced the release of dopamine from the

striatum, substantia nigra and tuberculum olfactorium,

noradrenaline from the locus coeruleus and serotonin

from the raphe in 0.5 and 0.1 mg/kg (1.78 and 0.36 nmol/

kg), respectively (Table 7). The release of dopamine

from the substantia nigra of rats treated for 3 weeks

with (À)IPAP or (À)BPAP demonstrates that the com-

pounds enhanced signi®cantly the release of dopamine

in rats treated with low or high doses, while the release

of noradrenaline from the locus coeruleus and serotonin

from the raphe was signi®cantly increased only in rats

treated with a low dose of (À)IPAP or (À)BPAP. In the

case of serotonin, high dose treatment even inhibited the

release of the transmitter.

Pharmacological Analysis

Enhanced activity of the catecholaminergic and

serotoninergic neurons in the brain of rats treated with

the new CAE/SAE substances

The brain amines, PEA and tryptamine, signi®cantly

enhance the electrical stimulation-induced release of

³H-noradrenaline (Fig. 1), ³H-dopamine (Fig. 2) and

³H-serotonin (Fig. 3) from the isolated brain stem of

rats. (À)Methamphetamine, (À)deprenyl, and (À)PPAP

act similarly and are more potent in this respect than the

corresponding (+)enantiomers.^2,3(À)PPAP strongly

enhanced the electrical stimulation-induced release of

³H-noradrenaline and ³H-dopamine in 2.5 mg/l

3

(9.6 mmol/l) concentration and that of H-serotonin in

10 mg/l (38.4 mmol/l) concentration. Table 6 shows the

eect of CAE/SAE substances on nerve stimulation-

3

induced release of H-noradrenaline, H-dopamine and

³H-serotonin from isolated rat brain stem in comparison

Figure 1. Release of [³H]-noradrenaline from isolated rat brain stem before (open column) and after (®lled column) electrical stimulation, in the

absence and presence of PEA (N=8) and tryptamine (N=8), respectively. Each column represents the amount of [³H]-noradrenaline in picomoles

released in a 3 min collection period. Vertical lines show SEM Paired Student's t-test. Measured according to Knoll et al.²Note the signi®cantly

enhanced release of transmitter to electrical stimulation in the presence of PEA and tryptamine, respectively.

1202

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

The newly synthesized compounds exerted their CAE/

SAE eect in ex vivo experiments too. The eect of the

most potent compound, (À)BPAP, on the release of

noradrenaline from the locus coeruleus (Fig. 5), dopa-

mine from the substantia nigra (Fig. 6) and serotonin

from the raphe (Fig. 7) in a concentration range from

10^À4to 10^À17M, illustrates the ex vivo eects of this

substance. (À)BPAP enhanced signi®cantly the release

of noradrenaline, dopamine and serotonin even in the

lowest, 10^À14±10^À16concentrations. Regarding the

enhancement of the release of noradrenaline from the

locus coeruleus, we found two peaks (at 10^À6M and at

the 10^À13M concentration). Regarding the enhance-

ment of the release of serotonin from the raphe the

maximum eect was found at 10^À10À10^À12M con-

centrations.

(À)Deprenyl increases, in a small dose, the survival of

substantia nigra neurons after MPTP treatment.⁶The

neuroprotective, anti-apoptotic eect of (À)deprenyl

was con®rmed in dozens of papers.

The ability of neurons to change their activity between

broad limits according to the physiological need via

speci®c endogenous enhancer substances, is seemingly a

widely distributed regulation in the brain and this

mechanism is stimulated selectively by the CAE/SAE

substances. Accordingly, (À)deprenyl, (À)PPAP and the

newly synthesized CAE/SAE substances enhance the

activity of many other than the catecholaminergic and

serotoninergic neurons in the brain. We tested this

eect, using (Æ)BPAP, on cultured hippocampal neu-

rons according to Watt et al.¹⁷Thus the cultured cells

were exposed to the neurotoxic eect of b-amyloid.

Cells evidently mobilize their resources to survive the

attack, but only about 20% of the neurons proved to be

successful. (Æ)BPAP, which enhanced the performance

of the neurons, increased signi®cantly the rate of the

surviving cells (Table 8). Interestingly, (Æ)BPAP exerted

its neuroprotective eect on the hippocampal neurons

with a similar peculiar concentration dependency (one

peak at 10^À14M and another one at 10^À8M), to that

which characterized the (À)BPAP-induced enhanced

The neuroprotective eect of the new CAE/SAE

substances

(À)Deprenyl protects the nigrostriatal dopaminergic

neuron from the toxic eect of a couple of speci®c neu-

rotoxic agents (6-hydroxydopamine,¹³MPTP,¹⁴DSP-

4¹⁵) and an 18-month treatment with the drug protects

these neurons from the accumulation of neuromelanin,

the typical marker of aging in the substantia nigra.¹⁶

Figure 2. Release of [³H]-dopamine from isolated rat brain stem before (open column) and after (®lled column) electrical stimulation, in the absence

and presence of PEA (N=8) and tryptamine (N=8), respectively. Each column represents the amount of [³H]-dopamine in picomoles released in a

3 min collection period. Vertical lines show SEM Paired Students's t-test. Measured according to Knoll et al.²Note the signi®cantly enhanced release

of transmitter to electrical stimulation in the presence of PEA and tryptamine, respectively.

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

1203

release of noradrenaline from the locus coeruleus (see

Fig. 5).

learning ability of the rat. Tetrabenazine (1 mg/kg, sc)

administered 1 h prior to testing inhibits the aquisition

of CARs by decreasing the amount of catecholamines in

the brain. CAE substances, which enhance the impulse

propagation mediated release of catecholamines in the

brain, fully antagonize the eect of tetrabenazine.

The antidepressant eect of the new CAE/SAE

substances

(À)Deprenyl, though it was never registered for this

purpose, is an ecient antidepressant in humans.¹⁸

Considering the key role of the catecholaminergic sys-

tem in the activation of the mammalian brain and the

abundant clinical experience proving in depressed

patients the bene®cial eect of catecholaminergic stimu-

lants, there can be little doubt that each potent, selective

CAE substance must be an ecient antidepressant.

Table 9 shows that a very high dose of (À)deprenyl was

needed to antagonize the eect of tetrabenazine in the

shuttle box. (À)PPAP was about twice as potent as

(À)deprenyl in this test. Of the newly synthesized com-

pounds, NPAP was as active as PPAP, the further order

of potency was as follows: IPAP<MPAP<BPAP. The

most eective CAE substance of the series, (À)BPAP,

was on a molecular weight basis about 130 times more

potent than (À)deprenyl. Considering the high potency

of (À)BPAP in this test, we may look upon (À)deprenyl

and (À)PPAP as relatively weak CAE substances.

The ability of (À)deprenyl and (À)PPAP to normalize in

the shuttle box the learnig performance of rats pre-

treated with 1 mg/kg tetrabenazine, was of crucial

importance in realizing that these PEA derivatives act

by enhancing exocytosis in the catecholaminergic neu-

rons of the brain.³We analyzed in the shuttle box the

acquisition of a two-way conditioned avoidance re¯ex

(CAR) during 5 consecutive days. The total number of

CARs displayed during the 5-day training and the

number of failures to response within 5 s to the uncon-

dition stimulus (escape failure, EF) characterize the

Discussion and Conclusion

Members of the described new family of highly selective,

potent CAE/SAE substances, of which (À)BPAP was

selected as the candidate compound for further studies,

stimulate brain activity via a previously unknown

Figure 3. Release of [³H]-serotonin from isolated rat brain stem before (open column) and after (®lled column) electrical stimulation, in the absence

and presence of 2.5 mg/ml PEA (N=8) and 0.25 and 2.5 mg/l tryptamine (N=8 each), respectively. Each column represents the amount of [³H]-

serotonin in picomoles released in a 3 min collection period. Vertical lines show SEM Paired Students's t-test. Measured according to Knoll et al.²

Note the signi®cantly enhanced release of transmitter to electrical stimulation in the presence of 2.5 mg/l PEA and 0.25 mg/l tryptamine and the

unchanged release of serotonin to stimulation in the presence of 2.5 mg/l tryptamine.

1204

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

mechanism. They enhanced the impulse propagation

mediated release of dopamine, noradrenaline and ser-

otonin in the brain without changing anything on the

catecholaminergic and serotoninergic receptors, without

inducing a non-speci®c release of noradrenaline, dopa-

mine and serotonin from the neuron, without inhibiting

the uptake system and without inhibiting MAO.

Table 10 shows the relation between structure and

pharmacological spectrum illustrating the development

Table 6. The eect of CAE/SAE substances on nerve stimulation induced release of [³H]-noradrenaline, [³H]-dopamine and [³H]-serotonin from

isolated rat brain stem in comparison to some drugs acting on the catecholaminergic and/or serotoninergic neurons^a

Concentration (mg/L) of the compound which strongly enhanced the nerve stimulation-induced release of:

(³H)-noradrenaline

(³H)-dopamine

(³H)-serotonin

PEA

1.0

0.5

2.5

1.0

2.5

1.0

2.5

1.0

2.5

0.25

I

2.5

I

Tyramine

Tryptamine

(À)Deprenyl

(À)PPAP

(À)MPAP

(À)NPAP

(À)IPAP

0.1

(À)BPAP

0.05

2.5

I

0.05

0.25

0.5

I

0.01

I

5.0

I

Bromocryptine

Pergolide

Fluoxetine

Clorgyline

Lazabemide

Amantadine

I

^aCompounds were tested according to their eectiveness in decreasing concentration as follows: 5.0; 2.5; 1.0; 0.5; 0.25; 0.1; 0.05; 0.01; 0.005 mg/l.

Each concentration was tested on two brain stems. The concentration which increased on both preparation with at least 25% the amount of the

labelled transmitter released to nerve stimulation was taken as the minimum concentration which strongly enhanced transmitter release. Ineective

(I) means that the drug did not reach the required eectiveness in 5 mg/l concentration. Note that (À)BPAP is the most potent compound.

Figure 4. Release of labelled amines from isolated rat brain stem before (open column) and after (®lled column) electrical stimulation in the absence

and presence of (À)BPAP (N=8 each). Each column represents the amount of labelled amine in picomoles released in a 3 min collection period.

Vertical lines show SEM Paired Student's t-test. Measured according to Knoll et al.²Note the signi®cantly enhanced release of transmitter to

electrical stimulation in the presence of (À)BPAP.

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

1205

from PEA and the amphetamines to the new CAE/SAE

substances in this paper. Taking this relation and others

into consideration, we summarize at the moment the

developments leading from PEA to (À)BPAP as fol-

lows. PEA, a short acting brain amine metabolized by

MAO-B, is primarily endogenous CAE/SAE substance

and in higher concentration a releaser of catecholamines

and serotonin.^1,2The attachement of a methyl group to

the a-carbon of PEA leading to amphetamine converts

the MAO-B substrate to a weak reversible inhibitor of

both types of MAO. Accordingly, amphetamine and

then methamphetamine are long acting central nervous

system stimulants. It is still believed that amphetamine

and methamphetamine exert their stimulating eects in

the therapeutic dose-range exclusively by releasing cate-

cholamines (in higher doses also serotonin) from their

storage sites in nerve terminals.¹⁹

The unexpected ®nding that N-propargyl methamphet-

amine (named later deprenyl) lost the blood pressure

increasing eect of its parent compound⁴led us to the

selection of (À)deprenyl as a reference substance for the

development. The introduction of a propargyl group to

the nitrogen resulted in the loss of the noradrenaline

releasing property. Thus deprenyl was the ®rst PEA

derived CAE substance devoid of the catecholamine

Table 7. Eect of three-weak treatment of male rats with (À)IPAP and (À)BPAP, respectively, on the release of catecholamines and serotonin from

selected brain areas^a

Amount of biogenic amine (nmol/g wet weight Æ SEM) released from the tissue within 20 min

Dose mg/kg

Substantia nigra (dopamine)

Locus coeruleus (noradrenaline)

Raphe (serotonin)

Saline

(À)IPAP

Ð

0.5

50

0.1

0.5

50

5.8 Æ 0.18

3.9 Æ 0.10

0.403 Æ 0.01

7.2 Æ 0.15****

11.1 Æ 0.12****

8.8 Æ 0.28****

8.3 Æ 0.23****

9.4 Æ 0.13****

7.2 Æ 0.10***

4.4 Æ 0.05

1.086 Æ 0.08**

0.454 Æ 0.02

(À)BPAP

7.4 Æ 0.15***

4.1 Æ 0.05

0.870 Æ 0.02***

1.907 Æ 0.04****

0.136 Æ 0.01***

4.1 Æ 0.40

^aTreatment once daily, sc, for 3 weeks. Measurement 24 h after the last injection. Measured according to Knoll et al.^12cPaired Student's t-test.

*p<0.05 **p<0.02 ***p<0.01 ****p<0.001.

Figure 5. The ex vivo eect of (À)BPAP on the release of noradrenaline from the locus coeruleus isolated from the brain of male rats. The amount

of noradrenaline (nmol/g wet weight) released from the tissue within 20 min was measured according to Knoll and Miklya.¹²Vertical lines show

SEM Paired Student's t-test. *p<0.05, **p<0.02, ***p<0.01, ****p<0.001.

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F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

Figure 6. The ex vivo eect of (À)BPAP on the release of dopamine from the substantia nigra isolated from the brain of male rats. The amount of

dopamine (nmol/g wet weight) released from the tissue within 20 min was measured according to Knoll and Miklya.¹²Vertical lines show SEM

Paired Student's t-test. * p<0.05, **p<0.02, ***p<0.01, ****p<0.001.

Figure 7. The ex vivo eect of (À)BPAP on the release of serotonin from the raphe isolated from the brain of male rats. The amount of serotonin

(nmol/g wet weight) released from the tissue within 20 min was measured according to Knoll and Miklya.¹²Vertical lines show SEM Paired

Student's t-test. *p<0.05, **p<0.02, ***p<0.01, ****p<0.001.

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

1207

releasing eect. Because of the discovery that (À)-

deprenyl was a selective inhibitor of MAO-B,¹⁰atten-

tion has primarily been focused on this new property of

the compound. However the subsequent development of

(À)PPAP¹¹proved that the maintenance of the nigro-

striatal dopaminergic neurons on a higher activity level,

the most remarkable pharmacological eect of the small

dose administration of (À)deprenyl, is unrelated to the

selective inhibition of MAO-B.¹²Also, the discovery

that tryptamine is an enhancer of the impulse propaga-

tion mediated release of catecholamines and serotonin

as seen in Figs 1±3, prompted us to design and synthe-

size new compounds including (À)BPAP by changing

the benzene ring of PPAP. Thus, some of the new

comounds obtained have turned out to be the ®rst

potent and selective enhancers of exocytosis of

noradrenaline, dopamine and serotonin in the brain,

which are structurally unrelated to the amphetamines.

work for decades. It will improve the quality of life in

the latter decades, hopefully shifting the time of natural

death, probably decreasing the precipitation of age-

related depression, likely eliminating the precipitation of

Parkinson's disease and possibly reducing or delaying

the onset of Alzheimer's disease.

Peculiar eects of (À)deprenyl and (À)PPAP like the

ones shown on frog heart cells,^2,3the high number of

papers of papers on the neuroprotective eect of

(À)deprenyl, as well as, the eect of (Æ)BPAP on the

cultured hippocampal neurons shown in this paper (vide

infra) reveal that also the activity of other than the

catecholaminergic and serotoninergic neurons in the

brain is enhanced by very low concentration of a CAE

substance. Table 8 demonstrates the remarkable eect

of (Æ)BPAP on cultured rat hippocampal neurons. We

created a life or death situation for the cells by adding a

toxic agent to the tissue culture, an amount of b-amy-

loid. It is assumed that the endangered cell, compelled

to ®ght for survival, is immediately switching over, via

the previously unknown (Æ)BPAP-sensitive activation

mechanism, from the resting, `economy' state into the

hyperactive, exceptional, `emergency' state, in order to

mobilize all possible resources to cope with the perilous

situation. Due, however, to the huge individual dier-

ences between cells regarding any performance, only

about 20% of cells, the `high performing' ones, escaped.

(Æ)BPAP made the cells, in a concentration as low as

10^À14M, higher performing and about 70% of the neu-

rons survived. Thus, (Æ)BPAP inhibited signi®cantly

the b-amyloid-induced neurotoxicity in the cultured

hippocampal neurons in two distinct ranges of con-

centration, one with a peak of 10^À14M and one with a

peak of 10^À8M as seen in Table 8. This is surprisingly

From the data presented in this paper, it is reasonable

to expect that (À)BPAP will be more potent than

(À)deprenyl in slowing the progression of Parkinson's

disease and Alzheimer's disease. As (À)BPAP is 130

times more potent than (À)deprenyl in antagonizing

tetrabenazine-induced inhibition of performance in the

shuttle box, we may also anticipate that this compound

will be a much more ecient antidepressant in the clinic

than (À)deprenyl, which was shown to have this eect.²⁰

Life-long (À)deprenyl medication, due to its CAE eect,

slows the age-related decline of sexual and learning

performances of rats and prolongs their life.⁹The

unique eect of (À)deprenyl on longevity described by

us^6,21was unequivocally con®rmed in studies performed

with rats, hamsters and dogs.²²Considering the potency

dierence between (À)deprenyl and (À)BPAP, the

administration of a very low daily dose of this new

substance (e.g. 0.5±0.1 mg/kg) might be sucient for

keeping the catecholaminergic activity of the brain dur-

ing post-developmental (aging) longevity on a higher

basis activity level. This prophylactic medication will

Table 9. Minimum doses of the CAE compounds which antagonized,

in a statistically signi®cant manner, the depressant eect of tetra-

benazine on both the CARs and Efs in shuttle box^a

Compound

Dose (mg/kg)

(Æ)Deprenyl

(À)Deprenyl

(+)Deprenyl

(Æ)PPAP

(À)PPAP

(+)PPAP

(Æ)NPAP

(À)NPAP

(+)NPAP

(Æ)MPAP

(À)MPAP

(+)MPAP

(Æ)IPAP

5.0

2.5

5.0

2.5

>5.0

1.0

0.5

1.0

2.5

0.5

2.5

1.0

Table 8. Survival of cultured rat hippocampal neurons in the absence

and presence of b-amyloid and the protective eect of (Æ)BPAP on b-

amyloid induced neurotoxicity^a

Treatment

Survival (%)

MeanÆSD

p value

Dunnett's t-test

Control

b-amyloid (20 mmol)

100.00Æ14.35

22.41Æ7.20

b-amyloid (20 mmol)

+ (Æ)BPAP

(À)IPAP

10^À15

10^À14

10^À13

10^À12

10^À11

10^À10

M

58.97 Æ 4.75

66.97 Æ 7.08

43.79 Æ 6.79

25.79 Æ 6.32

31.33 Æ 6.81

36.56 Æ 8.63

29.44 Æ 7.51

58.15 Æ 11.72

42.10 Æ 6.30

16.41 Æ 3.71

<0.0001

>0.05

<0.001

<0.0001

>0.05

<0.0001

>0.05

(+)IPAP

(Æ)BPAP

(À)BPAP

(+)BPAP

0.05

1.0

^aMeasurement according to Knoll et al.³In order to ®nd the minimum

dose of a new compound which antagonized signi®cantly the eect of

tetrabenazine in the shuttle box, we started the experiments with 1 and

5 mg/kg and changed the dose according to the need as follows: 2.5,

0.5, 0.25, 0.1, 0.05 and 0.025 mg/kg. If a substance did not antagonize the

eect of tetrabenazine in 5 mg/kg, we did not raise the dose further. Note

that (À)BPAP (boldface) is the most potent compound.

10^À9

10^À8

10^À7

10^À6

M

^aMeasurement according to Watt et al.¹⁷

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F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

Table 10. The relation between structure and pharmacological spectrum illustrating the development from PEA and amphetamines to the most

representative new CAE substances

Compound

Ar

R¹

H

R²

H

R³

H

CAE eect

+

Catecholamine

releasing eect

Relation to MAO

Phenylethylamine (PEA)

+

À

Substrate to MAO-B

Amphetamine

CH₃

H

CH₃

H

+

Weak MAO inhibitor

Methamphetamine

(À)-N,1-Dimethyl-N-

propargylphenylethylamine ((À)-

Deprenyl)

CH₃

CH₂-CꢃCH

n-C₃H₇

H

Potent MAO-B

inhibitor

1-Phenyl-2-propylaminopentane

(PPAP)

n-C₃H₇

H

Ð

1-(3,4-Methylenedioxyphenyl)-2-

propylaminopentane

(MPAP)

H

1-(2-Naphthyl)-2-

propylaminopentane (NPAP)

H

Ð

Tryptamine

H

Substrate to MAO

Weak MAO inhibitor

1-(Indol-3-yl)-2-propylaminopentane

(IPAP)

n-C₃H₇

H

n-C₃H₇

1-(Benzofuran-2-yl)-2-

propylaminopentane (BPAP)

H

Experimental

identical with the mode of eect of (À)BPAP on the

noradrenergic neurons in Fig. 5, indicating the essential

identity of the BPAP-sensitive mechanism in the nor-

adrenergic and hippocampal neurons.

The purity of each product was checked by thin-layer

chromatography (TLC) on silica gel plates (Merck Kie-

selgel 60 F₂₅₄, thickness 0.25 nm). Column chromato-

graphy was performed on silica gel (Merck, particle size

0.063±0.200 mm for normal chromatography and 15 mm

for ¯ash chromatography). All melting points (mp) were

determined on a Yanagimoto micro-melting point

apparatus and are uncorrected. Electron ionization (EI)

mass spectra were recorded on a Hitachi M-80B mass

spectrometer. Proton nuclear magnetic reasonance (¹H

NMR) spectra were obtained with a JEOL GX-270

(270 MHz) spectrometer with TMS as internal standard

and chemical shifts are expressed as d values (in ppm).

Elemental analyses were carried out on a Yanagimoto

MT-3 elemental analyzer. Optical rotations were mea-

sured on a Horiba SEPA-200 digital polarimeter.

In conclusion, endogenous substances, capable to

enhance the activity of special groups of neurons

according to the physiological need, exist in the brain

and renders survival in life and death situation possi-

ble.^1a(À)BPAP, selected from our study as a promising

reference compound for further work, a highly selective,

potent stimulant of this previously unknown regulation,

is capable to enhance the activity of dopaminergic, nor-

adrenergic, serotoninergic and hippocampal neurons in

10^À14À10^À15M concentration. The high potency of

(À)BPAP could make the search in the brain after much

more potent endogenous neuronal activity enhancer

substances than PEA and tryptamine, reasonable.

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

1209

Reagents and solvents were purchased from common

commercial suppliers and used as received.

4H), 2.14 (q, J=7.7 Hz, 2H), 2.95 (d, J=6.4 Hz, 2H),

4.30 (m, 1H), 5.16 (d, J=8.7 Hz, JH), 7.33±7.88 (m,

7H). Anal. (C₁₈H₂₃NO) C, H, N.

1-(2-Naphthyl)-2-propylaminepentane (6a) hydrochloride

((Æ)-NPAP). LiAlH₄(0.50 g, 13.2 mmol) was sus-

pended in ether (7 mL) and a solution of AlCl₃(0.50 g,

3.75 mmol) in ether (5 mL) was added dropwise thereto

under ice cooling to prepare an ether solution of alumi-

num hydride (AlH₃). To this solution, an ether solution

(10 mL) of (3; Ar=2-naphthyl) (1.00 g, 3.7 mmol) was

added dropwise under ice cooling and the mixture was

stirred at 0 ^ꢀC for 0.5 h and then stirred overnight at

room temperature. The reaction mixture was diluted

with H₂O little by little, alkalized with 5 N NaOH, and

®ltered. The ®ltrate was concentrated and extracted

with ether. The ether extracts were dried over anhy-

drous MgSO₄, concentrated and puri®ed by column

chromatography using CHCl₃:MeOH (30:1) as eluting

solvent to give 6a (0.50 g, 52.8%) as light yellow oil. The

oil 6a was converted by the usual method to 6a hydro-

chloride ((Æ)NPAP) (0.47 g, 80.5%) as white crystalline

1-(2-Naphthyl)-2-nitro-1-pentane (1: Ar=2-naphthyl). A

mixture of 2-naphthaldehyde (3.25 g, 20.8 mmol) and

1-nitrobutane (5.04 g, 48.9 mmol) in AcOH (10 mL) was

heated at 100 ^ꢀC in the presence of AcONH₄(1.54 g,

20.0 mmol) for 10 h. The reaction mixture was diluted

with H₂O, basi®ed with 28% aqueous NH₃, and

extracted with CHCl₃. The CHCl₃extracts were dried

over anhydrous Na₂SO₄, concentrated in vacuo and

puri®ed by column chromatography using hexane-

CHCl₃(6:1) as eluant to give (1; Ar=2-naphthyl)

(2.78 g, 55.4%) as pale yellow oil: EI±MS m/e 241(M)⁺;

¹H NMR(CDCl₃) d 1.05 (t, J=7.4 Hz, 3H), 1.74 (tq,

J=7.4, 7.4 Hz, 2H), 2.91 (t, J=7.4 Hz, 2H), 7.42±8.10

(m, 7H), 8.20 (s, 1H). Anal. (C₁₅H₁₅NO₂) C, H, N.

1-(2-Naphthyl)-2-aminopentane (2: Ar=2-naphthyl). A

solution of (1; Ar=2-naphthyl) (2.78 g, 11.5 mmol) in

THF (20 mL) was added dropwise to a suspension of

LiAlH₄(0.88 g, 23.1 mmol) in THF (15 mL) and the

mixture was stirred overnight at room temperature.

After decomposition of excessive LiAlH₄with H₂O, the

mixture was ®ltered and the ®ltrate was concentrated.

Ether (30 mL) was added to the concentrate and the

ether solution was extracted with 0.5 N HCl (20 mL).

The obtained water phase was basi®ed with 28% aque-

ous NH₃and extracted with ether (30 mL). The ether

extracts were dried over anhydrous Na₂SO₄, con-

centrated and puri®ed by column chromatography

using CHCl₃-MeOH (10:1) for eluting solvent to give (2;

Ar=2-naphthyl) (1.27 g, 51.6%) a pale yellow oil: EI±

MS m/e 213(M)⁺; ¹H NMR(CDCl ₃) d 0.91 (t,

J=6.7 Hz, 3H), 1.20±1.65 (m, 4H), 2.96 (dd, J=13.4,

4.7 Hz, 2H) 3.10 (m, 1H), 7.27±8.03 (m, 7H). The oil

was dissolved in ether and treated with ether solution

saturated with HCl to give the hydrochloride as color-

powder: mp 183 ^ꢀC (from MeOH); EI±MS m/e

1

256(M+1)⁺; H NMR(CDCl ) d 0.86 (t, J=7.4 Hz,

3

3H), 0.94 (t, J=7.4 Hz, 3H), 1.34±1.67 (m, 2H), 1.70±

1.90 (m, 2H), 7.34±7.88 (m, 7H), 9.55 (br, 2H).

1-(3,4-Methlendioxyphenyl)-2-nitropentene (1;Ar=3,4-

methylenedioxyphenyl). This compound was obtained

by

the

condensation

of

3,4-methylenedioxy-

benzaldehyde (19.29 g, 128.5 mmol) with 1-nitrobutane

(9.88 g, 192.80 mmol) as pale yellow oil (10.56 g, 34.9%)

EI±MS m/e 235(M)⁺; ¹H NMR(CDCl ₃) d1.03 (t,

J=7.4 Hz, 3H), 1.66 (tq, J=7.4, 7.4 Hz, 2H), (t,

J=7.4 Hz, 2H), 6.04 (s, 2H), 6.85±7.07 (m, 3H), 7.94 (s,

1H). The oil was used without further puri®cation to the

next step.

1-(3,4-Methylenedioxyphenyl)-2-aminopentane (2; Ar=

3,4-methylenedioxyphenyl). A solution of (1; Ar=3,4-

methylenedioxyphenyl) (10.56 g, 44.9 mmol) in THF

(80 mL) was treated with LiAlH₄(3.17 g, 83.5 mmol) in

THF (60 mL) by the same way as described above to

give (2; Ar=3,4-methylenedioxyphenyl) (7.12 g, 76.6%)

as colorless oil: EI±MS m/e 207 (M)⁺. The oil was

converted by the usual method to the hydrochloride of

as colorless needles: mp 205 ^ꢀC (from CH₂Cl₂); EI±MS

m/e 208 (M+1)⁺; ¹H NMR(CDCl ₃) d 0.91 (t,

J=7.0 Hz, 3H), 1.41±1.72(m, 4H), 1.14 (q, J=5.7 Hz,

1H), 3.34±3.37 (m, 1H), 5.93 (s, 2H), 6.67±6.77 (m, 3H).

less crystalline powder: mp 175±176 ^ꢀC (from CH₂Cl₂);

1

EI±MS m/e 214 (M+1)⁺; H NMR(CDCl ) d 0.90 (t,

3

J=7.0 Hz, 3H), 1.30±2.10 (m, 4H), 2.50±4.00 (m, 3H),

7.20±8.10 (m, 7H), 8.20±9.30 (br, 3H). Anal.

(C₁₅H₁₉N^ꢁHCl) C, H, N.

N-(2-(1-(2-Naphthyl)pentyl)propionamide (3; Ar=2-

naphthyl). To a THF (15 mL) solution of (2; Ar=2-

naphthyl). (1.27 g, 6.0 mmol) and triethylamine (0.90 g,

8.9 mmol), a solution of propinyl chloride (1.10 g,

11.9 mmol) in THF (3 mL) was added dropwise under

cooling. The mixture was stirred for 5 h at room tem-

perature, evaporated to dryness, and the residue was

dissoleved in ether (15 mL). The ether solution was

washed with 5% aquious HCl, 5% aqueous Na₂CO₃

and then saturated aqueous NaCl. The organic layer

was dried over anhydrous Na₂SO₄, evaporate to dry-

ness, and puri®ed by column chromatography using

CHCl₃-hexane (5:2) for eluant to aord crude (3;

Ar=2-naphthyl). Recrystallization from ether-hexane

gave white crystalline powder (1.00 g, 62.4%): mp 92 ^ꢀC;

EI±MS m/e 269(M)⁺; ¹H NMR(CDCl ₃) d 0.88 (t,

J=7.1 Hz, 3H), 1.10 (t, J=7.7 Hz, 3H), 1.20±1.85 (m,

ꢁ

Anal. (C₁₂H₁₇NO₂HCl) C, H, N.

N-(2-(1-(3,4-Methylenedioxyphenyl))pentyl)propiona-

mide (3;Ar=3,4-methylenedioxyphenyl). To a THF

(60 mL) solution of (2; Ar=3,4-methylenedioxyphenyl)

(7.12 g, 34.4 mmol) and triethylamine (4.80 g,

47.4 mmol), a solution of propionyl chloride (5.85 g,

63.2 mmol) in THF (10 mL) was added and treated by

the same way as described above give (3; Ar=3,4-

methylenedioxyphenyl) (6.38 g, 70.3%) as colorless nee-

dles: mp 73 ^ꢀC (from CH₂Cl₂); EI±MS m/e 263 (M)⁺;

¹H NMR(CDCl ) d 0.91 (t, J=7.0 Hz, 3H), 1.12 (t,

3

J=7.4 Hz, 3H), 1.25±1.52 (m, 4H), 2.15 (q, J=7.4 Hz,

1210

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

2H), 2.75 (d, J=6.0 Hz, 1H), 4.05±4.20 (m, 1H), 5.21 (d,

J=9.0 Hz, 1H), 5.92 (s, 2H), 6.58±6.80 (m, 3H). Anal.

(C₁₅H₂₁NO₃) C, H, N.

triethylamine (1.90 g, 18.8 mmol) in THF (30 mL) under

ice cooling and the mixture was stirred for 3.5 h at room

temperature. The reaction mixture was concentrated

and the residue was dissolved in ether (150 mL). The

ether solution was washed with 5% HCl (50 mL), 5%

aqueous Na₂SO₃(40 mL) and then saturated aqueous

NaCl (40 mL). The organic layer was dried over anhy-

drous Na₂SO₄, concentrated and puri®ed with ¯ash

column chromatography using CH₂Cl₂as eluant to give

(2; Ar=indol-3-yl) (0.97 g, 30.0%) as colorless nee-

dless:mp 129 ^ꢀC (from CH₂Cl₂); EI±MS m/e 258(M)⁺;

1-(3,4-Methylenedioxyphenyl)-2-propylaminepentane (5a)

hydrochloride ((Æ)MPAP). Compound (3; Ar=methy-

lenedioxyphenyl) (6.38 g, 24.2 mmol) was treated with

ether solution of AlH₃prepared from LiAlH₄(3.02 g,

79.6 mmol) in ether (60 mL) and AlCl₃(3.03 g,

22.7 mmol) in either (40 mL) in the same way as descri-

bed above to aord 5a (4.24 g, 70.2%) as colorless oil,

which was converted by the usual method to 5a hydro-

chloride ((Æ)MPAP) (4.41 g, 90.6%) as colorless need-

¹H NMR(CDCl ) d 0.89 (t, J=7.1 Hz, 3H), 1.09 (t,

3

J=7.7 Hz, 3H), 1.30±1.64 (m, 4H), 2.12 (q, J=7.7 Hz,

2H), 2.95 (dq, J=12.8, 6.1 Hz, 2H), 4.31 (m, 1H), 5.23

(d, J=9.4 Hz, 1H) 7.02±7.67 (m, 5H), 8.19 (br, 1H).

less: mp 142 ^ꢀC (from MeOH); EI±MS m/e

1

250(M+1)⁺; H NMR(CDCl ) d 0.90 (t, J=7.1 Hz,

3

3H), 0.95 (t, J=7.4 Hz, 3H), 1.37±1.84 (m, 4H), 1.93 (tq,

J=7.4, 7.4 Hz, 2H), 2.75±2.98 (m, 3H), 3.26 (m, 2H),

5.95 (s, 3H), 6.72±6.88 (m, 2H).

1-(Indol-3yl)-2-propylaminopentane (7a) hydrochloride

((Æ)IPAP). LiAlH₄(0.50 g, 13.2 mmol) was suspended

in ether (20 mL) and thereto a solution of AlCl₃(0.51 g,

3.82 mmol) in ether (10 mL) was added dropwise under

ice cooling to give an ether solution of AlH₃. To this

solution, a solution of (3; Ar=indol-3-yl) (0.97 g,

3.75 mmol) in THF (20 mL) was added dropwise under

ice cooling. The mixture was stirred for 4 h at room

temperature. The reaction mixture was diluted with

H₂O, alkalized with 5 N NaOH and ®ltered. The ®ltrate

was concentrated and extracted with ether (300 mL).

The ether extracts were dried over anhydrous Na₂SO₄,

concentrated and puri®ed with ¯ash column chromato-

graphy using AcOC₂H₅as eluant to give a pale yellow

oil of 7a (0.57 g, 62.1%). The oil 7a was dissolved in

ether and treated with ether solution saturated with HCl

to give 7a hydrochloride ((Æ)IPAP) (0.46 g, 70.0%) as

1-(2-(Indol-3-yl)2-nitro-1-pentene (1; Ar=indol-3-yl). A

mixture of indol-3-carboxaldehyde (9.39 g, 64.7 mmol)

and 1-nitrobutane (10.00 g, 97.0 nmol) in AcOH (10 mL)

was heated at 100 ^ꢀC in the presence of AcONH₄

(3.08 g, 40.0 mL) for 8 h. The reaction mixture was

diluted with H₂O, basi®ed with 28% NH₃, and extrac-

ted with CHCl₃(150 mL). The CHCl₃extracts were

dried over anhydrous Na₂SO₄and concentrated. The

residue was puri®ed by ¯ash column chromatography

using CHCl₃as eluting solvent to give (1; Ar=indol-3-

yl) (4.37 g, 31.7%) as pale yellow crystalline powder: mp

139 ^ꢀC (from EtOH); EI±MS m/e 230(M)⁺; H NMR

1

(CDCl₃) d1.90 (t, J=7.4 Hz, 3H), 1.71 (m, 2H), 2.92 (t,

J=7.7 Hz, 2H), 7.25±7.36 (m, 2H), 7.47 (dd, J=7.0,

1.4 Hz, 1H), 7.55 (d, J=2.7 Hz, 1H) 7.81 (d, J=6.7 Hz,

1H), 8.50 (s, 1H), 8.90 (br, 1H). Anal. (C₁₃H₁₄N₂O₂) C,

H, N.

colorless needles: mp 151 ^ꢀC (from MeOH); EI±MS m/

1

e 245(M+1)⁺; H NMR(CDCl ) d (t, J=7.1 Hz, 3H),

3

1.09 (t, J=7.7 Hz, 3H), 1.30±1.64 (m, 4H), 2.12 (q,

J=7.7 Hz, 2H), 2.95 (m, J=12.8, 6.1 Hz, 2H), 4.31 (m,

1H), 5.23 (d, J=9.4 Hz, 1H) 7.02±7.67 (m, 5H), 8.19 (br,

1H).

1-(2-(Indol-3-yl))-2-aminopentane (2; Ar=indol-3-yl). A

solution of (1; Ar=indol-3-yl) (1.56 g, 41.0 mmol) in

THF (30 mL) was added dropwise to a suspension of

LiAlH₄(4.73 g, 20.5 mmol) in THF (30 mL) under ice

cooling and the mixture was stirred for 24 h at room

temperature. After decomposition of excessive LiAlH₄

with H₂O, the mixture was ®ltered and the ®ltrate was

concentrated. Ether (100 mL) was added to the con-

centrate and the ether solution was extracted with 1 N

HCl (300 mL). The obtained water phase was basi®ed

with 28% aqueous NH₃and extracted with ether

(450 mL). The ether extracts were dried over anhydrous

Na₂SO₄and concentrated to give crude (2; Ar=indol-3-

yl) (2.52 g, 61.0%) as red oil. The oil was converted by

the usual method to the hydrochloride as colorless nee-

1-(2-(Benzofuran-2-yl))-2-nitro-1-pentene (1; Ar=benzo-

furan-2-yl). A mixture of benzofuran-2-carboxyldehyde

(50.00 g, 342.1 mmol) and 1-nitrobutane (58.00 g,

562.4 mmol) in AcOH (50 mL) wad heated at 100 ^ꢀC in

the presence of AcONH₄(18.48 g, 240.0 mmol) for 1 h.

The reaction mixture was diluted with H₂O, basi®ed

with 28% aqueous NH₃, and extracted with CHCl₃. The

CHCl₃extracts were dried over anhydrous Na₂SO₄and

concentrated to give a residue, which was distilled under

reduced pressure (153±167 ^ꢀC, 1 mm Hg). The com-

pound (1; Ar=benzofuran-2-yl) was obtained as pale

yellow crystalline powder (58.61g, 74.1%): mp 47 ^ꢀC;

EI±MS m/e 231(M)⁺; ¹H NMR(CDCl ₃) d1.06 (t,

J=7.4 Hz, 3H), 1.72 (tq, J=7.4, 7.4 Hz, 2H), 3.18 (t,

J=7.4 Hz, 2H), 7.12 (s, 1H), 7.25±7.90 (m, 4H), 7.91 (s,

1H). Anal. (C₁₃H₁₃NO₃) C, H, N.

dles: mp 222 ^ꢀC (from MeOH); EI±MS m/e

1

203(M+1)⁺; H NMR(CD OD) d 0.98 (t, J=7.0 Hz,

3

3H), 1.40±1.75 (m, 4H), 3.00 (dd, J=14.3, 7.4 Hz, 1H),

3.14 (dd, J=14.8, 6.4 Hz, 1H), 3.40±3.50 (m, 1H), 7.00±

7.18 (m, 3H), 7.40 (d, J=8.1 Hz, 1H) 7.55 (d, J=7.7 Hz,

1H). Anal. (C₁₃H₁₈N₂HCl) C, H, N.

1-(2-(Benzofuran-2-yl))-2-aminopentane (2; Ar=benzo-

furan-2-yl). A solution of (1; Ar=benzofuran-2-yl)

(58.61 g, 253.4 mmol) in THF (150 mL) was added

N-(2-(1-(Indol-3-yl))pentyl)propionamide (3; Ar=indol-

3-yl).

25.0 mmol) in THF (10 mL) was added drop by drop to

a solution of (2; Ar=indol-3-yl) (2.52 g, 12.5 mmol) and

A

solution of 3-propionylchloride (2.31 g,

dropwise to

a

suspension of LiAlH₄(12.50 g,

329.4 mmol) in THF (150 mL) over 1 h under ice cool-

ing. The mixture was stirred for 15 h at room tempera-

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

1211

ture. After excessive LiAlH₄was decomposed with H₂O

under ice cooling, the mixture was basi®ed with 28%

aqueous NH₃, and ®ltered. After the ®ltrate was con-

centrated, ether (500 mL) was added to the concentrate

and the ether solution was extracted with 0.5 N HCl

(100 mL). The obtained water phase was basi®ed with

28% aqueous NH₃and extracted with ether (600 mL).

The ether extracts were dried over anhydrous Na₂SO₄

and concentrated to give crude (2; Ar=benzofuran-2-

umn, CHIRALPACK AD (20 mm fÂ250 mm); Eluant,

hexane DEA or TFA (100:0.1). The ®rst fractions were

the (À)-enantiomers for (Æ)NPAP and (Æ)MPAP and

were the (+)-enantiomers for (Æ)IPAP and (Æ)BPAP.

Each oily enantiomer was dissolved in ether and was

treated with ether solution saturated with HCl to aord

colorless needles of the respective enantiomer hydro-

chlorides (see Table 5).

yl) as coloreless oil (23.98 g, 46.6%): EI±MS m/e

Measurement of the release of radiolabelled noradrena-

line, dopamine or serotonin from the isolated brain stem

of rats. The method used was described in a previous

paper.²To measure drug eects on transmitter-release

from the brain stem we incorporated either (³H)-nor-

adrenaline (1-(7,8-³H)-noradrenaline; speci®c activity:

30±50 Ci/mmol), (³H)-dopamine ((2,5,6-³H)-dopamine;

speci®c activity: 5±15 Ci/mmol) or (³H)-serotonin (5-

hydroxy-(G-³H)-tryptamine creatinine sulphate; speci®c

activity: 10±20 Ci/mmol) (Amersham, Buckinghamshire,

UK), respectively, into the transmitter stores of the

brain stem slices by preincubation. The brain stem was

stimulated with rectangular pulses (3 Hz, 1 ms, 60 V) for

3 min. At the beginning of the experiment, three

consecutive 3-min resting periods preceded the ®rst sti-

mulation. Thereafter seven resting periods were allotted

between stimulations.

1

203(M)⁺; H NMR(CDCl ) d 0.92 (t, J=7.0 Hz, 3H),

3

1.37±1.64 (m, 4H), 3.46±3.51 (m, 1H), 5.52 (br, 2H),

6.55 (s, 1H), 7.15±7.49 (m, 4H). The crude moduct was

used without further puri®cation to the next step.

N-(2-(1-(Benzofuran-2-yl))pentyl)propionamide

(3;

Ar=benzofuran-2-yl). To a solution of (2; Ar=benzo-

furan-2-yl) (23.98 g, 118.0 mmol) and triethylamine

(15.52 g, 153.4 mmol) in THF (250 mL), a solution of

propionyl chloride (16.38 g, 177.00 mmol) in THF

(100 mL) was added dropwise and the mixture was stir-

red for 3 h at room temperature. After the reaction,

ether (500 mL) was added to the reaction mixture and

the ether solution was washed with 1 N HCl (400 mL),

5% aqueous Na₂CO₃(300 mL) and then saturated

aqueous NaCl (300 mL). The organic layer was dried

over anhydrous Na₂SO₄and concentrated to give crude

(3; Ar=benzofuran-2-yl) (16.35 g, 53.4%) as white

crystalline powder: mp 72 ^ꢀC (from CH₂Cl₂); EI±MS m/

e 259(M)⁺; ¹H NMR(CDCl ₃) d 0.91 (t, J=7.4 Hz, 3H),

1.15 (t, J=7.4 Hz, 3H), 1.32±1.66 (m, 4H), 2.20 (q,

J=7.4 Hz, 2H), 2.98 (dq, J=15.1, 5.7 Hz, 2H) 4.34 (m,

1H), 5.44 (d, J=8.1 Hz, 1H), 6.46 (s, 1H), 7.15±7.53 (m,

4H). Anal. (C₁₆H₂₁NO₂) C, H, N.

Measurement of the release of noradrenaline from the lo-

cus coeruleus, dopamine from the substantia nigra, stria-

tum and tuberculum olfactorium and serotonin from the

raphe. The release of noradrenaline, dopamine or ser-

otonin was measured from selected brain stem areas by

HPLC with electrochemical deteciton. After incubation

of the quickly removed brain samples for 20 min, the

tissue was soaked for 20 min in fresh Krebs solution and

the concentration of the biogenic amine released during

this period of time was estimated as described in

previous paper.^12c

1-(Benzofuran-2-yl)-2-propylaminopentane (7i) hydro-

chloride ((Æ)BPAP). LiAlH₄(8.37 g, 220.6 mmol) was

suspended in ether (100 mL) and thereto a solution of

AlCl₃(8.40 g, 63.0 mmol) in ether (50 mL) was added

drop by drop under ice cooling to prepare an ether

solution of AlH₃. To this solution, a solution of (3;

Ar=benzofuran-2-yl) (16.35 g, 63.0 mmol) in THF

(100 mL) was added dropwise under ice cooling and the

mixture was stirred for 24 h at room temperature. The

reaction mixture was diluted with H₂O little by little,

alkalized with 5 N NaOH, and ®ltered. The ®ltrate was

concentrated and extracted with ether (600 mL). The

ether extracts were dried over anhydrous MgSO₄, con-

centrated and distilled under reduced pressure (132±

139 ^ꢀC, 1 mm Hg) to give 7i (10.51 g, 67.9%) as pale

yellow oil. The oil 7i was dissolved in ether and treated

with ether solution saturated with HCl to give 7i

hydrochloride ((Æ)BPAP) (10.17 g, 84.3%) as colorless

Measurement of ꢀ-amyloid induced neurotoxicity in cul-

tured hippocampal neurons. Primary rat embryonic hip-

pocampal cultures were established according to Watt et

al.¹⁷with some modi®cation. The hippocampus was

dissected from the brains of embryonic day 18 rat

embryos. At the 10th day, the cultured cells were

ingured by exposing them for 3 days to a 20 mM con-

centration of b-amyloid 25-35 fragment(Ab) (Peptide

Inst., Osaka, Japan). This concentration of b-amyloid

decreased the survival of the neurons (control=100%)

to 22.4 Æ 7.20% (means Æ SD). For testing the neuro-

protective eect of (Æ)BPAP, concurrently with b-amy-

loid a selected concentration of the compound was

present in the culture well and the change in survival of

the cells was calculated. Statistical signi®cance of data

was evaluated by Dunnett's t-test. Signi®cance level was

set at p<0.05.

needles: mp 136 ^ꢀC (from MeOH); EI±MS m/e

1

246(M+1)⁺; H NMR(CDCl ) d 0.92 (t, J=7.4 Hz,

3

3H), 0.94 (t, J=7.4 Hz, 3H), 1.40±1.68 (m, 2H), 1.68±

2.07 (m, 4H), 2.89 (m, 2H), 3.28 (m, 1H), 3.55 (m, 2H),

6.65 (s, 1H), 7.17±7.38 (m, 4H), 9.50 (br, 2H).

Measurement of the performance of rats in the shuttle

box. The method was described in a previous paper.³

The acquisition of a two-way conditioned avoidance

reglex (CAR) was analyzed in the shuttle box during 5

consecutive days. The instrument consists of six boxes,

each one separated inside by a barrier with a small gate

The optical resolutions of the racemates. The optical

resolutions of the above racemates thus obtained were

carried out by HPLC on a chiral stationary phase.

Conditions for preparative HPLC are as follows: Col-

1212

F. Yoneda et al. / Bioorg. Med. Chem. 9 (2001) 1197±1212

in the middle. Animals were trained to cross the barrier

under the in¯uence of a conditioned stimulus (CS) (light

¯ash). If they failed to do so, they were punished with a

footshock (1 mA), that is an unconditioned stimulus

(US). If the rat failed to respond within 5 s to US, it was

noted as escape failure (EF). The rats were trained with

100 trials per day. One trial consisted of 15 s intertrial

interval, followed by 15 s CS. The last 5 s of CS over-

lapped the 5 s of US. At each learning session, the

number of CARs, EFs and intersignal reactions (Irs)

were automatically counted and evaluated by multi-way

analysis of variance (ANOVA). Performance of rats was

inhibited by the subcutaneous administration of 1 mg/

kg tetrabenazine 1 h prior to their testing in the shuttle

box. For measuring the antagonistic eect of a com-

pound against tetrabenazine induced inhibition of per-

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Article Doi

Structure - Activity studies leading to ( - )1-(Benzofuran-2-yl)-2-propylaminopentane, (( - )BPAP), a highly potent, selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain

DOI: 10.1016/S0968-0896(01)00002-5

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Article abstract of DOI:10.1016/S0968-0896(01)00002-5

Full text of DOI:10.1016/S0968-0896(01)00002-5

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