Racemic N1-norphenserine and its enantiomers: Unpredicted inhibition of human acetyl- and butyrylcholinesterase and β-amyloid precursor protein in vitro

Article abstract of DOI:10.3987/com-03-s15

The optically pure enantiomers of N¹-norphenserine (15, 16) were synthesized and its racemate 17 was prepared by mixing equal parts of each enantiomer. (-)-N¹-Norphenserine (15) was prepared by partial synthesis initiated from the natural product, (-)-physostigmine (1). (+)-N ¹-Norphenserine (16) was prepared by total synthesis using the Julian oxindole route, with modifications. The in vitro inhibitory activities of 15-17 were quantified against human erythrocyte AChE and plasma BChE as well as against human neuroblastoma cell β-amyloid precursor protein secretion in cell culture. All were active. Racemic compound (17) with a high AChE and β-amyloid precursor protein inhibitory action may warrant further assessment in Alzheimer's disease models.

Full text of DOI:10.3987/com-03-s15

HETEROCYCLES, Vol. 61, 2003
529
HETEROCYCLES, Vol. 61, 2003, pp. 529 - 539
Received, 8th May, 2003, Accepted, 30th June, 2003, Published online, 4th July, 2003
RACEMIC N1-NORPHENSERINE AND ITS ENANTIOMERS:
UNPREDICTED INHIBITION OF HUMAN ACETYL- AND
BUTYRYLCHOLINESTERASE AND β-AMYLOID
PRECURSOR PROTEIN IN VITRO
a
Qian-sheng Yu, Weiming Luo, Harold W. Holloway, Tada Utsuki, TracyAnn
a
a
a
a
b
a
c@
Perry, Debomoy K. Lahiri, Nigel H. Greig, * and Arnold Brossi
a) Drug Design & Development Section, Laboratory of Neurosciences, Intramural
Research Program, National Institute on Aging, National Institutes of Health, 5600
Nathan Shock Dr., Baltimore, Maryland 21224-6825.
b) Department of Psychiatry, Psychiatric Research Institute Indiana University
School of Medicine, Indianapolis, IN 46202.
c) School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599.
Abstract- The optically pure enantiomers of N1-norphenserine (15, 16) were
synthesized and its racemate 17 was prepared by mixing equal parts of each
enantiomer. (-)-N1-Norphenserine (15) was prepared by partial synthesis initiated
from the natural product, (-)-physostigmine (1). (+)-N1-Norphenserine (16) was
prepared by total synthesis using the Julian oxindole route, with modifications. The
in vitro inhibitory activities of 15-17 were quantified against human erythrocyte
AChE and plasma BChE as well as against human neuroblastoma cell β-amyloid
precursor protein secretion in cell culture. All were active. Racemic compound
(17) with a high AChE and β−amyloid precursor protein inhibitory action may
warrant further assessment in Alzheimer’s disease models.
The natural product (-)-physostigmine (1), derived from the Calabar bean: the dried ripe seed of the
Physostigma venenosum, has not only proved to be a useful clinical drug (e.g., Antilirium ) and
pharmacological tool, but has acted as the base pharmacophore in the development of several current
drugs (pyridostigmine bromide oral: Mestinon, parenteral: Renenol; neostigmine methylsulfate:
*Corresponding Author: Phone: 410-558-8278; Fax: 410-558-8323; E-Mail:GreigN@vax.grc.nia.nih.gov
@ In celebration of Arnold Brossi’s 80th birthday

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HETEROCYCLES, Vol. 61, 2003
Prostigmin) and experimental therapeutics for Alzheimer’s disease (e.g., (-)-phenserine tartrate).1-4 (-)-
Physostigmine (1) is a potent and short-acting (half-life 30 min)1,3-5 anticholinesterase that co-inhibits
the
enzymes acetylcholinesterase (AChE: EC 3.1.1.7)4,6 and butyrylcholinesterase (BChE: EC
3.1.1.8)2,4,6 with equal potency and with a brain/plasma concentration ratio of unity.1 In contrast, its
Table 1. Our Literature Values of Natural (-)- and Unnatural (+)- Physostigmine and its Analogues
Versus Human Erythrocyte AChE and Human Plasma BChE.
3
3
2
2
Me
H
N
H
N
Me
4
4
O
O
1
R
1
N
R
R
R
1
1
N
5
5
3
3
O
O
6
N 8
R
N 8
R
2
6
7
7
2
3aS
3aR
IC50* (nM)
No. Compound
R1 R2 R3
AChE
BChE
Selectivity Ref
1 (-)-3aS-Physostigmine
8 (+)-3aR-Physostigmine
Me Me Me
28 + 2
16 + 3
Me Me Me 9890 + 6 2490 + 290
2-fold BChE 5,12,13
4-fold BChE
9
3 (-)-3aS-N1-Norphysostigmine
Me Me Me
21 + 1
2 + 1
202 + 16
10-fold BChE 11
10 (+)-3aR-N1-Norphysostigmine Me Me Me 193 + 5
4 (-)-3aS-N8-Norphysostigmine
Me H Me 57 + 4
none
13
8
7 + 2
9-fold BChE
11 (+)-3aR-N8-Norphysostigmine Me H Me 2190 + 110 1120 + 23
2-fold BChE 13
5 (-)-3aS-N1N8-Norphysostigmine Me H
12 (+)-3aR-N1N8-Norphysostigmine Me H H 1490 + 120 235 + 55
H
11 + 1
2 + 1
5-fold BChE
6-fold BChE
9
9
2 (-)-3aS-Phenserine
9 (+)-3aR-Phenserine
Ph Me Me
Ph Me Me 3500 + 55 23500 + 300 7-fold AChE
22 + 2
1560 + 270 70-fold AChE 5,10,12
9
6 (-)-3aS-N8Norphenserine
13 (+)-3aR-N8Norphenserine
Ph H Me 41 + 3 516 + 61 13-fold AChE 8
Ph H Me 5650 + 610 1745 + 325 3-fold BChE
8
7 (-)-3aS-N1N8-Bisnorphenserine Ph
14 (+)-3aR-N1N8-Bisnorphenserine Ph
H
H
H
H
22 + 2 895 + 104 41-fold AChE 9
230 + 23 950 + 107 4-fold AChE 9
15 (-)-3aS-N1-Norphenserine
16 (+)-3aR-N1-Norphenserine
17 (+)-N1-Norphenserine
Me Me
Ph Me
Ph Me
H
H
H
14 + 1
64 + 4
47
612 + 1
5295 + 740 88-fold AChE 13
1660 35-fold AChE 10
25-fold AChE 13,14
* Concentration to inhibit 50% of enzymatic activity against enzyme obtained from the same individual
phenylcarbamate analogue, (-)-phenserine (2) is similarly a reversible potent anticholinesterase, but is
long-acting (half-life ~8 hours), selective for human AChE vs. BChE (70-fold) and has a brain/plasma
concentration ratio of 10:1.5 Both compounds, (-)-physostigmine (1) and (-)-phenserine (2), have a N1-
methyl substituent, and their respective (+)-enatiomers (8,9) possess minimal anticholinesterase activity
(Table 1).5,7 A similar behavior also is found in the N8-nor and N1,N8-bisnor series (4,5,6,7,11,12,13,14),8,9
wherein the (-)- and (+)-enantiomers are potent and essentially devoid of AChE action, respectively. A

HETEROCYCLES, Vol. 61, 2003
531
notable exception, however, is the enantiomers of N1-norphysostigmine (3,10), a putative metabolite of 1,
that, when prepared earlier, were both found to be potent inhibitors of human AChE and BChE10 (Table
1) as well as eel AChE.11
In assaying enantiomers of biologically active racemates, it is paramount to establish their optical purity
as a minor contaminant within a biologically inactive enantiomer by a highly potent optical isomer could
falsely mimic a positive biological response.7 In this regard, our recent studies with (-)- and (+)-
phenserines (2,9) have found the (+)-enantiomer to be devoid of anticholinesterase action15 rather than to
possess a minimal, but measurable activity found in earlier studies (Table 1).8,9 In light of our prior
finding that both (-)- and (+)-N1-norphenserine (15,16) were potent AChE inhibitors (Table 1),13 together
with our recent finding that both enantiomers of phenserine (2,9) possess inhibitory action on β–amyloid
precursor protein (APP),15-18 which is critical in Alzheimer’s disease,19 we decided to revisit 15,16. In the
current study, we report the resynthesis of these compounds with particular emphasis on both their optical
as well as chemical purity, together with their evaluation for both cholinesterase and APP inhibitory
action.
Chemistry: The total synthesis of racemic N1-norphenserine (17), together with it’s enantiomers (15, 16),
has previously been reported.10 The precursor in this synthesis is oxindole (27), which is similar to
Julian’s first total synthesis of physostigmine,20 but with additional improvements. Both the (-) and (+)
series of analogues were obtained based on the chemical resolution of amino alcohol (18) by (+)-2,3-di-
O-(p-toluoyl)-D-tartaric acid. Optically pure intermediates could additionally be obtained by the
Figure 1
Me
Me
N Me
Me
O
O
Me
N
Me
O
Me
N
HN
OH
N
Me
Me
19
18
chromatographic separation of urea diastereoisomer (19) that, after fragmentation, provided optically
pure 30 and it’s enantiomer (Figure 1).21,22

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HETEROCYCLES, Vol. 61, 2003
Schemes 1
Me
Me
H
2
1
HO
N
O
N
Me
N
Me
95%
Me
68%
O
N
N
Me
Me
20
1
Me
Me
3
4
MeO
HO
N
N
Bn
Bn
89%
90%
N
N
Me
Me
21
22
Me
H
Me
H
N
5
N
O
O
N
N
70%
Bn
H
O
O
N
N
15
Me
Me
[a] -50.9 (c=0.55, EtOH)*
23
o
D
1), BuOH, catalytic amount of Na; 2), a, MeI/DMSO/KOH; b,BnNH2; 3), BBr3/CH2Cl2; 4),
PhNCO/Et2O/catalytic amount of Na; 5), Pd(OH)2/C, H2.
* [a]D -50.4o (c=0.5, CH3Cl).14
In choosing a synthetic route for compounds (15, 16, 17) with a focus on optical purity, synthesis of (-)-
3aS-N1-norphenserine (15) was initiated from natural (-)-physostigmine (1) that bears a 3aS
configuration. All reactions avoided the 3a chiral center throughout the process (Scheme 1). Specifically,
(-)-physostigmine (1) was dissolved in BuOH with addition of a catalytic quantity of Na, and was
refluxed until 1 disappeared. An equivalent of fumaric acid then was added to this hot solution with
stirring. After cooling, the fumarate of 20 was given almost quantitatively.24 Subsequently, 20 was
reacted with MeI in the presence of KOH in DMSO, BnNH2 then was added and the reaction mixture
maintained at 100o C for 2 h. It then was partitioned between H2O and Et2O. The Et2O layer contained N-
benzyltricyclic methyl ether (21) with tiny remainder of BnNH2 that was easily removed by simple
chromatography.10,11,23 Demethylation of 21 by BBr3 in CH2Cl2 at room temperature gave the HBr salt of
22 in good yield and quality without further purification.10,11 To the ether solution of free base (22) in the
presence of a catalytic amount of Na, 1 equivalent of phenylisocyanate was added in one portion. After
22 had disappeared, as assessed by TLC, the reaction was quenched with H2O. The ether layer was dried,
evaporated to give crude phenyl carbamate (23), which then was directly crystallized from Et2O without
chromatography.8,9,14 Debenzylation of 23 gave 15 that then was transformed to its fumarate and
recrystallized from MeOH.8,9,14

HETEROCYCLES, Vol. 61, 2003
533
The total synthesis of (+)-3aR-N1-norphenserine (16) was initiated from N-methylphenetidine (24), which
was reacted with 2-bromopropionyl bromide to give compound (25) in the presence of triethylamine,
used as a HBr scavenger, and a catalytic amount of DMAP that proved helpful to increase yield. The
intramolecular Friedel-Craft’s reaction gave compound (26). Oxindole (27) was obtained by reaction of
26 with dimethyl sulfate in KOH aqueous solution. Unfortunately, the electro negative active site of the
carbonyl α-position caused the occurrence of side reactions; hence the yield of this reaction was not
particularly satisfactory. Phase-transfer asymmetric alkylation of oxindole (27) gave the 3aR
configuration enriched compound (29). From previous studies25-28 the separation of compound (29) from
it’s enatiomer has proven to be readily achievable. The absolute optical rotation values of compounds
(29,25 30,11,25, 31,11,25 32,11 34,14 and 1614 ) have been previously reported. As a consequence, we used the
3aR configuration enriched compound (29) as the key intermediate in the following known
procedure10,14,27 to synthesize final product (16). Reductive cyclization of 29 by LiAlH4 gave a crude
residue of hexahydropyrroloindole (30), which was transformed to its fumarate by adding an equivalent
methanol solution of fumaric acid. Further recrystallization provided chemically pure compound (30),
whose ee% value increased to 71.5%. In the same manner, the final crude product (16) was purified by
formation and recrystallization of it’s di-p-toluoyl-L-tartrate. Comparing the optical rotation (determined
on a JASCO, model DIP-370, Spectroscopic Co., Ltd., Japan) of the free base of compound (16) with that
of its enantiomer (15) provided 16 with an ee% value of 98.2% (from Schemes 1 and 2). To secure
optical purity prior to biological assessment, we transformed the free base of 16 to it’s fumarate,
recrystallized it again, and then compared its optical rotation with that of the fumarate of its enantiomer
(15) (Table 2). The fumarate of racemic 17 was produced by mixing exact equivalents of fumarates (15
and 16) followed by recrystalization from MeOH.
We attempted to measure the optical purity of 15, 16 and 17 by a chiral HPLC, but streaking occurred
and the results proved unsatisfactory, as in the measurement of methyl ether (29).26 Taking an alternative
1
approach, we utilized (S)-(+)-2,2,2-trifluoro-1-(9-anthryl)ethanol as a chemical shift reagent in the H-
NMR spectral measurement of the optical purity of 15, 16, and 17 and, as illustrated in Figure 2, we
determined 15 and 16 to possesses an optical purity of 100% based on both optical rotation and 1H-NMR
spectral assessment.29

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HETEROCYCLES, Vol. 61, 2003
Scheme 2
Me
EtO
Br
Me
1
2
HO
EtO
3
O
73%
85%
51%
N
O
N
NH
24
Me
Me
25
Me
26
N+
Br-
HO
Me
Me
CN
MeO
MeO
5
CF3
O
O
28
N
N
N
50%
Me
Me
27
ee=56.2%
29
87%
Me
Me
Me
MeO
MeO
7
6
HO
N
N
H
N
Bn
Bn
N
N
90%
80%
N
30
ee=71.5%
Me
Me
31
Me
32
Me
H
Me
H
N
O
O
8
9
N
O
N
N
Bn
H
90%
N
35%
O
N
34
Me
16
Me
[a]D +50.0o (c=0.2, EtOH)
ee=98.2%
1),2-Bromopropionyl bromide, triethylamine, DMPA; 2), ALCl3; 3), (MeO)2SO2/KOH; 4), ClCH2CN,
(-)-Cinchonidine p-trifluorobenzyl bromide/NaOH; 5),LiALH4/THF; 6),BnBr/K2CO3/DMF;
7),BBr3/CH2Cl2; 8),PhNCO/Et2O/Na; 9),Pd(OH)2/C, H2; di-p-toluoyl-L-tartaric acid.
Biological evaluation and discussion: Compounds (15, 16 and 17) were tested for in vitro inhibitory
activity of human erythrocyte AChE and plasma BChE by previously published methodology.5,8-15 The
results, shown in Table 2, in general, are in accord with our studies (Table 1, bottom).10,13 However, in
our current assessment with an emphasis on optical purity, the (-)-enantiomer (15) proved to be
approximately 2-fold more AChE potent and 10-fold more BChE than our previous data suggested. The
AChE selectivity provided by the phenylcarbamate in (-)-phenserine (2) (70-fold, Table 1, top),5,12 having
a N1-methyl substituent, was dramatically diminished in 15; which possessed significant and, likely,
clinically relevant BChE inhibitory activity when compared to the first approved Alzheimer’s disease
drug, tacrine (Cognex, IC50: AChE 190 + 40 nM, BChE 47+ 10 nM).1,2,5,12 Particularly interesting are the
anticholinesterase activities of (+)-enantiomer (16). These are similar to the unpredicted ones found in
our prior synthesis, and verify that (+)-N1-norphenserine (16) is, indeed, a potent inhibitor of AChE with

HETEROCYCLES, Vol. 61, 2003
535
1
Figure 2: Partially Expanded H-
NMR (CDCl3) (Varian 400) (δ 1.00
- δ 3.00) Spectrum. An equivalent
of chemical shift reagent (S)-(+)-
2,2,2-triflioro-1-(9-anthryl)ethanol
was added to each compound.29
(A) (-)-N1-Norphenserine (15)
(B) (+)-N1-Norphenserine (16)
(C) (+)-N1-Norphenserine (17).

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HETEROCYCLES, Vol. 61, 2003
Table 2. (-)-3aS-N1-Norphenserine (15), (+)-3aR-N1-Norphenserine (16) and N1-Norphenserine (17):
Melting Point, Optical Rotation, and Their Inhibitory Activity Versus Human Erythrocyte AChE and
Human Plasma BChE.
IC50 IC50
(nM)AChE (nM)BChE Selectivity
Compound
mp
[α]D
Fumarate of
(-)-N1-Norphenserine (15) 187-188oC -44.4o (c=0.3 EtOH)
8.1 + 0.7
69 + 24
54 + 4.6
7-fold AChE
-38.6o (c=0.4 DMSO)
Fumarate of
(+)-N1-Norphenserine (16) 187-188oC +44.4o (c=0.3 EtOH)
5895 + 830 86-fold AChE
177 + 37 11-fold AChE
+38.6o (c=0.5 DMSO)
Fumarate of
(+)-N1-Norphenserine (17) 191-192oC
none
18.5 + 0.3
activity comparable to several currently used anticholinesterases (e.g., galantamine, Reminyl, IC50: AChE
800 + 60 nM BChE 7,300 nM; huperzine A AChE 47 + 22 nM, BChE >10,000 nM; and tacrine:
ibid).1,2,5,12 In light of the AChE activity of (+)-N1-norphenserine, its inactivity against BChE is
unexpected, particularly as the (-)-enantiomer (15) has reasonable BChE potency. The mechanisms
underpinning this are one focus of our current studies and likely relate to the 3-dimensional interaction
between the compounds and their binding domains within the cholinesterase proteins.30 This occurs
within a narrow gorge of approximately 20 Å depth that extends into the center of these highly
homologous (65%) enzymes.2,4,6 The N1 position of (-)-physostigmine (1) and analogues interfaces with
the choline binding site within both AChE and BChE and binding likely involves both hydrophobic
interactions and hydrogen bonding.2 Minor differences in the amino acid sequence that define the
differential three-dimensional structures of both enzymes, particularly in the choline binding area, where
the binding pocket is known to be physically restrictive,2 likely account for the ability of the less size-
constrained (-)- and (+)-N1-norphenserine (15, 16) to bind versus active (-)-phenserine (2) and inactive
(+)- phenserine (10). The activity of the racemate (17) which demonstrated approximately half the
potency of the (-)-enantiomer (15), is in accord with expectations. Compounds (15 and 16) were
additionally assessed for activity to reduce extracellular levels of APP by previously published
methodology.15,16,31 One of the critical hallmarks of Alzheimer’s disease is the presence of extraneuronal
senile plaques whose core constituent is amyloid β–peptide,19 a toxic peptide that is generated from APP
processing.19,32-34 A reduction in the level of amyloid β–peptide in the brain of afflicted individuals is a

HETEROCYCLES, Vol. 61, 2003
537
primary focus in the development of drugs to halt or slow the course of neurodegeneration in Alzheimer’s
disease.32-34 Our previous studies have demonstrated that this can be achieved by lowering APP.15 As
illustrated in Figure 3, both 15 and 16, similar to our prior studies with phenserine (2, 10),15,31 decreased
secreted APP levels in cultured human neuroblastoma cells; a widely used model of human neural cells.15-
18,31
We predict that the mechanism underpinning this accomplishment is unrelated to the
anticholinesterase action of 15 and 16, and, as recently demonstrated with phenserine (2, 10),15 likely
relates to interaction in the post-transcriptional events controlling APP synthesis.
Figure 3: Compound (15) and (16) treatment of SH-SY-5Y human neuroblastoma cells (10 and 50 µM,
16 h) decreased APP protein levels versus untreated controls (CNT) as assessed by Western blot analysis
probed by an N-terminal anti-APP antibody (mAb 22C11, Roche Molecular Biochemicals). Two high
molecular mass bands were observed that corresponded to alternate forms of APP (100-125 kDa).
kDa
-250
-148
- 98
- 64
15 16 CNT 15 16
10 µM 50 µM
In conclusion, the optically pure enantiomers of N1-norphenserine (15, 16), together with racemate (17),
were synthesized and demonstrated potent AChE inhibitory action that was unexpected for the (+)-
enantiomer (16). Both enantiomers (15, 16) additionally lowered APP levels in a well-established
neuronal cell culture model and, together with racemate (17) that can be inexpensively synthesized, may
warrant further assessment as experimental therapeutics for Alzheimer’s disease.
ACKNOWLEDGEMENT
The authors are indebted to Dr. Amy Newman, Medicinal Chemistry Section, National Institute on Drug
Abuse, NIH, for use of NMR and polarimeter equipment. D.K.L. is
supported in part by NIH grants (AG18379 and AG18884).
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1
29. Partially expanded H-NMR (CDCl3) (Varian 400) (δ 1.00 - δ 3.00) spectrum with an equivalent of
chemical shift reagent (S)-(+)-2,2,2-triflioro-1-(9-anthryl)ethanol):

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Compound (15), δ 1.285 (s, 3H, 3aS-CH3), 2.685 (s, 3H, N8-CH3)
Compound (16), δ 1.223 (s, 3H, 3aR-CH3), 2.518 (s, 3H, N8-CH3); )
Compound (17), δ 1.258 (s, 1 and 1/2H, 3aR-CH3), 1.278 (s, 1 and 1/2 H, 3aS-CH3), 2.595 (s, 1 and
1/2 H, N8-CH3), 2.665 (s, 1 and 1/2 H, N8-CH3).
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