Current Medicinal Chemistry, 2000, 7, 355-374 355

Huperzine A, A Potential Therapeutic Agent for Treatment of

Alzheimer’s Disease

D.L. Bai*, X.C. Tang and X.C. He

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 294 Taiyuan

Road, Shanghai 200031, China

Abstract: HupA is a potent, reversible and selective inhibitor of AChE with a

rapid absorption and penetration into the brain in animal tests. It exhibits
memory-enhancing activities in animal and clinical trials. Compared to tacrine
and donepezil, HupA possesses a longer duration of action and higher
therapeutic index, and the peripheral cholinergic side effects are minimal at
therapeutic doses. This review article deals with a comprehensive survey of
the progress in chemical and pharmacological studies of HupA including the isolation and
structure elucidation, pharmacological actions, total synthesis, SAR studies and the future
development of HupA.

Recently, it has been reported that HupA could reduce neuronal cell death caused by
glutamate. The dual bio-activities of HupA would further enhance its value and potentiality as the
therapeutic agent for Alzheimer’s disease.

Introduction Most recently, it was reported that HupA could

In recent decades, some few of bio-active
ingredients and promising lead compounds have been
discovered from traditional Chinese medicinal plants.
artemisinin and huperzine A (1 , HupA) are the most
famous ones among them [1].
Alzheimer’s disease (AD) is one of the major
diseases affecting the elderly people in the world. The
disease is characterized by a gradual and progressive
memory impairment. There is considerable evidence
that the memory deficits are due to the impairment of
cholinergic neurotransmission in the central nervous
system. Therefore, the cholinergic enhancement
strategy has been the major efforts to palliate the
cognitive symptoms. The well-known naturally
occurring acetylcholinesterase (AChE) inhibitors
physostigmine and galanthamine have already shown
their utilities in the symptomatic treatment of AD.
Pharmacological studies demonstrated that HupA is
a potent, selective and reversible inhibitor of AChE,
and showed memory-enhancing effects on a range of
behavioral models in animals. Now, it has been
approved and clinically used as a palliative agent for AD
in China.
*Address correspondence to this author at the Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, 294 Taiyuan Road,
Shanghai 200031, China
reduce neuronal cell death caused by glutamate. The
pharmacologically dual actions would further enhance
the value of HupA as the therapeutic agent for the
treatment of AD.
A number of brief accounts and reviews dealing with
HupA covering the period 1986-1993 by Kozikowiski
[2,3], Ayer [4], Ji [5] and Tang [6] have been published.
In this review, we would like to comprehensively survey
the progress of chemical and pharmacological studies
of HupA, and the literature in Chemical Abstracts is
covered up to the end of 1998.
Isolation and Structure Elucidation
The new Lycopodium alkaloid HupA was isolated
from Huperzia serrata (Thunb) Trev. by Liu and his co-
worker in the early 1980’s. This club moss was used as
a folk medicine in China for the treatment of contusion,
strain, swelling and schizophrenia.
The dried powdered herb was extracted with 95%
ethanol and the crude alkaloids were extracted with 1%
aqueous solution of sodium hydroxide. The alkali
solution was then neutralized with hydrochloric acid
and brought back to pH>10 with concentrated
ammonia. The aqueous solution was extracted with
chloroform and the residue of the phenolic alkaloids
from the extracts was chromatographed on basic silica
gel column eluted with chloroform-methanol. The

0929-8673/00 $19.00+.00 © 2000 Bentham Science Publishers B.V.

356 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

Me

7 H
8
Huperzine A
9 10 N
Me 11 1
O [5R-(5α,9β,11E)]-5-amino-11-ethlidene-5,6,9,10-tetrahydro-
5
7-methyl-5,9-methanocycloocta [b] pyridin-2(1H)-one
13 NH 2
4 3

crude HupA was crystallized from acetone to give HupA
d
with a mp 229-30C and [α]25 –150.4 (c 0.498, MeOH).
The average yield of HupA in plants is 0.011% [7, 8, 9].
The structure and absolute configuration of HupA
were determined on the basis of NMR, IR, UV, CD data
and chemical transformations, and it was further
confirmed by X-ray crystallographic analysis [10]. The
structure of HupA is very similar in many aspects to that
reported for selagine and isoselagine. Reinvestigation
of these structures has revealed that the earlier
structural assignments for selagine and isoselagine are
incorrect. In fact, they are identical with HupA [11].
donepezil. HupA has the highest specificity for AChE.
A Lineweaver-Burke plot for HupA indicated a pattern
of AChE inhibition of the mixed competitive type, as
the intersection of the lines occurred in the second
quadrant [13,15]. The Ki value of HupA was 24.9 nM.
HupA was about 4-fold and 9-fold as potent as tacrine
and galanthamine, respectively, and was about 2-fold
less potent compared with donepezil [15]. The AChE
activity did not exhibit progressive decrease with the
prolongation of incubation with HupA in vitro, and the
AChE activity recovered to 94% of the control after 5
times washing, indicating the inhibitory manner of
HupA was reversible [13,14].

Significant inhibition of the brain AChE activity was

Pharmacological Actions
Effects on Cholinesterase Activity
The cholinesterase (ChE) inhibition of HupA was
evaluated in vitro and in vivo using
spectrophotometric method [12] with slight
modification. HupA inhibited the activity of AChE in the
rat cortex with the effect being initiated at 10 nM. The
concentration of inhibitor yielding 50% inhibition of
enzyme activity (IC50) of HupA on AChE and
butyrylcholinesterase (BuChE) compared with other
ChE inhibitors are listed in Table 1 . The inhibition of
AChE activity induced by HupA was more potent than
that of tacrine and galanthamine, but less pronounced
than that of donepezil in vitro [13,14,15]. In contrast,
HupA inhibited BuChE at much higher concentration
than needed for inhibition of AChE compared with
demonstrated in rat following administration of HupA at
doses of 0.12 - 0.5 mg/kg in a dose-dependent
manner[16-19]. In contrast to the AChE inhibition in
vitro, the relative inhibitory effect of oral HupA on cortex
AChE was found to be about 24-fold and 180-fold, on
a an equimolar basis, potent than donepezil and tacrine,
respectively. [18] HupA injected intraperitoneally
exerted similar efficacy of AChE inhibition in rats as
observed following oral administration, while tacrine
and donepezil resulted in a greater inhibition not only
on AChE activity, but also on serum BuChE [18]. The
inhibitory potency of HupA on brain AChE was less
than that of donepezil after the intraventricular
injection[19]. These findings indicated that HupA has
higher bio-availability and penetrates the blood brain-
barrier easier than that of donepezil and tacrine.
Maximal AChE inhibition in rat cortex and whole brain

Table 1. Anticholinesterase Effects of Huperzine A, Donepezil, Tacrine and Galanthamin In vitro

IC50 µM

Cholinesterase inhibitors AChE (rat cortex) BuChE (rat serum) Inhibitory pattern Ki * (nM)

Huperzine A 0.082 74.43 mixed competition 24.9

Donepezil 0.010 5.01 noncompetition 12.5

Tacrine 0.093 0.074 noncompetition 105.0

Galanthamine 1.995 12.59 competition 210.0

*assayed with rat erythrocyte membrane AChE.

Treatment of Alzheimer’s Disease
reached at 30 - 60 min and maintained for 360 min
following administration of HupA (Fig.1 ) [16,17,18].
Repeated doses of HupA showed no significant
difference on the AChE inhibition as compared to that
of single dose, indicating no tolerance to HupA
occurred [18,20].
Fig. (1). Time course of ChE inhibition following oral
administration of HupA (1.5 µmol/kg), donepezil (16
µmol/kg), and tacrine (120 µmol/kg) in rats. Values are
expressed as % inhibition (vs saline control). n = 4 – 6.
Effects on Neurotransmitter Level
HupA caused a significant increase in acetylcholine
(ACh) level in rat brain. The rats treated with HupA at
doses of 0.3, 0.5 or 2 mg/kg increased ACh levels for
about 6 hr after administration [16,17,21]. HupA
produced a more prolonged increase of ACh levels in
whole brain than that of tacrine, heptylphysostigmine,
physostigmine and metrifonate, respectively[22].
There was considerable regional selectivity in the
degree of ACh elevation after HupA administration, the
maximal increase was seen at 60 min in frontal and
Current Medicinal Chemistry, 2000, Vol. 7, No. 3 357
parietal cortex [16,17,18]. Considering that ACh level is
particularly low in the cerebral cortex of patients with
AD, this particularly regional specificity produced by
HupA may constitute a therapeutic advantage. HupA
also caused significant increase of brain
norepinephrine and dopamine levels following either
systemic administration or local administration through
the microdialysis [21]. These effects may involved in
memory improvement of HupA, since there was
evidence of interaction between cholinergic and
monoaminergic system in the control of cognition [23].
Effects on Brain Receptor
Pretreatment of cultured brain neurons with HupA
(100 µM) reduced neuronal cell death caused by toxic
levels of glutamate, and reduced glutamate-induced
calcium mobilization, but did not affect the increase in
intracellular free calcium channel induced by exposure
to high KCl or a calcium activator Bay-K-8644[24],
suggesting that HupA might act on glutamate receptors
to exert its neuroprotective effects .
HupA (0.1 - 300 µM) reversibly inhibited NMDA (100
µM)-induced current in acutely dissociated
hippocampus pyramidal neurons in a concentration-
dependent manner with IC50 of 0.49 µM. The results in
binding assay clearly demonstrated that HupA acted
directly on NMDA receptor [25].
The studies on displacement of 3H-QNB and 3H-
(-)nicotine specific binding showed that HupA generally
had little direct effect on cholinergic receptors as
compared to other ChE inhibitors [16,22].
Enhancement of Learning and Memory
HupA has been found to be an effective cognitive
enhancer in a number of different animal species.
Beneficial effects were seen not only in intact adult
rodents [14], aged rodents and monkeys (Fig.2 )
[26,27], but also in cognitively impaired rodents (Fig.3 )
and monkeys [26-31]. The duration of improving
effects with oral HupA on learning and memory
retention were longer than that of physostigmine,
galanthamine and tacrine, respectively[32]. The
improving effect on memory of aged monkey remained
for about 24 hr after a single injection of HupA 10 µg/kg
[27]. The magnitudes of improving effects produced by
oral or intraperitoneal administration of HupA at a dose
of 0.2 mg/kg were nearly equivalent, indicating HupA
had a higher efficacy with oral route than that of tacrine
and donepezil, respectively[29]. HupA induced no
significant tolerance in the cognitive improvement after
oral once daily for 8 d [31]. HupA did not show any
significant affinity for muscarinic and nicotinic receptors
[16], was devoid of pre- and post-synaptic actions [33],
358 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

as well as was devoid of inhibition on choline Total Synthesis

acetyltransferase activity [17,20], indicating the
improving effect of HupA on cognition was due
primarily to its inhibition of brain AChE.
The pronounced bioactivity of HupA and its low
yields in plants have provided the impetus for renewed
interests in the synthesis of this target molecule.
Several groups have devoted intensive efforts to the
chemical synthesis of HupA. HupA possesses the rigid
bicyclo [3.3.1]nonene skeleton and fused pyridone
ring. The retro-synthetic analysis of this molecule is
shown in Scheme 1 .
tandem Michael-aldol
reaction and dehydration
Me

Wittig Me
H
olefination
N H
Me O
N
NH 2 O
Curtius O
1 rearrangement COOMe
O
Fig. (2). Effects of huperzine A in aged monkeys. Saline or
Me
huperzine A was administered intramuscularly 20 min before
H
testing. Huperzine A produced a dose-related improvement in N OMe
the delayed response performance of aged monkeys (n = 4).
Values represent mean + S.E.M. number of trials correct out N OMe
of a possible 30 trials. O

Clinical trials have been demonstrated that HupA
significantly improves cognition in junior middle school
students complaining of memory inadequacy [34],
patients suffering from aged related memory
dysfunction [35] or AD [36]. Peripheral cholinergic side
effects at cognitively efficacious dose of HupA are not
detected.
H
O O OMe
COOMe
2 3
Scheme 1.
The β−keto ester functionalities in key intermediate
2 would provide not only an activating group for the
construction of three-carbon bridge by tandem

Fig. (3). Effects of ChE inhibitors on AF64A-induced working memory deficit in a partially baited radial maze paradigm in rats.
** P < 0.01 vs non-lesioned group, ++ P < 0.01 vs AF64A-lesioned, saline drug control.
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 359

Me O
Me
R
R2 O
O
NH
Me O
O O O
NH 2 R1 R
1 4 5
Scheme 2.

Michael-aldol reaction, but also the latent groups for the
formation of both ethylidene substitution by Wittig
reaction and amino group via Curtius rearrangement of
the acid from ester 2 . Endocyclic double bond may be
formed by dehydration of the aldol product. The
pyridone ring could be protected as a stable methoxy
pyridine. Based on the synthetic strategy in Scheme 1 ,
the total synthesis of racemic and natural HupA was
accomplished starting from β−keto ester 2 which
usually occurs in enol form 3 .
Another approach to HupA is to construct the
functionalized bicyclo [3.3.1]nonane derivative 4 first,
further employed to prepare some pyridone ring
opening analogues. However, there is no report on the
total synthesis of HupA itself and the preparation of the
desired heterocyclic analogues using functionalized
bicyclo [3.3.1]nonane derivatives as key intermediates.
Synthesis of β−Keto Ester
A number of methods have been developed for the
preparation of the key intermediate, β−keto ester 2 ,
from different starting materials.

Me Me Me
CH 2
HO
O O
O O

O O
Me Me Me
O
NHCOOMe COOMe SOPh COOMe
6 7 8 9

followed by the formation of the fused pyridone ring From 1,4-Cyclohexanedione Mono-ethylene
(Scheme 2 ). Compound 4 could be obtained from α− Ketal 10
carbomethoxy- or phenylsulfinyl cyclohexane-1,4-
dione monoketal 5 . A couple of bicyclo [3.3.1]nonane The fused 2−pyridone l2 was prepared by heating
or nonene compounds 6, 7, 8 and 9 were prepared the enamine1 1 derived from mono-ethylene ketal 1 0
[37, 38, 39, 40]. These intermediates have been with acrylamide followed by hydrolysis. The resulting

H
N OMe
O O HC CCOOCH 3 N O Ag2CO3
O
NH3 / MeOH O MeI
O
O
13
O 10 12
I, H+ 2, (MeO) 2 CO
KH
pyrrolifdine
p-TsOH HC CCONH2
N OMe
N

O O
11
O COOMe
2
Scheme 3.
360 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

Me N O Me N OMe N OMe
1, MeI/Ag2 CO3 1, PhLi/HCHO ClCH2

2, LAH 2, SOCl2
EtOOC HOCH2 ClCH2
14
MeO 2C N OMe N OMe
1, NaCN/DMSO
NaH
2, HCl/MeOH MeO 2C
THF O

COOMe
15 O3 /NaOH 2
O Br
N N OMe
Me N OMe
N O Me N OMe
1, LDA/AllyIBr
Br

2, n-BuLi/CuI/AllyIBr
Br
16 17

Scheme 4.

mixture of unsaturated lactams was N-benzylated and
then dehydrogenated by α−selenation and oxidative
elimination, yielding N-benzylpyridone which was
debenzylated by hydrogenolysis to afford pyridone
1 2 . The pyridone ring in 1 2 proved to be sensitive in
subsequent steps, and was protected by conversion to
methoxypyridine 1 3 with methyl iodide and silver
carbonate. Hydrolysis of the ketal and α−
carbomethoxylation with potassium hydride and
dimethyl carbonate produced β− keto ester 2 [41]. This
route was somewhat laborious. Therefore, Kozikowski
and his coworkers examined alternate routes to 2−
pyridone l2 and found that a one-pot, three-
component condensation was the best one. By simply
admixing mono-ethylene ketal 10 and methyl
propiolate in ammonia-saturated methanol in a Parr
reactor at 100 C, 2−pyridone l2 was obtained in a yield
of 70% (Scheme 3 ) [42]. Compound 1 2 was also
produced by the reaction of enamine 1 1 and
propynamide in THF or DMF at 80C in a yield of 75-80%
[43].
From Pyridine Derivatives
Ji group reported that β−keto ester 2 was obtained
from ethyl 2-methyl-6-hydroxy-nicotinate 1 4 . Selective
O-methylation and reduction of 1 4 afforded alcohol,
which reacted with phenyllithium and formaldehyde
yielding diol followed by conversion of diol into
dichloride. Treatment of dichloride with sodium cyanide
and methanolysis of the resulting dicyanide gave
diester 1 5 . Dieckmann condensation of 1 5 afforded
the key intermediate 2 . The overall yield was 39% via 7
step sequence of reactions (Scheme 4 ) [44]. The key
step in this approach is to construct two appending
carbomethoxy side chains. 2-Methoxy-6-
methylpyridine 1 6 was also used as a starting material
for the preparation of diester 1 5 . Bromination of 1 6
with 1, 3-dibromo-5.5-dimethyl hydantoin gave desired
bromide. Twice allylation of bromide formed compound
1 7 . An oxidative cleavage of two double bonds in 1 7
followed by esterification gave rise to diester 1 5 in an
overall yield of 41% (Scheme 4 ) [45].
From Dimethyl 4-oxopimelate
Yang et al developed a four step approach to β−keto
ester 2 from readily available dimethyl 4-oxopimelate
1 8 (Scheme 5 ). The enamine 1 9 was directly
condensed with propynamide to give pyridone 2 0 with
two desirable carbomethoxy side chains. After O-
methylation, Dieckmann condensation of diester 1 5
gave 2 in 39% overall yield [46].

O
pyrrolidine HC CCONH2
N
MeOOC COOMe TiCl4
18 MeOOC COOMe
19

H MeO 2C N OMe N OMe

MeO 2C N O Ag2CO3 NaH

MeO 2C MeI MeO 2C

O
20 15
COOMe
2
Scheme 5.
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 361

Me Me
RO
N OMe 1, methacrolein AcONa
MeONa AcOH
N N
2, MsCl OMe 130o C OMe
O Et3N O O
COOMe COOMe
COOMe
21 R = H 23
2 22 R = Ms

Me Me

Ph3p +C2 H5 Br-

BuLi KOH N
N
Me OMe Me OMe
MeOH

COOMe COOH
24 25

Me
(PhO)2 P(O)N 3 1, Me3SiCl, Nal
CH3 CN
EtOH (±)-1
N 2, KOH, toluene
Me OMe 18-crown-6 ether

NHCO 2Et
26
Scheme 6.

Synthesis of Racemic HupA and only E-isomer could be hydrolyzed to acid 2 5 .

Total synthesis of (±)-HupA was first accomplished
independently by both Ji and Kozikowski groups in
1989. Almost the same synthetic strategy was adopted
starting from β−keto ester 2 (Scheme 1 ). The earlier
works of Kozikowski group have been surveyed [2-4].
The following is the synthetic approach to (±)-HupA
reported by Ji et al. [44,47] (Scheme 6 ).
The tandem Michael-aldol reaction of β−keto ester 2
and methacrolein was catalyzed with sodium methoxide
in methanol to generate stereoisomeric aldol mixture
2 1 , which was converted into corresponding
mesylates 2 2 . Endocyclic alkene 2 3 was given in 30%
low yield, possibly only axial mesyl group could be
eliminated. Wittig reaction of 2 3 produced a Z- and E-
alkene mixture 2 4 in a ratio of 19:1 to 17:3, which was
treated with potassium hydroxide in refluxing methanol
Curtius rearrangement of acid 2 5 gave urethane 2 6 .
(±)-HupA was obtained after deprotection.
The yields of E-isomer of 2 4 was greatly improved
by Kozikowski et al. [48] through the isomerization of
the mixture by heating with thiophenol and 2,2’-
azobisisobutyronitrile, giving E- and Z- alkene in 9:1
ratio. Furthermore, in the study of pyrimidone
analogues of HupA, Kozikowski et al. utilized the
Danheiser methodology to obtain solely E-olefine
(Scheme 7 ) [49]. Ketone 2 7 was transformed into a
sterically less crowded β−lactone 2 8 with complete
stereoselectivity by the addition of the anion of
thiophenol ester of propionic acid. Upon heating of β−
lactone 2 8 in the presence of silica gel in toluene,
[2+2] cycloreversion reaction occurred to afford the
desired E-olefinic product 2 9 in a total yield of 70%.
This stereoselective construction of ethylidene moiety

CH 2 CH 2 CH 2

EtC(O)SPh Me
N LDA N SiO2 N
OMe H OMe Me OMe
O toluene
O O
COOMe COOMe COOMe

27 28 29
Scheme 7.
362 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

R1

R
N
30 R = OH, R1 = H
N OMe
31 R = H, R1 = OH
OMe 33 R = OAc, R1 = H O
O COOMe
COOMe
32

via [2+2] cycloreversion reaction may represent a
practical method instead of Wittig reaction and double
bond isomerization in the synthesis of HupA.
The low yield of endocyclic alkene 2 3 is related to
the configurations of mesylates 2 2 , since the mesyloxy
group and the adjacent hydrogen in 2 2 may not adopt
a trans-diaxial orientation for base-induced elimination
to alkene2 3 [38]. In the preparation of HupA
analogues by author’s group, acrolein was used in
Michael-aldol reaction, two aldol products were
separated by column chromatography to produce axial
hydroxyl compound 3 0 and equatorial hydroxyl
compound 3 1 . They were converted into mesylates,
which were eliminated in acetic acid with sodium
malonate at 130 C respectively. The expected product
3 2 was only formed from axial mesylate. However, SN2
reaction occurred with equatorial mesylate to give axial
acetate 3 3 [50]. Now it is evident that only axial
mesylate in the mixture of 2 2 could be eliminated to
form olefine 2 3 , and the equatorial mesylate would be
substituted in acetic acid to form axial acetate.
the mixture of mesylates of resultant aldol adducts
obtained by this reaction in the presence of different
bases was subjected to the elimination respectively,
the endocyclic olefine was obtained in a wide range of
19-68% yields. The big difference in yields probably
depends upon the different ratio of four diastereomers
in aldol adducts formed under different reaction
conditions [51].
Based on the elimination mechanism, Yang et al.
reported that when mesylates 2 2 were treated with
1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) and
sodium iodide in DMF at 100-110C, the yields of the
desired alkene 2 3 was increased to 47-57%[52].
To avoid the low yielding step of elimination in
Scheme 6 , a palladium-catalyzed bicycloannulation of
1-carbomethoxy-2-tetralone with a bifunctional allylic
agent, 2-methylene-1, 3-propanediol diacetate 3 4 was
reported by Gravel et al. [53]. The regioselective
double bond migration completed the construction of
three-carbon bridge in 70-80 yields.

In the course of the studies of stereoselective On the basis of this report, the palladium-catalyzed
synthesis of HupA via Michael-aldol reaction, a variety bicycloannulation of β−keto ester 2 using
of chiral bases were tested by Terashima et al.. When tetramethylguanidine (TMG) as a base and 2-

CH 2
CH 2
CH 2
N OMe
AcO OAc
34 N
N
Me OMe
OMe
O Pd(OAc) 2, Ph3 P
TMG O
COOMe NH 2
COOMe
2 27 35

TfOH TfOH

Me

(±)-1

N
OMe
O
COOMe
23
Scheme 8.
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 363

Me Me

1, Et3N
(S)-MTPA-Cl 1, Jones oxidation
N MeO N
2, column MeO 2, MeI, DBU
chromatography CH 3CN
3, LAH
Me
CH 2OH CH 2OH
Me

(Z)-(±)-36 (Z)-36

Me
Me

1, olefin Me
isomerization
N H
N 2, hydrolysis MeO Me
MeO
N
O Me
Me COOH
COOMe
(Z)-37 NH 2
(E) -25

(+)-1
Scheme 9.

methylene-1,3-propanediol diacetate 3 4 as the bis- via Stereoselective Michael-aldol Reaction

electrophile in the presence of tetrakis
(triphenylphosphine)-palladium(0) in refluxing dioxane
was first achieved by Kozikowski et al. (Scheme 8 ). The
resulting methylene-bridged compound 27 was in
92% yield. The double bond migration was pursued
with triflic acid in dioxane at 93C, affording endocyclic
olefine 2 3 in 90% yield. Since the double bond
migration could be performed in last step, an isomer of
HupA, compound 3 5 , was obtained. The overall yield
of (±)-HupA was 40% from β−keto ester 2 [54, 55].
Synthesis of Natural (-)-HupA and
Enantiomer
For the preparation of natural (-) HupA and its
enantiomeric (+) HupA, three methodologies have
been developed by now.
via Diastereomeric Separation
Kozikowski et al. reported the diastereomeric
separation to obtain (+)-HupA [56] (Scheme 9 ). Z - (±) -
Alcohol 36 reacted with (S)-α-methoxy-α-
(trifluoromethyl) phenylacetyl chloride (MTPA-Cl) to
form diastereomeric esters, which were separated by
column chromatography. The resulting optically active
ester was reduced with lithium aluminium hydride (LAH)
to give 3 6 . Jones oxidation of 3 6 followed by
esterification afforded ester 3 7 , which was isomerized
to E-olefine and then hydrolyzed to optically pure E-
acid 2 5 . E-Acid 2 5 was further transformed into (+)-
HupA according to the reaction sequence as shown in
Scheme 6 .
Using Chiral Auxiliary
The first molecular modeling-based synthesis of
natural (-)-HupA was achieved by Kozikowski group in
1991 (Scheme 1 0 ). β-Keto ester 2 was transesterified
with (-)-8-phenylmenthol, giving ester 3 8 , which was an
effective substrate for the stereoselective Michael-aldol
addition of methacrolein in the presence of TMG at –20
C. After dehydration, the ratio of two diastereomers was
9:1 with the major isomer possessing the desired
absolute configuration. The chromatographically
its separable major compound 3 9 was subjected to
further transformation. Reduction of ester 4 0 with LAH
and re-oxidation of the resulting alcohol gave (+)- acid
2 5 . Subsequent Curtius rearrangement of (+)-2 5 and
deprotection led to the natural (-)-HupA [56].
Using Chiral Catalyst
Yang et al. first reported the stereoselective
Michael-aldol reaction of β-ketoester 2 catalyzed by (-)-
quinine at room temperature for 10 days. After
elimination of adducts, alkene 2 3 was obtained in 52%
ee. The final product was afforded in 62.8% optical
purity in comparison with the natural (-)-HupA [52].
Chiral guanidines 4 1 and 4 2 were also tested for the
stereoselective Michael-aldol addition of β-keto ester 2
with methacrolein. Disappointedly, the ee values of the
products were insignificant [57].
Terashima et al. also reported the (-)-cinchonidine
promoted stereoselective Michael-aldol reaction of β-
ketoester 2 with methacrolein. The reaction took place
364 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

Ph
Me
N OMe N OMe
1, Michael-aldol reaction,
R*= R*OH TMG

O O N
H+ 2, dehydration, OMe
3, chromatographic
COOMe H separation O
O OR*
COOR*
Me
2
38 39
Me
Me

1, Wittig reaction 1, LAH

N
OMe 2, Jones reagent N
2, isomerization Me OMe (-)-1
Me
COOR*
COOH
40
(+)-25

Scheme 10.

smoothly at –10 C for 10.5 days . After dehydration of
the adducts, endo-olefine (+)-2 3 was obtained in 64%
ee. The enantiomerically pure (+)-2 3 was readily
obtained by recrystallization of the product from
hexane, and converted into (-)-HupA according to the
reaction sequence reported for (±)-HupA. Enantiomer
(-)-2 3 was similarly prepared in 61% ee using (+)-
cinchonine as chiral catalyst [51, 58].
via Enantioselective Palladium-catalyzed
Bicycloannulation
Terashima and his coworkers investigated the
bicycloannulation of the β-keto ester 2 with 2-
methylene-1,3-propanediol diacetate 34 in the
presence of palladium catalyst with chiral ligands
(Scheme 1 1 ). They first examined various
bisphosphines as chiral ligands of palladium, but no
promising results were observed. When a couple of
ferrocenylphosphine ligands carrying appropriate linker
chains were tested, they found the most effective
ligand (R)-(S)-4 3 . The reaction of 2 and 3 4 was carried
out in the presence of palladium diacetate, the chiral
ligand (R)-(S)-4 3 and TMG in 1,2-dimethoxyethane at –
30C followed by gradual warming to 15C. The bridged
compound (+)-27 was yielded in 64% ee and 92%
yield. The enantiomer (-)-2 7 with 63% ee was also
afforded using the enantiomeric ligand (S)-(R) –4 3 .
Both enantiomers were converted to the endocyclic
olefines 2 3 by double bond migration with triflic acid.
Enantiomeric enrichment was performed by
recrystallization of optically crude product from hexane
to give optically pure (+)-2 3 and (-)-2 3 respectively.
Following the reaction steps reported for the
preparation of (±)-HupA, the natural (-)-HupA and
unnatural (+)-HupA were finally furnished [51,59].
The author’s group has been also interested in the
synthesis of (-)-HupA by palladium-catalyzed
asymmetric bicycloannulation. Different bifunctional
allylic esters 4 4 and chiral ligands were tested. A 52%
ee of 2 7 induced by (S)-2,2’-bis(diphenylphosphino)-
1,1’- binaphthyl (BINAP) was observed [60]. More
recently, a few of new chiral ferrocenylphosphine
ligands have been developed for the allylic
bicycloannulation. Compound (+)-2 7 has been

NH
OH
R R
N
N N RO OR1
Me
44
Ph Ph PPh2
Fe
R= R1
= COPh
PPh2
41 R = Me (R)-(S)-43 R = Ac, R1 = CO2 Me
42 R = 2,4,6-trimethyl-benzyl-
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 365

Me
CH 2
CH 2
N OMe
AcO OAc
1, TfOH
34 N N
O OMe OMe
2, recryst.
Pd(OAc)2, TMG
O O
COOMe (R)-(S)-43
COOMe COOMe

2
(+)-27 (+)-23

(-)-1

Scheme 11.

obtained in 80% ee and 90% chemical yield. Optically
pure (+)-2 3 was furnished in 70% yield after
recrystallization. The synthetic (-)-HupA obtained from
(+)-2 3 possesses the same anti-AChE potency as
natural HupA [61].
Studies on the Structure-activity
Relationships
The stereoselectivities of the inhibition of rat cortical
AChE by two enantiomers of Hup A were first
determined by McKinney et al. [62]. The natural (-)-Hup
A is the more potent one with a Ki value of 8nM, and
(+)-HupA is 38-fold less potent with a Ki value of 300
nM. Racemic Hup A is about 2-fold less potent than (-)-
Hup A. The comparison of the inhibitory effects of two
enantiomers and racemate of HupA on AChE was
made by Tang et al. [17]. The (±)-Hup A is 3 times less
potent than (-)-HupA in vitro using rat hippocampal
crude homogenates as the enzyme resource. The ratio
of potencies for (-), (±), and (+)-HupA is 70:3:1, which is
comparable to those reported by MicKinney et al.
As a promising lead compound, scientists have
been much interested in the chemical modification of
Hup A for the search of new analogues or derivatives,
which may possess high activity, longer duration of
action, less toxicity, and could be prepared by simpler
and efficient approaches as compared with HupA itself.
In recent 10 years, major efforts have been devoted to
the preparation of both structurally simplified analogues
and derivatives with the tricyclic skeleton of Hup A.
In comparison with the flexible chain structure of
ACh, there is no conformational isomers of HupA
because of the rigidity of the bridged ring structure.
However, the computer-generated superposition of
Hup A and ACh suggested that HupA possessed the
basic structural features of ACh. The amino nitrogen
atom in HupA would attract a proton in vivo to form a
positively charged center, it is as distant from the
carbonyl of the pyridone ring as the quaternary
nitrogen atom is from the ester carbonyl in ACh. It is
apparent that a reasonable structural similarity can be
found between the nitrogen, oxygen and carbonyl in
ACh with the corresponding amino nitrogen, nitrogen
and carbonyl in HupA.. Therefore, 5-aminomethyl-2
(1H)-pyridone part of HupA may constitute a
pharmacophoric moiety [1,48].
Structurally Simplified Analogues of HupA
Several types of the simplified analogues were
designed and prepared. All of them possess the
supposed pharmacophoric moiety of HupA.
5-Substituted aminomethyl-2(1H)-pyridones 4 5
[3,48,63,64], 5-substituted amino-5,6,7,8-
tetrahydroquinolinones 4 6 , 47 [63,64,65,66]and 5-
substituted aminoquinolinones 4 8 [67] were prepared
and tested successively. None of the conformationally

H R
N O N O

CH 2CH=CMe2
N N O
R3 N R = H, alkyl
R1 46a: Ar=3,4-dichlorophenyl
R R1 R = H, alkyl R1 = H, alkyl 46b: Ar=2-chlorophenyl
R2
R2 = H, alkyl, arylakyl 46c: Ar=3-chlorophenyl
R1 = H, alkyl 46
45 46d: Ar=4-hydroxyphenyl
R3 = H, alkyl NH(CH2) 2 Ar
366 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

R Me
H N O
N O R
Me
CH2
Me R2
R1
Me NH 2 NHR1
49a: R 1=R 2=H, R=OAc
47 48 49b: R1 =R2 =H, R=NH2
R = H, CH 2Ph 49c: R 1=R 2=Me, R=nicotinoyloxy
R1 = (CH 2) nAr 49d: R1 =H, R2 =C(O)Bu-n,
R=OCONMe2

more flexible aminomethypyridones 4 5 was found to
be an AChE inhibitor. Analogues 4 6 were designed as
simplified analogues of HupA without bridge moiety.
Among them compounds 4 6 a -d inhibited AChE in
vitro markedly, whereas they were less active than (±)-
HupA. Compound 4 6 a and 4 6 d also showed the
ability to reverse scopolamine-induced memory
impairment in mice. Because these compounds lack
the unsaturated three-carbon bridge of HupA, and
both the quinolinone nitrogen atom and the amino
group must be substituted, it is unlikely that they bind
to AChE in a similar fashion as HupA [65].
The single ring analogues 4 9 derived from HupA by
the removal of the bridge and the opening of pyridone
ring, and by the replacement of amide group with the
isosteric ester group as well were also reported [68].
The anti-AChE activities of these compounds were at
least 1000 times less active than (-)-Hup A.
In addition, several bridged ring analogues 50a-c
[40,37] derived from the opening of pyridone ring of
HupA were prepared, and the inhibitory activities of
5 0 a and 5 0 b dropped down dramatically. The
bioactivity of 5 0 c is not reported yet.

Compound 4 7 is derived from the removal of the
three-carbon bridge of HupA, and was at least 4727-
fold less active than (±)-HupA [66]. It clearly
demonstrates the necessity of the bridge ring in Hup A,
since compound 4 7 mimics the quinolinone portion of
HupA quite well. The aminoquinolinones 48 showed
no inhibitory activity at all.
All these structurally extensively simplified
analogues mentioned above were found to be inactive
or less active in the inhibition of AChE. It means that the
conformational constraints, hydrophobic binding, steric
and electrostatic fields provided by the unsaturated
bridge and fused pyridone ring in HupA must involve in
its AChE inhibitory activity.

Me
CH 2

OCONMe2
R
Me
CH2 NH 2
NMe2
50c
50a: R=nicotinoyloxy
50b: R=nicotinamido
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 367

Derivatives from Natural (-)-Hup A
Some dozens of derivatives using (-)-Hup A as the
starting material were prepared, i.e. alkylamino-,
acylamino-,1-alkyl-,3-bromo-,dihydro-and tetrahydro-
derivatives, and Schiff base of Hup A. (Fig. 4 )
AChE
All these optical active derivatives were tested for
their inhibitory activities of AChE from rat erythrocyte
membrane over a concentration range from 1 nmol. L-1
to 10 mmol. L-1. The IC50 for AChE by these
compounds revealed that most of them were less
active than (-)-HupA itself, except a few of Schiff bases
AChE BuChE
BuChE

NH 25.1 /
NH
O
O 0.0631 63.1
NH 2
NHCOCH2

NH NH
O 158 11000 O 1.58 56.2

CH 3
N
NHCH2
CH 3

CH 3
NH
N 0.631 200
O 316 11000 O
N

N CH 3 CH C3H7
CH 3

NH
O 1.58 /
NH
NH
O 0.2 15.8
NH 2
H2C
O

NH NH
O O 0.891 398
3.16 50.1
NH 2 NH
CH 2OH

CH 2 N

NH
HO CH 2
O 3160 11000

NHCOCH3

NH
1.00 100
O

NH
NHCH2
O 7.94 166
N
NH 2
Br
Fig. (4). IC50 (µM) of anti-ChE activities by (-)-HupA analogues on rat erythrocyte membrane AChE and rat serum BuChE.
368 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

5 1 . The anti-AChE activities and selectivities between ring skeleton and only to alter or remove certain atoms
AChE and BuChE of 5 1 a and 5 1 b are comparable to or groups in Hup A. The first example was a benzene
(-)-Hup A [69,70], Hydrogenation of the exocyclic isostere 5 2 , which was at least 1000-fold less potent
double bond or both exo- and endocyclic double than (-)-Hup A. (±)-Z-HupA 5 3 , a geometric isomer of
bonds in Hup A led to a diminution of anti-AChE HupA was 60-fold less potent than (-)-Hup A [71,72].
activity.
In order to make an evaluation of the contribution of
the endocyclic double bond, C-14 and C-12 methyl
Me groups to the activity of HupA, compounds 5 4 a -f and
Me
5 5 a -i were prepared by Kozikowski group and author’s
group, respectively [48,50,55]. Unfortunately, none of
NH these compounds is an effective AChE inhibitor. The
Me O dinor HupA 5 5 a and nor HupA 5 5 b are 3500- and 30-
Me fold less potent than (-)-HupA respectively. The
N=CHR ethylidene moiety modifiers 55c, 55g and 5 5 h were
NH 2
51a: R=C3 H7 much less active too, and 5 5 f showed a 40-fold lower
52 potency than (±) Hup A. The 12-phenyl 5 5 d ,
51b: R=
dimethylamino 5 5 e and aminomethyl 5 5 i analogues
were all poorly active.

Based on the above-mentioned results, it suggests

R1 Me
that the structural requirements for high anti-AChE
activity of Hup A appear to include a free amino group
or protected amino group which can be easily
NH NH
regenerated, two double bonds and a unsubstituted O O
nitrogen atom in pyridone ring. R2 Me

NR 2
NH 2
Analogues Bearing the Bridged Tricyclic 1
55a: R=R =R =H 2
Skeleton of Hup A 55i
55b: R=R 2=H, R 1 =Me
1 2
55c: R=H, R =Me, R =Et
Although a number of the chemical modification to 55d: R=H, R 1 = Ph, R2=Me
Hup A and the anti-AChE activities of these 55e: R=R1 =R2 =Me
compounds have been reported including alterations 55f: R=H, R 1=Me, R2 =CH2 OH
of amino group, three-carbon bridge, ethylidene side 55g: R=H, R 1 =Me, R2= CO2 Et
chain, and the endocyclic double bond, no analogues 55h: R=H, R 1 =Me, R2= CN
thus obtained has achieved the anti-AChE potency as
parent compound HupA. It is unlikely that a more
potent analogue could be found by means of the The demethylated and hydroxylated analogues
extensively structural simplification of HupA. 5 6 a , 56b [73], and 1-methyl pyridone analogues 5 7 a
and 5 7 b were much less effective on anti-AChE
R activity than (-)-HupA [74]. 1-Methyl substitution of
Me
HupA would impede the formation of H-bond of the
pyridone nitrogen atom to the protein residue. Actually,
1-methyl- (-)-HupA was less active by two order of
NH
NH
O
magnitude than (-)-HupA [75].
O R1

NH 2 HO R
NH 2
Me 54a: R=ax Me, R =Me 1
53
54b: R=eq Me, R 1=Me
NH
54c: R=H, R 1=Me NMe
O
1
54d: R=R =H O
CH2 R1
54e: R=ax NH2 , R1=Me
NH 2
54f: R=eq NH2, R 1=Me NH 2
56a: R=ax OH 57a: R=R1 =H
56b: R=eq OH
57b: R=Me, R1 =H
In the meantime, many attempts have been made in
57c: R=R1 =Me
the chemical modification of Hup A to retain the tricyclic
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 369

Me CH 2

NMe NH
O O
NH Me
O
Me

NH 2 NH 2
58 59 60

A cyclopropane bearing analogue 5 8 was
synthesized, but its biological activity is not reported
yet [76]. Diamine 5 9 and di-exo-double bonds
analogue 6 0 were 88- and 22-fold less potent than (±)-
HupA respectively [55].
The replacement of the pyridone ring in HupA by a
pyrimidone or other heterocyclic ring may lead to an
analogue having improved AChE inhibitory potency
due to its ability to form an additional H-bond.
Kozikowski group prepared compound 6 1 , 6 2 , the
surprising to find that they are less active than (±)-
HupA. However, it is unexpected that 6 1 is even more
less active than 6 2 . In fact, the structure of 6 1 is more
closely related to HupA. The studies on the pyrimidone
analogues revealed that even a minor alteration of the
HupA structure via the replacement of CH in pyridone
ring by N caused a major decrease in anti-AChE activity.
A thiazolone analogue 6 4 was ineffective either [78].
The phenol and catechol analogues of HupA,
compounds 6 5 a , 6 5 b , were recently reported by

Me Me
Me

NH NH
NH H2N H2N
O
O N
Me
N
N
NH 2
Me Me
61 62 63

pyrimidone analogues of both HupA and iso-HupA,
and 6 3 , the pyrazole analogues of iso-HupA as well
[49,77]. None of these analogues was found to be an
active AChE inhibitor. In view of the relatively dramatic
structural alterations in compound 6 2 and 6 3 , it is not
Kozikowski group [79]. Surprisingly, no AChE
inhibition of both compounds was observed.
It is well recognized that introduction of fluorine
atom into bio-active compounds frequently improves
their therapeutic profiles. Five fluorinated analogues

Me R1
Me

R
NH
NH Me R1 O
R
Me O
S NH 2 NH 2
NH 2
65a: R=H, R 1=OH
66a: R=Me, R1 =CH2F
64 65b: R=R 1=OH
66b: R=Me, R1 =CF3
66c: R=CF3 , R1 =Me
66d: R=CF 3, R 1=CF3
66e: R=CH2 F, R1=Me
370 Current Medicinal Chemistry, 2000, Vol. 7, No. 3
Me
N N
Me
NH 2
67
6 6 a - e were reported by Terashima group and Ji group
respectively [80,81,82,83,84]. However, all the
fluorinated analogues had the inhibitory activities
inferior to that of HupA.
Seventeen polycyclic compounds structurally
related to tacrine and HupA were prepared as the
hybrids of both molecules by Camps et al. [85]. Hybrid
compound 6 7 exhibited an AChE inhibitory activity
approximately 2.5 times lower than tacrine. The
configuration of the ethylidene group is critical, since
the Z-isomer of 6 7 showed a much lower activity.
Compound 6 8 a and 6 8 b derived from 6 7 by
elimination of the ethylidene unit were 2 and 4 times
more potent than tacrine respectively. Their inhibitory
activity was not directly compared with HupA. Camps et
al. [86] have also developed an enantioselective
approach to these molecules, but the bio-assay data of
the enantiomers of compound 6 8 a and 6 8 b have not
been reported yet.
Although quite a number of analogues and
derivatives of HupA have been successively reported
during last 10 years, but none of these compounds is
able to compete with natural (-)-HupA in its AChE
inhibitory activity until 1996.
Recently, in the continuation of structure-activity
relationships studies on HupA, Kozikowski and his
coworkers have identified several C-10 methyl
analogues that showed the similar or even better anti-
AChE activities than (±) HupA. 10.10-Dimethyl HupA
Table 2. Ki Values for the Inhibition of FBS AChE and Equine BuChE by HupA and Its
Analogues
Compound
(+)-HupA
ax Me 69c
eq Me 69b
dimethyl 69a
*dissociation constant
Bai et al.
R
NH 2
68a: R=Me
68a: R=Et
69a was obtained from 2.2-dimethyl-1.4-
cyclohexanedione mono-ethylene ketal via the
reaction sequence reported for the preparation of
HupA. Using AChE purified from FBS, the kinetic and
inhibition parameters demonstrated that 10.10-
dimethyl analogue of HupA was comparable in the
inhibitory activity to (±) HupA, but less active than
natural (-) HupA [87]
The C-10 axial methyl compound 6 9 c showed 8-
fold potency for AChE inhibition, and the equatorial
methyl compound 6 9 b is about 1.5-fold less active
than (±) HupA (Table 2). The axial alkyl groups larger
than methyl, for example, the ethyl analogue was 100-
fold less active than (±) HupA. The C-10 n-propyl
analogue was inactive at all [88]. The C-10 methyl HupA
was prepared from 2-methyl-1.4-cyclohexanedione
mono-ethylene ketal via the previously reported route
to HupA. The axial and equatorial methyl isomers could
be separated by fractional crystallization of the acid 7 0
[88].
In exploring further modification at this position of
HupA, a 10-spirocyclopropyl analogue 7 1 was
prepared in an optically active form [89]. Compound (-)-
7 1 was found to be nearly as active as natural (-)-HupA
(Table 3 ). According to the synthetic route to (-)-HupA,
(-)-10-spirocyclopropyl HupA 7 1 was synthesized from
2-spirocyclopropyl-1,4-cyclohexanedione mono-
ethylene ketal by a diastereoselective Michael-aldol
reaction, using (-)-8-phenylmenthol as a chiral auxiliary.
C-10 Methyl
Ki*(Μ) AChE Ki(Μ) BuChE
0.024 24
0.003 5.8
0.035 5.5
0.017 9.5
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 371

Table 3. Ki Values for the Inhibition of FBS The interaction between fluorinated analogues 6 6 b
AChE by HupA and Its C-10 and the active site of TcAChE was simulated by
Analogues
molecular dynamic simulation method. It was found that
C-12 methyl group in HupA could form weak H-bonds
Compound Ki*/nM
with the phenol oxygen of Tyr 121 and the main-chain
oxygen of Gly 118 in the active site of AChE [92].
(-)-HupA 3.9

(+)-10,10-dimethyl HupA 13.2 The difference of structure between HupA in

(+)-10-spirocyclopropyl HupA 14.0
(-)-10-spirocyclopropyl HupA 6.4
* Ki= koff / kon
In order to understand the differences in
bioactivities between axial methyl compound 6 9 c and
equatorial methyl compound 6 9 b , Kozikowski and his
coworkers carried out molecular modeling studies
using the refined binding site for HupA in TcAChE by
including crystallographic waters [88,90]. The modeling
studies of C-10 methylated analogues of HupA
demonstrated that either one or two methyl groups at
this position did not change their binding mode nor
cause any significant conformational change of the
protein residues.
TcAChE-(-)-HupA complex and in gas phase was
studied by Jiang et al. using ab inito method of
quantum chemistry [93]. The results indicated that the
binding conformer of HupA did not adopt its lowest
energy one. Because of the formation of H-bonds
between HupA and AChE in complex, great changes
took place in the total atomic charges, bond lengths
and the orientation of the atoms and bonds of HupA.
Ashani et al. reported the biochemical constants
K on, K off and Ki of complexes formed between HupA
and mutants of rHuAChE, which were compared with
wild-type rHuAChE and TcAChE. The results
demonstrated that inhibition of AChE by HupA
occurred via association with residues located inside
the active site gorge rather than at the rim of the gorge.
Tyr 337 was essential for inhibition of rHuAChE by
HupA, an aromatic lining constituted from Tyr 337, Phe

Me
Me
Me
R
NH
O Me NH
Me O
NH Me
R1 O
Me
NH 2
NH 2
69a: R=R1 =Me COOH
70 71
69b: R=Me, R1 =H
69c: R=H, R 1=Me

The molecular modeling and docking studies of the
C-10 methyl analogues to the X-ray crystal structure of
TcAChE revealed that the C-10 axial methyl pointed to
a hydrophobic region of the residues, while the
equatorial methyl was directed to a less favorable
hydrophilic region. The hydrophobic-hydrophilic
contact of C-10 equatorial methyl group was not
beneficial to the overall binding energy.
A 3D-QSAR analysis for 10 analogues of HupA by
molecular modeling and comparative molecular field
analysis method indicated a good conventional
statistical correlation between the 3D-structures of
these compounds and their anti-AChE activities [91].
295 and probably Trp 86 was likely to offer a multi-
contact sub-site that interacted with the ammonium
group and both exo- and endocyclic double bonds of
HupA [94].
Hitherto, a variety of HupA analogues have been
designed and prepared so far using trivial and rational
structural modification by several groups, the docking
studies of these analogues to the X-ray crystal structure
of TcAChE by Kozikowski et al. demonstrated the
importance of lipophilic substituents capable of
providing additional hydrophobic contacts, which could
increase the anti-AChE potency of the analogues [88].
372 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Bai et al.

More recently, Sussman and his coworkers [95] Acknowledgment

solved the 3-D structure of a TcAChE-(-)-HupA
complex by X-ray diffraction at 2.5 Å resolution. The We are grateful to the State Key Laboratory of Drug
results showed an unexpected orientation of HupA Research for financial support.
with surprisingly few strong direct interactions with the
protein residues in the active site gorge of the enzyme.
List of Abbreviations
The principle protein-ligand interactions include: 1)
a strong H-bond of the carbonyl of (-)-HupA to the HupA = Huperzine A
hydroxyl oxygen of Tyr130 ; 2) in the active-site gorge,
AD = Alzheimer’s disease
the interactions of (-)-HupA and protein residues are
mediated by one or two molecules of water ; 3) ChE = Cholinesterase
interaction of the positively charged amino group of the
ligand with the aromatic rings of Trp 84 and Phe 330 ; 4) ACh = Acetylcholine
an unusually short H-bond between the ethylidene
methyl group and the main chain oxygen of His 440 ; 5) AChE = Acetylcholinesterase
several hydrophobic contacts of (-)-HupA with the side-
BuChE = Butyrylcholinesterase
and main-chain atoms of Trp 84, His 440 and with
residues Gly118 through Ser 122 ; 6) a significant rHuAChE = Recombinant human AChE
change in the main-chain conformation of the protein.
TcAChE = Torpedo californica AChE
A correlation of the affinities of some analogues with
their interactions with the protein by docking studies FBS = Fetal bovine serum
suggested the importance of individual hydrophobic
interactions between (-)-HupA and aromatic residues in IC50 = Concentration of inhibitor yields 50%
the active-site gorge of the enzyme. inhibition of enzyme activity

NMDA = N-Methyl-D-aspartate
Future Directions TMG = Tetramethylguanidine
At present, many analogues of HupA have been MTPA-Cl = α-Methoxy-α−(trifluoromethyl)phenyl-
prepared, but neither the simplified analogues nor the acetyl chloride
derivatives from the natural HupA possess the anti-
AChE potency as HupA itself. Although C-10 axial LAH = Lithium aluminium hydride
methyl, C-10 dimethyl and C-10 spirocyclopropyl
analogues of HupA have been found to have DBU = 1,8-Diazabicyclo [5.4.0] undec-7-ene
comparable or somewhat more potent anti-AChE
ee = Enantiomer excess
activities than HupA, the preparation of these
analogues are quite laborious, and the costs will be BINAP = 2, 2’- Bis(diphenylphosphino)-1, 1’-
even more expensive than natural HupA. binaphthyl
The structure-based SAR studies of HupA should R = rectus (right)
be valuable for the rational design of new analogues of
HupA with improved therapeutic profile in future. S = sinister (left)

Several stereoselective syntheses of natural (-)- E = Entgegen (opposite)

HupA have been developed, however they have to be
further improved in many aspects such as ee, chemical Z = Zusammen (together)
yield, reaction duration, and operational simplicity. The
stereoselective bicycloannulation methodology
utilizing chiral palladium catalysts seems to be more References
feasible. [1] Bai, D.L. Pure & Appl. Chem., 1993, 65, 1103.

In view of the potent, highly selective anti-AChE [2] Kozikowski, A.P. J. Heterocyclic Chem., 1990, 27, 97.
activity and its chemical stability, HupA is likely to be
[3] Kozikowski, A.P.; Thiels, E.; Tang, X.C.; Hanin, In Advances in
used as a safe and long-lasting prophylactic treatment
Medicinal Chemistry ; Maryanoff, B.E.; Maryanoff, C.A., Eds.;
against organophosphate nerve agents in human [96]. JAI Press: Greenwich, 1992, Vol. 1, pp175-205.
In United Sates, HupA will be accessible to AD patients
as a nutraceutical or dietary supplement [97].
Treatment of Alzheimer’s Disease Current Medicinal Chemistry, 2000, Vol. 7, No. 3 373

[4] Ayer, W.A.; Trifonov, L.S. In The Alkaloids, Chemistry and [31] Xiong, Z.Q.; Tang, X.C. Pharmacol. Biochem. Behav., 1995, 51,
Pharmacology; Cordell, G.A.; Brossi, A., Ed.; Academic Press: 415.
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