Current Medicinal Chemistry Donepezil Hydrochloride (E2020 ... · Current Medicinal Chemistry, 2000...

Current Medicinal Chemistry, 2000, 7, 303-339 303

Donepezil Hydrochloride (E2020) and Other AcetylcholinesteraseInhibitors

Hachiro Sugimoto1, Yoshiharu Yamanishi2, Youichi Iimura1 and Yoshiyuki Kawakami1*

1Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan

2Eisai London Research Laboratories, Ltd., Gower Street, London WC1E6BT, UK

Abstract: A wide range of evidence shows that acetylcholinesterase (AChE) inhibitors caninterfere with the progression of Alzheimer's disease (AD). The successful development ofthese compounds was based on a well-accepted theory that the decline in cognitive and mentalfunctions associated with AD is related to the loss of cortical cholinergic neurotransmission. Theearliest known AChE inhibitors, namely, physostigmine and tacrine, showed modestimprovement in the cognitive function of Alzheimer's patients. However, clinical studies showthat physostigmine has poor oral activity, brain penetration and pharmacokinetic parameterswhile tacrine has hepatotoxic liability. Studies were then focused on finding a new type ofacetylcholinesterase inhibitor that would overcome the disadvantages of these twocompounds. Donepezil hydrochloride inaugurates a new class of AChE inhibitors with longerand more selective action with manageable adverse effects. Currently, there are about 19 newAlzheimer's drugs in various phases of clinical development.

Introduction Study Concept

A number of studies has confirmed that the averagelife expectancy for individuals has increased from 50years to about 75 years. Corollary with this increase,however, is the probability of mental health decline ordementia. Alzheimer's disease (AD) is said to be theleading cause of dementia in elderly individuals. Withthe continuing increase of the elderly population, theprevalence of AD is likely to increase.

Aging is often regarded as the main factor inmemory impairment and decline in other mentalfunctions or dementia. In this context, dementia isviewed as an inevitable phenomenon in old age.However, the presence of memory impairments insome elderly individuals does not necessarily makethem demented. Some memory impairments arenormal part of the aging process ("normal aging").Therefore, memory loss and other neuropsychologicsymptoms such as impairments of judgment, language,learning and abstract thinking, which are descriptive ofAD, may be attributed to normal aging. In this context,the relationship between normal aging and AD is adebatable concept [1]. This relationship makes thediagnosis of AD most uncertain until the disease hasprogressed to a moderate to severe stage whentreatment prospects become more formidable [2].

AD individuals exhibit retrogression in mental healthfunctions rendering them incapacitated and unable toperform normal daily activities. Elderly persons are theones commonly afflicted with this disease. However,evidence show that it can also afflict even individuals asyoung as 40 years of age. Ironically, the true nature orcause of the disease is still unknown making thedevelopment of treatment drugs a complex endeavor.Currently, the loss of cholinergic function is the onlyevidentiary finding responsible for cognitive decline.Hence, therapeutical development has focused on thistheory.

Alzheimer’s Disease

Definition

Alzheimer's disease, discovered by Dr. AloisAlzheimer in 1907, is described as a degenerativedisease of the central nervous system (CNS)characterized especially by premature senile mental

*Address correspondence to this author at Tsukuba ResearchLaboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635,Japan; Phone number +81-298-47-5653; Fax number +81-298-47-5771;E-mail: [email protected]

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

304 Current Medicinal Chemistry, 2000, Vol. 7, No. 3 Kawakami et al.

deterioration. AD patients exhibit marked decline incognitive ability and severe behavioral abnormalitiessuch as irritability, anxiety, depression, disorientation,and restlessness. AD is a progressive disease, i.e., theonset of the disease may show mild symptoms butthese symptoms will sooner or later become more andmore severe until the patient loses his or her capacity tohandle normal daily activities. While AD is commonlyregarded as a senile disease, the symptoms can alsomanifest in presenile individuals.

pathological hallmarks of the disease have beenidentified, i.e., neuritic plaques and neurofibrillarytangles (NFTs). Amyloid protein is a major componentof the neuritic plaques. These plaques accumulateextracellularly in the brain. The neuritic senile plaqueconsists of a fibrillar amyloid core surrounded bydystrophic neurites and reactive microglia. NFTsdevelop within the soma of the neuron and afterdegeneration of the parent cell convert intoextraneuronal structures, and are finally engulfed anddegraded by astrocytes. The NFTs are composed ofpaired helical filaments microtubule. There is a majordebate on whether amyloid plaques are the cause ofneurodegeneration or simply an end-stage of amyloidfibril build-up [7].

Prevalence

In Japan, the most prevalent type of dementia iscaused by cerebro-vascular diseases (CVD) affectingabout 42% of the population. Alzheimer's Diseaseranks only second, affecting about 32% of theJapanese population [3].

At the cellular level, there is a marked reduction inthe levels of neurotransmitters, e.g., acetylcholine,serotonin, noradrenaline, dopamine, glutamate andsubstance P. In this dramatic and global reduction ofneurotransmitters, the depletion of acetylcholine is themost important event [8].

In the United States, however, statistics show thatAlzheimer's Disease is the leading cause of dementiaaffecting about four million of the U.S. population or10% of Americans over the age of 65 [4].

Causes of Alzheimer's Disease

Impact on Health Care At present AD is considered a multifactorial diseasewith a combination of aging, genetic and/orenvironmental factors triggering the pathologicaldecline. However, the precise mechanisms causing thedisease are still unknown. Clinically, increasing age anda positive family history of dementia are the onlydefinite risk factors for AD. And recent studies haveshown that the presence of Apoliprotein E (ApoE) type4 allele is also a potential risk factor [9,10].

The annual healthcare costs of AD in the UnitedStates have been estimated to be as high at $100billion [5]. In Japan, the annual healthcare cost of AD isprobably much smaller compared to the U.S. However,with the increasing elderly population in Japan, thenumber of elderly individuals with AD may also beincreasing in frequency. It is therefore inevitable thatsooner or later, Japanese health authorities will have toconfront increasing healthcare figures related to AD.

Development of Treatment DrugsIn addition to the impact on healthcare budget,

there is also the emotional as well as physical stressbrought to the family of the AD patient. While relativesand caregivers of elderly AD patients have the will tocare for them, family care seems to break down sooneror later. Traditionally, the Japanese have had a uniqueattitude toward misfortune and burden. As a result,many caregivers in Japan endured the care burdenbecause most have accepted it as their fate. But thistraditional attitude is slowly fading as more and moreyounger caregivers in modern Japanese society arechoosing not to undertake home care but opt forinstitutionalization [6].

Numerous drugs have been studied as potentialtreatments for AD. But with the actual cause of thedisease yet to be identified, drug development in thisarea has been confined to limiting the progression ofthe disease. At the moment, drug development hasfocused on (i) symptomatic treatments aimed atrepleting deficient neurotransmitters, and (ii)etiologically based treatments aimed at slowing orhalting the rate of progression [11].

Cholinergic System and Alzheimer’sDisease

Pathogenetic Aspects The most consistent neurotransmitter-relatedchange in the brain of an AD patient is the dramaticdecrease in cholinergic innervation in the cortex andhippocampus due to the loss of neurons in the basal

Because the origin of AD is still unknown, auniversal concept on the pathophysiology of thedisease remains a debatable issue. However, two

Donepezil Hydrochloride (E2020) Current Medicinal Chemistry, 2000, Vol. 7, No. 3 305

forebrain. This fact has been confirmed in a largenumber of animal and human studies. This loss ofcholinergic neurons and the associated decrease inlevels of cholinergic neurotransmission have beenassociated with the cognitive impairment seen in ADpatients [4].

neurotransmitter ACh. AChE tends to becomedeposited within the neurofibrillary tangles and amyloidplaques associated with Alzheimer's Disease. In a studyconducted by Inestrosa et al. [13] several cellularproteins, including AChE, have been found to co-localize with β-amyloid (Aβ) deposits and promote theassembly of Aβ peptide into amyloid fibrils. In thiscontext, preventing the aggregation of Aβ into plaquesis another way to combat AD (see Section - Anti-Amyloid Strategy).

Cholinergic Hypothesis

The above findings led to the development of thecholinergic hypothesis. Simply stated, the cholinergichypothesis proposes that the cognitive loss associatedwith AD is related to decreased cortical cholinergicneurotransmission. Therefore, it is presumed thatincreasing cholinergic transmission may enhancecognitive function [4,10].

In a related study by Alvarez et al. [13] it is reportedthat the incorporation of AChE into Alzheimer's amyloidaggregates results in the formation of stable complexesthat change the biochemical and pharmacologicalproperties of the enzyme and cause an increase in theneurotoxicity of the β-amyloid fibrils, suggesting thatAChE could play a pathogenic role in AD by influencingthe process leading to amyloid toxicity and theappearance of AD. Analysis of the catalytic activity ofthe AChE incorporated into these complexes showsanomalous behavior reminiscent of the AChEassociated with senile plaques, which includes aresistance to low pH, high substrate concentrations,and lower sensitivity to anti-acetylcholinesteraseagents.

Cholinergic Enhancement Therapy

The cholinergic theory has provided the rationalbasis for therapeutic developments in AD. Based onthis theory, six classes of drugs have been developedto enhance cholinergic deficit in AD patient. These are:

a) Cholinesterase inhibitors (ChEI), which block theAChE enzyme thereby invigorating cholinergicactivity to enhance cognitive function.

AChE Inhibitorsb) Choline precursors, such as

phosphatidylcholine, aimed at increasing thebioavailability of choline.

Different types of AChE inhibitors have beenstudied for the treatment of AD. Some of the AChEinhibitors described below differ in their mechanism ofaction, metabolism and brain selectivity. Donepezilhydrochloride (E2020) is discussed in detail in Section5.

c) ACh releasers, which should facilitate the releaseof ACh from presynaptic end terminals.

d) M1 and M3 receptor agonists, which mimic AChon postsynaptic end terminal receptors. Physostigmine

Physostigmine (PHY) (Fig. (1 )), a cholinesteraseinhibitor developed by Pfizer, is one of the first drugs toshow cognitive improvements for some AD patients in11 studies conducted. The results of an early review ofthese studies showed certain degree of improvementsin 5 investigations using parenteral administration andin 4 of the 5 using oral administration [15]. However,further studies show that this is a short-acting AChEinhibitor. In November 1998, the US FDA issued a non-approval letter for PHY's Alzheimer's indication basedmainly on a lack of efficacy as shown from results inPhase II and Phase III studies [16].

e) M2 and M3 receptor antagonists, generallypresynaptic (autoreceptors), which regulate AChrelease via negative feedback.

f) Nicotinic agonists or substances having nicotinic-like effects, which should enhance ACh release.

Among the above pharmacological agents, AChEinhibitors seem to be the most effective method toimprove cholinergic deficit thus reducing the symptomsof the disease [12].

AChE Mechanism of Action Tacrine

ACh is the most abundant neurotransmitter in thebody and the primary neurotransmitter in the brainwhich is responsible for cholinergic transmission. Theenzyme AChE plays a key role in the hydrolysis of the

Tacrine (THA/Cognex) (Fig. (1 )) is the first drugapproved by the U.S. FDA for the palliative treatment ofmild to moderate AD. Tacrine is an aminoacridinecompound which is centrally active and a reversible


H3CHN

O

O

N

NCH3

CH3

H3C

H

H3C(H2C)6HN

O

O

N

NCH3

CH3

H3C

H

N

NH2

N

NH2OH

N

NH2

Physostigmine(Forest)

Eptastigmine(Mediolanum Farmaceutici)

Tacrine(Warner-Lambert)

Velnacrine Amiridine(Nikken Chem)

Fig. (1) . AChE inhibitors-1.

AChE inhibitor with a moderately long duration ofaction.

monitored for ALT levels in order to prevent thepossibility of hepatotoxicity.

In an early study conducted by Summers et al. [17]in 1986, the patients receiving tacrine showed a small,but nonetheless significant reduction in the decline ofcognitive performance (2.4 points in ADAS-cog)compared to placebo. These results led to a largermulticenter study carried out during the period 1990-1994 involving a total of 1905 patients enrolled in sixmajor double-blind, placebo-controlled trials usingsimilar methods of assessment of cognitive function[18-20]. On the basis of comparisons with placebo incognitive scoring, mental deterioration was arrested by2 to 12 months according to Mini Mental StateExamination (MMSE) and 5 to 6 months according tothe Alzheimer's Disease Assessment Scale (ADAS)when tacrine treatments continued for 2 to 6 months. Adose-response effect (20 to 80 mg per day) wasdescribed with a 4-point improvement on the ADAScognitive scale after 3 months' treatment [21]. In a 30-week, randomized, controlled trial carried out withincreasing doses of tacrine (up to 160 mg/day) [22], theresulting observation was a significant, dose-relatedimprovement in objective performance-based tests,clinician- and caregiver-rated global evaluation andmeasures of quality of life. However, a high proportionof patients showed increased levels of serum alanineaminotransferase which is indicative of hepatocellularinjury [23]. In summary, tacrine can modestly improvecognitive function in a portion of patients who are ableto tolerate it. Given the propensity of this drug to causemarked increases on alanine aminotransferase levels,patients receiving this compound must be carefully

Because of the positive effects observed in Tacrine,this compound has been used as the reference drug inthe clinical development of other AChE inhibitors forboth clinical efficacy and side effects [12]. Severaltacrine analogs are under preclinical or clinicalevaluation and some of these are presented below.

Velnacrine

Velnacrine (HP-029) (Fig. (1 )) is a hydroxymetabolite of tacrine, with a shorter half-life. Thiscompound acts biochemically as a potent AChEinhibitor. Pharmacodynamic trials with single doses ofthis compound in both healthy young individuals andpatients with AD demonstrated statistically significantcognitive improvements compared to placebo [24].However, as much as 20-30% of patients exposed tothis drug developed hepatotoxicity [25].

Amiridine

Amiridine (NIK-247, Senita) (Fig. (1 )), anothertacrine-derivative AChE inhibitor, is now under Phase IIIstudy in Japan. Animal studies of this compound showincrease acetylcholine levels as measured during invitro and in vivo experiments [26-28]. In a passiveavoidance test on rats with experimentally inducedamnesia, Amiridine improved cognitive function atdifferent levels of learning and memory processes. Thecompound was administered orally before or aftertraining at doses ranging from 0.1 to 3 mg/kg. Theresults of this study show that amiridine may have more


than one memory-enhancing mechanism, one at lowdoses and the other at high doses.

Phase III study carried out using doses up to 15 mg.t.i.d. for 25 weeks, the drug is still shown to be welltolerated in AD patients and appears to affect theircognitive and functional performance. The positiveeffects of eptastigmine compared to placebo appear tobe greater in the more severely impaired patients.

A clinical study of this compound was conducted ina group of 88 patients with AD [28]. Amiridine wasadministered to 74 patients (at doses of 20, 40, or 60mg/day) and the remaining 14 patients make up thecontrol group. The treatment period lasted for 14months, with a follow-up period that included clinicalobservation and testing. Results of this study show thatamiridine had well-defined therapeutic effects in 30-40% of patients treated. Cognitive functions andgeneral mental status of the patients continued toimprove or were stabilized in 14 months while speechimproved for a shorter period of time. Beneficial effectswere produced at both the initial and marked stages ofthe disease, although the most significant results wereobtained in patients with unmarked dementia.

Galanthamine

Galanthamine (Fig. (2 )) is a tertiary alkaloidoriginating from botanical sources. As early as the1950s, this compound was used by Bulgarian andRussian scientists in postsurgery reversal oftubocurarine-induced muscle relaxation, musculardystrophy and traumatic brain injury. In 1972, Sovietresearchers had demonstrated that galanthamine couldreverse scopolamine-induced amnesia in mice. Thisfinding was later on extended to humans. Despite thesubstantial and long-lasting history of clinical use of thiscompound in humans, it was not until 1986 when thiscompound was studied for the treatment of AD [30,31].The limited use of this compound in clinical studies maybe attributed to the fact that all supplies came fromnatural extracts and were only available in very limitedamounts. It was not until 1960 when the chemicalsynthesis of this compound was accomplished leadingto the industrial scale production and thus allowingdrug development without the limitations imposed bythe natural sources [32,33].

Eptastigmine

Eptastigmine (heptylphysostigmine, MF 201, L-693487) (Fig. (1 )) is an alkaloid derivative ofphysostigmine. Compared to physostigmine andtacrine, eptastigmine has a much longer duration ofaction. In a study conducted by Troetel et al. [29] it isfound that the drug inhibits red blood cell AChE in adose-dependent fashion. The mean AChE recoveryhalf-life was about 10h, with a mean residual inhibitionof 13% 24h after a 30 mg dose. Phase II studies inAlzheimer's patients indicate that doses of 40-60 mgper day of eptastigmine are relatively safe and welltolerated and that moderate AChE inhibition (30-40%)is associated with maximal cognitive efficacy. In the first

Animal studies identify galanthamine as a reversibleAChE inhibitor [34] with differential activity on thecentral nervous system and hence an ability to crossthe blood-brain barrier [35]. In human studies, the drug

N

O

OH

O

CH3

H3C

N

CH3 O

O NH3C

CH3

CH3

CH3

H3C

H3CO

PO

Cl

OCl

Cl

OH

NH

H3C

CH3

O

H

NH2

Galanthamine(Waldheim Pharm.)

Rivastigmine(Novartis)

Metrifonate(Bayer-Miles)

Huperzine A(Chinese Acad. Med. Sci)



has shown promise in the treatment of patients withsenile dementia of Alzheimer type (SDAT) [36]. Theinterim results obtained from Phase II study confirmearlier reports that galanthamine holds promise as aneffective and well-tolerated treatment for cognitiveimpairment in patients with SDAT [37].

inhibition over time [38]. Metrifonate has a short plasmahalf-life but has a long duration of action in the brain.These characteristics make it a unique compoundamong the AChE inhibitors being used or studied forthe treatment of AD.

Studies performed on laboratory animals show thatmetrifonate improve the cognitive performance ofanimals in various behavioural models. This compoundis observed to readily enter the animal brains and dose-dependently inhibit AChE activity. As a consequenceof AChE inhibition, metrifonate increases extracellularacetylcholine levels in the brain, local cerebral glucoseutilization, and the cortical EEG activity in rat models[39-41].

Galanthamine is approved in Austria for ADindication. It is currently in Phase III studies in Europe,USA, Australia, Canada and other countries [16].

Rivastigmine

Rivastigmine (SDZ-ENA-713/Exelon) (Fig. (2 )) is amiotine derivative of physostigmine. This compound isa carbamate type AChE inhibitor with a short kinetichalf-life and a duration of action of approximately 10h[11]. This carbamate inhibit AChE by carbamoylatingthe serine residue of the catalytic triad in apseudoirreversible manner.

The first application of metrifonate in AD therapy wasstudied by Becker et al. [42]. In this study, it wasreported that metrifonate significantly improvedcognitive function on the ADAS-cog scale andinhibited the erythrocyte AChE activity to 55.9% ofcontrol levels. In a 3-month dose-finding study [43],metrifonate improved the cognitive and global functionof probably AD patients. In this study, a once-dailymetrifonate dose of 30-60 mg, based on weight,improved ADAS-cog and CIBIC-Plus scores in theintent-to-treat patient population by 2.94 and 0.34points, respectively, at 12 weeks of treatment incomparison with placebo. Adverse reactions observedduring this study were generally transient and mild inintensity. A prospective 6-month study conducted byMorris et al. [44] confirms the finding of the earlier studyand extends it by demonstrating that the efficacy ofmetrifonate was sustained over the longer treatmentperiod.

In Phase III clinical studies conducted in the U.S., itwas reported that patients receiving the highest dosesof this compound (6-12 mg/day) showed averageimprovement of 0.79 points in the ADAS-cog scale,compared with an average decline of 4.15 points in theplacebo group. The low-dose group showed a declineof 2.2 points, intermediate between placebo and high-dose groups. In the CIBIC-Plus scale, improvement wasobserved in 24% of the patients receiving the highdose, 25% on the low dose and 16% in the placebogroup. About 25% of drug-treated patients showedside effects such as nausea, vomiting, diarrhea, loss ofappetite, dizziness and fatigue. Based on thesefindings, a regimen of increasing dosage seems to benecessary in order to achieve maximum clinical benefitswith minimal side effects [12]. The new drug application for this compound was

filed in late 1997 in US and Europe. However, thePhase III trials being conducted in the US was halted forat least 3 months in September 1998 in agreement withthe US FDA after some patients experienced muscleweakness with some requiring respiratory support. TheEuropean launch is also expected to be delayedbecause of this finding [16].

Rivastigmine is approved in more than thirtycountries worldwide under the trade name Exelon. Ithas been launched in Europe, Latin America, Asia,Afria and some Middle East countries. In July 1998, theUS FDA requested additional analyses of data toconfirm safety but no additional trials were requested.The launching of this product in the US is expected bythe middle of this year (1999). (-)-Huperzine A

Metrifonate (-)-Huperzine A (HupA) (Fig. (2 )) is another kind ofAChE inhibitor derived from natural extracts. It is analkaloid isolated from the club moss, Huperzia serrata,which has gained popularity in Chinese herbalmedicine. The compound's unique pharmacologicalfeatures and relative lack of toxicity [45] makes it apotent compound for AD treatment. The structure ofracemic Huperzine A shows some similarity to otherknown AChE inhibitors [46]. Animal studies revealsignificant cognitive enhancement [47]. Initial clinical

Metrifonate (Fig. (2 )) is an organophosphorouscompound originally used as an antihelmintic drug. It isnot a AChE inhibitor in itself but is time-dependentlytransformed into a physiologically active AChE inhibitorby dehydrochlorination. This compound is convertednonenzymatically to the active metabolite 2,2-dimethyldichlorovinylphosphate (DDVP). DDVP is shown toincrease ACh levels by stably binding to the active siteof the AChE enzyme, leading to a sustained enzyme


trials have established the safeness of HupA andprovided preliminary evidence for significant effects onpatients exhibiting dementia and memory disorders[48]. In a recent study conducted by Koenig et al. [49],it was demonstrated that HupA decreases neuronal celldeath caused by glutamate, particularly in primarycultures derived from hippocampus and cerebellum ofembryonic rat. Because this compound is shown toincrease acetylcholine levels in the brain and at thesame time decrease neuronal death, HupA may be animportant and promising drug for the treatment of AD.

N

O

H3CO

H3CO HCl

Fig. (3) . Donepezil hydrochloride.

The research on E2020 started in 1983. Followingresearch developments of tacrine, our group in Eisaistarted to develop tacrine derivatives. However, wefailed to develop a non-toxic tacrine derivative.Through random screening, we encountered N-benzylpiperazine derivative 1 which was then originallybeing synthesized in the study of anti-arterial sclerosis.Our tests showed that the acetylcholinesterase activityof N-benzylpiperazine derivative was 12.6µM at IC50 inrat brain homogenate. This was not very strong but thecompound's novel structure was very promising. Wedecided to use N-benzylpiperazine derivative as theseed compound and synthesized about 700derivatives.

Donepezil Hydrochloride (E2020)

Discovery of Donepezil Hydrochloride

Donepezil hydrochloride (E2020) (Fig. (3 )) is thesecond drug approved by the U.S. FDA for thetreatment of mild to moderate AD. It is a new class ofAChE inhibitor having an N-benzylpiperidine and anindanone moiety which shows longer and moreselective action. It is now marketed in the U.S. and insome European and Asian countries under the tradename of Aricept. In Japan, Aricept is now underapplication to the Ministry of Welfare.

Our succeeding experiments showed a dramaticincrease in anti-acetylcholinesterase activity when N-benzylpiperazine was replaced with N-benzylpiperidine(Fig. (4 )). It was a quantum leap in our investigation. Our

O2N O N N

O2N O N

O2NN

NH

O

CH2SO2

NN

O

CH3

1

2

3

4

lC50 =12600 nM

lC50 = 340 nM

lC50 = 55 nM

lC50 = 0.6 nM

Fig. (4) . Leading compounds.


next challenge was to replace the ether moiety with anamide moiety. This process also increased anti-acetylcholinesterase activity. We thought that theintroduction of a functional group at the para-position ofthe benzamide group might increase potency butremoval of the nitro group at the para-positiondecreased the potency of the compound.

Our next strategy in drug design was thereplacement of the amide moiety with ketone moiety 7 .This approach maintained the AChE activity of thecompound. Furthermore, this cyclic-amide derivative 6showed enhanced inhibitory action. On the basis ofthese results, an indanone derivative 8 was designed.The resulting AChE activity was moderate, but weachieved longer duration of action. Subsequently,various indanone derivatives were synthesized andtested for anti-AChE activity. Among the indanonederivatives that were developed, donepezil was foundto be the best balanced compound (Fig. (5 ))[52].

On the basis of these findings, we synthesizedbenzamide derivatives. We later on discovered thatbenzylsulfonyl derivative 4 was the most potent AChEinhibitor with an anti-AChE activity 21,000-fold greatercompared to the seed compound 1 [50,51]. However,our excitement was shortlived because we found thatthis compound has a very poor bioavailability rate andhas a short duration of action and therefore could notbe a candidate for clinical testing. But thisbenzylsulfonyl derivative has a novel chemical structureand has a selective affinity to AChE, making it a veryattractive lead compound. Immediately after thesefindings, we started the screening process again.

Structure-Activity Relationships (SAR)

The indanone derivatives were tested for in vitroinhibition of AChE. A rat brain homogenate was usedas the AChE source and the activities were measuredaccording to the method of Ellman et al.[53].

The indanone derivative was divided into four partsas shown in Fig. (6 ): Part 1 (indanone moeity), part 2

NH

N

O

NN

O

N

O

N

O

5

6

7

8

lC50 = 560 nM

lC50 = 98 nM

lC50 = 530 nM

lC50 = 230 nM

Donepezil

lC50 = 5.7 nM

Fig. (5) . New leading compounds.


(linkage moiety), part 3 (piperidine moiety), and part 4(benzyl moiety). All data were obtained from theracemic compounds.

(Table 1). contd.....

CompoundNo.

X Inhibition ofAChE IC50 [nM]a

CH2 N CH2

O

Part 1 Part 2 Part 3 Part 4

E2020

O

MeO

MeO

5.7

12

OH

MeO

MeO

300

13

MeO

MeO

4400Fig. (6) . Four parts of indanone-piperidine derivative.

Part 1. Table 1 shows the anti-AChE activity ofderivatives from the indanone moiety with variousbicyclic rings. The effect of replacement of indanonering with α-tetralone, 1-benzosuberone, 5,6-dimethoxy-1-indanone, 5,6-indanol, 5,6-dimetoxyindene was measured. The ring expansion ofcyclic ketone (i.e. compounds 1 0 , 1 1 ) greatlydecreased the activity. But the introduction of themethoxy group to the 5,6-position of the indanonemoiety increased the activity by 25-fold (E 2 0 2 0 ). Thecarbonyl group of the indanone moiety is essential tothe activity since the indanol (1 2 ) and indene (1 3 )derivatives both showed decreased potency.

a: Deviation of measurement of IC50 value is 10-20%.

The effect of introducing one or more methoxygroups in the indanone moiety is shown Table 2. Weobserved that the introduction of a methoxy group atthe R3-position increased the activity by 20-fold (1 5 ). Amethoxy substituent at the R4-position increased theactivity by 10-fold (1 6 ) while substitution at the R2-

Table 2. Anti-AChE Activity of Part 1Substituted Compounds (2)

CH2

R2

R3

R1 O

N CH2

R4

CompoundNo.

R1 R2 R3 R4 Inhibition ofAChE IC50 [nM]a

9 H H H H 150

14 H OMe H H 81

15 H H OMe H 6.4

16 H H H OMe 12

E2020 H OMe OMe H 5.7

17 OMe OMe H H 85

18 OMe H OMe H 25

19 OMe H H OMe 36

20 H H OMe OMe 20

21 OMe OMe OMe H 13


Table 1. Anti-AChE Activity of Part 1Substituted Compounds (1)

NCH2X CH2

CompoundNo.

X Inhibition ofAChE IC50 [nM]a

9

O

150

10

O

2100

11

O

15000


position (1 4 ) results in slightly increased activity. Theseresults suggested that the methoxy group at the para-position in the carbonyl group of the benzoyl moietygreatly enhanced binding to the active site of the AChEenzyme. Among these derivatives, 5,6-dimethoxyl-indanone derivative or the E2020, showed the highestactivity.

Table 4. Anti-AChE Activity of Part 3Substituted Compounds

CH2

MeO

MeO

O

Z CH2

Part 2. Various bridging groups between theindanone moiety and the piperidine moiety weretested. The results are shown in Table 3. Directconnection of the indanone and the piperadine ringsdramatically decreased potency(2 2 ). The effect of thelength of the bridging moiety on potency varied in thefollowing order: propylene(2 6 ) > methylene(E 2 0 2 0 ) >pentylene(2 8 ) > ethylene(2 5 ) > butylene(2 7 ). Theintroduction of an exo-methylene double bond on boththe indanone and piperidine moiety decreased theactivity(2 3 , 2 4 ).

CompoundNo.

Z Inhibition of AChEIC50 [nM]a

E2020 N 5.7

29 N 480

30 N N 94Table 3. Anti-AChE Activity of Part 2

Substituted Compoundsa: Deviation of measurement of IC50 value is 10-20%.

Y

MeO

MeO

O

N CH2Part 4. Table 5 shows the relationships between

the benzyl moieties. The 3-position-substituted benzylderivatives showed the highest potency among the 2-,3-, and 4-substituted regioisomers. Substitution of thebenzene ring with an electron-donating methyl groupand an electron-withdrawing nitro group showed asimilar effect. The basicity of the nitrogen atom in thepiperidine ring appear to have an important effect inincreasing activity since the N-benzoylpiperadinederivative(3 4 ) was almost inactive. Removal of thebenzyl group (3 8 ) caused a great reduction in thepotency of the compound but the activity was retainedafter replacement with cyclohexylmethyl group(3 9 ).The replacement of benzyl moiety with phenethyl (4 0 )and 2-naphthyl group (4 1 ) decreased potency. Amongthe indanone derivatives, compound E 2 0 2 0 is one ofthe most potent compounds in terms of anti-AChEactivity.

Compound No. Y Inhibition of AChEIC50 [nM]a

E2020 CH2 5.7

22 - 3300

23 =CH 13

24 CH= 90

25 CH2CH2 30

26 CH2CH2CH2 1.5

27 CH2CH2CH2CH2 35

28 CH2CH2CH2CH2CH2 14

Rationale for Donepezil as a Selective AChEInhibitor


Part 3. Table 4 shows the relationships betweenthe location and the number of nitrogen atom andactivity. The nitrogen atom at 1-position of thebenzylpiperidine moiety was very important since theactivity of 4-benzylpiperidine derivative(2 9 ) largelydecreased activity. Replacement of the piperidinegroup with a piperazine group(3 0 ) also resulted indecreased potency. The distance between thecarbonyl group in indanone ring and the nitrogen atomin piperidine ring might be critical for anti-AChE activity.

The reversible inhibitor donepezil is the leadcompound in a new class of AChE inhibitors having anN-benzylpiperidine and an indanone moiety whichshows a greater selectivity for AChE than forbutyrylcholinesterase, and is not expected to have anyperipheral effects [50]. The enantiomers of donepezilexhibit near identical pharmacological profiles,including inhibitory effects, and the two enantiomersinterconvert readily in aqueous solution, via a ketoenol


Table 5. Anti-AChE Activity of Part 4Substituted Compounds

CH2

MeO

MeO

O

N R

CompoundNo.

R Inhibition ofAChE IC50 [nM]a

E2020 CH2 5.7

31 CH2

Me

10

32CH2

Me

2.0

33 CH2 Me 40

34 CO >10000

35CH2

O2N

160

36CH2

NO2

4.0

37 CH2 NO2 100

38 H 5400

39 CH2 H 8.9

40 CH2CH2 180

41CH2

2900


intermediate [54]. Consequently, donepezil is beingdeveloped as a racemic mixture. Tacrine became thefirst available agent for the treatment of Alzheimer'sdisease in the United States. However, theaminoacridines, in general, suffer from dose-limitinghepatotoxicity which is believed to be structure related[55]. Donepezil appears to be devoid of such anunfavorable side-effect profile probably because of itsnovel benzylpiperidine structure.

A number of compounds have been made andtested by Eisai in the quest for a potent and selectiveAChE inhibitor which has been achieved withdonepezil. The corresponding structure-activityrelationship (SAR) data has been used in computer-assisted molecular design (CAMD) studies [56-58] todevelop guidelines for target synthesis and,retrospectively, to explain unusual SAR behavior.Some groups have undertaken the design andsynthesis of donepezil-like compounds whichincorporate bioisosteric replacements for the indanonering [59,60]. A 3D-QSAR using comparative molecularfield analysis, CoMFA, have been reported for a set ofAChE benzylpiperidine inhibitors [61]. All of the CAMDstudies have been receptor-independent in that thegeometry of AChE had not been determined and wasavailable for structure-based design studies. Over thecourse of this drug discovery study, we havedeveloped a image of the active site of AChE based ona common active shape-pharmacophore hypothesis.We have postulated similar structural features betweenACh and donepezil such as the carbonyl group and thetertiary nitrogen adjacent to bulky groups. Weproposed an active conformation which is characterizedby the indanone and piperidine rings being"perpendicular" to one another, with respect to a planealigned along the larger of the two thicknessdimensions of the piperidine ring [56, 62].

Five years after the discovery of donepezil,Sussman et al. have reported the crystal structure ofAChE from Torpedo californica electric organ [63]. Itwas found that the active site of AChE lies close to the"bottom" of a deep and narrow aromatic gorge which islined with the side chain rings of fourteen aromaticamino acid residues. It appears that the size of theactive-site pocket is too small for donepezil, and itsrelated analogs, to mimic the binding geometry of ACh.The availability of the geometry of an AChE presentsthe opportunity to extend the CAMD studies toinhibitor-enzyme docking analysis and to support,refute, and/or refine previous CAMD studies. Thecrystal structure of AChE has, in fact, already beenused in nonenergetic docking studies of donepeziland some indanone isosteric analogs of donepezil andin the prediction of donepezil binding site on AChE[64,65].


Docking simulations may provide some guidelinesfor structural changes in the four features of donepezil.A primary target for structural changes may be theindanone ring. There are other bicycles that lead toactive analogues [54,60] suggesting a structure-activitymapping may be possible by docking simulation.

Over the course of the lead optimization it appearedthat methyl substitution on the amide nitrogenenhanced activity. Thus, trans and cis forms ofbenzamide were considered as models since methylsubstitution at the amide nitrogen may change the ratioof cis and trans isomers of the amide. Overall,conformation was considered an important componentin a QSAR analysis used to guide the initial leadoptimization process. A good correlation equation,QSAR, was found between activity, as measured byIC50, and the difference in energy between the cis andtrans isomers, ∆E(C-T) and the bulkiness ofsubstituents (see Fig. (7 )). The QSAR suggests thatthe intrinsic activity of cis isomers of benzamide arehigher than the trans isomers, since there is a negativecorrelation between IC50 and ∆E(C-T).

Implementation of docking simulations in a designmode is predicated upon reliable calculations. Theability to predict reliable and accurate ligand-receptorbinding energies remains elusive in intermolecularmodeling. Perhaps the best chances for success in thedonepezil analogue - AChE docking simulations is tocombine X-ray studies to elucidate AChE-inhibitorcomplex geometries with free energy force fieldrefinement [66] docking simulations. The X-raystructures can provide realistic initial geometries to thedocking simulations for a training set of inhibitors forwhich the inhibition potencies have been measured.The terms in the force field used to compute thebinding energies of the inhibitors are then scaled, in aQSAR fashion, to establish a correlation betweeninhibition potency and binding free energy.

log 1/C = 0.019 MR - 2.372 ∆E(C-T) + 22.94

n = 19, s = 0.256, r = 0.833

log 1/C : negative lograithm of IC50 of AChEMR : molar refractivity∆E(C-T) : energy difference between cis and

trans isomer (kca/mol) by CNDO/2

Lead Identification and OptimizationFig. (7) . QSAR of para-substituted benzamides.

Table 6 contains the structure-activity behavior of N-alkylated amides which have higher activity comparedto non-N-alkylated analogs. Conformational analysis ofthe N-alkyl substituted benzamides was performedusing the MNDO quantum chemical method [67]. Non-substituted amides exist almost completely in the transform, but N-substitution makes the cis conformationmore stable. These results again indicate that AChEactivity increases as the population of the cis isomer ofthe benzamide increases.

A benzylpiperazine compound 1 (Fig. (4 )) whichdemonstrated weak, (its IC50 value is 12.6 µM), butdefinite inhibitory activity against AChE was identified inblind screening. Continued screening was performedaround compounds of similar structures. Another typeof "seed" compound 3 was found which has bothbenzylpiperidine and benzamide moieties in itsstructure. This compound has strong AChE activity, itsIC50 being in excess of two orders of magnitude that ofthe original benzylpiperazine lead. Lead optimizationsynthesis, mainly involving modification of thebenzamide moiety, led to N-[4-(1-benzyl)piperi-dinyl]ethyl-N-methyl-(4-benzylsulfonyl)benzamide 4 .Compound 4 has an AChE IC50 five orders ofmagnitude smaller (higher inhibition potency) ascompared to the original benzylpiperazine compound,as well as selective AChE inhibitory activity. However,compound 4 has a very short half-life due tohydrolyzation by proteases in the liver, which isbelieved to lead to an N-disubstituted amidemetabolite.

Table 6. N-Alkyl Analogs of N- [4- (1-Benzyl)piperidinyl]ethylbenzamide

R

N

O N

R IC50 (nM)

H 560Metabolic resistance was sought by incorporating

bioisosteric replacements for the N-disubstituted amidemoiety. A series of benzamide analogs possessinghigh AChE activity and which are readily transportedinto brain became the focus in the lead optimizationprocess.

Benzyl 180

Methyl 170

Ethyl 130

Phenyl 35


NCH3

N

O

N

O

R

O

N

O

O

ONH3CO

H3CO

O

cis form

rigid

flexible

Donepezil

Fig. (8) . Lead evolution of AChE inhibitor from benzamide.

The QSAR analysis provided the structuralrequirements for lead evolution. The specific structuralrequirements identified in the QSAR analysis are asfollows:

and physostigmine show poor selectivity having thesame order of magnitude IC50s for both AChE andBuChE. Inhibition of BuChE, which is abundant inplasma, may be associated with potentiating peripheralside effects [68]. Therefore, an AChE inhibitor which isessentially devoid of BuChE activity may display highertherapeutic indices than those which are also activeBuChE inhibitors.

1) The cis conformation of the benzamide is theactive conformation.

2) Bulky groups at the para position of thebenzamide increase activity. X-ray Crystallography of Donepezil

3) The carbonyl oxygen of the amide is a protonacceptor for an intermolecular hydrogen bond.

Fig. (9 ) shows the ORTEP [69] drawing of theracemic donepezil hydrochloride crystal structure. TheORTEP drawing shows a pattern of irregular thermalellipsoids. There is an asymmetric carbon atom at the 2-position of indanone ring and the irregular thermalmotions phenomenon occurs around this chiral center.This thermal behavior is observed both at 23 and -20 Cand corresponds to two puckering structures of theindanone ring. Thus, the crystal structure of theindanone ring consists of two conformers (Fig. (1 0 )).One conformer has a relative R configuration and theother has a relative S configuration. The mostremarkable feature of R and S enantiomers is that theyhave the same molecular-shape except for smallportions of the indanone and piperidine rings. The Rand S enantiomers are so similar that they are not evenrecognized in the crystalline state. These twoconformers also have almost equal heats of formationas computed by MNDO, and the crystal structure is oneof the most stable intramolecular conformations of each

A cis conformation rigid isostere by cyclization wasattempted without success. However, flexibleanalogues having the cis conformation were made.One of these was donepezil which possessbenzylpiperidine and indanone moieties (Fig. (8 )).Donepezil was the first compound in which anindanone ring was part of the enzyme inhibitor.

Donepezil as a Selective AChE Inhibitor

Donepezil was initially thought to be a mimic of AChby structural similarity, and, therefore, a competitiveinhibitor of AChE. The N-benzylpiperidines showoutstanding in vitro selectivity for AChE. The IC50 ofdonepezil for AChE is 5.7 nM while the IC50 for BuChE(butyrylcholinesterase), is about 7000 nM, a selectivityratio in excess of three orders of magnitude. Tacrine


Fig. (9) . X-ray crystal structure of racemic donepezil hydrochloride at -20 C. Irregular thermal ellipsoids observed both at 23oC and at -20 oC. (bottom) View into the plane of the indanone ring. (middle) View along the plane of the indanone ring. X-raycrystal structure of racemic donepezil hydrochloride discreting peaks with occupancy 0.6 and 0.4 found from differential Fouriermap.

donepezil enantiomer. This high intramolecular stabilitysuggests that these two conformations may not onlyexist in the crystal state, but also in solution.

the aromatic ring of the benzylpiperidine substructure.The most significant QSAR involves a representation ofmolecular shape, the largest principal moment ofinertia, and the HOMO of the substituted aromatic ring.It appears that upon binding the receptor "wall" isclosely fit around the benzyl ring, especially near thepara position. Overall, the QSAR analysis suggestsinhibition potency can be better enhanced bysubstitution on the indanone ring, as compared to thearomatic sites of the benzylpiperidine ring. Moreover,inhibition potency can be rapidly diminished,presumably through steric interactions with the

Receptor Independent Molecular Modelingand QSAR Analysis

QSAR analyses have been performed on thesubstituted indanone and benzylpiperidine ringsubstructures of a set of donepezil. A set of QSARswere constructed and evaluated for substituents on


Fig. (10). Two conformations in crystal structure of racemic donepezil hydrochloride. The mirror image of a conformer setexists in an asymmetric unit. (left) R-form in the conformation A. (right) S-form in the conformation B.

receptor surface of AChE, by substitution of moderateto large groups on the benzyl ring, particularly at thepara position.

indanone-benzylpiperidine inhibitors of AChE. It waspossible to define an active conformation with respectto the flexible geometry of the benzylpiperidine moiety,as well as an active conformation of the indanone ring-piperidine ring substructure for analogues having asingle spacer group between these rings(Fig. (1 1 )). No

Conformational analyses and molecular-shapecomparisons were carried out on an analogue series of

Fig. (11) . Molecular superposition of the active analogues under the criterium of maximizing overlap steric volume of eachanalogue with R-form of donepezil in its active conformation. R is a postulated anionic receptor site, which is estimated to be6.7 Å from each of the nitrogens of the compounds.


Fig. (12) . Global view of the AChE molecule looking down into the active site gorge.

active conformation could be postulated for analogueshaving two or three spacer units between the indanoneand piperidine rings. Still, a receptor binding model canbe constructed for all indanone and piperidine ringsubstructures. The postulated active conformation fordonepezil is close to the crystal structures with respectto the indanone-piperidine substructure, but differsfrom the crystal structures for the benzylpiperidinemoiety. However, the crystal conformations and thepostulated active conformation of the benzylpiperidineportion of the AChE inhibitor are estimated to be aboutequally stable.

Sussman and coworkers have reported the crystalstructure of AChE from Torpedo californica electricorgan [63]. The atomic coordinates of AChE have beendeposited in the Brookhaven Protein Data Bank (PDBentry: 1ACE).

Receptor Dependent (Docking) MolecularModeling

Structure of AChE

AChE which contains 537 amino acids is shown inFig. (1 2 ) as ribbon model. AChE belongs to the class

Fig. (13) . Close-up view of the AChE active site from the enzyme furface. The cylindrical atomic representations in the centerof the protein are the residues Ser-200, His-440, and Glu-327.


of α/β proteins and consists of 12 β-sheets surroundedby 14 α helices. A close-up view of the enzyme activesite from the enzyme surface is shown in Fig. (1 3 ). Theactive site serine residue can be seen from the surfaceof the enzyme. The active site consists of a catalytictriad composed of Ser-200, His-440 and Glu-327. Thecatalytic triad lies near the bottom of a long and narrowgorge, which is lined with the side chain rings offourteen aromatic amino acid residues. These aromaticresidues are color-coded orange in Fig. (1 3 ).

They rationalize this result by noting that the substrate,by virtue of Brownian motion, will make repeatedattempts to enter the gate each time it is near the gate.If the gate is rapidly switching between the open andclosed states, one of these attempts will coincide withan open state, and then the substrate succeeds inentering the gate. However, there is a limit on theextent to which rapid gating dynamics can compensatefor the small equilibrium probability of the open state.Thus the gate is effective in reducing the binding ratefor a ligand 0.4 Å bulkier by three orders of magnitude.This relationship suggests a mechanism for achievingenzyme specificity without sacrificing efficiency.

AChE catalysis involves an active serine residue,and the overall catalytic process proceeds in a three-step mechanism, as defined in Fig. (1 4 ), like transferwithin a catalytic triad [61, 70]. The activated serineresidue enables a nucleophilic attack on ACh, resultingin hydrolysis and acetate products being released.Some reversible AChE inhibitors show a mixed type ofinhibition by blocking the deacetylation process [71-73]. The acetyl group is introduced into the esteraticsite in this catalytic process. Therefore, acylated AChEcan accept an external charged molecule at this vacantanionic site to form an EA-inhibitor complex (EAI).Donepezil appears to bind to both the free enzyme andthe acylated enzyme to block both Michaelis complexformation and deacetylation [74].

It is known that anionic surface residues play a role inthe long-range electrostatic attraction between AChEand cationic ligands [77]. Tara et al. [78] show thatanionic residues also play an important role in thebehavior of the ligand within the active site gorge ofAChE. Negatively charged residues near the gorgeopening not only attract positively charged ligands fromsolution to the enzyme, but can also restrict the motionof the ligand once it is inside of the gorge. They useBrownian dynamics techniques to calculate the rateconstant kon, for wild type and mutant AChE with apositively charged ligand. These calculations areperformed by allowing the ligand to diffuse within theactive site gorge. Although a number of residuesinfluence the movement of the ligand within the gorge,Asp-74 is shown to play a particularly important role inthis function. Asp-74 traps the ligand within the gorge,and in this way helps to ensure a reaction.

E + S E S E A

P1

E + P2

I

Ki

E I

I

Ki*

E A I

+

++

Hydrated molecular dynamics simulations of isolatedAChE (no inhibitor or substrate present) have identifieda "backdoor" opening to the active site located alongthe active site gorge [79]. Our water-independentsimulation studies of AChE, in the presence of ACh, orinhibitors, does not indicate that a backdoor can form alarge-enough opening for a long-enough time periodto permit the entry, or exit, of ligands and/or products tothe active site [80]. However, an open "side door" inone of the AChE was recorded at 152 picoseconds of alarge molecular dynamics simulation [75].

Fig. (14) . Reaction scheme in which the inhibitor combineswith E and EA. E, free enzyme; S, substrate; ES, enzymesubstrate complex; EA, acylated enzyme; P, product; I,inhibitor; EI, enzyme inhibitor complex; EAI, acylatedenzyme inhibitor complex; Ki, dissociation constant forenzyme and inhibitor; Ki*, dissociation constant for EA andinhibitor.

Recent molecular dynamics simulations showed thatreorientation of five aromatic rings leads to rapidopening and closing of the gate to the active site. Thesimulations watch the dynamic fluctuations in thechannel. The channel walls can have openings largeenough to admit solvent molecules, while the channelitself can open widely enough to admit substrate [75].In other study the molecular dynamics trajectory is usedto quantitatively analyze the effect of the gate on thesubstrate binding rate constant [76]. For a 2.4-Å probemodeling acetylcholine, the gate is open only 2.4% ofthe time, but the quantitative analysis reveals that thesubstrate binding rate is slowed by merely a factor of 2.

Dipole Moments of ACh and Inhibitors

Ripoll et al. [81] have reported that AChE has aremarkably large dipole moment which is aligneddirectly along the axis defining the center of thearomatic gorge. Dipole-dipole interactions may play animportant role in the long-range molecular recognitionof AChE ligands. Molecules with complementingdipoles to AChE can be drawn toward, and down, thearomatic gorge, leading to the active site in anorientation in which a matching dipole-dipoleinteraction is formed. Thus, inhibitors that interact with


the enzyme in this orientation are expected to havepotent activities, and some groups have proposedmodels for the binding of large dipole inhibitors toAChE [81]. It shows that electrostatic steering ofligands contributes to the high rate constants that areobserved experimentally for AChE.

treated as united atoms. Docking studies for anacylated form of the enzyme, EA, were also carried out[80]. The three-dimensional structure of EA has notbeen determined so that the EA model used in thisstudy was generated from the X-ray structure of AChE.

The docking simulations show that stableconformations are adopted by donepezil and itsderivatives upon binding to both AChE and the EA.Donepezil and its derivatives considered dock to bothAChE and the EA in a similar manner. The dockingsuggests two common features of binding for thesecompounds;

The calculated dipole of ACh, in its boundconformation, is about 23 Debye, while the extendedconformation dipole moments of donepezil and itsderivatives are on the order of 35-50 Debye. Thedirection of the dipole in each inhibitor runs throughthe molecule from the benzylpiperidine moiety to themethoxy groups on the indadone ring. Thus, thecalculated large dipoles of Ach, donepezil and itsderivatives support the dipole-dipole recognitionmodel mentioned above [81, 82]. In essence, one hasa tube (the active-site gorge) which possesses apositive charge on one end and a negative charge onthe other. A successful inhibitor has a cylindrical shapewhich can fit into and slide along the inner surface ofthe tube. The cylindrical (inhibitor) possesses positiveand negative charges on its ends so that the preferredorientation and position of the cylinder in the tube isdictated by electrostatic interactions.

1) The N-benzyl substituent forms a π-stackinginteraction with the indole side chain of Trp-84.

2) The piperidine ring locates on the narrowest partof active-site cavity which is formed by four aminoacid residues, Tyr-70, Asp-72, Tyr-121, and Tyr-334.

Donepezil exhibits five key binding interactions (KBI1 to 5) which is identified in the binding models basedon the interactions among AChE and five partialstructures of donepezil. The definition of five KBIs areas follows;

Binding Models of Donepezil to AChE andEA KBI 1: the interaction between the indanone ring

and the indole side chain of Trp-279.The atomic coordinates of AChE were obtained

from the Brookhaven Protein Data Bank (PDB entry:1ACE). Hydrogen atoms were added after deleting thecrystalline water molecules present in the X-raystructure, and aliphatic carbon-hydrogen groups were

KBI 2: the interaction between the methoxy group inthe indanone and the main-chain carbonyl ofArg-289.

Fig. (15) . (left) Superposition of donepezil-AChE binding models in the active-site cavity with the five key binding interactions(KBIs) residues defined. (right) Superposition of the docking models of donepezil in the acylated enzyme cavity shown in gridsurface form.


KBI 3: the interaction between the carbonyl group inth eindanone and the hydroxy group of Tyr-121 or Tyr-70.

of the two thickness dimensions of the piperidine ringcontains its plane relative to the indanone ring [56, 62].The preferred mode of binding of donepezil to AChE isshown in Fig. (1 6 ). Overall, these results suggest thatdonepezil, and related analogs, are noncovalent AChEinhibitors which bind in the aromatic gorge, but do notinteract with the catalytic residues. Consequently, thedonepezil analogs can inhibit the Michaelis complexformation step and/or the deacylation step of thehydrolytic reaction of AChE.

KBI 4: the interaction between the NH group in theprotonated piperidine and the carboxyl groupof Asp-72, the phenyl ring of Phe-330 or thehydroxy group of Tyr-121.

KBI 5: the interaction between the phenyl ring in thebenzyl piperidine and the indole ring of Trp-84.

Structure of Acetylcholinesterase Complexwith Donepezil

The carbonyl oxygen interacts with Tyr-121 (KBI 3a)and Tyr-70 (KBI 3b). However, the S enantiomer ofdonepezil only adopts the KBI 3b binding mode. Crystal Structure

Superposition of the models reveals that the threebinding classes, with respect to the piperidine ring, aresimilar in that they share five key interactions which areshown in Fig. (1 5 ). In a different structural model theindanone ring is parallel to the indole side chain of Trp-84 at a distance of 3.6 Å. The geometric bindingdifference between enantiomers is in the direction ofthe carbonyl in the indanone ring. The carbonyl groupinteracts in the KBI 3a mode (R form only), and in theKBI 3b mode (both forms). The other four keyinteractions, namely the indanone ring with Trp-279,the methoxy group with Arg-289, the protonatedpiperidine NH with Asp-72 and the phenyl ring with Trp-84, are observed in all model.

Sussman and coworkers have reported the crystalstructure of a complex of donepezil with Torpedocalifornica acetylcholinesterase (TcAChE) [83]. Theatomic coordinates of AChE have been deposited inthe Brookhaven Protein Data Bank (PDB entry: 1EVE).The X-ray structure, at 2.5 Å resolution, shows that theelongated donepezil molecule spans the entire lengthof the active-site gorge of the enzyme (Fig. (1 7 )). Itthus interacts with both the "anionic" subsite, at thebottom of the gorge, and with the peripheral anionicsite, near its entrance, via aromatic stacking interactionswith conserved aromatic residues. It does not interactdirectly with either the catalytic triad or with the"oxyanion hole".

The results of the docking study support the activeconformation model discussed above which ischaracterized by the indanone and piperidine ringsbeing "perpendicular" to one another, where the larger

They have also reported only the R enantiomer isbound within the active-site gorge when the racemateis soaked into the crystal inspite of racemic compound

Fig. (16) . Lowest-energy binding model of the R enantiomer of donepezil with AChE in the active-site cavity.


Fig. (17) . Active-site cavity of TcAChE with donepezil molecule obtained from the Brookhaven Protein Data Bank (PDB entry:1EVE). Green grid, solvent-accessible surface of active-site cavity; pink balls, water molecules. Space-filling atoms areshown for donepezil. Residues which participate in interactions are purple. Residues involved in the catalytic triad are orange.

whose enantiomers have similar affinity for the enzyme.The R and S enantiomers have the same molecular-shape except for small portions of the indanone andpiperidine rings as described at X-ray crystallography ofdonepezil section. Therefore, this behavior may beassumed to be caused by undistinguishablenessbetween the R and S enantiomers in the crystalstructure at 2.5 Å resolution.

Although docking simulation predicted two morepossible interactions between the charged nitrogen ofthe piperidine ring and the carboxyl group of Asp-72and the hydroxy group of Tyr-121, the ring nitrogenmakes an in-line hydrogen bond with WAT1159.

At the top of gorge, the indanone ring stacksagainst the indole ring of Trp-279 in a parallel π−πinteraction. But, none of binding partner to themethoxy group at the indanone moiety is found in thecrystal structure.

Comparison of Binding Modes in SimulationModel with That in Crystal

In a docking study in donepezil, we proposed fivekey binding interactions (KBI 1 to 5) which is identifiedin the binding models based on the interactions amongAChE and five partial structures of donepezil asdescribed before. Three major KBIs are observed inthe crystal structure of TcAChE complex withdonepezil(Fig. (1 8 )).

Modeling of BuChE

Torpedo acetylcholinesterase (TcAChE) and humanbutyrylcholinesterase (hBuChE) have 73% similarityand 53% identity of their amino acid sequences [85].Both enzymes contain three internal disulfide loopswith exactly the same chain lengths. Kyte-Doolittleamino acid hydropathy plots are almostindistinguishable and the known structure of AChEsuggests that cholinesterase are α/β-proteins withstructural similarity to other crystallized proteins. Thispermitted modeling hBuChE (Fig. (1 9 )) on the basis ofthe three-dimensional structure of TcAChE.

One face of the benzyl benzene ring stacks againstthe indole ring of Trp-84 at the anionic subsite near thebottom of the gorge in both model and crystalstructure. On the opposite face, it makes a classicaromatic hydrogen bond with a water molecule(WAT1160). This water is held firmly by a hydrogenbond to another water molecule (WAT1161), in the"oxyanion hole", and to WAT1159. The modeled hBuChE [86] has a remarkably large

dipole moment which is aligned directly along the axisdefining the center of the aromatic gorge. Thecalculated dipole is about 354 Debye while that of

In the constricted region, halfway up the gorge, thecharged nitrogen of the piperidine ring makes a cation-π interaction [84] with the phenyl ring of Phe-330.


Fig. (18). Binding modes of donepezil to TcAChE. (left) Simulated binding modes of donepezil with KBIs defined. (right)Experimental binding modes of donepezil to TcAChE [80]. Donepezil is displayed as a ball-and-stick model ( chiral centermarked with black star); direct binding residues are represented as dark sticks; water-mediated binding residues as graysticks; water molecules as light gray balls; standard H-bonds as heavy dashed lines; aromatic H-bonds, -cation and -stackingas solid lines.

TcAChE is about 361 Debye. The modeled hBuChEstructure closely resembled that of TcAChE in overallfeatures. However, six conserved aromatic residuesthat line the active-site gorge are absent in hBuChE.Modeling showed that two such residues, Phe-288and Phe-290, replaced by leucine and valine,respectively, in BuChE. In the hBuChE model, thecatalytic triad and Trp-84, the "anionic" site, do notmove relative to its position in TcAChE. Hence theanionic-site-directed inhibitor tacrine can bind toBuChE s well as AChE.

of donepezil is strongly dependent on interaction withTrp-279 and Phe-330, which are absent in hBuChE,may explain its high relative specificity for AChE versusBuChE. So tacrine or other small inhibitors whichposition in the anionic site near the catalytic triad couldnot acquire the selectivity against AChE.

Preclinical Pharmacology of Donepezil(E2020)

The following experiments were designed toevaluate the properties of donepezil, a newcholinesterase inhibitor, with respect to its effect on thecentral cholinergic system. The conventional ChEinhibitors such as tacrine and physostigmine were usedas reference compound in some experiments.

The accessible surface areas at the active site cavitywhich includes the aromatic gorge and the catalytic triadin the hBuChE is 952.1 Å2, and is larger than that ofTcAChE, 694.9 Å2 [84]. The waist of active site cavity inAChE have disappeared from the BuChE active sitecavity (Fig. (2 0 )). Consequently, the goodness of fit ofdonepezil into the active site cavity may decrease. Effects on Cholinesterase Activity

(a) The comparative specificity of donepezil, tacrine,and PHY for brain AChE activity

Trp-279, at the entrance of the active-site gorge inTcAChE, is absent in hBuChE. Modeling designated itas part of the "peripheral" anionic site, which is lackingin hBuChE (Fig. (2 1 )). The indanone ring of donepezilstacks against the indole moiety of Trp-279, in theperipheral binding site, in a π-π interaction. The binding

The initial experiments were designed to determinethe relative in vitro inhibitory effects of donepezil on theactivities of AChE and butyrylcholinesterase (BuChE,pseudocholinesterase) in comparison with two


Fig. (19) . Global view of the modeled hBuChE molecule looking down into the active site gorge.

recognized cholinesterase inhibitors, physostigmine(PHY) and tacrine. Rat brain homogenates were usedas the source of AChE and rat plasma served as thesource of BuChE. ACh was used as the substrate forAChE and butyrylthiocholine(BuCh) was the substratefor BuChE. Both enzyme preparations were incubatedwith several concentrations of each inhibitor. The

results, expressed as IC50 values, are shown in Table 7.More extensive kinetic studies indicated that

donepezil, like tacrine, is a reversible noncompetitiveinhibitor of AChE [74]. On the basis of this study, it isclear that, in vitro, donepezil is a much more selectiveinhibitor of AChE than either tacrine or PHY [87]. Sincedonepezil was a more specific inhibitor of AChE in vitro

Fig. (20). Comparison of solvent-accessible surfaces of TcAChE active-site cavity with that of modeled hBuChE. Acalculation of the solvent accessible surface area at the active site cavity was made using the program GRASP. (Upper left)Side view of the active site cavity of AChE. (Upper right) Top view of the active site cavity of AChE. (Bottom left) Side view ofthe active site cavity of hBuChE. (Bottom right) Top view of the active site cavity of hBuChE.


Fig. (21) . Close-up view of the modeled hBuChE active site from the enzyme furface.

than the reference standards, tacrine and PHY, furtherexperiments were conducted to determine the effectof oral administration of these agents on cholinesteraseactivity in the brain and in other tissues from treatedanimals.

In order to test the relative tissue specificity of theinhibitors, the compounds were administered orally torats, the animals were sacrificed one hour afteradministration, and the blood and other tissues wereremoved and analyzed for ChE activity as in previousexperiments. Since the peripheral tissues containprimarily BuChE and the brain contains AChE, thisstudy provides evidence for the relative tissuespecificity of the inhibitors given in vivo. The resultsindicate that PHY and tacrine inhibit the ChE from bothbrain and peripheral tissue at all doses tested. Incontrast, donepezil significantly inhibits the AChE ofbrain, but does not inhibit the BuChE from heart andsmall intestine, and has only a marginal effect on theChE from pectoral muscle (Fig. (2 3 )). Althoughcomplete dose-response curves were not run for eachagent, it is still clear that donepezil, when given orally, ismore specific for AChE than either PHY or tacrine. Inview of the clear-cut ability of donepezil to inhibit braincholinesterase activity in vitro and in vivo, it isreasonable to assume that donepezil can increase the

(b) The effect of oral administration of donepezil andother cholinesterase inhibitors on the cholinesteraseactivity of treated animals

In order to determine the comparative effect ofcholinesterase inhibitors in vivo, the compounds wereadministered orally at doses from 1 to 60 mg/kg to maleWistar rats. One hour after administration, the rats weresacrificed and the brain (excluding the cerebellum) wasfrozen. AChE activity was later measured in a brainhomogenate using acetylthiocholine as a substrate.The results of this in vivo treatment (Fig. (2 2 )) indicatethat all three compounds caused a dose-dependentinhibition of brain AChE activity, and that donepezilappeared to be a more potent inhibitor than either PHYor tacrine [88].

Table 7. Inhibitory Effects of E2020 and Reference Compounds on Rat Brain AChE and Rat PlasmaBuChE in vitro

IC50 (nM)

Compound AChE Activity BuChE Activity Ratio of EC50s (BuChE/AChE)

E2020 5.7 ± 0.2 5.7 ± 0.2 1252.0

Physostigmine 0.68±0.02 8.1 ± 0.3 11.9

Tacrine 80.6 ± 2.5 73.0 ± 0.9 0.9

*Values represent the mean + S.E. from 4 dose-response curves for each test drug.


Fig. (22) . Effects of oral administration of E2020, physostigmine (PHY) and tacrine on rat brain AChE activity ex vivo. Eachcolumn denates ± S.E., *, **: p<0.05, p<0.01. The top numbers in each column represent the percent inhibition relative tocontrol. The lower numbers represent the number of animals used.

concentration of ACh in the brain. This was tested inthe following two different experiments (Fig. (2 3 )).

measured using an HPLC technique. The results(Table 8) indicate that oral administration of donepezilcaused a dose-dependent increase in ACh in all threeareas of the brain tested.Effects of Donepezil on Brain Acetylcholine

ConcentrationsBy using microdialysis technique, it was possible to

carry out the effect of donepezil on the concentrationof ACh in the extracellular space of the rat brain cortex[89]. A microdialysis probe was implanted into thecerebral cortex of conscious rats and perfused withbuffer. The acetylcholine which was released into theextracellular space was collected with the perfusingbuffer and analyzed by HPLC. Baseline release of AChwas measured for at least 60 minutes, and the amountof ACh in the perfusate was determined at 20-minuteintervals. Donepezil was given intraperitoneally atdoses of either 1 or 3 mg/kg, and 20-minute sampleswere analyzed over the next 2 or 3 hours. The resultswere expressed as a percentage of the average

In view of this clear-cut ability of donepezil to inhibitbrain cholinesterase activity in vitro and in vivo, it isreasonable to assume that donepezil can increase theconcentration of ACh in brain. This was tested in twodifferent systems.

(a) The effect of donepezil on the concentration of AChin rat brain

In the first set of experiments, donepezil was givenorally to rats, and the animals were sacrificed bymicrowave irradiation of the head one hour later. Thecerebral cortex, hippocampus and striatum weredissected, and acetylcholine was extracted and

Table 8. Effects of E2020 on ACh Concentrations of the Cerebral Cortex, Hippocampus andStriatum of Rats

ACh concentration (nmole/g)

Treatment Dose mg/kg n Cortex Hippocampus Striatum

E2020 - 6 15.8 ± 0.79 23.5 ± 0.74 67.7 ± 3.52

5 6 20.4 ± 0.69* 25.6 ± 0.78 87.8 ± 3.99

20 6 21.8 ± 1.44** 28.7 ± 1.14** 99.3 ± 8.95**

Figures are mean ± S.E.*,**: p<0.05, 0.01 vs. respective control (Dunnett's t-test).


release measured during the control period (Fig. (2 4 )).From this experiment, it is clear that E2020 increases

ACh in the extracellular space of the rat cerebral cortexin a time- and dose-dependent fashion.

Fig. (23) . Effects of E2020, Physostigmine and tacrine on ChE Activity in the Brain and Peripheral Tissues ex vivo .

Each column indicates mean, the vertical line indicating ± S.E.

*, **: p<0.05, P<0.01 vs saline control (Dunnett's t-test).

The number of animals is 6, except for that indicated by @ (n=5).


Fig. (24) . Effects of E2020 on extracellular ACh levels in the cerebral cortex of rats.

*,**: p<0.05, 0.01 vs. baseline release (paired t-test, n=4-5).

It was concluded from the preceding experimentsthat donepezil is capable of decreasing AChE activityand increasing the ACh concentration in the cerebralcortex of normal animals.

below normal level, it was tested, along with tacrine, in aseries of in vivo model systems in which the corticalcholinergic system is impaired.

In the first study, the neurotoxin ibotenic acid wasinjected into the nucleus basalis magnocellularis regionof the rat brain. Destruction of this region, whichinnervates the cerebral cortex, causes a decrease inthe concentration of acetylcholine in the cerebralcortex. Two to three weeks after injection, the animals

(b) The effect of donepezil and tacrine on AChconcentration in the cerebral cortex of animals withcerebral cholinergic hypofunction

Since donepezil was designed for use undercircumstances in which the concentration of ACh is

Fig. (25) . Effects of E2020 and tacrine on ACh concentration in the cerebral cortex of rats with ibotenic acid-induced NBMlesion.*,**: p<0.05, 0.01 vs. NBM-lesioned rats (Dunnett's t-test).S: Saline; Sham: Sham operation. The number in each column represents the number of animals.


were given an oral dose of either donepezil or tacrine.One hour later, the animals were sacrificed by whole-head microwave irradiation, and the concentration ofACh in the cerebral cortex was determined by HPLCanalysis. The results (Fig. (2 5 )) indicate that exposureto ibotenic acid significantly decreased theconcentration of ACh in the cerebral cortex, and thattreatment with either donepezil (1.25 to 10 mg/kg) ortacrine (5 to 20 mg/kg), caused a dose-dependentincrease in cortical ACh. In this model system,donepezilappears to be a more potent agent thantacrine.

given an oral dose of either donepezil or tacrine, andsacrificed one hour later by whole-head microwaveirradiation. The concentration of ACh in thehippocampus was then determined by HPLC analysis.The hippocampus was chosen for analysis in this studybecause of its involvement in memory-related functions[88]. The results (Fig. (2 7 )) indicate that the AF64Ainjection produced a decrease in the concentration ofACh in the hippocampus, and that an oral dose (5mg/kg) of donepezil significantly reversed this effect.

On the basis of these three studies, it has beenestablished that an oral dose of donepezil can increasethe concentrations of ACh in the brain of animals withabnormally low level of ACh.

In the second model, a peripheral injection of thecholinergic blocker scopolamine was used to cause atemporary decrease in the concentration of ACh in thebrain. For this study, the test compounds, eitherdonepezil or tacrine, were given orally, and 30 minuteslater the animals were injected intraperitoneally withscopolamine. Thirty minutes after scopolamine, theanimals were sacrificed by whole-head microwaveirradiation, and the ACh concentration in the cortex wasdetermined. The results (Fig. (2 6 )) indicate thatscopolamine produced the expected decrease in theACh concentration of the cortex, and that this decreasewas prevented by prior administration of eitherdonepezil or tacrine. As in the first model, donepezilwas more potent than tacrine under the conditions ofthis experiment.

Effect of Donepezil in Behavioral Models ofCholinergic Hypofunction

On the basis of the previous studies, it is apparentthatdonepezil is a relatively specific inhibitor of AChEwhich can increase the concentration of ACh in bothnormal and ACh-deficient animals. The final set ofstudies was designed to determine the ability ofdonepezil to alter behavior which is impaired due to adeficiency in cortical ACh. Accordingly, donepezil wastested for its effect on several model systems ofabnormal animal behavior.

(a) The effect of treatment with donepezil on thebehavior of functionally impaired animals

In a third study, the neurotoxin AF64A was injectedinto both lateral ventricles of the brains of anesthetizedrats. Two to four weeks after injection, the animals were

The first study was designed to evaluate the relativeeffects of donepezil and tacrine on scopolamine-

Fig. (26) . Effects of E2020 and tacrine on ACh concentration in the cerebral cortex of rats treated with scopolamine.*,**: p<0.05, 0.01 vs. Scopolamine alone (Dunnett's t-test). S: saline. The number in each column represents the number ofanimals.


Fig. (27) . Effects of E2020 and Tacrine on ACh Concentrations in the Hippocampus of AF64A-treated Rats.

*: p<0.05 vs. AF64A saline-treated group (Dunnett's test).

n=6. S: saline. i.c.v.: intracerebroventricular injection.

induced impairment of 8-arm radial-maze performance,a spatial memory task in rats. A chocolate chip wasplaced at the end of each of the radial arms of the maze,and the rats were trained to find and eat the chips. Oncompletion of training and subsequent initiation of thestudy, performance is rated by measuring the timerequired for the animal to find 8 out of 8 chips (totalrunning time) and by noting the number of incorrectmaze-arm selections (error number). When animalswere pretreated with scopolamine, performance, asmeasured by running time and number of errors) wassignificantly impaired. However, when scopolamine-

treated animals were pretreated orally with donepezil ortacrine, maze performance was improved (Table 9).Under these experimental conditions, donepezilappears to be more potent than tacrine in improvingmaze performance. These results indicate that E2020can reverse the effects of central cholinergicimpairment in the maze-running performance task inrats.

The second study was designed to evaluate theeffects of donepezil and tacrine on a passive avoidancetask in animals with lesions in the nucleus basalis

Table 9. Effects of E2020 and Tacrine on Errors and Running Time in the Performance of the RadialMaze Task

Treatment n Error Number (X_

± SE) Total Running Time (seconds ( X_

± SE)

E2020 Saline / pre-scopolamine 10 0.7 ± 0.3 65.6 ± 6.78

Saline / scopolamine 10 7.5 ± 1.57## 194.1 ± 18.62##

E2020 0.52 mg/kg / Scopolamine 10 5.2 ± 1.29 147.5 ± 21.12*

E2020 0.50 mg/kg / Scopolamine 10 3.3 ± 0.67* 127.5 ± 18.33**

Tacrine Saline / pre-Scopolamine 13 0.5 ± 0.14 47.2 ± 5.85

Saline / Scopolamine 13 7.0 ± 1.17## 194.5 ± 21.40##

Tacrine 1.0 mg/kg / Scopolamine 13 5.3 ± 1.53 154.8 ± 22.68

Tacrine 2.0 mg/kg / Scopolamine 13 4.5 ± 1.28* 123.5 ± 18.88*

##: p < 0.01 when compared with value before scopolamine treatment.*: p < 0.05 vs. saline control (Student's t-test).


magnocellularis (NBM) [90]. The NBM was destroyed intest animals by bilateral injection of ibotenic acid. Afterone week, NBM-lesioned and sham-operated animalswere placed in a passive avoidance box consisting oflight and dark compartments, and trained, using electricshock, to avoid entry into the dark compartment. Onehour prior to training, they were given either donepezil,tacrine or saline orally, and tested 24 hour later todetermine whether they remembered their training.Retention (memory) was measured by the amount oftime each animal waited before entering the darkcompartment (response latency). Animals whichretained the training, i.e., memory of the electric shock,had longer latency times. As shown in Fig. (2 8 ), sham-operated animals had a response latency ofapproximately 400 seconds, and lesioned animalstreated with saline had a latency of approximately 100seconds, indicating a decrease in their ability to retainthe training. Lesioned animals treated with donepezil atdoses from 0.125 to 1 mg/kg showed a statisticallysignificant increase in latency. Animals treated withtacrine at doses from 0.25 to 1 mg/kg showedincreases in response latency at 0.5 mg/kg, but thisincrease did not achieve statistical significance. Theseresults indicate that donepezil is capable of enhancingthe retention of training (memory) in animals withcholinergic hypofunction.

were tested for motor activity 2 weeks later. NBM-lesioned animals become exceptionally active at nightor when placed in a new environment. When shamoperated animals are placed in a new environment,they are most active in the first 20 minutes, andgradually become less active over the course of twohours. In contrast, when NBM-lesioned animals areplaced in an identical environment, their activity is muchgreater than that of sham operated animals, and theincreased activity is maintained for a longer period oftime. When lesioned animals were given subcutaneousinjections of either 2 mg/kg donepezil or 5 mg/kgtacrine immediately before placement in a newenvironment, their activity demonstrated a responsepattern closer to that of the unlesioned animals. Whileinitial activity remained high for the first 40 minutes inthe observation chamber, donepezil animals acclimatedquickly thereafter and showed statistically significantimprovement over the untreated, lesioned animals from40 to 120 minutes. Tacrine, while somewhat effective,did not produce a statistically significant reduction inlocomoter activity until 80 minutes after placement ofthe animal in the chamber. Further, the average degreeof inhibition of hyperlocomotion produced by tacrinewas less than that for donepezil until the 120 minuteobservation point. NBM lesioning also resulted innocturnal hyperlocomotion, even in a familiarenvironment. donepezil (2 mg/kg, s.c.) had a significantinhibitory effect [91].The third study was designed to determine the

effect of donepezil and tacrine on the hyperlocomotionwhich occurs in animals with a lesion in the nucleusbasalis magnocellularis. The NBM of the test animalswas lesioned by electro-coagulation and the animals

These results indicate that both donepezil andtacrine can partially reverse the hypermotility shown byNBM-lesioned rats in a new environment and at night.

Fig. (28) . Effects of E2020 and tacrine on the latency of passive avoidance response in NBM-lesioned rats. *,**: p<0.05,p<0.01 (Mann-Whitney's U-test). S: Saline, Sham: Sham-operated rats, NBM Lesion: NBM lesioned rats. Number of animals isgiven in each column.


Fig. (29) . Effects of cholinergic drugs on hyperlocomotionÅ@induced by a new environment in rats with NBM lesions.

*: p<0.05, **: p<0.01 vs. saline control Dunnett's multiple range test. (n=5)

However, as in the other model systems studied,donepezil appears to be more potent than tacrine (Fig.(2 9 )).

and Mini-mental State Exam (MMSE) scores werereported; no changes were found in this study of shortduration on the clinical global impression of change.However, a statistically significant correlation betweenplasma concentrations of donepezil and AChEinhibition was demonstrated. Moreover, thereappeared to be a possible correlation between plasmadrug concentrations and cognitive scores. Treatment-related side effects were comparable with all threedoses.

Discussion

The neurochemical studies show that donepezil is ahighly selective, reversible and noncompetitve inhibitorof acetylcholinesterase in vitro and the inhibitory effectof donepezil is relatively selective for brain and serumcholinesterase, i.e. AChE in vivo. In the systems usedfor these studies, donepezil was a more selectiveinhibitor of brain AChE than the reference compound,tacrine. donepezil is capable of increasingacetylcholine not only in the brain of normal rats and inthe extracellular space of rat cerebral cortex, but also inthe rat models of cholinergic hypofunction. In thebehavioral studies, donepezil is capable of partiallyreversing the impairment in retention of a passiveavoidance task produced by brain lesion and in mazeperformance caused by scopolamine. These resultssuggest that donepezil may be a candidate drug forAD.

15-Week Phase III Study

In a Phase III study, approximately 150 patients eachreceived either donepezil 5 mg/day, donepezil 10mg/day or placebo once daily for 12 weeks followed bya single-blind placebo washout for 3 weeks [92]. The10 mg/day dose was titrated using a blinded schedulein which subjects received 5 mg doses of donepezil forthe first 7 days. Consistent with FDA guidelines, theprincipal outcome measures were ADAS-cog andCIBIC-Plus (Clinician's Interview-Based Impression ofChange-Plus). Statistically significant improvements inADAS-cog score were seen within 3 weeks continuingto study endpoint and were significantly different fromthe placebo group. Significant improvements in thisstudy were also seen in the CIBIC-Plus at both 5 and 10mg doses.

Clinical Studies of Donepezil

U. S. Multicenter Study Phase II

A double blind, placebo-controlled, randomized trial1, 3 and 5 mg donepezil in 141 patients was reported in1996. A 12-week double-blind phase was followed bya-week single-blind placebo washout. Improvements inAlzheimer's Disease Assessment Scale (ADAS-cog)

30-Week Phase III Study

In this study, which was similar in design to the 15-week study, approximately 150 patients each wereentered into donepezil 5 mg, donepezil 10 mg or


Fig. (30) . Effect of E2020 on cognitive function measured using ADAS-cog.

placebo. The patients were followed for 24 weeks,followed by a 6-weeks washout [93]. Once again, therewere statistical improvements in ADAS-cog in patientstreated with both drug doses at 12 and 18 weeks.CIBIC-Plus scores also improved in both groupscompared to placebo; Fig. (3 0 ), Fig. (3 1 ).

Safety

A high proportion of patients completed both of thePhase III studies [93]. Five percent of patients droppedout due to adverse events in placebo and low-dosedonepezil groups, increasing to 13% in the higher, 10mg dose groups. This greater drop-out at the 10 mg

Fig. (31) . Effect of E2020 on global function measured using CIBIC Plus.


Table 10. Number (%) of Patients with Treatment-emergent Signs or Symptoms

Adverse Event Placebo (n = 162) Donepezil 5 mg/d (n = 154) dopenezil 10 mg/d (n = 157)

Fatigue 3 (2) 8 (5) 12 (8) *

diarrhea 11 (7) 14 (9) 27 (17)*

Nausea 6 (4) 6 (4) 26 (17)*

Vomiting 3 (2) 5 (3) 16 (10)*

Anorexia 3 (2) 3 (2) 11 (7)

Muscle cramps 1 (1) 9 (6) 12 (8)*

Dizziness 9 (4) 15 (10) 13 (8)

Rhinitis 4 (2) 1 (1) 9 (6)

dose was thought to be due to the rapid titration, sincein open-label studies, the frequency of drop-out at 10mg is much lower if the titration is taken over 4-6 weeks.Most of the treatment-emergent adverse events weremild in intensity and lasted less than 2 days. As withother cholinesterase inhibitors, the most common sideeffects were nausea, diarrhea, insomnia, musclecramps, vomiting and fatigue (Table 10).

plasma with an IC50 value of 19.0 nM [60]. It is alsoshown to have superior potency over tacrine, PHY anddonepezil in inhibiting noradrenaline (IC50 =4.02 µM)and serotonin uptake (IC50 = 1.35 µM) intosynaptosomal fractions of rat cerebral cortez andhippocampus.

An evaluation of the central cholinergic activity andselectivity of TAK-147 have been evaluated and thecompound is shown to significantly inhibitapomorphine-induced circling behavior in rats withunilateral striatal lesions at a dose of 3 mg/kg p.o.(31.1% inhibition) with no significant peripheralcholinergic effects. It is also shown to significantlyimprove diazepam-induced memory impairment [60].Using other animal models, TAK-147 was furtherevaluated for its enhancing effect on learning andmemory. These studies show TAK-147 improvedscopolamine-induced impairment of a delayedmatching to sample task and in impaired differentialreinforcement at low rate performance in AF64A-treated rats at doses of 0.3 ∼ 3 mg/kg p.o. devoid ofsignificant behavioral and peripheral effects [95].Studies in aged rats indicated that the improvementsobserved in water maze and passive avoidancelearning after repeated oral administration of TAK-147are associated with improvement in impairedmetabolism in brain regions involved in learning andmemory processes.

N-Benzylpiperidine Derivatives

The N-benzylpiperidine moiety originally found inthe donepezil structure is shown to have superiorefficacy, minimal side effect, and high brain selectivitycompared to tacrine and PHY. This makes donepezil avery novel compound. Donepezil and other N-benzylpiperidine derivatives are shown to reversiblyand specifically inhibit AChE by forming a complex inwhich the N-benzylpiperidine group presumablyinteracts with the anionic site which recognizes thequaternary ammonium group of ACh.

Clinical studies have shown that the inhibition ofbutyrylcholinesterase (BuChE) which is abundant inhuman plasma may be linked with potentiating sideeffects. N-benzylpiperidine compounds are shown tohave high selectivity for AChE over BuChEdemonstrating an exceptional safety profile of thesecompounds. Because of these findings, recent studieshave focused on N-benzylpiperidines [50, 59, 94]. TwoN-benzylpiperidine derivatives are presented below: T-82 (SS Pharm.)

TAK-147 (Takeda) In vitro studies show that the AChE activity of T-82 is9.5 times higher than tacrine. In a two-compartment testwith 8-week-old male Wistar rats which received oraldoses of the compound 15 minutes after scopolamine(0.5 mg/kg i.p.), 24-h memory retention was improvedin a dose-dependent manner, i.e. 0.03 mg/kg. : 50%improvement; 0.1 mg/kg : 92%; 0.3 mg/kg : 96%).

TAK-147 is a 4-substituted benzylpiperidinecompound currently in Phase II clinical trials in Japan. Invivo studies show that TAK-147 is more potent thantacrine and PHY in inhibiting AChE activity in ratcerebral cortex homogenates with an IC50 value of 51.2nM. It is least potent in inhibiting BuChE activity in rat


N

HN

O

TAK-147

N

N

NOOMe

T-82


There are strong indications that T-82 is also a 5-HT3receptor antagonist. As a consequence of the tonicinhibitory control which the serotonin system exertsover acetylcholine release, this type of activity couldlead to enhanced presynaptic discharge ofacetylcholine in cholinergic neurons. Because this typeof serotonergic receptor does not seem to besignificantly impaired in AD, a very high synergisticpotential could result if AChE inhibition and 5-HT3antagonist could be combined in one molecule [96-98](Fig. (3 2 )).

progression of the associated inflammation in AD [99-102].

Anti-oxidants

Several central nervous system (CNS) disordersincluding AD are thought to be related to changes inoxidative metabolism. Normally, the interaction of thesecompounds with molecules crucial for cellular viability isinhibited by endogenous antioxidant enzymes andfree radical scavengers. However, when anti-oxidantdefenses are inadequate, free radicals will producecytotoxic damage to the cell membrane, enzymes andDNA by altering the electron number, the structure andthe function of lipids, proteins, nucleic acids and othercellular constituents [8]. Studies show that oxygenradicals initiates amyloid build-up leading toneurodegeneration [103,104]. On the basis of thisobservation, anti-oxidants as a therapeutic approach toAlzheimer's disease has been pursued.

Other Approaches in Alzheimer’s DiseaseTreatment

While AChE inhibitors can alleviate the symptoms ofAD, one should give some thought on the limitations ofthese therapeutic agents. What is more important is tobe able to develop a therapeutic agent that can actuallycure the disease. However, the development ofcurative compounds is markedly limited by the fact thatthe etiology and pathogenesis of AD is still unclear. Yetthe research should continue.

Vitamin compounds with anti-oxidant propertiessuch as retinol (Vitamin A), ascorbic acid (Vitamin C) andtocopherol (Vitamin E) are now being studied as apotential therapeutic approach to AD.

Briefly discussed below are other approachescurrently being pursued in the treatment of AD, such asanti-inflammatory agents, anti-oxidants, estrogenreplacement therapy, galanin receptor antagonist, andthe anti-amyloid strategy.

Estrogen Replacement Therapy

Animal studies have provided evidence thatestrogen stimulates nerve growth production. Inoveriectomized rats, ERT has been shown to: (1)restore and even enhance learning abilities; (2) preventthe decrease of neuronal choline uptake and cholineacetyltransferase; (3) reduce the decline in nervegrowth factor and brain-derived neurotrophic factormRNA in those brain areas, and (4) exertneuroprotective effects in a tissue culture model [105].

Anti-inflammatory Agents

The use of anti-inflammatory agents in the treatmentof AD was pursued based on the large body ofpathological evidence suggesting the presence ofinflammations in AD brain. These inflammations cangenerate a self-propagating process in which differentmolecules (complement, cytokines, acute phaseproteins) act, potentiating β-amyloid toxicity. Asignificant observation derived from a study conductedon the use of non-steroidal anti-inflammatory drugs(NSAIDs) in treating elderly arthritic patients, is the low-incidence of AD among these patients suggestingNSAIDs may delay neuronal degradation and limit the

These observations led to the application ofestrogen replacement therapy (ERT) to preventneuronal degeneration and cognitive decline. Studiesconducted by Honjo et al. and Hagion et al. [106, 107]all confirmed the evidence that estrogen-responsivesubjects exhibited osteoporosis and lower serumestrogen levels prior to beginning estrogenreplacement, which suggests that AD in some women


may be associated with systemic estrogen deficiency.Long-term prospective trials of estrogen in normalperimenopausal women and in women with AD are nowneeded to substantiate these hypotheses.

aid in neural repair but more importantly to prevent theformation of additonal β-amyloid [114].

Conclusions

Galanin Receptor Antagonist Given the fact that the etiology and pathogenesis ofAD is still unclear, the development of a curativecompound is markedly limited. This limitation is evenmore compounded by the unavailability of a true animalmodel of AD. The screening of new AChE inhibitors isperformed with a battery of pharmacological testings.But the question remains on whether the animalmodels are valid [1]. Appropriately validated animalmodels are important in the efficient and rationaldevelopment of AChE inhibitors and other treatmentcompounds for AD.

Galanin is a 29-amino acid peptide abundant in thebrain and peripheral tissues. This peptide and itsreceptors have been implicated in a variety ofphysiological processes such as food intake, pain,anxiety and depression, memory and neuroendocrinefunctions. Tatemoto et al. [108] were the first to isolatethis polypeptide from porcine intestine. Galaninappears to co-exist with several other neurotransmittersincluding ACh in the ventral forebrain [109]. Theinteraction between galanin and acetylcholine hasbeen a major focus in galanin research. Axonalplexuses innervating the ventral hippocampus andnucleus basalis have been described in rat, monkeyand human [110] and galanin inhibits the release ofacetylcholine in these regions [111]. In AD brain tissue,galanin hyperinnervation of cholinergic neurons in thenucleus basalis has been observed suggesting thatcholinergic dysfunction in AD patients may bedepressed further by plasticity in the galanin system[112]. It is therefore attractive to speculate that galaninhas a crucial role in the regulation of cholinergicfunction in forebrain pathways relevant to memory andmay be involved in the cognitive dysfunctionsassociated with Alzheimer's disease [113].

Acknowledgments

The authors wish to thank Professor A. J. Hopfingerand Dr. Mario Cardozo of Department of MedicinalChemistry and Pharmacognosy, the University of Illinoisat Chicago, for their contribution and help this study.We also appreciate the helpful discussions withProfessor J. L. Sussman, the Weizmann Institute ofScience, and Dr. G. Kryger for providing Fig. (1 8 ). Wethank Drs. T. Kawai and A. Inoue for their activecollaboration in the study of x-ray crystallography anddocking simulation. We wish to give special thanks toMs. Mary Alpa Cornelio of Eisai Co. Ltd. for her valuableassistance in compiling this paper.

Anti-Amyloid Strategy

ReferencesAs discussed in Section - AChE Mechanism ofAction, β-amyloid deposits are found to co-localize withcellular proteins leading to the assembly of Aβ peptidesand its aggregation into amyloid plaques. Because ofits close relationship with the plaques andneurofibrillary tangles, β-amyloid has long beenconsidered to be responsible for the development andthe progression of the neurodegeneration in AD. Butthe processes that transform β-amyloid into potentiallyneurotoxic amyloid and its accumulation as senileplaques are still unclear. However, the discovery of itspresence in AD brains, provides insight as to possibletherapeutic targets for AD. Research has focused oninterventions to prevent the destruction of neuronsand the disruption of brain function by β-amyloid.These include the administration of antioxidants andfree radical scavengers to reduce further neuraldamage from deposits of β-amyloid, the activation ofvarious growth factors to repair damaged cells andrestore their functions, and the stimulation of thenormal processing of the precursor protein not only to

[1] Moos, W.; Hershenson, F. Drug News &Perspectives 1989, 2,397.

[2] Cutler N.; Sramek J.; Veroff A. Alzheimer’s disease: Optimizingdrug development strategies, 1994.

[3] New Current, 1996, 7, 4.

[4] Evans, D.A.; Funkenstein, H.; Albert, M.S.; Scherr, P.A.; Cook,N.R.; Chown, M.J.; Hebert, L.E.; Hennekens, C.H.; Taylor, J.O.JAMA, 1989, 262, 2551.

[5] Aricept: Product Monograph, 1997.

[6] Asada, T.; Motonaga, T. In Alzheimer’s Disease: Biology,Diagnosis and Therapeutics; Iqbal, K.; Winblad B.; Nishimura, T.;Takeda, M.; Wisniewski, H. Eds.; John Wiley & Sons Publishers,1997, pp. 790.

[7] Laakso M. In Incipient Alzheimer’s Disease, Series of Reports,Univ of Kuopio, Finland, 1996.

[8] Parnetti, L.; Senin, U.; Mecocci, P. Drugs, 1997, 53, 752.

[9] Helisalmi, S. Molecular Genetics of Alzheimer’s Disease, Seriesof Reports, Univ of Kuopio, Finland, 1998.


[10] Poirier, J. In Alzheimer’s Disease: Biology, Diagnosis andTherapeutics, Iqbal, K.; Winblad, B.; Nishimura, T.;Takeda, M.;Wisniewski, H. Eds.; John Wiley & Sons Publishers, 1997, pp.93.

[31] Kobayashi, S.; Shingu, T.; Uyeo, S. Chem. Ind., 1956, 147.

[32] Barton, D.H.R.; Kirby, G.W. Proc. Chem. Soc. 1960, 392.

[33] Akhnazarova, S.L.; Tolsykh, L.P., Rusakova; S.V. et al. FzvUyssh Uchebn Zaved Khim Tekhnol, 1987, 93.[11] Felician, O.; Sandson, T. Drugs of Today, 1997, 33, 665.

[34] Institute for Drug Research, Budapest. 14-day repeated dosecomparative oral toxicity study with galanthamine in rats.Protocol 9625, sponsored by Waldheim Pharmazeutika, 1996.

[12] Brufani, M.; Filocamo, L.; Lappa, S.; Magg, A. Drugs of theFuture, 1997, 22, 397.

[13] Inestrosa, N.; Alvarez, A. In Alzheimer’s Disease: Biology,Diagnosis and Therapeutics, Iqbal, K.; Winblad, ; Nishimura, T.;Takeda, M.; Wisniewski, H. Eds.; John Wiley & Sons Publishers,1997, pp. 500.

[35] Irwin, H.L.; Smith, H.J. Biochem. Pharmacol., 1990, 147.

[36] Kewitz, H.; Wilcock, G.; Davis, B. In Alzheimer Disease:Therapeutic Strategies, Becker, R.; Giacobini, E. Eds.;Birkhauser: Boston, 1994, pp. 141.[14] Alvarez, A., Alarcon, R., Opazo, C.; Campos, E.O.; Munoz, F.J.;

Calderon, F.H.; Dajas, F.; Gentry, M.K.; Doctor, B.P.; De Mello,F.G.; Inestrosa, N.C. J. Neurosci., 1998, 18, 3213. [37] Wilcock, G.; Wilkinson, D. In Alzheimer’s Disease: Biology,

Diagnosis and Therapeutics, Iqbal, K.; Winblad, B.; Nishimura,T.;Takeda, M.; Wisniewski, H. Eds.; John Wiley & Sons Publishers,1997, pp. 661.

[15] Mohs, R.C.; Davis, K.L. In Psychopharmacology: ThirdGeneration of Progress. Meltzer, H.Y., Ed.; Raven Press: NewYork, 1987, 921.

[38] Holmstedt, B.; Nordgren, I.; Sandoz, M.; Sundwall, A. Arch.Toxicol., 1978, 41, 3.[16] 1998 Alzheimer’s Disease Cooperative Study.

[39] Hinz, V.C.; Grewig, S.; Schmidt, B.H. Neurochem. Res., 1996,21, 331.

[17] Summers, W.K.; Majovski, L.V.; Marsh, G.M.; Tachiki, K.; Kling,A. N. Engl. J. Med., 1986, 315, 1241.

[40] Hinz, V.C.; Grewig, S.; Schmidt, B.H. Neurochem. Res., 1996,21, 339.

[18] Eagger, S.A.; Levy, R.; Sahakian, B.J. Lancet, 1991, 337(8748),989.

[41] Schmidt, B.H.; Hinz, V.C.; Blokland, A. et al. In Alzheimer’sdisease: From molecular biology to therapy, Becker, R.;Giacobini, E.; Robert, P. Eds.; Birkhauser: Boston, 1996, pp. 217.

[19] Davis, K.L.; Thal, L.J.; Gamzu, E.R.; Davis, C.S.; Woolson, R.F.;Gracon, S.I.; Drachman, D.A.; Schneider, L.S.; Whitehouse, P.J.;Hoover, T.M. et al. N. Engl. J. Med., 1992, 327, 1253.

[42] Becker, R.E.; Colliver, J. et al. Drug Develop. Res., 1990, 35,425.

[20] Farlow, M.; Gracon, S.J.; Hershey, L.A.; Lewis, K.W.; Sadowsky,C.H.; Dolan-Ureno, J. JAMA, 1992, 268, 2523.

[43] Cummings, J.L.; Cyrus, P.A.; Biever, F.; Mas, J.; Orazem, J.;Gulanski, B. Neurology, 1998, 50, 1214.

[21] Wilcock, G.K.; Surmon, D.J.; Scott, M.; Boyle, M.; Mulligan, K.;Neubauer, K.A.; O’Neill, D.; Royston, V.H. Age Aging, 1993, 22,316.

[44] Morris, J.C., Cyrus, P.A., Orazem, J., Bieber, F.; Ruzicka, B.B.;Gulanski, B. Neurology, 1998, 50, 1222.[22] Knapp, M.J.; Knopman, D.S.; Solomon, P.R.; Pendlebury, W.W.;

Davis, C.S.; Gracon, S.I. JAMA, 1994, 271, 985.[45] Laganiere, S.; Corey, J.; Tang, X.C.; Wulfert, E.; Hanin, I.

Neuropharmacology, 1991, 30, 763.[23] Watkins, P.B.; Zimmerman, H.J.; Knapp, M.J.; Gracon, S.I.;Lewis, K.W. JAMA, 1994, 271, 992.

[46] Geib, S.J.; Tuckmantel, W.; Kozikowski, A.P. Acta. Crystallogr.C., 1991, 47, 824.[24] Siegfred, K. In The management of Alzheimer’s disease,

Wilcock, G.K. Ed.; Wrightson Biomedical Publishing, 1993, pp.189. [47] Xiong, Z.Q.; Tang, X.C. Pharmacol. Biochem. Behav., 1995, 51,

415.

[25] Murphy, M.F.; Hardiman, S.T.; Nash, R.J.; Huff, F.J.;Demkovich, J.J.; Dobson, C.; Knappe, U.E. Ann N Y AcadSci,1991, 640, 253.

[48] Zhang, R.W.; Tang, X.C.; Han, Y.Y.; Sang, G.W.; Zhang, Y.D.;Ma, Y.X.; Zhang, C.L.; Yang, R.M. Chung Kuo Yao Li Hsueh Pao,1991, 12, 250.

[26] Shibanoki S.; Ishii Y.; Kubo, T.; Kogure, M.; Asai, S.; Ishikawa, K.Pharmacol. Biochem. Behav., 1991, 39, 499. [49] Ved, H.S.; Koenig, M.L.; Dave,J.R.; Doctor, B.P. Neuroreport,

1997, 8, 963.

[27] Ishii Y.; Sumi, T. Neuropharmacology,1992, 31, 61.[50] Sugimoto, H.; Tsuchiya, Y.; Sugumi, H.; Higurashi, K.; Karibe,

N.; Iimura, Y.; Sasaki, A; Kawakami, Y.; Nakamura, T.; Araki, S.;Yamanishi, Y.; Yamatsu, K. J. Med. Chem., 1990, 33, 1880.

[28] Moskovskij, N.L.; Grigoryeva, I.V.; Sokolchik, E.I. The efficacy ofamiridine in Alzheimer’s type senile dementia. Zu NevropatolPsikhiatr Im SS Korsakova, 1991, 53.

[51] Sugimoto, H.; Tsuchiya, Y.; Sugumi, H.; Higurashi, K.; Karibe,N.; Iimura, Y.; Sasaki, A.; Araki, S.; Yamanishi, Y.; Yamatsu, K.J. Med. Chem., 1992, 35, 4542.

[29] Troetel, W.M.; Imbimbo, B.P. In Alzheimer’s Disease: Biology,Diagnosis and Therapeutics, Iqbal, K.; Winblad, B.; Nishimura,T.;Takeda, M.; Wisniewski, H. Eds.; John Wiley & Sons Publishers,1997, pp. 672. [52] Sugimoto, H.; Iimura, Y.; Yamanishi, Y.; Yamatsu, K.

J.Med.Chem., 1995, 38, 4821.

[30] Mucke, H. Drugs of Today, 1997, 33, 259.


[53] Ellman, G.L.; Courtney, D.; Andres, V.Jr.; Featherstone, R.M.Biochem. Pharmacol. 1961, 7, 88.

[78] Tara, S.; Elcock, A.H.; Kirchhoff, P.D.; Briggs, J.M.; Radic, Z.;Taylor, P.; McCammon, J.A. Biopolymers, 1998, 46, 465.

[54] Matsui, K.; Oda, Y.; Ohe, H.; Tanaka, S.; Asakawa, N. J.Chromatogr., 1995, A 694, 209.

[79] Gilson, M.K.; Strastsma, T.; McCammon, J.A. Science, 1994,263, 1276.

[55] Summers, W.K.; Koehler, A.L.; Marsh, A.M.; Tachiki, K.; Kling,A. Lancet, 1989, 1(8640), 729.

[80] Inoue, A.; Kawai,T.; Wakita, M.; Iimura, Y.; Sugimoto, H.;Kawakami, Y. J. Med. Chem., 1996, 39, 4460.

[56] Cardozo, M.; Kawai, T.; Iimura, Y.; Sugimoto, H.; Yamanishi, Y.;Hopfinger, A. J. Med. Chem., 1992, 35, 590.

[81] Ripoll, D.R.; Faermam, C.H.; Axelsen, P.H.; Silman, I.;Sussman, J.L. Proc. Natl. Acad. Sci. USA, 1993, 90, 5128

[57] Kawakami, Y.; Kawai, T.; Inoue, A.; Iimura,Y.; Sugimoto, H.;Yoshida, Y.; Sato, T. In Proceedings of the 14th Symposium onMedicinal Chemistry (Japan), 1993. Pp. 19.

[82] Raymond, C.T.; Thanh, N.T.; McCammon, J.A. Biochemistry,1993, 32, 401.

[83] Kryger, G.; Silman, I.; Sussman, J.L. J. Physiol. Paris, 1998, 92,191.[58] Kawakami, Y.; Inoue, A.; Kawai, T.; Wakita, M.; Sugimoto, H.;

Hopfinger, A. BioOrg. Med. Chem., 1996, 4, 1429.[84] Harel,M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.;

Hirth, C.; Axelsen, P.; Silman. I.; Sussman, J.L. Proc. Natl. Acad.Sci. USA, 1993, 90, 9031.

[59] Villalobos, A.; Blake, J. F.; Biggers, C. K.; Butler, T. W.; Chapin,D. S.; Chen, Y. L.; Ives, J. L.; Jones, S. B.; Liston, D. R.; Nage, A.A.; Nason, D. M.; Nielsen, J. A.; Shalaby, I. A.; White, W. F., J.Med. Chem., 1994, 37, 2721. [85] Taylor, P. J. Biol. Chem., 1991, 266, 4025.

[86] Inoue, A., Kawakami, Y. unpublished results.[60] Ishihara, Y.; Hirai, K.; Miyamoto, M.; Goto, G. J. Med. Chem.,1994, 37, 2292.

[87] Yamanishi, Y.; Ogura, H.; Kosasa, T.; Araki, S.; Sawa,Y.;Yamatsu, K. In Basic, Clinical, and Therapeutic Aspects ofAlzheimer’s and Parkinson’s Diseases; Nagatsu, T.l.; Fisher,A.;Yoshida, M., Eds.; Plenum Press: New York, 1990, Vol. 2, pp.409.

[61] Tong,W.; Collantes, E.R.; Yu, C.; Weish, W.J. J. Med. Chem.,1996, 39, 380.

[62] Cardozo, M.G.; Iimura, Y.; Sugimoto, H.; Yamanishi, Y.;Hopfinger, A.J. J. Med. Chem., 1992, 35, 584.

[88] Yamanishi,Y.; Ogura, H.; Kosasa, T. et al., Eur. J. Pharmacol.,1990, 183, Abst. 1935.[63] Sussman, J.L.; Harel,M.; Frolow, F.; Offner, C.; Goldman, A.;

Toker, L.; Silman, I. Science, 1991, 253, 872.[89] Kosasa, T.; Yamanishi, Y.; Ogura, H. et al. Eur. J. Pharmacol.,

1990, 183, Abst. 1936.[64] Yamamoto, Y.; Ishihara, Y.; Kuntz, I.D. J. Med. Chem., 1994, 37,3114.

[90] Rogers, S.L.; Yamanishi, Y.; Yamatsu, K. In Cholinergic basis forAlzheimer therapy, Becker, R.; Giacobini, E. Eds.; Birkhauser:Boston, 1991, pp. 314.

[65] Pang, Y.P.; Kozikowski, A.P. J. Comp. Aid. Mol. Des. , 1994, 8,683.

[66] Hopfinger, A.J.; Tokarski, J. J. Chem. Inf. and Computer Sci.,1997, 37, 792.

[91] Ogura, H.; Kosasa, T.; Araki, S.; Yamanishi, Y. et al. Soc.Neurosci. 1988, 14, Abstr., pp. 60.

[67] Dewar, M.J.S.; Thiel, W. J. Am. Chem. Soc., 1977, 99, 4899. [92] Rogers, S.L.; Friedhoff,L.T. Dementia, 1996, 7, 293.

[68] Hulme, E.C.; Birdsall, N.J.M.; Buckley, N.J. Annu. Rev.Pharmacol. Toxicol., 1990, 30, 633.

[93] Rogers, S.L.; Farlow,M.R.; Doody, R.S.; Mohs, R.; Friedhoff,L.T. Neurology, 1998, 50, 136.

[69] Johnson, C.K. ORTEP II, Report ORNL-5138. Oak RidgeNational Laboratory, TN.,

[94] Thomsen, T.; Zendeh, B.; Fischer, J.P.; Kewitz, H. Biochem.Pharmacol., 1919, 41, 139.

[70] Quinn, D.M. Chem. Rev., 1987, 87, 955. [95] Miyamoto, M.; Takahashi, H.; Nishiyama, M.; Ishihara, Y.; Goto,G. Japan J. Pharmacol., 1994, 64 (Suppl. 1): Abst. pp. 644.

[71] Krupka,R.M.; Laidler, K.J. J. Am. Chem. Soc., 1961, 83, 1445.[96] Carli, M.; Luschi, R.; Samanin, R.; Behav. Brain Res., 1997, 82,

185.[72] Krupka, R.M.; Laidler, K.J. J. Am. Chem. Soc., 1961, 83, 1454.

[73] Krupka, R.M. Biochemistry, 1965, 4, 429. [97] Barnes, N.M.; Costall, B.; Naylor, R.J.; Williams, T.J.; WischikC.M. Neuroreport, 1990, 1, 253.

[74] Nochi,S.; Asakawa, N.; Sato, T. Biol. Pharm. Bull., 1995, 18,1145. [98] Mucke, H.A.M.; Castaner, J. Drugs Fut., 1998, 23, 1075.

[75] Wlodek, S.T.; Clark, T.W.; Scott, L.R.; McCammon, J.A. J. Am.Chem. Soc., 1997, 119, 9513.

[99] McGeer, P.L.; McGeer, E.; Rogers, J.; Sibley, J. Lancet, 1990,335, 1037.

[76] Zhou, H.X.; Wlodek, S.T.; McCammon, J.A. Proc. Natl. Acad.Sci., USA, 1998, 95, 9280.

[100] Rogers, J.; Kirby, L.C.; Hempelman, S.R. et al. Neurology, 1993,43, 1609

[77] Radic, Z.; Kirchhoff, P.D.; Quinn, D.M.; McCammon, J.A.;Taylor, P. J. Biol. Chem., 1997, 272, 23265.


[101] Breitner, J.C,; Gau, B.A.; Welsh, K.A.; Plassman, B.L.;McDonald, W.M.; Helms, M.J.; Anthony, J.C. Neurology, 1994,44, 227.

[108] Tatemoto, K.; Rokaeus, A.; Jornvall, H.; McDonald, T.J.; Mutt, V.FEBS Lett., 1983, 164, 124.

[109] Melander. R.Y.; Hokfelt, T.; Rokaeus, A.; Cuello, A.C.; Oertel,W.H.; Verhofstad, A.; Goldstein, M. J. Neurosci., 1986, 6, 3640.[102] Rich, J.B.; Rasmusson, D.X.; Folstein, M.F.; Carson, K.A.;

Kawas, C.; Brandt, J. Neurology, 1995, 45, 51.[110] Merchenthaler, I.; Lopez, F.J.; Negro-Vilar A. Prog.Neurobiol.

1993, 40, 711.[103] Subbarao, K.V.; Richardson, J.S.; Ang, L.C. J. Neurochem., 1990,55, 342.

[111] Fisone, G.; Wu, C.F.; Consolo, S.; Nordstrom, O.; Brynne, N.;Bartfai, T.; Melander, T.; Hokfelt, T. Proc. Nat.l Acad. Sci. USA,1987, 84, 7339.

[104] Gotz, M.E.; Freyberger, A.; et. al. Dementia, 1992, 213.

[105] Simpkins, J.W.; Singh, M.; Bishop, J. Neurobiol. Aging, 1994, 15Suppl. 2, 195. [112] Chan-Palay, V. J. Comp. Neurol., 1988, 273, 543.

[106] Honjo, H.; Ogino, Y.; Naitoh, K.; Urabe, M.; Kitawaki, J.; Yasuda,J.; Yamamoto, T.; Ishihara, S.; Okada, H.; Yonezawa, T. et al. J.Steroid. Biochem., 1989, 34, 521.

[113] Crawley, J.N.; Wenk, G.L. Trends Neurosci., 1989, 12, 278.

[114] Nikolov, R.; Richardson, J.S. Drugs of Today, 1998, 34, 673.

[107] Hagion, N.; Ohkura, T. et al. 3rd International Symposium onAlzheimer’s and Parkinson’s Disease, Chicago, November 1993

Current Medicinal Chemistry Donepezil Hydrochloride (E2020 ... · Current Medicinal Chemistry, 2000...

Documents

Transcript of Current Medicinal Chemistry Donepezil Hydrochloride (E2020 ... · Current Medicinal Chemistry, 2000...