WO2006060082A1 - Tricyclic compounds, preparation thereof and use thereof as cholinesterase activity inhibitors - Google Patents

Abstract

Described herein are tricyclic compounds of formula (I) and (II) and methods of making them and using them as cholinesterase activity inhibitors.

Description

TRICYCLIC COMPOUNDS , PREPARATION THEREOF AND USE THEREOF AS CHOLINESTΞRASE ACTIVITY INHIBITORS

CROSSREFERENCETORELATEDAPPLICATION

5

This application claims priority to U.S. Provisional Application Serial No. 60/621,287, filed October 22, 2004, which application is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

Physovenine (1), with an ether linkage in ring-C of natural physostigmine (2), instead of a N-Me group (Figure 1), is a further alkaloid present in Physostigma venenosum, whose chemistry and biological properties have been extensively reviewed.¹ Physostigmine and its analogues represent the backbone of current Alzheimer's disease (AD) treatment.⁴

Physostigmine has served as a template in the development of several agents for AD, including a slow-release formulation of the parent compound (synapton, Forest Laboratories, St Louis, MO), its heptyl-carbamate (eptylstigmine, Mediolanum, Italy), both withdrawn from clinical development due to efficacy/toxicity issues, and its phenyl-carbamate (phenserine, National Institute on Aging, Baltimore, MD, and Axonyx, New York, NY) that currently is in phase 3 clinical assessment in mild to moderate afflicted patients. The pharmacodynamics that underscores the action of this class of drugs is the elevation of brain acetylcholine (ACh) levels that is achieved by inhibition ofits hydrolyzing enzymes, AChE and BChE.⁴ A hallmark of AD is synaptic loss, particularly of the forebrain cholinergic system that is pivotal in higher brain functions associated with learning, memory and behavior.⁴

Extensive structural information is available on the interaction of AChE and BChE with ACh and related substrates thanks to extensive X-ray crystallography and mutagenesis studies.^5"7 The catalytic subunit of both enzymes has a molecular mass of 60 to 80 kDa, depending on its level of glycosylation and the species from which it derives.⁶ The active site for ACh hydrolysis is buried within a primarily hydrophobic 20 A deep gorge.⁷ In its interaction and inhibition of AChE and BChE, it is generally believed that, under physiological conditions, the basic N^CH₃) group of physostigmine (PZa = 8.46)⁸ and analogues gain a H⁺ to form a quaternary ammonium group. In a manner similar to ACh, positively charged physostigmine is drawn into the enzyme by electrostatic field forces. Thereafter, it interacts with specific binding domains to form an inhibitor-enzyme complex. Whereas the quaternary ammonium group plays an important role in this process, physovenine (1), that bears a neutral ring- C oxygen instead of basic ring-C nitrogen, appears to be equipotent as an anticholinesterase, as is the ring-C thia analogue of 1, thiaphysovenine.⁹ This phenomenon can be potentially explained by the common capacity of the ether oxygen and amino nitrogen of 1 and 2 to form H-bonding with amino acids, providing the basis of the interaction between the compounds and enzyme. However, the common ring-B (CH₃) in both provides a weaker basic group (PKa = 3.7),⁸ and hence reintroduces the potential involvement of charge into the compound-enzyme interaction.

Thus, it would be advantageous to easily synthesize compounds that are more active with respect to treating diseases such as, for example, dementia. Described herein are tricyclic compounds that can be used in a number of different applications including the treatment of dementia.

SUMMARY

Described herein are tricyclic compounds and methods of making and using thereof. The advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. Like numbers represent the same elements throughout the figures. Figure 1 shows chemical structures of Physovenine- and physostigmine- based carbamates.

Figure 2 shows a general reaction scheme for producing compounds having the formula II and IV.

Figure 3 shows a reaction scheme for preparing racemic compounds described herein.

Figure 4 shows the X-ray crystallographic structures of two compounds described herein.

Figure 5 shows the action of carbamates described herein on cell viability (upper) as assessed by MTT assay and cell number (lower) as assessed by trypan blue cell count (mean + standard error of the mean).

Figure 6 shows the dipole moments and superimposed picture of four compounds described herein.

Figure 7 shows the docking model of a compound described herein in the binding domain of TcAChE (EC3,1 ,1 ,7). Figure 8 shows the synthesis of optically active compounds described herein.

Figure 9 shows the synthesis of optically active compounds described herein.

Figure 10 shows a reaction scheme for preparing compounds described herein.

Figure 11 shows a reaction scheme for preparing compounds described herein.

Figure 12 shows a reaction scheme for preparing compounds described herein. Figure 13 shows a reaction scheme for preparing compounds described herein. Figure 14 shows X-ray crystallo graphic picture and corresponding chemical structure of a compound described herein.

Figure 15 shows a Chiral HPLC analysis²¹ of two enantiomers described herein. Figure 16 shows the union of molecular volume maps for compounds described herein.

Figure 17 illsutrates a model of a TcAChE complex showing its covalent bonding with Ser₂₀₀.

Figure 18 shows the chemical structure of compounds described herein.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

"Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase "optionally substituted lower alkyl" means that the lower alkyl group can or can not be substituted and that the description includes both unsubstituted lower alkyl and lower alkyl where there is substitution.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. Variables such as R¹ -R⁵, n, X, and Y used throughout the application are the same variables as previously defined unless stated to the contrary.

The term "substantially pure" with respect to enantiopurity refers to greater than 95%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100% of one enantiomer with respect to the other enantiomer. The term "alkyl group" as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 25 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, /z-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Examples of longer chain alkyl groups include, but are not limited to, an oleate group or a pahnitate group. A "lower alkyl" group is an alkyl group containing from one to six carbon atoms.

The term "alkenyl group" as used herein is a hydrocarbon group of from 2 to 24 carbon atoms and structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (AB)C=C(CD) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C. The term "alkynyl group" as used herein is a hydrocarbon group of 2 to 24 carbon atoms and a structural formula containing at least one carbon-carbon triple bond.

The term "aryl group" as used herein is any carbon-based aromatic group including, but not limited to, benzene, naphthalene, etc. The term "aromatic" also includes "heteroaryl group," which is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy.

The term "cycloalkyl group" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "heterocycloalkyl group" is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulphur, or phosphorus.

The term "aralkyl" as used herein is an aryl group having an alkyl, alkynyl, or alkenyl group as defined above attached to the aromatic group. An example of an aralkyl group is a benzyl group.

The term "ether" as used herein is represented by the formula ROR', where R and R' can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term "carboxylate" as used herein is represented by the formula -C(O)OH or the ester thereof.

The term "amide" as used herein is represented by the formula -C(O)NR, where R can alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

By "subject" is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. The term "subject" can include domesticated and/or companion animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).

"Treatment" or "treating" means to administer a composition to a subject or a system with an undesired condition. The effect of the administration of the composition to the subject can have the effect of but is not limited to reducing or preventing the symptoms of the condition, a reduction in the severity of the condition, or the complete ablation of the condition.

By "prevent" or "preventing" means the administration of a composition to a subject or a system at risk for an undesirable condition. The condition can include a disease or a predisposition to a disease. Prevention can range from a reduction in the severity of the condition to the complete ablation of the condition.

By "effective amount" is meant a therapeutic amount needed to achieve the desired result or results, e.g., inhibiting enzymatic activity.

Herein, "inhibition" or "inhibiting" means to reduce activity as compared to a control. It is understood that inhibition can mean a slight reduction in activity to the complete ablation of all activity. An "inhibitor" can be anything that reduces the targeted activity.

Disclosed are compounds, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

I. Compounds

In one aspect, described herein are compounds having the formula I

I wherein each R¹ is, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group,

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, and n is from 1 to 4, or the pharmaceutically-acceptable salt or ester thereof wherein the compound having the formula I is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the

(-)-enantiomer and (+)-enantiomer. The R¹O group denoted in formula I can be present at any position of the aryl ring. It is contemplated that when two or more R O groups are present, each and every combination OfR¹O groups with respect to their position on the aryl ring are included in formula I.

In one aspect, compounds having the formula I have the formula II

II wherein R¹ is hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group, and

R is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula II is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer. ha one aspect, R² in formulae I or II is a C₁-C₄ branched- or straight-chain alkyl group, hi another aspect, R² in formulae I or II is methyl, hi a further aspect, R¹ in formulae I or II is hydrogen. In another aspect, R¹ is hydrogen and R² is methyl in formula I or II. hi another aspect, R¹ in formulae I or II is C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group. These compounds are referred to as carbamate compounds, hi one aspect, R³ and R⁴ can be, independently, a lower alkyl group such as, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, and the like, hi another aspect, R³ is hydrogen and R⁴ is a lower alkyl group, hi a further aspect, R³ is hydrogen and R⁴ is ethyl, o-tolyl, orp-isopropyl phenyl. hi another aspect, with respect to formulae I and II, R¹ is C(O)NR³R⁴, wherein

R³ is hydrogen and R⁴ is ethyl, o-tolyl, orp-isopropyl phenyl, and R² is methyl. In another aspect, described herein are compounds having the formula III

III wherein each R¹ is, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group,

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, and n is from 1 to 4, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula III is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer. Similar to above for formula I, the R¹O group (single or multiple) denoted in formula III can be present at any position of the aryl ring.

In one aspect, compounds having the formula III have the formula IV

IV wherein R¹ is hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group, and R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula II is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer.

In one aspect, R² in formulae III or IV is a C₁-C₄ branched- or straight-chain alkyl group. In another aspect, R² in formulae III or IV is methyl. In a further aspect, R¹ in formulae III or IV is hydrogen. In another aspect, R¹ is hydrogen and R² is methyl in formula III or IV.

In another aspect, R¹ in formulae III or IV is C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group. These compounds are referred to as carbamate compounds, hi one aspect, R³ and R can be, independently, a lower alkyl group such as, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, and the like. In another aspect, R³ is hydrogen and R⁴ is a lower alkyl group. In a further aspect, R³ is hydrogen and R⁴ is ethyl, o-tolyl, or j^-isopropyl phenyl.

In another aspect, with respect to formulae III and TV, R¹ is C(O)NR³R⁴, wherein R³ is hydrogen and R⁴ is ethyl, o-tolyl, or j^-isopropyl phenyl, and R² is methyl.

The compounds having the formulae I-IV can be racemic or the substantially pure (+)- or (-)-enantiomer. Depending upon the selection of the starting materials used to produce the compounds and experimental procedures employed, it is possible to produce substantially pure enantiomeric compounds. Methods for producing racemic and substantially pure enantiomeric compounds are presented below, hi one aspect, compounds having the formulae I-IV are the substantially pure (+)-enantiomer. In another aspect, compounds having the formulae I-IV the substantially pure (-)- enantiomer. In a further aspect, compounds having the formulae I-IV are racemic. Any of the compounds described herein can be the pharmaceutically acceptable salt or ester thereof. Pharmaceutically acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base. Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like.

In another aspect, if the compound possesses a basic group, it can be protonated with an acid such as, for example, HCl or H₂SO₄, to produce the cationic salt. In one aspect, the compound can be protonated with tartaric acid to produce the tartarate salt. In one aspect, the reaction of the compound with the acid or base is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0 ⁰C to about 100 °C such as at room temperature, hi certain aspects where applicable, the molar ratio of the compounds described herein to base used are chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material, the starting material can be treated with approximately one equivalent of pharmaceutically-acceptable base to yield a salt.

Ester derivatives are typically prepared as precursors to the acid form of the compounds and accordingly can serve as prodrugs. Generally, these derivatives will be lower alkyl esters such as methyl, ethyl, and the like. Amide derivatives -(CO)NH₂, -(CO)NHR and -(CO)NR₂, where R is an alkyl group defined above, can be prepared by reaction of the carboxylic acid-containing compound with ammonia or a substituted amine. It is contemplated that the pharmaceutically-acceptable salts or esters of the compounds described herein can be used as prodrugs or precursors to the active compound prior to the administration. For example, if the active compound is unstable, it can be prepared as its salts form in order to increase stability. Prior to administration, the salt can be converted to the active form. For example, the salt can be added to a saline solution to produce the active compound, followed by administration of the saline solution containing the active compound to the subject. II. Methods for Preparing Compounds

Described herein are methods for producing compounds having the formulae I and III. In one aspect, a general reaction scheme for producing compounds having the formula II and IV is depicted in Figure 2. Referring to Figure 2, the first step involves synthesizing a compound having the formula VIII

VIII wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group. In one aspect, R² in formula VIII is a C₁-C₄ branched- or straight-chain alkyl group. In another aspect, R² is not a methyl group, which is formula V referred to herein.

In one aspect, a method for making compounds having the formula VIII involves reacting 1,4-cyclohexandione with a compound having the formula VI

wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group. In one aspect, R² is a C₁-C₄ branched- or straight-chain alkyl group. In another aspect, R² is methyl, hi one aspect, the reaction can be conducted at an elevated temperature (i.e., above room temperature) in order to facilitate or expedite the reaction. For example, the reaction temperature can occur at the boiling point of the solvent used in the reaction. In one aspect, the reaction is refluxed in toluene.

Once a compound having the formula VIII is produced, it can be converted to a compound having the formula VII

VII wherein R² and R⁵ are, independently, a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula VII is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer. hi one aspect, R² and R⁵ are, independently, a C]-C₄ branched- or straight-chain alkyl group, hi another aspect, R² and R⁵ are methyl. hi one aspect, compounds having the formula VII can be prepared by (a) reacting a compound having the formula VIII with a base to produce a first product; and

(b) reacting the first product with a compound having the formula XCH₂COOR⁵, wherein R⁵ is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, and X is a leaving group. It is believed that treatment of compound VIII with a base deprotonates the α- hydrogen of the lactone to produce an enolate, which then reacts with XCH₂COOR⁵ to produce VII. Any base that can deprotonate an α-hydrogen of an ester or lactone is suitable in this aspect. In one aspect, the base is a hydride, a hydroxide, a carbonate, or an amide. In another aspect, the base is sodium hydride. hi general, after treatment with base, an enolate is produced in situ. The enolate is reacted with XCH₂COOR⁵, where X is a leaving group. The term "leaving group" is any group bonded to a compound that can be readily displaced from the compound when the compound is reacted with an anion. For example, the enolate that is produced after base treatment of VIII produces a carbanion, which then reacts with XCH₂COOR and displaces X^". In one aspect, the leaving group is a sulfonate, acetate, or carbonate. In another aspect, the X is a halide such as, for example, fluoride, chloride, bromide, or iodide.

Parameters such as, for example, reaction temperatures, can vary depending upon the selection of compound VIII, base, and XCH₂COOR⁵. In one aspect, the reaction is conducted at reduced temperature.

In one aspect, it is possible to produce the substantially pure enantiomeric forms of compound VII. For example, the hydroxyl group of formula VII can be deprotonated with a base followed by treatment with a chiral auxiliary. Chiral auxiliaries are well-known in the art for resolving and separating enantiomers. In one aspect, the chiral auxiliary is an optically active carbonate such as, for example, (+)- or (-)-menthylchloroformate. Once the chiral auxiliary is bonded to the compound, it is possible to resolve and separate each enantiomer of formula VII using techniques known in the art such as, for example, recrystallization or chromatography. Methods for producing substantially pure enantiomers of formula VII are discussed in detail below.

Referring to Figure 2, in one aspect, compounds having the formula X or XI

X or

XI wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, wherein the compound having the formula X or XI is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)- enantiomer and (+)-enantiomer, can be prepared by (a) reacting a compound having the formula VII

VII wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group,

R⁵ is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, wherein the compound having the formula VII is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer, with a reducing agent to produce a first product; and

(b) reacting the first product with an acid to produce a compound having the formula X, XI, or a mixture thereof, wherein when a mixture of compounds X and XI are present,

(c) separating compound X from compound XI.

Not wishing to be bound by theory, it is believed that the reducing agent can form either one reduction product (X or XI) or a mixture of reduction products (X and XI) from formula VII. The amount and nature of the reduction products will vary depending upon the selection of the starting material, reducing agent and reductive conditions (e.g., reaction temperature and time). Examples of reducing agents include, but are not limited to, substituted or unsubstituted aluminium hydrides and borohydrides, where the aluminium hydride and borohydride can be AlH₄ ^", BH₄ ^" or one or more the hydrogen atoms can be substituted. In one aspect, the reducing agent can be lithium aluminium hydride (LiAlH₄), Red- Al, BINAl-H, DIBAL-H, lithium borohydride (LiBH₄), sodium borohydride (NaBH4), LiAlHRR'R", LiBHRR'R", or NaBHRR'R", where R, R', and R" can be, independently, alkyl, alkoxy, aryl, aryloxy, alkenyl, aralkyl, alkynyl, cycloalkyl, cycloalenyl, heterocycloalkyl, heterocycloalkenyl, heteroaryl, substituted amino and mercapto, or cyano. After formula VII is reduced by the reducing agent, the resultant anionic species is protonated with an acid, hi another case, hydrogen transfer can be achieved by catalytic hydrogenation.

If a mixture of X and XI is present, it is possible to separate the compounds using techniques known in the art such as, for example, recrystallization or chromatography. In another aspect, if it is difficult to separate X from XI, the hydroxyl group on the aryl ring of X and VI can be converted to another group that can facilitate the separation of the two compounds. For example, a mixture of X and XI can be reacted with a base (e.g., a hydride, a hydroxide, a carbonate, or an amide) to deporotonate the aryl hydroxyl group followed by reacting the alkoxide with a compound having the formula R¹ -Y, wherein R¹ is a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group, and Y comprises a group capable of reacting with the deprotonated hydroxyl group to produce compounds II and W. In one aspect, R¹ -Y is a branched- or straight-chain alkyl isocyanate or a substituted or unsubstituted aryl isocyanate. In this aspect, compound II and IV are carbamates. In one aspect, the isocyanate is ethyl isocyanate, o-tolyl isocyanate, oτp- isopropyl phenyl isocyanate. In this aspect, once the carbamate analogs of II and IV are produced, they can be separated from one another using techniques known in the art.

III. Methods of Use Delivery

As used throughout, administration of any of the compounds described herein can occur in conjunction with other therapeutic agents. Thus, the compound can be administered alone or in combination with one or more therapeutic agents. For example, a subject can be treated with a compound alone, or in combination with chemotherapeutic agents, antibodies, antivirals, steroidal and non-steroidal antiinflammatories, conventional immunotherapeutic agents, cytokines, chemokines, and/or growth factors. Combinations may be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term "combination" or "combined" is used to refer to either concomitant, simultaneous, or sequential administration of two or more agents.

The compounds can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intracutaneous, intraperitoneal or intramuscular injection. In one aspect, the compounds can be delivered intrathecally. It is also contemplated that the compounds can be administered transdermally via, for example, a patch or ionotophoresis. The disclosed compounds can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, intraocularly (e.g., intravitreally), transdermally, intratracheally, extracorporeally, or topically (e.g., topical intranasal administration or administration by inhalant). As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism. The latter can be effective when a large number of subjects are to be treated simultaneously. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying mechanism or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.

Parenteral administration of the compound, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. Alternatively, parenteral administration can involve the use of a slow release or sustained release system such that a constant dosage is maintained.

The exact amount of the compounds described herein required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular neurological disorder to be targeted, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every compound. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. The time at which the compounds can be administered will also vary depending upon the subject, the disorder, mode of administration, etc. The compound can be administered to the subject prior to the onset of the muscle disorder or during a time when the subject is experiencing symptoms of the muscle disorder. The compound can be administered over several weeks or months at varying intervals depending upon the subject and disorder to be treated.

Pharmaceutically-Acceptable Carriers

In one aspect, any of the compounds described above can be combined with at least one pharmaceutically-acceptable carrier to produce a pharmaceutical composition. The pharmaceutical compositions can be prepared using techniques known in the art. In one aspect, the composition is prepared by admixing the compound with a pharmaceutically-acceptable carrier. The term "admixing" is defined as mixing the two components together. Depending upon the components to be admixed, there may or may not be a chemical or physical interaction between two or more components. Pharmaceutically-acceptable carriers are known to those skilled in the art. These most typically would be standard carriers for administration to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

Molecules intended for pharmaceutical delivery may be formulated in a pharmaceutical composition. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally).

Preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles, if needed for collateral use of the disclosed compositions and methods, include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles, if needed for collateral use of the disclosed compositions and methods, include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

It will be appreciated that the actual preferred amounts of active compound in a specified case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, and the particular situs and mammal being treated. Dosages for a given host can be determined using conventional considerations, e.g. by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate conventional pharmacological protocol. Physicians and formulators, skilled in the art of determining doses of pharmaceutical compounds, will have no problems determining dose according to standard recommendations (Physicians Desk Reference, Barnhart Publishing (2004).

Therapeutic Uses

The compounds described herein can be used to treat a number diseases in a subject. In one aspect, described herein is a method of treating dementia in a subject diagnosed with dementia, comprising administering to the subject an effective amount of a compound described herein, whereby the compound treats dementia in the subject. As used herein, the term "dementia" describes a neurodegenerative disorder that results from an organic brain disease in which a subject experiences usually irreversible deterioration of intellectual faculties with accompanying emotional disturbances. In one aspect, the dementia can be Parkinson's disease, vascular dementia, schizophrenia, or Lewy body dementia. In another aspect, the dementia is Alzheimer's Disease or Mild Cognitive Impairment (MCI), whichis often depicted as a pre- Alzheimer's state. In one aspect, the compounds described herein can be used for improving a symptom associated with the dementia, stabilizing the symptom, or delaying the worsening of the symptom. In another aspect, the compound increases the life-span of a subject compared to the average life-span of corresponding subjects not administered the compound.

In another aspect, the compounds described herein can be used to treat (1) Down's syndrome or (2) glaucoma and other opthalmological indications exemplified, but not limited to accommodative esotropia, myasthenia gravis confined to the extraocular and eyelid muscles, Adie (or tonic pupil) syndrome, and louse or mite infestation of lashes). In a further aspect, the compounds described herein can be used to treat abdominal or urinary distension from a variety of medical or surgical causes (paralytic ileus and atony of the urinary bladder), stroke, head trauma, myasthenia gravis, Sjorgrens syndrome, and type 2 diabetes in a subject diagnosed with such a malady. In another aspect, the compounds described herein can be used to reduce pain in subject.

Any subject suffering from dementia can be treated with the compounds described herein. In one aspect, the subject is a mammal such as, for example, a human or domesticated animal. In one aspect, compounds having the formula II, IV, or a mixture thereof can be used to treat dementia or the other diseases described herein in a subject. In another aspect, when the compound is II, IV, or a mixture thereof, R³ is hydrogen and R⁴ is ethyl, o-tolyl, or j?-isopropyl phenyl, and R² is methyl.

In one aspect, described herein is a method for inhibiting cholinesterase activity in a subject, comprising administering an effective amount of one or more compounds described herein. Not wishing to be bound by theory, it is believed that inhibiting cholinesterase activity reduces the production of amyloid precursor protein that ultimately produces the neurotoxic peptide β-amyloid protein. Elevated levels of β- amyloid protein have been associated with dementias such as, for example Alzheimer's disease, hi one aspect, the cholinesterase that is inhibited comprises acetylcholinesterase or butyrylcholinesterase. It is contemplated that different isotypes or isoforms of the cholinesterase (e.g., Gl, G2, G4 forms) can be inhibited, hi one aspect, compounds having the formula II, FV, or a mixture thereof can be used to inhibit cholinesterase activity. In another aspect, when the compound is II, IV, or a mixture thereof, R³ is hydrogen and R⁴ is ethyl, o-tolyl, orp-isopropyl phenyl, and R² is methyl.

In one aspect, described herein is a method of inhibiting production of amyloid β-peptide in a subject, comprising administering to the subject an effective amount of one or more compounds described herein. As described above, elevated levels of amyloid β-peptide have been associated with dementia, hi another aspect, described herein are methods of inhibiting production of a C-terminal fragment produced from cleavage of amyloid β-peptide from an amyloid precursor protein, comprising administering to the subject an effective amount of one or more compounds described herein. Not wishing to be bound by theory, it is believed that CTFβ and CTFγ, which are C-terminal fragments produced from cleavage of amyloid β-peptide, can be toxic to cells if they remain in a cell after cleavage of an amyloid β-peptide. The compounds described herein can be either delivered to the subject in vivo using the techniques described above or the cells of the subject can be contacted directly with the compound in order to inhibit production of amyloid β-peptide. In the case when the cells are contacted directly, the contacting step can be performed in vivo or in vitro. In another aspect, described herein are methods for screening a compound for the ability to inhibit cholinesterase activity in a subject, comprising:

(a) administering to the subject a compound described herein; and

(b) detecting a decrease in the cholinesterase activity as compared to the cholinesterase activity in a control subject not administered the compound, whereby decreased cholinesterase activity identifies the compound as having the ability to inhibit cholinesterase activity in a subject. The detection of cholinesterase activity can be carried out by methods standard in the art. In one aspect, compounds having the formula II, IV, or a mixture thereof can be screened to determine if they inhibit cholinesterase activity. In another aspect, when the compound is II, IV, or a mixture thereof, R³ is hydrogen and R⁴ is ethyl, o-tolyl, orp-isopropyl phenyl, and R² is methyl.

In another aspect, any of the compounds described herein can be used as an imaging agent for the diagnosis of dementia in a subject. For example, the compounds described herein can be used as imaging agents in the brain to image plaque morphology for diagnosing Alzheimer's. In another aspect, the compounds described herein can ebe used to label AChE and BuChE in living animals and tissue in order to quantify these enzymes in a healthy and diseased subject. For example, in Alzheimer's disease and Lewy body disease, AChE is known to decline and BuChE to increase early during the disease process.

In another aspect, the compounds described herein can be used as a prophylactic and/or post treatment for exposure to a nerve gas (e.g., sarin, soman, Tabun, VX), an insecticide (e.g., parathion, fenthion, dimpylate, malathion, TEPP), an anticholinergic poison (e.g., atropine), or excessive echothiophate (e.g., phospholine iodide). EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

Example I

Melting points (uncorrected) were measured with a Fisher- Johns apparatus; ¹H- NMR were recorded on a Bruker (Bellevica, MA) AC-300 spectrometer; MS (m/z) were recorded on a Hewlett-Packard 5973 GC-MS (CI). HRMS were performed by the UCR Mass Spectrometry Facility, Department of Chemistry, University of California. Optical rotations were measured by JASCO, Model DJJP-370 (Japan, Spectroscopic Co., LTD.); Chiral HPLC analysis: ChiraxDex (5μm) colume (part No: 79925CB-584) provided by Agilent Technologies, eluted by MeOH/H₂O in different ratio, UVD 254 nm. AU reactions involving non-aqueous solutions were performed under an inert atmosphere.

I. Synthesis and Characterization of Racemic Compounds (Figure 3)

5-Hydroxy-3-methyIbenzofuran-2(3iϊ)-one (3). Pyruvic acid (8.81g, 0.10 mol) was added to 1 ,4-cyclohexanedione (11.2 g, 0.10 mol). The reaction mixture was heated to 160-170 ⁰C and then stirred for 3 hours. The mixture was maintained at room temperature to allow crystallization. Filtration gave a crude product that then was recrystallized from ethanol to provide product 3 as pale-yellow crystals (6.5O g, 40%): mp. 167.0-167.5⁰ C ; ¹H NMR (CDCl₃) δ 6.80-6.51 (m, 3 H, Ar-H), 4.69 (s, IH, OH), 3.55 (q, IH, C3-H) and 1.38 (d, 3H, C3-CH₃) ppm; CI-MS (CH₄), m/z : 165 (MH⁺) and 137. 5-Hydroxy-3-methyl-3-methoxycarbonylmethylenebenzofuran-2(3H)-one

(4). Under a nitrogen atmosphere, sodium hydride (0.24 g, 0.01 mol) was added to a solution of compound 3 (1.64 g, 0.01 mol) and methyl chloroacetate (1.19 g, O.Olmol) in 6 mL of dry DMF at 0 ⁰C in portions over one hour. The mixture was stirred for another hour at 0 ⁰C, and then overnight at room temperature. The reaction mixture was poured over 25 g of ice, and extracted with ether (20 mL x 3). The extracted solution thereafter was washed with brine (20 mL x 2) and dried over magnesium sulfate. The final concentrated solution was maintained at room temperature and crystallized to provide a crude product that then was recrystallized from EtOAc to give product 4 as white needle-like crystals (1.09 g, 46.1%): m.p. 153.5-155.0 ⁰C; ¹H NMR (CDCl₃) δ 6.92-6.61 (m, 3 H, Ar-H), 5.07 (s, IH, OH), 3.44 (s, 3H, OCH₃), 3.02, 2.84 (AB,

Jgem=18.0Hz, 2H, C3-CH₂CO) and 1.41 (s, 3H, C3-CH₃) ppm; ¹³C-NMR (CDCl₃) δ 179.7, 170.0, 154.0, 144.9, 132.1, 114.6, 110.6, 110.2, 51.5, 44.5, 40.9 and 24.7 ppm; CI-MS (CH₄), m/z : 237 (MH⁺), 205, and 177; HR-MS m/z: Calcd for C₁₂H₁₂O₅: 236.0685; Found: 236.0673. 5-Hydroxy-3a-methyl-2,3,3a,8a-tetrahydrofuro[2,3-6]benzofuran (5) and 7- hydroxy-4,5-dihydro-2,5-methano-5-methyl-l,3-benzodioxepine (6). Under a nitrogen atmosphere, a solution of compound 4 (1.04 g, 4.40 mmol) in 20 mL of THF was added dropwise to lithium aluminium hydride (0.33 g, 8.70 mmol) in THF (20 mL) at room temperature. The mixture was stirred for one hour at room temperature, and then oxalic acid (1.97 g, 21.90 mmol) was added and stirred for another 0.5 hour, likewise, at room temperature. The mixture was filtered and a solid material partitioned between the ether and aqueous solution of oxalic acid. Thereafter, the combined THF and ether solution was evaporated to remove solvents. The resulting residue was chromatographed on silica gel (EtOAc / Hexane = 1 /3) to provide a mixture of 5 and 6 as a gum (84.8 mg, 10.0%): ¹H NMR (CDCl₃) δ 6.75-6.60 (m, 6 H, Ax-H), 5.85 (s, IH, C8a-H of 5), 5.78(d, J=I.80Hz, IH, C2-H of 6), 1.55 (s, 3H, C3a-CH₃ of 5), 1.50 (s, 3H, C5-CH₃ of 6)ppm; GC-MS (CI): two peaks (1 : 1), m/z 193 (MH⁺) respectively. It was directly used as a reactant in the following reactions without separation.

3a-Methyl-2,3,3a,8a-tetrahydrofuro[2,3-6]benzofuran-5-yI iV- ethylcarbamate (7) and 4,5-dihydro-2,5-methano-5-methyl-l,3-benzodioxepin-7-yl iV-ethylcarbamate (8). Under a nitrogen atmosphere, two small pieces of sodium (lmg cα.per piece) were added into a solution of compound 5 and 6 (32.0 mg, 0.166 mmol) in 5 niL of dry ether at room temperature. The mixture was stirred for 2 minutes, and then ethyl isocyanate (24.8 mg, 0.3489 mmol) was added in one portion. The reaction was continued for another one hour at room temperature, then 5 mL of water was added and the ether layer was separated. After drying over sodium sulphate and filtering, the filtrate was evaporated to provide a residue that then was chromatographed (preparative TLC: silica gel, EtOAc / Hexane = 1/3 ). This latter procedure was repeated on three occasions to afford product 7 (11.3 mg, 51.7%) and 8 (13.2 mg, 60.4%.) as colorless gums. Product 7: ¹H NMR (CDCl₃) δ 6.87-6.60 (m, 3H, Ar-H), 5.77 (s, IH, C8a-H), 4.89 (s, br, IH, NH), 4.06-3.56 (m, 2H, C2-H), 3.23 (q, 2H, CH₂ of EtNH-), 2.13-1.91 (m, 2H, C3-H), 1.47 (s, 3H, C3a-CH₃) and 1.14 (t, 3H, CH₃ of EtNH-) ppm; ¹³C-NMR (CDCl₃) δ 155.2, 153.9, 144.4, 131.9, 120.6, 115.9, 115.8, 108.4, 67.6, 52.9, 40.1, 35.1, 23.2 and 14.1 ppm; CI-MS (CH₄), m/z: 264 (MH⁺), 193, 175 and 72; HR-MS m/z: Calcd for C₁₄Hi₇NO₄: 263.1158; Found: 263.1162. Product 8: ¹H NMR (CDCl₃) δ 6.82-6.67 (m, 3H, Ar-H), 5.69 (d, J=I.80Hz, IH, C2-H), 4.88 (s, br, IH, -NH-), 4.17, 3.70 (AB, Jgetn= 7.20Hz, 2H, C4-H₂), 3.22 (q, 2H, CH₂ of EtNH-), 2.19-1.91 (m, 2H, ClO-H₂), 1.41 (s, 3H, C5-CH₃) and 1.16 (t, 3H, CH₃ of EtNH-) ppm; ¹³C-NMR (CDCl₃) δ 155.3, 149.3, 145.0, 132.7, 121.7, 117.3, 117.0, 100.8, 84.8, 40.0, 36.5, 21.6, 17.2 and 15.5 ppm; CI-MS (CH₄), m/z: 264 (MH⁺), 246, 220, 193, 175, 149 and 72; HR-MS m/z: Calcd for Ci₄H_nNO₄: 263.1158; Found: 263.1164.

3a-Methyl-2,3,3 a,8a-tetrahydrofuro [2,3-b] benzofuran-5yl JV-(2 '- methylphenyl)- carbamate (9) and 4,5-dihydro-2,5-methano-5-methyl-l,3- benzodioxepin-7-yl iV-(2'-methylphenyl)-carbamate (10). Under a nitrogen atmosphere, two small pieces of sodium (lmg ca. per piece) were added into a solution of 5 and 6 (29.5 mg, 0.153mmol) in 5 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes, and then o-tolyl isocyanate (20.4 mg, 0.153 mmol) was added in one portion. The reaction was continued for 30 minutes at room temperature. Thereafter 5 mL of water was added and the ether layer was separated. After drying over sodium sulphate and filtering, solvent was removed from the filtrate by evaporation. The residue was chromatographed on a preparative TLC silica gel plate (EtOAc/Hexane=l/3) to afford products 9 (18.6mg, 74.9%), and 10 (16.1 mg, 64.4%) as a colorless gum. Product 9: ¹H-NMR (CDCl₃) δ 7.84 (s, br, IH, -NH-), 7.32-6.70 (m, 7H, Ar-H), 5.89 (s, IH, C8a-H), 4.18-3.68 (m, 2H, C2-H₂), 2.39-2.05 (m, 2H, C3-H₂), 2.31 (s, 3H, Ar-CH₃) and 1.56 (s, 3H, C3a-CH₃) ppm; CI-MS (CH₄), m/z: 326 (MH⁺), 193 and 134; HR-MS m/z: Calcd for C₁₉H₂₀NO₄: 326.1392; Found: 326.1402. Product 10: ¹H-NMR (CDCl₃) δ 7.87 (s, br, IH, -NH-), 7.33-6.70 (m, 7H, Ar-H), 5.79 (d,

J=1.80Hz, IH, C2-H), 4.22, 3.79 ( AB, J_g8M=6.84Hz, 2H, C4-H₂), 2.37-2.01 (m, 2H, ClO-H₂), 2.31 (s, 3H, Ar-CH₃) and 1.51 (s, 3H, C5-CH₃) ppm; CI-MS(CH₄), m/z: 326 (MH⁺) 193 and 134; HR-MS m/z: Calcd for C₁₉H₂₀NO₄: 326.1392; Found: 326.1387.

3a-Methyl-2,3,3a,8a-tetrahydrofuro[2,3-£]benzofuran-5yl iV-(4^»- isopropylphenyl)- carbamate (11) and 4,5-dihydro-2,5-methano-5-methyl-l,3- benzodioxepin-7-yl JV-(4'-isopropylphenyl)-carbamate (12). Under a nitrogen atmosphere, two small pieces of sodium (lmg ca. per piece) were added into a solution of compound 5 and 6 (18.0 mg, 0.094 mmol) in 5 mL of dry ether at room temperature. The mixture was stirred for 2 minutes, and then/»-isopropylphenyl isocyanate (15. lmg, 0.094 mmol) was added in a single portion. The reaction was continued for a further 45 minutes at room temperature, and then 3 mL of water was added and the ether layer was separated. After drying over sodium sulphate and filtering, the filtrate was subjected to evaporation to remove solvent. The resulting residue was chromatographed on a preparative TLC silica gel plate (EtOAc/Hexane = 1/3) to afford separate products 11 (11.6mg, 70.0%) and 12 (lO.Omg, 60.0%) as colorless gums. Product 11 : the gum- like material then was crystallized from the mixed solvents of EtOAc and hexane to provide cubic transparent crystals, mp. 156-158⁰C; ¹H-NMR (CDCl₃) δ 7.31-6.71 (m, 8H, Ar-H and -HN-), 5.80 (s, IH, C8a-H), 4.09-3.59 (m, 2H, C2-H₂), 2.82 (septet, J=5.24Hz, IH, -CHMe₂), 2.19-1.96 (m, 2H, C3-H₂), 1.49 (s, 3H, C3a-CH₃) and 1.18 (d, J=5.24Hz, 6H, -CHMe₂) ppm; CI-MS(CH₄), m/z: 354 (MH⁺); HR-MS m/z: Calcd for C_2JH₂₄NO₄: 354.1705; Found: 354.1716. Product 12: the gum-like material was crystallized from mixed solvents of EtOAc and hexane to afford needle-like transparent crystals, mp. 149-15O⁰C; ¹H-NMR (CDCl₃) δ 7.30-6.72 (m, 8H, Ar-H and -HN-), 5.71 (d, J=1.80Hz, IH, C2-H), 4.17, 3.70 (AB, J_gem=5.40Hz, 2H, C4-H₂), 2.81 (septet, J=5.22 Hz, IH, CHMe₂), 2.19-1.94 (m, 2H, ClO-H₂), 1.48 (s, 3H, C5-CH₃) and 1.19 (d, J=5.22Hz, 6H, CH₃CCH₃) ppm; CI-MS(CH₄), m/z: 354 (MH⁺); Calcd for C₂₁H₂₄NO₄: 354.1705; Found: 354.1695.

X-ray Crystallography (Figure 4). Single-crystal X-ray analysis of 11 and 12. A colorless crystal of 11 with dimensions 0.22 x 0.26 x 0.26 mm^32b was mounted on glass fiber using a small amount of epoxy. A colorless crystal of 12 was mounted in an identical manner and had the dimensions of 0.18 x 0.21 x 0.40 mm.³² Data for compounds 11 and 12 were collected on a Bruker three-circle platform diffractometer equipped with a SMART 6000 CCD detector. Both crystals were irradiated using a rotating anode CuKpSource (D= 1.54178) with incident beam Gδbel mirrors. Data collection was performed and the unit cell was initially refined by using SMART [v5.625].^32a

Compounds 11 and 12 were both non-merohedral twins with the approximate ratios of 70:30 and 75:25, respectively. This required an orientation matrix to be found for each component. This was done and the data reduction for both components were performed using SAINT [v6.36A]^32b and XPREP [v6.12]^32c. Corrections were applied for Lorentz, polarization, and absorption effects using TWINABS vl.05.^32d Each structure was solved and refined with the aid of the SHELXTL-plus [v6.10] system of programs.³²⁶ The full-matrix least-squares refinement on F² included atomic coordinates and anisotropic thermal parameters for all non-H atoms. The H atoms were included using a riding model. The final refinements included the structure factors from both twin components.

The two racemates of compound 11 occupy the same crystallo graphic site in the lattice. This appears as a superposition of the two molecules resulting in a disorder around C3A and C8A. The disorder was modeled with a 50:50 ratio of occupancies between the racemates. The results of the X-ray studies are illustrated in Figure 4. Compound 11 : The four atoms of carbamoyl moiety -N-CO-O- compose a plane. The angle between planes N-plienyl and carbamoyl is 10.95 degrees. The angle between the carbamoyl and O-phenyl (ring- A) is 95.75 degrees. These two aromatic rings are almost perpendicular to each other (84.80 degrees). The terminal five-membered ring-C has an envelope conformation with C3 being out of plane atoms. The central five- membered ring-B is coplanar with its adjoining phenyl ring. The fused ring system is folded at the bond in common to the two five-membered rings (C3a-C8a). The angle of O8-C8a-Ol is 108.32 degrees.

Compound 12: The four atoms of carbamoyl -N-CO-O- also are in a plane. The angle between planes N-phenyl and carbamoyl is 4.33 degrees. The angle between carbamoyl and O-phenyl (ring-A) is 72.27 degree. The N-phenyl ring is at 67.94 degrees to the O-phenyl (ring-A). The 1,3-dioxepine is folded at the bridgehead C2- C5. The angle of C10-C2-O1 is 113.17 degree, the angle of C10-C2-O3 is 117.8 degrees, and the angle of O1-C2-O3 is 108.68 degrees.

II. Evaluation of Racemic Compounds (Table 1)

Compounds 13 and 14 were synthesized using the following procedures and tested below.

(-)-(3aS)-3a,8-Dimethyl-2,3,3a,8a-tetrahydrofuro[2,3-b]indoI-5-yliV- ethylcarbamate (13). It was made according to known procedure^ of making (-)- physovenine. In this case the mole ration (physovenol : ethyl isocyanate) was 1 : 3 and the reaction time was increased to about 3 hours (monitored by TLC ). The product was obtained quantitatively as a gum: [α]_D-97.4° (c=0.2, CHCl₃); ¹H NMR (CDCl₃): δ 6.77 (m, 2H, C4-H and C6-H), 6.22 (d,2H, C7-H), 5.10 (s, IH₃ C8a-H), 4.85 (s, br., IH, N- H), 3.90 (m, IH, C2-H_a), 3.40 (m, IH, C2-H_b), 3.30 (q, 2H, -CH₂-), 2.88(s, 3H, N⁸- CH₃), 2.00(m, 2H, C3-H₂), 1.40(s, 3H, C3a-CH₃), 1.20(t, 3H, -CH₃); CI-MS (CH₄), m/z: 277 (M⁺H); HR-MS m/z: Calcd for C₁₂H₂₀N₂O₅: 277.1552; Found: 277.1563.

(-)-(3aS)-l,3a,8-Trimethyl-l,2,3,3a,8,8a-hexahydropyrrolo[2,3,b]indol-5-yl iV-ethylcarbamate (14). It was made according to known procedure¹⁹ by using ethyl isocyanate as alkylisocyanate. The product was obtained quantitatively as crystals: mp 92-93 ⁰C; [α]_D-60.9° ( c=0.1, CHCl₃); ¹H NMR (CDCl₃): δ 6.72 (m, 2H, C4-H and C6- H), 6.29 (d,2H, C7-H), 4.85 (s, br., IH, N-H), 4.10 (s, IH, C8a-H), 3.00 (q, 2H, CH₂), 2.90 (s, 3H, N⁸-CH₃), 2.60 (m, 2H, C2-H_a), 2.52 (s, 3H, N^CH₃), 1.90 (m, 2H, C3-H₂), 1.45 (s, 3H, C3a-CH₃), 1.20 (t, 3H, -CH₃); CI-MS (CH₄), m/z: 290 (M⁺H); HR-MS m/z: Calcd for C₁₆H₂₃N₃O₂: 290.1869; Found: 290.1865.

Quantitation of anticholinesterase activity. The action of compounds 7-14 to inhibit the ability of freshly prepared human AChE and BChE to enzymatically degrade their respective specific substrates, acetyl-(β-methyl)thiocholine and s- butyrylthiocholine (0.5 mmol/L) (Sigma Chemical Co., St. Lois, MO), was quantified.³'⁷'^16"19'²³ Samples of AChE and BChE were derived from whole red blood cells and plasma, respectively. Compounds were dissolved in Tween SCVEtOH 3:1 (v:v; <150 μL total volume) and were diluted in 0.1 M Na3P04 buffer (pH 8.0) in half-log concentrations to provide a final concentration range that spanned 0.3 nM to 30 uM. Tween 50/EtOH was diluted to in excess of 1 in 5000 and no inhibitory action on either AChE or BChE was detected in separate prior experiments.

For the preparation of BChE, freshly collected blood was centrifuged (10,000 x g, 10 min, 4° C) and plasma was removed and diluted 1 : 125 with 0.1 M Na3Pθ4 buffer (pH 7.4). For AChE preparation, whole red blood cells were washed five times in isotonic saline, lysed in 9 volumes of 0.1 M Na3P04 buffer (pH 7.4) containing 0.5% Triton-X (Sigma) and then were diluted with an additional 19 volumes of buffer to a final dilution of 1:200. Analysis of anticholinesterase activity was undertaken by utilizing a 25 μL sample of each enzyme preparation, and was undertaken at their optimal working pH, 8.0, in 0.1 M Na3P04 buffer (0.75 mL total volume). Compounds were preincubated with enzymes (30 min, at room temperature.) and then were incubated with their respective substrates and with 5,5'-dithiobis-2-nitrobenzoic acid (25 min, 37° C). Substrate/enzyme interaction was immediately halted by the addition of excess enzyme inhibitor (physostigmine 1 x 10^"5M) and production of a yellow thionitrobenzoate anion was then measured by spectrophotometer at 412 nm λ. To correct for nonspecific substrate hydrolysis, aliquots were co-incubated under conditions of absolute enzyme inhibition (by the addition of 1 xlO^"5 M physostigmine (2)), and the associated alteration in absorbance was subtracted from that observed through the concentration range of each test compound. Each agent was analyzed on four separate occasions and assayed along-side physostigmine (2), as a control and external standard whose activity we have previously reported.^3>7'^16"19'²³ The enzyme activity at each concentration of test compound was expressed as a percent of the activity in the absence of compound. This was transformed into a logit format (logit = ln(%activity/100 minus %activity)) and then was plotted as a function of its log concentration. Inhibitory activity was calculated as an ICso, defined as the concentration of compound (nM) required to inhibit 50% of enzymatic activity, and determined from a correlation between log concentration and logit activity. Only results obtained from correlation coefficients of r²>-0.98 were considered acceptable. Studies that did not obtain this threshold were repeated.

Quantitation of proliferation and action on β-amyloid precursor protein (APP) levels. Human neuroblastoma SK-N-SH cells were seeded onto 60mm culture plates supplemented with 10% FBS (Gibco/ InVitrogen) in MEM Eagle (Mediatech/ Cellgro) and grown to 80% of confluence. Compounds 9 and 10 were dissolved in 100% DMSO to a concentration of 0.01M and were further diluted with media to obtain final treatment levels of lOμM and 30μM. A final sample size of 20 plates and a treatment/control number of 4 per group was utilized as a study design: vehicle control (0.3% DMSO) and 9 and 10 both at lOμM and 30μM. At the initiation of the study, low serum media (0.05% FBS in MEM Eagle) and compound or control vehicle was added to cells. At 48 hours thereafter, the conditioned media and cell pellet were collected for analysis. Specifically, i) a MTT assay was performed on live cells to measure cell viability at the 48 hour harvest time (n=4). ii) Levels of lactate dehydrogenase (LDH) were assayed in the conditioned media for assessment of cellular toxicity (n=4)³³'³⁴. iii) A small aliquot of cells from each group was removed and stored on ice for cell counting by the Trypan Blue method (n=l) as a secondary measure of cellular viability, and iv) conditioned media samples were subjected to Western blot analysis using a primary antibody against total secreted APP (sAPP). This was undertaken on a 10% SDS-PAGE gel, and samples (n = 4 per treatment/control group) were loaded at equal protein (30ug), based on the Bradford Micro assay Protein Estimation. Primary antibodies werethal 22Cl 1 (APP) at 1 :500 dilution and B-Actin at 1 :2000 dilution, both probed for 3 hours at room temp. Biological Evaluation. Table 1 illustrates the biological activity of compounds 7—12 against freshly prepared human AChE and BChE, in comparison to the corresponding carbamates of physovenol (1, 13, 15, 17) and eseroline (2, 14, 16, 18), whose measured values were similarly obtained. As previously demonstrated, physostigmine analogues with N-straight chain alkyl (e.g., methyl, butyl and octyl) carbamoyl moieties have similar anticholinesterase activities,^19"21 N-ethyl carbamates (7, 8, 13 and 14) as the representatives of this class were synthesized. Ethyl isocyanate is commercially available and safer than the methyl analogue to both transport and handle. All compounds (7 - 12) are racemic.

Table 1. 50% Inhibitory Concentration (IC₅₀, nM, ± SEM) of Compounds versus Human Erythrocyte AChE and Plasma BChE

Tetrahydrofurobenzofuran series: The tetrahydrofurobenzofuran carbamates (7, 9, 11) proved to be potent anticholinesterases. Like physovenine (1), the ethyl carbamate (7) possessed potent BChE inhibitory action, but surprisingly low AChE activity to provide it a BChE selectivity of 60-fold. The 2'-methylphenylcarbamate, which for both the physostigmine and physovenine series (16, 15) provided AChE potency and selectivity, similarly provided the tetrahydrofurobenzofuran series (9) high AChE potency. Interestingly, the compound was devoid of BChE action at the highest concentration assessed, 30 uM, providing it a remarkable and unexpected AChE selectivity. In contrast, the 4'-isopropylphenyl carbamate, which for both the physostigmine and physovenine series (18, 17) reverses the potency and selectivity to favor BChE, correspondingly provided the tetrahydrofurobenzofuran series (11) a high BChE potency.

Dihydrobenzodioxepine series: The dihydrobenzodioxepine carbamates (8,10,12) mirrored the selectivity described for the tetrahydrofurobenzofurans, but with slightly less potency. This potency was favorable in comparison to many of the clinically available anticholinesterases (Table 2), and the enzyme subtype selectivity achieved by 10 and 12 were superior to those of the more classical physostigmine series.

Table 2. Comparison of the IC_5O Values (j^SEM) of Anticholinesterases of Clinical Interest Against Human Erythrocyte AChE and Plasma BChE

IC so Value* (ΏM)

Compound AChE BChE Selectivity

Tacrine (Cognex) 190 + 40 47.0 ± 10 4-fold BChE Donepezil (Aricept) 22 + 8 4150 + 1700 188-fold AChE Galanthamine (Reminyl) 800 ± 60 7300 ± 830 9-fold AChE Rivastigmine (Exeloή) 4150 ± 160** 37 ± 5 122-fold BChE Phenserine 22 ± 1.4 1560 ± 45 70-fold AChE

2'Ethylphenyl geneserine 125 + 23 1700 + 385 14-fold AChE -N-oxide HCl (CHF2819) Heptylphysostigmine 22 + 2 5.0 + 0.1 4-fold BChE (Eptastigmine)

Huperzine A 47 + 22 >10,000 >212-fold AChE

*IC₅o values were determined in duplicate on a minimum of 4 different occasions **Rivastigmine is atypical in that its activity against brain derived AChE is far more potent than against red blood cell derived enzyme³⁵, the measured value may therefore noticeably underestimate its activity in brain and the agent has been reported to be nonselective between AChE and BChE.

Actions on cellular viability and toxicity and secretion of amyloid-β precursor protein levels in a neuronal cell line: As phenserine has demonstrated the ability to lower APP, the precursor to the putative Alzheimer toxin, amyloid-β peptide (Aβ), in neuronal cell lines,¹⁵ representative carbamates, 9 and 10, were assessed for actions on APP in human neuroblastoma SK-N-SH cells in culture. Cell viability and toxicity were additionally quantified by MTT assay and measurement of lactate dehydrogenase levels, respectively, at 48 hours. Neither compound altered secreted levels of APP at the concentrations assessed (10 and 30 μM), which were well tolerated. As illustrated in Figure 5, however, there was a significant increase in cell viability induced by 10 at the lower concentration. This was validated by quantifying viable cell number by trypan blue cell count (Figure 5).

Enzyme/inhibitor binding interactions: Based on extensive X-ray crystallography and mutagenesis studies, AChE appears to have three binding domains that interact separately or in a combined manner with divergent enzyme inhibitors.²⁴'²⁵ Their binding affinities are dependent, in part, with their 3-dimensional fit within the substrate gorge of the enzyme, alignment with one or more of the binding domains and the chemical basis underpinning the interaction between inhibitor and binding domain. Physostigmine analogues access the same two binding domains as does the natural substrate, Ach.⁶

Table 1 illustrates that the potency and AChE versus BChE selectivity of the four series of carbamates depends on the N-substituted group of the carbamoyl moiety. Specifically, the N-alkyl and N-4'-isopropylphenyl carbamates are BChE selective, whereas the N-2'-methylphenyl carbamate is AChE selective, irrespective of the phenol: physovenines (1, 13, 15, 17), physostigmines (2, 14, 16, 18), tetrahydrofurobenzofuran (7, 9, 11) or bridged benzodioxepine (8, 10, 12). This is illustrated in Figure 6, in which the four AChE active compounds, 15, 16, 9 (in a 3a-S configuration) and 10 (in a 5-S configuration), are superimposed in an optimized conformation. The carbamoyl moieties perfectly overlap, suggesting a similar inhibitory mechanism and in accord with similar inhibitory activities, but slight deviations exist in the overlay of the phenols. Specifically the N¹ -methyl group of tolserine (16) protrudes away from the rest. This may explain the enantiomeric selectivity found with the physostigmine series, with the (+)-enantiomers being cholinergically inactive, whereas both enantiomers are active for the physosvenine as well as N'-norphysostigmine series.³'²³'²⁶

Chemical interactions underpinning activity: The tetrahydrofurobenzofuran (7, 9, 11) and dihydrobenzodioxepine (8, 10, 12) carbamates, unlike physostigmine (2), lack a ring-C amino group to capture a proton and bear a positive charge. Hence, to account for their enzyme interaction, the dipole-dipole electrostatic actions and H- bonding rather than on ion-ion attraction was examined.

Figure 6 shows that the vectors of the dipole moments of the AChE active compounds 9, 10, 15 are very similar, only the direction of the dipole moment of tolserine (16) deviates. The biological data (Table 1) indicates that any differences in the vectors of the dipole moments have relatively little impact on AChE activities. In assessing H-bonding, the binding interaction between compound 9 and Torpedo California* AChE (TcAChE, EC 3,1,1,7) was modeled (Figure 7). Specifically, the crystal structure of IcAChE, carbamylated by the physostigmine analogue, 8-(cis-2,6-dimethylmorpholino)octyleseroline (MF 268), was utilized for this purpose.²⁷ The carbamoyl moiety was removed from this complex to allow the carbamoyl moiety of optimized compound 9 to be superimposed and docked to TcAChE. The dynamic H-bonds between ligand and enzyme were then highlighted (Figure 7).

As illustrated and similar to studies of Ach,⁵'²⁸ there appears to be three H-bond possibilities between the carbonyl oxygen and the main chain N-H OfGIy₁₁₈, GIy₁₁₉ and AIa₂₀] . However, disparate from the ACh model, an extra H-bond appears to exist between the N-H of the carbamoyl group and the Oγ atom of Ser_2Oo (Figure 7). With two points (N-H and C=O) fixed to the backbone of the binding domain, the freedom degree of the N-phenyl group is tremendously decreased. In synopsis, this part of each compound is relatively fixed and tolerates little structural change without a loss of binding and resulting inhibitory action (hence the overlay of the phenylcarbamate moieties in Figure 6).

In contrast, keeping the orientation of the carbamoyl portion fixed, manual rotation of the remaining tricyclic phenol portion around the C5-O10 or OlO-Cl 1 bonds resulted in no preferred bonding between the inhibitor and enzyme, indication that modifications to the tricyclic structure can be tolerated and will allow specific interactions to differentiate the four series of analogues.²³'²⁹

Action on cellular viability and amyloid-β precursor protein levels: Human SK-N-SH cells in culture express cholinergic markers, synthesize and secrete APP, and have proven to be useful in defining the action of experimental compounds on AD amyloidogeneis.¹⁵'¹⁶ hi contrast to (-)-phenserine (10 and 30 μM) that lowers secreted APP levels by 50%,¹⁵ 9 and 10 lacked APP action. Such action for (-)-phenserine, however, is not cholinergically mediated, as its (+)-enantiomer that lacks anticholinesterase activity lowers APP and, as a consequence, Aβ levels.¹⁵ Furthermore, the inhibition of cholinergically-mediated biochemical pathways, specifically those involving MAP kinase and PI kinase, whose activation are associated with anticholinesterase action and muscarinic receptor stimulation,¹⁶ without the loss of action on APP confirmed the disassociation between phenserine's action as an anticholinesterase and as an APP inhibitor,.¹⁵ The APP lowering activity appears to be post-transcriptionally mediated via the 5 '-untranslated region of APP niRNA¹⁵ through a specific region containing a CAGA box that is conserved in mammals that deposit Aβ.³⁰ Like 9 and 10, (-)-physotigmine (2) also lacks this APP action,³¹ and the structure/activity relationship underpinning it is the focus of ongoing studies.

In contrast, an increase in cell viability was never previously observed, as induced by 10, despite the fact that MTT assays were performed to dissociate between drug-induced biochemical changes and toxicity. The mechanism underpinning this activity is being investigated and, as neither 2 nor prior carbamates have demonstrated this action, it likely is non-cholinergically mediated.

III. Synthesis and Characterization of Optically Active Compounds (Figures 8 and 9)

Melting points (uncorrected) were measured with a Fisher- Johns apparatus; ¹H- NMR were recorded on a Bruker (Bellevica, MA) AC-300 spectrometer; MS (m/z) were recorded on a Hewlett-Packard 5973 GC-MS (CI). HRMS were performed by the UCR Mass Spectrometry Facility, Department of Chemistry, University of California. Optical rotations were measured by JASCO, Model DEP-370 (Japan, Spectroscopic Co., LTD.); Chiral HPLC analysis: ChiraxDex (5μm) colume (part No: 79925CB-584) provided by Agilent Technologies, eluted by MeOH/H₂O in different ratio, UVD 254 nm. AU reactions involving non-aqueous solutions were performed under an inert atmosphere. (35) and (3R)-MenthyI carbonates of 5-hydroxy-3-methyl-3-methoxy- carbonyl methylenebenzofuran-2(3H)-one (2) and (3). Under a nitrogen atmosphere, a solution of 5-hydroxy-3-methyl-3~methoxycarbonyl- methylenebenzofuran-2(3H)~one (1) (1.16 g, 4.91mmol) in 0.6 mL of triethylamine and 15mL of benzene was dropwise added into (-)-menthyl chloroformate (1.18 g, 5.39 mmol) at room temperature. The mixture was stirred for 1.5 hours at the same temperature. Evaporation of solvent gave a crude product that was chromatographed on silica gel (EtOAc/Hexane=l/3) to give a mixture of stereomer 2 and 3 (1.56 g , 76.2%) as a white crystals: ¹H-NMR (CDCl₃) δ 7.20-7.02 (m, 3H, Ar-H), 4.68-4.52 (m, IH, CH-OC=O), 3.53 (s, 3H, CH₃O), 3.10, 2.96 (AB, J_gem=17.6Hz, 2H, C3-CH₂COO), 2.21-2.11 (m, IH, CH-¹Pr), 2.10-1.98 (m, IH, CH-Me), 1.80-1.65 (m, IH, CHMe₂), 1.51(s, 3H, C3-CH₃), 1.59-1.41 (m, 2H, CH₂COC=O), 1.26-0.80 (m, 4H, CH₂CH₂), 0.97 (d, 6H, CH₃CCH₃) and 0.85 (d, 3H, CH₃CCCOC=O) ppm; CI-MS(CH₄), m/z: 419(MH⁺), 418, 417, 371, 237, 181, 153 and 109; HR-MS m/z: Calcd for C₂₃H₃₄NO₇ ^' MNH₄ ⁺: 436.2335; Found: 436.2326. The above crystalline mixture was recrystallized in hexane to give two different crystals, needle and flaky, which precipitated in different batches. They were recrystallized in ethanol several times until the optical rotation and melting point would not change. Product 2: needle crystals; mp. 106.8-107.4⁰C; [α]_D ²⁵- 60.8°(c=0.53,CHCl₃). Product 3: flaky crystals; mp. 148.0-148.8⁰C; [α]_D ²⁶-19.7 °(c=0.74,CHCl₃). The X-ray crystallographic structure of compound 3 is depicted in Figure 14.

(-)-(5Sj-5-Hydroxy-3-methyl-3-methoxycarbonylmethylenebenzofuran-2- one (4). Under a nitrogen atmosphere, a mixture of compound 2 (493 mg, 1.178 mmol) and sodium hydroxide (200mg, 5.0 mmol) in 18 mL of methanol was stirred for 1.5 hours at room temperature. Thereafter, it was neutralized with IN HCl. After removing the solvent, the residue was chromatographed on silica gel

(CH₂Cl₂/MeOH=10/l) to give product 4 (168.2 mg, 60.4%) as white needle crystals: mp. 157⁰C - 159⁰C; [α]_D ²⁶ -2.6 ° (c=0.62,CHCl₃); e.e.100%, was measured by chiral HPLC analysis: eluted by MeOH/H₂O = (45/55), 0.5 ml/min, UVD 254 nm, and at room temperature. The ¹H-NMR and CI-MS are the same as reported for racemic compound synthesized above.

(+)-(3R)-5-Hydroxy-3-methyl-3-methoxycarbonylmethylenebenzofuran-2- one (5). Compound 5 was prepared from 3 by the procedure described for 4, above, in a yield of 66.0%: white needle crystal; mp.l56.0°C - 158.6⁰C; [α]_D ²⁷+2.2 °(c=0.45, CHCl₃); e.e.100%, was measured in the same way, as described above. The ¹H-NMR and CI-MS are as same as that of compound 4.

(5αS)-5-Hydroxy-3a-methyI-2,3,3a,8a-tetrahydrofuro[2,3-6]benzofuran (6) and (5S)-7-hydroxy-5-methyl-4,5-dihydro-2,5-methano-l,3-benzodioxepine (7). The mixture OfLiAlH₄ (48.6 mg , 1.28 mmol) and Et₂O (10 ml) under a nitrogen atmosphere was cooled by an ice water bath to O⁰C. Then a solution of compound 2 (134 mg, 0.32 mmol) in 15mL OfEt₂O was dropwise added into the mixture. The reaction mixture was stirred for 0.5 hour at 0 ⁰C, then 1 hour at room temperature. Afterwards, the mixture was acidified by 1 M HCl anhydrous Et₂O solution to pH 3 - 4 and stirred for another 0.5 hour at room temperature. The mixture then was filtered and the filtrate was evaporated to remove solvent and excess HCl. The residues were chromatographed on silica gel (EtOAc/Hexane=l/3) to give a mixture of compound 6 and 7 in a molar ratio of approximately 1 : 1 ( 43 mg, 69.9%). The ¹H-NMR and CI- MS were the same as that of the racemic compounds synthesized above.

(3αR)-5-Hydroxy-3a-methyl-2,3,3a,8a-tetrahydrofuro[2,3-ό]benzofuran (8) and (5R)-7-hydroxy-5-methyl-4,5-dihydro-2,5-methano-l,3-benzodioxepine (9). Compounds 8 and 9 were prepared from compound 3 according to the procedures described above for 6 and 7.

(-)-(JαS)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-A]benzofuran-5yliV-ethyI carbamate (10) and (-)-(5iS)-5-methyl-4,5-dihydro-2,5-methano-l,3-benzodioxepin- 7yl iV-ethyl carbamate (II,). Under a nitrogen atmosphere, two small pieces of sodium (1 mg ca. per piece) were added into a solution of a mixture of compounds 6 and 7 (8.8mg, 0.0458 mmol) in 1.5 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes, and then ethyl isocyanate (11.1 μL, 0.137 mmol) was added in one portion. After the reaction mixture was stirred for another one hour at room temperature, 1.4 mL of water was added to quench the reaction. Thereafter, the ether layer was separated. After drying over sodium sulphate and filtering, filtrate was evaporated to remove solvent. The residue was chromatographed on silica gel plate (EtOAc/Hexane =1/3) to afford product 10 (4.2mg, 69.7%) as gel: [αj_D ²⁶- 92.2°(c=0.09, CHCl₃), e.e.100%; and product 11 (5.1mg, 84.6%) as gel: [α]_D ²⁵- 10.0°(c=0.12, CHCl₃), e.e.100%. The e.e values of both compounds, 10 and 11, were measured by chiral HPLC analysis: eluted by MeOH/H₂O = 65/35, 1.2 ml/min, UVD 254 nm and at room temperature. The ¹H-NMR and CI-MS of 10 and 11 are as same as that of racemic compounds synthesized above.

(+)-(5flR)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-6]benzofuran-5yliV- ethyl carbamate (16) and (+)-(5R)-5-Methyl-4,5-dihydro-2,5-methano-l,3- benzodioxepin-7yl iV-ethyl carbamate (17). The two chiral compounds were prepared from the mixture of compounds 8 and 9 in the same way described above for enantiomers 10 and 11. Product 16, as gel: [α]_D ²⁶+92.5°(c=0.08, CHCl₃), e.e. 100% and product 17 as gel: [α]_D ²⁶+10.0°(c=0.05, CHCl₃), e.elOO%. The e.e values of both compounds, 16 and 17, were measured in the same manner as for enantiomers 10 and 11. The ¹H-NMR and CI-MS of products 16 and 17 were the same as that of the racemic compounds synthesized above. (-)-(3«S)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-6]benzofuran-5yI N-o- tolyl carbamate (12) and (+)-(5S)-5-Methyl-4,5-dihydro-2,5-methano-l,3- benzodioxepin-7yl JV-ø-tolyl carbamate (13). Under a nitrogen atmosphere, two small pieces of sodium (1 mg ca. per piece) were added into a solution of mixture of compound 6 and 7 (9.3 mg, 0.0484 mmol) in 2.8 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes, and then o-tolyl isocyanate (6.3 μL, 0.0498 mmol) was added to the reaction mixture in one portion. After the reaction mixture was stirred for another 35 minutes at room temperature, 1.6 mL of water was added and ether layer was separated. After drying over sodium sulphate and filtering, filtrate was evaporated to remove solvent. The residue was chromatographed on silica gel plate (EtOAc/Hexane = 1/3) to afford product 12 (5.2mg, 66.1%) as a gel: [α]_D ²⁷- 103.0°(c=0.10, CHCl₃), e.e.100% and product 13 (5.0mg, 63.5%) as a gel: [α]_D ²⁷ +30.4°(c=0.135, CHCl₃), e.e.100%. The e.e values of both compounds 12 and 13 were measured by chiral HPLC analysis: eluted by MeOH/H₂O = 55/45, 1.6 ml/min, UVD 254 nm and at room temperature. The ¹H-NMR and CI-MS of 12 and 13 are as same as that of racemic compounds synthesized above.

(+)-(3flR)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-6]benzofuran-5yl iV-o- tolyl carbamate (18) and (-)-(5R)-5-Methyl-4,5-dihydro-2,5-methano-l,3- benzodioxepin-7yl iV-ø-tolyl carbamate (19). The two chiral compounds were prepared from the mixture of compounds 8 and 9 in the same manner as described above for the enantiomers 12 and 13. Product 18, as gel:

[α]_D ²⁶+104.2°(c=0.095,CHCl₃), e.e. 100% and product 19 as a gel: [α]_D ²⁷-29.0°(c=0.10, CHCl₃), e.e. 100%. The e.e values of both compounds 18 and 19 were measured in the same way as 12 and 13. The ¹H-NMR and CI-MS of product 18 and 19 were also the same as that of the respective racemic compounds synthesized above.

(-)-(3αS)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-6]benzofuran-5yl N-p- isopropylphenyl carbamate (14) and (+)-(5S)-5-Methyl-4,5-dihydro-2,5-methano- l,3-benzodioxepin-7yl iV-/?-isopropylphenyl carbamate (15). Under a nitrogen atmosphere, two small pieces of sodium (1 mg ca. per piece) were added into a solution of mixture of compounds 6 and 7 (16 mg, 0.083 mmol) in 5 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes, and then/?-isopropylphenyl isocyanate (14 μL, 0.086 mmol) was added to the reaction mixture in one portion. After the reaction mixture was stirred for another 45 minutes at room temperature, 3 mL of water was added and the ether layer was separated. After drying over sodium sulphate and filtering, filtrate was evaporated to remove solvent. The residue was chromatographed on silica gel plate (EtOAc/Hexane = 1/3) to afford product 14 (8 mg, 54.4%) as a gel: [α]_D ²⁶-91.4°(c=0.35, CHCl₃), e.e.100% and product 15 (9 mg, 61.2%) as a gel: [α]_D ²⁶ +50.0°(c=0.2, CHCl₃), e.e.100%. The e.e values of both compounds 14 and 15 were measured by chiral HPLC analysis: eluted by MeOH/H₂O = 62/38, 1.6 ml/min, UVD 254 nm and at room temperature. The ¹H-MVIR and CI-MS of 14 and 15 were as same as that of their respective racemic compounds synthesized above.

(+)-(3αR)-3a-Methyl-2,3,3a,8a-Tetrahydrofuro[2,3-6]benzofuran-5yI _J/V-p- isopropylphenyl carbamate 20 and (-)-(51?)-5-Methyl-4,5-dihydro-2,5-methano- l,3-benzodioxepin-7yl JV-/Msopropylphenyl carbamate 21. These two chiral compounds were prepared from the mixture of compounds 8 and 9 in the same manner as described above for enantiomers 14 and 15. Product 20, as gel: [CC]_D ²⁷+91.4°(C=0.07, CHCl₃), e.e. 100% and product 21 as a gel: [α]_D ²⁷ -48.0°(c=0.05, CHCl₃), e.e. 100%. The e.e values of both compounds 20 and 21 were measured in the same way as enantiomers 14 and 15. The ¹H-NMR and CI-MS of product 20 and 21 were also as same as that of their enantiomers 14 and 15. Quantification of anticholinesterase activity. Quantification of anticholinesterase activity of optically active products 10- 21 was undertaken against freshly prepared enzymes according to the procedure reported above for the racemic compounds. Table 3 illustrates the biological activity of compounds 10-21 against human AChE and BChE derived from erythrocyte and plasma, respectively. For comparison, the biological data of racemic compounds 22-27, (which are the racemic compounds 7-12 synthesized above) ar also listed. Table 3. IC₅₀(nM) OF CARBAMATES OF 5-HYDROXY-3a-METHYL-2,3,3a,8a- TETRAHYDROFURO [2,3-6]BENZOFURAN AND ITS BRIDGED ISOMER 7- HYDROXY-S-METHYL-^S-DIHYDRO-l.S-METHANO-l.S-BENZODIOXEPINE

Example II

This Example provides further information pertaining to the compounds disclosed herein, including their synthesis, biological activity, specificity, and enantiomeric properties. More specifically, this example includes synthesis and activity of their optically pure enantiomers with the dual purpose of (i) developing new AD drug candidates and (ii) utilizing them as tools to elucidate the molecular interactions required by the tricyclic skeleton of this important class of ChEI to achieve enantio- selectivity within AChE and BChE.

Enantiomeric synthesis utilizing classical resolution provided two novel series of anticholinesterase active and optically active compounds: (-)- and (+)- 0-carbamoyl phenols of tetrahydrofurobenzofuran and methanobenzodioxepine. An additional two series of (-)- and (+)-0-carbamoyl phenols of pyrroloindole and furoindole were obtained by known procedures, whose biological activity were similarly quantified against human acetyl- (AChE) and butyrylcholinesterase (BChE). Based on the biological data of these four series of compounds, a SAR analysis was provided by molecular volume calculations; thereafter, a transition state model was established according to the known X-ray structure of a transition state complex of Torpedo californica AChE-m-(N,N,N,trimethylammonio)-2,2,2-trifluoroacetophenone (JcAChE-TMTFA). Each of the four carbamates demonstrated relatively high anticholinesterase potency, whose enantio-selectivity and enzyme subtype selectivity can be explained by the use of the model.

These carbamates provide not only potent cholinesterase inhibitors of potential clinical relevance but also pharmacological tools to define the drug-enzyme binding interactions within an enzyme crucial in the maintenance of cognition and numerous other physiological functions in, for example, health, aging and disease.

Chemistry: The starting material, 5-hydroxy-3-methyl-3-methylacetate- benzofuran-2-one (101), was produced from C3-alkylation of the condensation product of 1,4-cyclohexandione and pyruvic acid, according to a previously reported procedure.⁴¹ First, compound 101 was reacted with (-)-menthyl chloroformate to provide a mixture of the stereoisomers of (3S) and (3i?)-menthylcarbonates of 5- hydroxy-3-methyl-3-methylacetate-benzofuran-2-one 102 and 103 (Figure 10). Since it proved difficult to isolate stereoisomer 102 and 103 with chromatography, they were separated by repeated crystallization. This was performed from hexane, at first, and then from ethanol to eventually yield two separate crystals, 102 and 103, with different crystalline forms: needle and flaky. The former crystals, 102: mp. 106.8-107.4⁰C;

[α]²⁵-60.8°(c=0.53,CHCl₃), and the flaky crystals 103: mp. 148.0-148.8⁰C; [α]²⁶-19.7° (c=0.74,CHCl₃).

The absolute configuration of the C-3 position was determined based on the known configurations of C- 19, C-21 and C-24 from compound 103. Figure 14 shows that compound 103 has a R configuration at its 3-position. As a consequence, compound 102 should possess a S configuration at its 3-position.

The use of chiral HPLC to measure the optical purity of compounds 102 and 103 proved unsuccessful. The hydrolysis of stereoisomer 102 and 103 provided (-)- enantiomer 104, [α]_D ²⁶-2.6° (00.62, CHCl₃), and (+)-enantiomer 105, [α ]_D ²⁷+2.2° (C=0.45, CHCl₃) (Figure 10), respectively. The enantiomeric excesses value (ee%) of each was measured by chiral HPLC analysis (Figure 15). The two compounds were at least substantially optically pure. In the process of reduction, the stereochemistry at the 3-position of compounds 102 and 103 was unchanged. Hence, 102 gave 106a (3aS- confϊguration) and 107a (5S-configuration); whereas, 103 gave 106b (3aR- configuration) and 107b (5R-configuration). The reductive lithium-aluminum complexes of compound 102 and 103 were acidified by IM HCl in anhydrous Et₂O, instead of oxalic acid, to elevate the yield to approximately 30% (Figure 11).

Phenols 106a and 107a, as well as 106b and 107b were reacted with different isocyanates to give 6 pairs of corresponding carbamates: 108a and Ilia, 109a and 112a, HOa and 113a, as well as 108b and 111b, 109b and 112b, HOb and 113b, respectively. Thereafter, each of these pairs of compounds was separated by preparative TLC.

The syntheses of carbamates of the physostigmine and physovenine series of analogues are shown in Figure 12. Compounds 115a and b, 117a, 118a, 120a and b, 122a and 123a are known compounds.³⁹'⁴⁰ Compounds 116a and 121a were synthesized from compounds 114a and 119a, which were obtained from natural physostigmine according to a known procedure.⁴¹ Compounds 117b, 118b, 122b and 123b were synthesized from (+)~3a(i?)-eseroline (114b) and (+)-3a(i?)-physovenol (119b). These phenols, (114b) and (119b), were obtained by asymmetric syntheses starting from N-methylphenetidine. X-ray Crystallography. The results of the X-ray studies of compound 103 are illustrated in Figure 14. The chirality of the asymmetric center are as follows: C3-i?, C19-i?, C21-i? and C24-S. The six-membered ring of the menthyl moiety has a chair conformation; in contrast, the benzofuran system is flat.

Biological Evaluation: Table 4 shows the anticholinesterase activity of enantiomers 108-113, 115-118 and 120-123 against freshly prepared human AChE and BChE, derived from erythrocytes and plasma, respectively. The concentration of compound required to inhibit 50% enzyme activity (IC₅₀ value) was quantified by a modified Ellman technique,^37"41'^42"44 and values for ^-configuration versus S- confϊguration are represented by the symbol R/S. A smaller IC₅₀ is associated with a lower Ki value and a higher affinity of inhibitor-enzyme binding.^45"47 A R/S value of 1 is indicative of similar inhibitory activity for the R- and iS-configurations. For a R/S value >1, the compound with a .^-configuration has a lower potency than its enantiomer.

Table 4. 50% Inhibitory Concentration (IC₅₀, nM, ± SEM) of Compounds Versus Freshly Prepared Human Erythrocyte AChE and Plasma BChE

*R/S: IC₅₀ ofi?-configuration/ IC₅Q of ^-configuration. AfB: selectivity for AChE or BChE from ICs₀ values. **The IC₅₀ data of compounds 115a, 115b are from reference 14, the data of compound 120a, 123a, 120b are from reference 15, the data of 117a, 122a, 118a are from reference 16, and the data of 116a, 121a are from reference 17. hi each case, however, enzyme was obtained from the same individual and assays were performed similarly.

#None: Insufficient activity in the range of 0.3 nM to 30 uM to calculate an IC_5O value, and hence considered to be inactive. With the exception of the compound pair, 117a and 117b, that possessed low and approximately similar BChE inhibitory activity (R/ S=O.6), all compounds with a S- configuration proved to be more potent than their corresponding enantiomer (R/S > 1). This was most evident in the physostigmine series, where AChE inhibitory activity was achieved with a R/S value > 353 and BChE inhibitory action was achieved with a R/S value > 102. Hence, in the physostigmine series, carbamates of (+)-physostigmine (115b, 117b, 118b) possess minimal cholinergic activity, quite the opposite of their potent (-)-enantiomers.

In contrast with the physostigmine series, both enantiomers of the novel tetrahydrofurobenzofuran (108a and b, 109a and b, 110a and b) and dihydrobenzodioxepine (Ilia and b, 112a and b, 113a and b) series possessed potent anticholinesterase action for AChE, BChE or both, with R/S values of <9.3. This activity is in accord with the congener, physosvenine carbamates (120a and b, 121a and b, 122a and b, 123a and b) that are similarly active in both enantiomeric forms with a preference for the ^-configuration. As described, N-demethylation of physostigmine (115a) minimizes the enantio-selectivity of this series,³⁸ which suggests that the N¹ -substitution of the tricyclic ring can be a determinant regarding enantio- selectivity. Additionally, the development of members of the tetrahydrofurobenzofuran and dihydrobenzodioxepine series to clinical development could be undertaken with either the potent S- (-)-enantiomers or the racemates, whose synthesis would be far easier and cheaper (avoiding optical resolution) and whose anticholinesterase activity is almost equal. A comparison of Tables 2 and 4, the former defining the anticholinesterase activity of agents of current and recent clinical interest against enzyme drawn from the same individual and analyzed similarly, suggests that compounds 109a, 112a and 110a, 113a possess sufficient potency and selectivity for AChE and BChE, respectively, to be of clinical interest.

In each of the four series of compounds detailed in Table 4, the differential selectivity for AChE or BChE is determined by the structure of N-substituted moiety of the carbamate. Specifically, the N-methyl carbamates provide minimal enzyme subtype selectivity. The N-ethyl carbamates have a moderate BChE preference. The N-2'- methylphenylcarbamates have high AChE selectivity and N-4'- isopropylphenylcarbamates reverse this and demonstrate a high BChE preference. Of particular note, the AChE and BChE selectivities of the novel tetrahydrofurobenzofuran and dihydrobenzodioxepine series are amongst the greatest found of all ChEIs yet synthesized on the tricyclic backbone of physostigmine. Molecular comparison by volume calculations: Since the exemplified carbamates possess structural similarity as well as a similar inhibitory mechanism, they can initially be compared by means of superimposition to further elucidate the basis of their enantio-selectivity. This was undertaken for the four AChE potent inhibitors with ^-configuration carbamates (109a, 112a, 117a and 122a) by generating a 'molecular volume map' of each molecule that corresponds to their van der Waals surface.⁴⁸'⁴⁹

As illustrated in Figure 16, for each compound, the relative positions of their two phenyl moieties, on either side of the carbonyl group in their optimized conformation, were almost identical - closely overlaying one another. These four compounds could be closely superimposed, except for disparity in their tricyclic systems. The combined molecular volume map of compounds 109a, 112a, 117a and 122a provides an 'enzyme - excluded map' (green area in Figure 16), within which all active compounds should fit. The molecular volume of the i?-2'-methylphenyl carbamates, 109b, 112b, 117b and 122b, minus the enzyme - excluded volume provides an 'estimate volume' that represents the additional volume generated by that of each i?~isomer (yellow meshed area within Figure 16).

As shown in Table 5, the lowest estimate extra volume of compound 109b (7.1 A³) corresponds to the lowest enantio-selectivity (R/S = 1.8). The highest estimate extra volume (24.8 A³) of compound 117b corresponds to the highest enantio-selectivity (R/S = 551). Specifically, the larger estimate extra volume of compound 117b (meshed area in Figure 16) likely hinders the approach to and hence binding between the inhibitor and enzyme. Molecular modeling studies were then undertaken to elucidate how this additional estimate extra volume can hinder binding.

Molecular model of the transition state: The wide structural diversity of available ChEIs suggests that unlike types of inhibitors bind with AChE in different ways via disparate yet specific interactions between compound and enzyme.^50"52 Most anticholinesterases, such as those in Table 2 as well as the potent non-clinical tacrine- based triazoles and other hybrids, are cationic at physiological pH; whereas some, including those of the present example and arisugacin, are not.^53"55 Human cholinesterases are large complex molecules composed of catalytic subunits that can accommodate a variety of specific binding interactions associated with these diverse inhibitors. They can contain up to 583 amino acids, a Mr of 70-80 kDa, and are variably glycosylated.⁵²'⁵⁶ Three-dimensional analyses of AChE and BChE, based on x-ray crystallography, have provided structural information regarding the positioning of the catalytically important amino acid residues within these proteins.^57"64 Thus, three major binding domains have been described within AChE and two within BChE in an internalized, primarily hydrophobic gorge of some 20 A length, but as narrow as 0.5 A wide. Deepest within this gorge is a catalytic 'acyl' binding domain, which hydrolyses choline esters through electron transfer within a catalytic triad, termed a 'charge relay system. The triad includes a Ser₂o_O, the imidazole group of a HiS₄₄₀, and the carboxylic acid moiety of a GIu₃₂₇ (JcAChE numbering). A 'choline' binding domain resides midway along the gorge, and a 'peripheral' anionic site exists at the gorge mouth for AChE but not BChE,^53"64 and this latter site can be involved in the complexing of AChE with amyloid-β peptide in the Alzheimer brain.⁶⁵ Notably, the neurotoxicity associated with Aβ-AChE complexes has been shown to be greater than that induced by the Aβ peptide alone in both cell culture and animal experiments.⁶²

On the basis of calculations and electro-optical measurements, it has been suggested that electrostatic charges associated with seven negatively charged amino acids residues close to the entrance of the gorge trap and steer charged ligands into its mouth.⁶⁶'⁶⁷ In the case of ACh, the quaternary choline moiety interacts with Trp₈₄ of TcAChE and, to a lesser extent, with Phe₃₃o within the choline binding domain. This orientates the compound to allow the approach and nucleophilic attack of the catalytic triad on its carbonyl group. A quaternary transition state between Ser₂₀o and ACh momentarily exists that then collapses into an acylated-enzyme intermediate and released choline. Thereafter, hydrolysis of the acetylester reactivates the enzyme to allow efficient cleavage of as many as 10⁴ ACh molecules per second.⁶⁰'⁶¹ (-)- Physostigmine (115a) and analogues interact similarly with the same two binding domains. However, nucleophilic attack of its carbonyl moiety results in a transient (-)- physostigmine-AChE intermediate, in a tetrahedral conformation, that then swiftly collapses to a carbamylated drug-enzyme complex that is dramatically more stable than the acetyl enzyme one associated with ACh; enzyme inhibition occurs consequent to the slow rate of enzyme decarbamylation.

The inhibition process, hence, involves AChE-catalyzed hydrolyses of inhibitors.³⁷'³⁸ In such a chemical reaction, if the formation of a transition state is not an equilibrium process and is fast enough relative to the interaction between AChE and ACh, the highest energy transition state controls the overall rate of the reaction. A catalytic enzyme can lower this energy barrier by specific interaction with the inhibitor in the transition state (124 - 125).⁶⁸ (Figure 13). As shown in Table 4, each of the eight different carbamates with a same N-substituted side chain (e.g., 108a and b, 111 a and b, 116 a and b, 121 a and b) should generate identical carbamylated enzyme structures after their reaction with either AChE or BChE. Hence their different IC₅₀ values should be related to their affinities and rates of carbamylation, and not to decarbamylation — which would impact the time-dependence of the inhibition associated with each. As a consequence we can focus on carbamylation due to the short time frame of our biological studies. Additional insight into the molecular interactions of the enzyme and inhibitor can be obtained at the transition state. Whereas it is not readily feasible to produce a crystal structure of an enzyme complexed with its substrate in the transition state, due to the short transition state lifetime versus the time required for X-ray data collection, one can exploit the high affinity of transition state analogues. In this regard, the analogue, m-(N,N,N-trimethylarnmonio)-2,2,2-trifluoroacetophenone (TMTFA), possessing a low Zi(15 fM) for inhibition of TcAChE,⁷⁰ was utilized to aid model the interaction of our compounds within the active site of AChE.

An investigation of the X-ray crystallographic structure of JcAChE complexed with the known physostigmine analogue, MF268 (8-(cis-2,6-dimethylmoφholino) octylcarbamoyleseroline), demonstrated the presence of a covalent bond between the carbonyl carbon and γ-0 of Ser_2Oo, and that the Ν-substituted side chain of the carbamoyl moiety is located inside the acyl-binding pocket and extends to the rim of the active site gorge.⁷⁰'⁷¹ The X-ray crystallographic transition state structure of TcAChE-TMTFA (code 1 AMΝ) was obtained from the RCSB protein databank. After extraction of TMTFA, the (-)-(3aS>Ν-2'-methylphenylcarbamoyl phenol of furobenzofuran (109a) was docked, in accord with knowledge gained from the binding ofMF268.⁷⁰'⁷¹

As shown in Figure 17, six hydrogen bonds are apparent within the model (yellow lines): the NH OfGIy₁₁₈, Glyng and AIa_20I with the oxygen of the ligand carbonyl, the γ-0 of Ser₂₀₀ with a single H, the hydrogen with N of His₄₄₀, and the NH ofHis₄₄₀ with the oxygen OfGIu₃₂₇. Together with the covalent bond of the γ-0 of Ser_2Oo with the C of the ligand's carbonyl group, the illustrated hydrogen bonds comprise an acylation site. This site is considered as a crucial part of the transition state. A transition state model was developed by (i) keeping the acylation site intact (by constraining the relative distances between the described atoms) and, (ii), minimizing the complex energy, after automatic adjustment of the conformation of the remaining complex.

This model resembles a 'beam balance'. The central supporting point is a covalent bond between the γ-0 of Ser_2Oo and the C of the ligand's carbonyl group. The π-π interaction system between the N-phenyl group of carbamate (109a) and the phenyl group of PlIe₂₈₈ of the enzyme comprises one side of this balance. The π-π or lipophilic (C-H ^" π) interaction system of the furobenzofuran moiety of compound 109a and the indole system of Trp₈₄ of the enzyme forms the other side of the balance. Such C-H' π interactions have been observed in the x-ray crystal structure of other neutral molecules with TbAChE. Any change on either side of this balance can adversely affect the formation of the transition state. Utilizing this model, it is possible to evaluate the basis of enantio-selectivity and enzyme subtype selectivity.

Enantio-selectivity: As shown by molecular volume calculations (Figure 16, Table 5), for the exemplified carbamates having an ^-configuration, with the exception of the physostigmine series, their molecular volumes are not significantly different. This can support the lipophilic interaction between the tricyclic ring system of the ligands and the indole system of Trp₈₄ of the enzyme. Hence, it can be shown that these compounds retain substantial anticholinesterase activity compared to their iS-isomers.

As stated above, the exception involves the ^-configuration compounds having a N^!-methyl group (the physostigmine series). The model predicts that the N¹ -methyl group will become inserted between the tricyclic ring system and the indole system of Trp₈₄. The lipophilic interaction will consequently disappear, and a transition state between ligand and enzyme complex would not be readily formed without this lipophilic interaction. As a result, the (+)-physostigmine analogues 115b, 117b and 118b can be predicted to possess a poor AChE and BChE inhibitory action, in accord with their measured poor ChEI activity.

Enzyme subtype selectivity: The acyl-binding pockets within AChE and BChE differ from one another in size and shape. The pockets can be differentiated on the basis of two amino acid residues located at the bottom of the acyl-loop. These residues are aromatic in AChE (Phe₂₈₈ and Phe₂go) but aliphatic in BChE (Leu₂₈₆ and VaI₂₈₈).⁵⁶'⁵⁸'⁶⁰'⁶¹'⁶⁹ The latter pocket, hence, is slightly larger due to the smaller protruding side chains associated with Leu and VaI.³⁶ Previous studies have shown that the conformational energy barrier of the N- phenyl group of compound 117a and its analogues, rotated around Cl'-N bond, is correlated to the IC₅₀ of AChE. A higher energy barrier is associated with a lower inhibitory activity (a larger IC₅₀); although, inexplicably, the energy barrier is as low as 0-0.7 kcal/mol. ⁴⁴

According to the exemplified transition state model (Figure 17), the π-π interaction system between the N-phenyl group of the carbamate (117a) and the phenyl group of Phe₂₈₈ of the enzyme comprises one side of this balance. Rotation of the N- phenyl group can decrease this π-π interaction and directly influence the stability of the transition state; consequently lowering the affinity between ligand and enzyme.⁴⁴

Utilizing the same model, it is also possible to propose a basis for the cholinesterase subtype selectivity of the different carbamates moieties, (i) The small methyl group of N-methylcarbamates has almost no lipophilic interaction with residues of the acyl pocket, it thus has little influence on this balance and hence there is minimal subtype selectivity between AChE and BChE for N-methyl carbamates of all series, (ii) The ethyl group of N-ethylcarbamates, likewise, has relatively meager, but, nevertheless, greater lipophilic interaction than N-methylcarbamates. The direction of this interaction is more favorable to form a stable transition state within BChE than AChE. Hence, the N-ethyl carbamates possess a realtively moderate BChE selectivity. (iii) The phenyl group of N-phenylcarbamates provides a significant lipophilic interaction with the aromatic ring of Phe₂₈₈. This interaction together with the lipophilic interaction between the tricyclic ring system and indol moiety of Trp₈₄ provides a relatively highly stable transition state. Thus, all N-phenyl and N-methylphenyl carbamates possess potent AChE activity. Within BChE, however, PlIe₂₈₈ can be replaced by an amino acid with an aliphatic residue. A similar π-π interaction cannot be formed and leads to an unfavorable stability in the transition state, and consequent weak BChE inhibitory activity. N-phenyl and N-methylphenyl carbamates hence have high AChE selectivity. Finally, (iv) the 4'-isopropylphenyl moiety of N-4'~ isopropylphenyl carbamates cannot achieve a π-π interaction within the acyl-binding pocket of AChE consequent to the bulk and hindering action of the isopropyl moiety.

Within BChE, however, the lipophilic interaction of the isopropylphenyl group with residues of BChE in its larger acyl-binding pocket is favored (consequent to replacement of AChE Phe₂₈₈ and Phe₂₉₀ by VaI and Leu), which stabilizes the transition state. Thus the N-4'-isopropylphenyl carbamate series have a potent and selective BChE inhibitory action. hi accord with this, one of the most potent and selective BChE inhibitors in the physostigmine series, (-)-N¹-phenethylnorcymserine (126) (IC₅₀: AChE >30,000 nM, BChE 6.0+1.0 nM, selectivity >5000-fold BChE) (Figure 18),⁴² similarly can fit within the elongated acyl binding pocket associated with BChE. However, as the N^phenethyl moiety is directed outside the lipophilic interaction system, activity is not perturbed. Indeed, it is augmented, likely, as a consequence of hydrophobic interactions close to the gorge mouth. Interestingly, the elongated (-)-4'-benzoxyphenyl carbamate of physostigmine, 127 (Figure 18),⁷³ that lacks a hindered 4'-isopropyl group, can maintain the π-π interaction associated with potent AChE binding, but make use of the larger acyl pocket associated with BChE to achieve BChE inhibitory potency (IC₅₀: AChE 58±4 nM, BChE 7.0+1.0 nM, selectivity 7-fold BChE).⁷³

Chemistry. Melting points (uncorrected) were measured with a Fisher- Johns apparatus; ¹H NMR and ¹³C NMR were recorded on a Bruker (Bellevica, MA) AC-300 spectrometer; MS (m/z) were recorded on a Hewlett-Packard 5973 GC-MS (CI); HRMS were performed by the UCR Mass Spectrometry Facility, Department of Chemistry, University of California; Optical rotations were measured by JASCO, Model DIP-370

(Japan, Spectroscopic Co., LTD.); Elemental analyses were performed by Atlantic Microlab, Inc.; The ee% value of optically active compounds was determined by HPLC analyses using a HPl 100 instrument and a chiral column (ChiraDex^® 5μm) (Agilent Technology), mobile phase MeOH/H₂O = 45/55, elution rate 0.5 ml/min., UVD 254 nm - all at room temperature. All reactions involving non-aqueous solutions were performed under an inert atmosphere.

(3S) And (3R)-Menthyl carbonates of 5-hydroxy-3-methyl-3-methylacetate- benzofuran-2 -one (102) and (103). Under a nitrogen atmosphere, a solution of 5-hydroxy- 3-methyl-3-methoxycarbonylmethylenebenzofuran-2-one (101) (116mg, 0.491mmol) in 0.06 mL of triethylamine and 1.5mL of benzene was dropwise added into (-)-menthyl chloroformate (118mg, 0.539mmol) at room temperature. The mixture was stirred for 1.5 hours at the same temperature. The crude product was subjected to chromatography on silica gel (EtOAc/Hexane=l/3) to give 156.5 mg of white crystalline product, yield 76.2%: ¹H- NMR (CDCl₃) δ 7.20-7.02 (m, 3H, Ar-H), 4.68-4.52 (m, IH, CH-OC=O), 3.53 (s, 3H, CH₃O), 3.10, 2.96 (AB, J_gew=17.6Hz, 2H, C3-CH₂COO), 2.21-2.11 (m, IH, CH-¹Pr), 2.10- 1.98 (m, IH, CH-Me), 1.80-1.65 (m, IH, CHMe₂), 1.51 (s, 3H, C3-CH₃), 1.59-1.41 (m, 2H, CH₂COC=O), 1.26-0.80 (m, 4H, CH₂CH₂), 0.97 (d, 6H, CH₃CCH₃) and 0.85 (d, 3H,

CH₃CCCOC=O) ppm; CI-MS (CH₄), m/z: 419(MH⁺), 418, 417, 371, 237, 181, 153 and 109; HR-MS m/z: Calcd for C₂₃H₃₄NO₇(MNH₄ ⁺): 436.2335; Found: 436.2326. The above white crystal product was, thereafter, recrystallized from both hexane and ethanol, separately, several times, until the optical rotations and melting points of the two different isolated crystalline forms did not change. One, a needle crystalline form, was product 102: m.p. 106.8-107.4⁰C; [α]_D ²⁵-60.8°(c=0.53,CHCl₃). Anal. (C₂₃H₃₀O₇) C, H. The other, a flaky crystalline form, was product 103: m.p.l48.0-148.8°C; [α]_D ²⁶-19.7 °(c=0.74,CHCl₃). Anal. (C₂₃H₃₀O₇) C₅ H.

(-)-(3S)-5-Hydroxy-3-methyl-3-methyIacetate-benzofuran-2-one (104). Under a nitrogen atmosphere, a mixture of reactant 102 (0.493g, 1.178 mmol) and sodium hydroxide (0.2g, 5.0 mmol) in 18 niL of methanol was stirred for 1.5 hours at room temperature. It was then neutralized with IN HCl. After removing solvent, the residue was chromatographed on silica gel (CH₂Cl₂/MeOH=10/l) to give 168.2 mg of product 104, yield 60.4%: m.p. 157.0-159.0⁰C; [α]_D ²⁶ -2.6 °(c=0.62,CHCl₃); ee%=100% (HPLC); ¹H-NMR (CDCl₃) δ 6.92-6.61 (m, 3H, Ar-H), 5.07 (s, IH, OH), 3.44 (s, 3H, OCH₃), 3.02, 2.84 (AB, J_gem=18.0Hz, 2H, C3-CH₂CO) and 1.41(s, 3H, C3-CH₃) ppm; CI-MS (CH₄), m/z: 237(MH⁺), 205, 177 and 163.

(+)-(3R)-5-Hydroxy-3-methyI-3-methylacetate-benzofuran-2-one (105). Compound 105 was prepared as described above, yield 66.0%: m.p. 156.0-158.6 ⁰C; [α]_D ²⁷+2.2 °(c=0.45,CHCl₃); ee%=100%. The ¹HNMR and CI-MS (CH₄) are the same as that of compound 104.

(3aS)-5-Hydroxy-3a-methyl-2, 3,3a, 8a-tetrahydrofuro[2,3-#] benzofuran (106a) and (5S)-7-Hydroxy-5-methyI-4, 5-dihydro-2, 5-methano-l, 3- benzodioxepine (107a). Under a nitrogen atmosphere, a solution of compound 102 (177 mg, 0.423mmol) in 2mL of ether was dropwise added to lithium aluminum hydride (32 mg, 0.843 mmol) in ether at O⁰C. The mixture, after stirring at O⁰C for 0.5h, was stirred for another one hour at room temperature. Thereafter, IM HCl ether solution and was to provide a mixture of pH 3-4, which then was stirred for 0.5 hour at room temperature. Next, the mixture was filtered and the filtrate was concentrated. The residue was subjected to chromatography on silica gel (EtOAt/Hexane=l/3) to give 24.4 mg of a mixture of compounds 106a and 107a. The GC-CI-MS and ¹H-NMR of this mixture were the same as that of the racemic⁴¹ and showed an approximate 1:1 molar ratio of (3a5)-5-hydroxy-3a-methyl-2, 3,3a, 8a-tetrahydrofuro[2,3-δ] benzofuran (106a) and (5S)-7-hydroxy-5-methyl-4, 5-dihydro-2, 5-methano-l, 3-benzodioxepine (107a). The yields were 30% and the compounds were directly used as starting material for the next reactions.

(3aR)-5-Hydroxy-3a-methyl-2, 3,3a, 8a-tetrahydrofuro [2,3-6] benzofuran (106b) and (5R)-7-Hydroxy-5-methyl-4, 5-dihydro-2, 5-methano-l, 3- benzodioxepine (107b). Compounds 106b and 107b were prepared from compound 103, according to the procedure described for compounds 106a and 107a. (-)-(3aS)-3a-Methyl-2, 3,3a, 8a-tetrahydrofuro [2,3-6] benzofuran-5yl JV- ethyl carbamate (108a) and (-)-(5S)-5-Methyl-4, 5-dihydro-2, 5-methano-l, 3- benzodioxepin-7yl iV-ethyl carbamate (Ilia). Under a nitrogen atmosphere, two small pieces of sodium (about 1 mg) were added into a solution of mixture of 106a and 107a (24.4 mg, 0.127 mmol) in 5 niL of anhydrous ether at room temperature. This mixture was then stirred for 2 minutes, and ethyl isocyanate (30.1 μL, 0.381 mmol) was next added to the reaction mixture in one portion. An hour into the reaction at room temperature, 1.4 mL of water was added and the ether layer was separated. After drying over sodium sulphate and filtering, the filtrate was evaporated to remove solvent. The residue was chromatographed on silica gel plate (EtOAc/Hexane =1/3) to afford products 108a and Ilia. Product 108a (11.6 mg, 69.7%): [α]_D ²⁶ -92.2°(c=0.09,

CHCl₃); e.e.100%; ¹H-NMR (CDCl₃) δ 6.87-6.60 (m, 3H, Ar-H), 5.77 (s, IH, C8a-H), 4.89 (s, br, IH, NH), 4.06-3.56 (m, 2H, C2-H), 3.23 (m, 2H, C5-CH₂NHCOO), 2.13- 1.91 (m, 2H, C3-H), 1.47 (s, 3H, C3a-CH₃) and 1.14 (t, 3H, C5-CH₃CH₂NHCOO) ppm; CI-MS (CH₄), m/z: 264 (MH⁺), 193, 175 and 72. Anal. (C₁₄H₁₇NO₄) N, H; C: Calcd. 63.87; Found 63.14. Product Ilia (14.1 mg, 84.6%): [α]_D ²⁵-10.0°(c=0.12, CHCl₃), e.e.100%. ¹H-NMR (CDCl₃) δ 6.82-6,67(m, 3H, Ar-H), 5.69 (d, J=1.80Hz, IH, C2-H), 4.88 (s, br, IH, C7-NHCOO), 4.17, 3.70 (AB, l_gem=7.20Uz, 2H, C4-H), 3.22 (m, 2H, C-7CH₂NHCOO), 2.19-1.19 (m, 2H, ClO-H), 1.41 (s, 3H, C5-CH₃) and 1.16 (t, 3H, C7-CH₃CH₂NHCOO) ppm; CI-MS (CH₄), m/z: 264 (MH⁺), 246, 220, 193, 175, 149 and 72. Anal. (C₁₄H₁₇NO₄) C, H; N: Calcd. 5.32; Found 4.91. (+)-(3aR)-3a-Methyl-2, 3,3a, 8a-Tetrahydrofuro[2,3-δ] benzofuran-5yl N- ethyl carbamate (108b) and (+)-(5R)-5-Methyl-4, 5-dihydro-2, 5-methano-l, 3- benzodioxepin-7yl N-ethyl carbamate (111b). Under a nitrogen atmosphere, two small pieces of sodium (about 1 mg) were added into a solution of mixture of 106b and 107b (26 mg, 0.135 mmol) in 5 niL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes and, thereafter, ethyl isocyanate (32.8 μL, 0.408 mmol) was added to the reaction mixture in one portion. The reaction was continued for an hour at room temperature, 4.0 mL of water was then added and the ether layer was separated. After drying over sodium sulphate and filtering, the filtrate was evaporated to remove solvent. Thereafter, the residue was subjected to chromatography on silica gel plate (EtOAc/Hexane = 1/3) to afford products 108b and 111b. Product 108b (14.8 mg, 83.1%):

[α]_D ²⁶+92.5°(c=0.08, CHCl₃); e.e. 100%; ¹H-NMR (CDCl₃) δ 6.87-6.60 (m, 3H, Ar- H), 5.77 (s, IH, C8a-H), 4.89 (s, br, IH, NH), 4.06-3.56 (m, 2H, C2-H), 3.23 (m, 2H, C5-CH₂NHCOO), 2.13-1.91 (m, 2H, C3-H), 1.47 (s, 3H, C3a-CH₃) and 1.14 (t, 3H, C5-CH₃CH₂NHCOO) ppm; CI-MS (CH₄), m/z: 264 (MH⁺), 193, 175 and 72. Anal.

(Ci₄H₁₇N O₄) C, H; N: Calcd. 5.32; Found 4.78. Product 111b (10.4 mg, 58.4%): [α]_D ²⁶ +10.0°(c=0.05, CHCl₃); e.e. 100%. ¹H-NMR (CDCl₃) δ 6.82-6,67(m, 3H, Ar-H), 5.69 (d, J=1.80Hz, IH, C2-H), 4.88 (s, br, IH, C7-NHCOO), 4.17, 3.70 (AB, J_gβB=7.20Hz, 2H, C4-H), 3.22 (m, 2H, C-7CH₂NHCOO), 2.19-1.19 (m, 2H, ClO-H), 1.41 (s, 3H, C5- CH₃) and 1.16 (t, 3H₅ C7-CH₃CH₂NHCOO) ppm; CI-MS (CH₄), m/z: 264 (MH⁺), 246, 220, 193, 175, 149 and 72. Anal. (C₁₄H₁₇NO₄) C, H, N.

(-)-(3aS)-3a-Methyl-2, 3,3a, 8a-Tetrahydrofuro[2,3-6] benzofuran-5yl N-o- tolyl carbamate (109a) and (+)-(5S)~5-Methyl-4, 5-dihydro-2, 5-methano-l, 3- benzodioxepin-7yl N-ø-tolyl carbamate (112a). Under a nitrogen atmosphere, two small pieces of sodium (about 1 mg) were added into a solution of mixture of 106a and 107a (27.9 mg, 0.145 mmol) in 5 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes, and then o-tolyl isocyanate (18.9 μL, 0.149 mmol) was added to the mixture in one portion. The reaction was continued for 35 minutes at room temperature, 4 mL of water then was added and the ether layer was separated. After drying over sodium sulphate and filtering, the filtrate was evaporated to remove solvent. The residue was chromato graphed on silica gel plate (EtOAc/Hexane = 1/3) to afford products 109a and 112a. Product 109a (15.6 mg, 66.1%): [α]_D ²⁷-103.0°(c=0.10, CHCl₃); e.e.100%; ¹H-NMR (CDCl₃) δ 7.84 (s, br, IH, NH), 7.32-6.70 (m, 7H, Ar-H), 5.89 (s, IH, C8a-H), 4.18-3.68 (m, 2H, C2-H), 2.39-2.05 (m, 2H, C3-H), 2.31 (s, 3H₅ Ar-CH₃) and 1.56 (s, 3H, C3a-CH₃) ppm; CI-MS (CH₄), m/z: 326 (MH⁺). Anal. (Ci₉Hi₉N O₄.1/4H₂O) C, H; N: Calcd. 4.25; Found 3.71. Product 112a (15.0 mg,

63.5%): [α]_D ²⁷+30.4°(c=0.135, CHCl₃), e.e.100%; ¹H-NMR (CDCl₃) δ 7.87 (s, br, IH, C7-NHCOO), 7.33-6.70 (m, 7H, Ar-H), 5.79 (d, J=1.80Hz, IH, C2-H), 4.22, 3.79 (AB, J_gem=6.84Hz, 2H, C4-H), 2.37-2.01 (m, 2H, ClO-H), 2.31 (s, 3H, Ar-CH₃) and 1.51 (s, 3H, C5-CH₃) ppm; CI-MS (CH₄), m/z: 326 (MH⁺). Anal. (C₁₉Hi₉NO₄) C; H: Calcd. 5.89; Found 6.36; N: Calcd. 4.31; Found 3.57.

(+)-(3ai?)-3a-Methyl-2, 3,3a, 8a-Tetrahydrofuro [2,3-6] benzofuran-5yl N-o- tolyl carbamate (109b) and (-)-(5R)-5-Methyl-4, 5-dihydro-2, 5-methano-l, 3- benzodioxepin-7yl N-ø-toIyl carbamate (112b). Under a nitrogen atmosphere, two small pieces of sodium (about 1 mg) were added into a solution of mixture of 106b and 107b (18 mg, 0.09 mmol) in 5 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes, and then o-tolyl isocyanate (18.3 μL, 0.145 mmol) was added in one portion. The reaction was continued for a further 35 minutes at room temperature, 4 mL of water then was added and the ether layer was separated. After drying over sodium sulphate and filtering, the filtrate was evaporated to remove solvent. The residue was chromatographed on silica gel plate (EtOAc/Hexane^l/S) to afford products 109b and 112b. Product 109b (11.0 mg, 74.9%), [α]_D ²⁶+104.2°(c=0.095, CHCl₃); e.e. 100%; ¹H-NMR (CDCl₃) δ 7.84 (s, br, IH, NH)₅ 7.32-6.70 (m, 7H₅ Ar-H), 5.89 (s, IH₅ C8a-H)₅ 4.18-3.68 (m, 2H₅ C2-H), 2.39-2.05 (m, 2H₅ C3-H)₅ 2.31 (s, 3H, Ar-CH₃) and 1.56 (s, 3H, C3a-CH₃) ppm; CI-MS (CH₄), m/z: 326 (MH⁺). Anal. (C₁₉Hi₉NO₄) C₅ H; N: Calcd. 4.31; Found 3.78. Product 112b (9.5 _mgj 64.4%), [αj_D ²⁷-29.0°(c=0.10₅ CHCl₃); e.e. 100%; ¹H-NMR (CDCl₃) δ 7.87 (s, br, IH, C7-NHCOO), 7.33-6.70 (m, 7H, Ar-H), 5.79 (d, J=I.80Hz, IH, C2-H), 4.22, 3.79 (AB, J_gew,=6.84Hz, 2H, C4-H), 2.37-2.01 (m, 2H, ClO-H), 2.31 (s, 3H, Ar-CH₃) and 1.51 (s, 3H, C5-CH₃) ppm; CI-MS (CH₄), m/z: 326 (MH⁺). Anal. (C₁₉H₁₉N 04.1/15H₂O) C H, N. (-)-(3aS)-3a-Methyl-2, 3,3a, 8a-Tetrahydrofuro[2,3-A] benzofuran-5yl N-/?- isopropylphenyl carbamate (110a) and (+)-(55)-5-Methyl-4, 5-dihydro-2, 5- methano-1, 3-benzodioxepm-7yl 7V-p-isopropylphenyl carbamate (113a). Under a nitrogen atmosphere, two small pieces of sodium (about 1 mg) were added into a solution of mixture of 106a and 107a (16 mg, 0.083 mmol) in 5 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes andp-isopropylphenyl isocyanate (14 μL, 0.086 mmol) was added to the mixture of 106a and 107a in one portion. The reaction was continued for an additional 45 minutes at room temperature; thereafter, 4 mL of water was added and the ether layer separated. After drying over sodium sulphate and filtering, the filtrate was evaporated to remove solvent. The residue was chromato graphed on silica gel plate (EtOAc/Hexane = 1/3) to afford products HOa and 113a. Product HOa (8 mg, 54.4%): [α]_D ²⁶-91.4°(c-0.35, CHCl₃); e.e.100%; ¹H-NMR (CDCl₃) δ 7.31-6.71 (m, 8H, Ar-H, HNCOO), 5.80 (s, IH, C8a-H), 4.09-3.59 (m, 2H, C2-H), 2.82 (septet, J=5.24Hz, IH, CHMe₂), 2.19-1.96 (m, 2H, C3- H), 1.49 (s, 3H₃ C3a-CH₃) and 1.18 (d, J=5.24Hz, 6H, CH₃CCH₃) ppm; CI-MS (CH₄), m/z: 354 (MH⁺). Anal. (C₂₁H₂₃N O₄. 1/8H₂O) C, H; N: Calcd. 3.94; Found 3.30. Product 113a (9 mg, 61.2%): [α]_D ²⁶+50.0°(c=0.2, CHCl₃); e.e.100%; ¹H-NMR (CDCl₃) δ 7.30-6.72 (m, 8H, Ar-H, HNCOO), 5.71 (d, J=I.80Hz, IH, C2-H), 4.17, 3.70 (AB, J_gem=5.40Hz, 2H, C4-H), 2.81 (septet, J=5.22 Hz, IH, CHMe₂), 2.19-1.94 (m, 2H, ClO-H), 1.48 (s, 3H, C5-CH₃) and 1.19 (d, J=5.22Hz, 6H, CH₃CCH₃) ppm; CI- MS (CH₄), m/z: 354 (MH⁺). Anal. (C₂₁H₂₃N O₄) C, H; N: Calcd. 3.96; Found 4.56.

(+)-(3aR)-3a-Methyl-2, 3,3a, 8a-Tetrahydrofuro [2,3-b] benzofuran-5yl N-p- isopropylphenyl carbamate (HOb) and (-)-(5R)-5-Methyl-4, 5~dihydro-2, 5- methano-1, 3-benzodioxepin-7yl iV-/7-isopropylphenyl carbamate (113b). Under a nitrogen atmosphere, two small pieces of sodium (approx.l mg) were added into a solution of mixture of 106b and 107b (18 mg, 0.094 mmol) in 5 mL of anhydrous ether at room temperature. The mixture was stirred for 2 minutes, and then p-isopropylphenyl isocyanate (14.6 μL, 0.096 mmol) was added in one portion. The reaction was continued for a further 45 minutes at room temperature, and then 4 mL of water was added and the ether layer separated. After drying over sodium sulphate and filtering, filtrate was evaporated to remove solvent. The residue was chromatographed on silica gel plate (EtOA/Hexane = 1/3) to afford products HOb and 113b. Product HOb (13.7 mg, 82.1%): [α]_D ²⁷+91.4°(c=0.14, CHCl₃); e.e. 100%; ¹H-NMR (CDCl₃) δ 7.31-6.71 (m, 8H, Ar-H, HNCOO), 5.80 (s, IH, C8a-H), 4.09-3.59 (m, 2H, C2-H), 2.82 (septet, J-5.24Hz, IH, CHMe₂), 2.19-1.96 (m, 2H, C3-H), 1.49 (s, 3H, C3a-CH₃) and 1.18 (d, J=5.24Hz, 6H, CH₃CCH₃) ppm; CI-MS (CH₄), m/z: 354 (MH⁺). Anal. (C₂₁H₂₃N O₄.I/8H₂O) C, H, N. Product 113b (10.1 mg, 60.4%): [α]_D ²⁷-48.0°(c=0.1, CHCl₃); e.e. 100%; ¹H-NMR (CDCl₃) δ 7.30-6.72 (m, 8H, Ar-H, HNCOO), 5.72 (d, J=I.80Hz, IH, C2-H), 4.17, 3.70 (AB, J_gm=5.40 Hz, 2H, C4-H), 2.81 (septet, J=5.22 Hz, IH, CHMe₂), 2.19-1.94 (m, 2H, ClO-H), 1.48 (s, 3H, C5-CH₃) and 1.19 (d, J=5.22Hz, 6H₅CH₃CCH₃) ppm; CI-MS (CH₄), m/z: 354 (MH⁺). Anal. (C₂₁H₂₃N O₄.1/8H₂O) C, H, N. (+)-(3aR)-l,3a, 8-TrimethyI-l, 2,3,3a, 8,8a-hexahydropyrrolo[2,3-b] indol-

5ol (14b). Compound 114b was synthesized, according to the procedure for its enantiomer, from N-methylphenetidine.³⁸

(+)-(3aR)-l,3a, 8-Trimethyl-l, 2,3,3a, 8,8a-hexahydropyrrolo[2,3-b] indol- 5yl N- (2'-methylphenyl) carbamate (117b). Compound 117b was made, according to the procedure for its antipode, from o-tolyl isocyanate and (+)-eseroline: ⁴⁴ [α]o²⁶ +68.2 °(c=0.2, CHCl₃); ¹H NMR and MS were the same as reported for its enantiomer.⁴⁴ Anal. (C₂₁H₂₅N₃ O₂.1/2H₂O) C, H, N.

(+)-(3aR)- 1, 3a, 8-Trimethyl-l, 2,3,3a, 8,8a-hexahydropyrrolo[2,3-b] indol- 5yl N- (4'-isopropylphenyl) carbamate (118b). Compound 118b was synthesized, according to the procedure for its antipode, from /Msopropylphenyl isocyanate and (+)- eseroline: ⁴⁴ [α]_D ²⁶+67.5 °(c=0.2, CHCl₃); ¹H NMR and MS were the same as reported for its enantiomer.⁴⁴ Anal. (C₂₃H₂₉N₃ O₂.1/4H₂O) C, H, N.

(+)-(3aR)-3a, 8-DimethyI-2, 3,3a, 8a-tetrahydrofuro[2,3-b] indol-5-ol (19b). Compound 119b was made according to the procedure for synthesis of its enantiomer.³⁸'⁴¹

(+)-(3aR)-3a, 8-Dimethyl-2, 3,3a, 8a-tetrahydrofuro[2,3-b] indol-5-yl JV- (2'- methylphenyl) carbamate (122b). Compound 122b was synthesized, according to the procedure for its antipode, from o-tolyl isocyanate and (+)-eseroline: ⁴⁴ [α]o² +27.5 °(c=0.2, CHCl₃); ¹H NMR and MS were the same as reported for its enantiomer.⁴³ Anal. (C₂₀H₂₂N₂ O₃) C, H; N: Calcd. 8.28; Found 7.84. (+)-(3aR)-3a, 8-Dimethyl-2, 3,3a, 8a-tetrahydrofuro[2,3-b] indol-5-yl N-

(4'-isopropylphenyl) carbamate (123b). Compound 123b was made, according to the procedure for its antipode, from^?-isopropylphenyl isocyanate and (+)-eseroline: [α]_D ²⁶ +32.0 °(c=0.2, CHCl₃); ¹H NMR and MS were the same as reported for its enantiomer.⁴¹ Anal. (C₂₂H₂₆N₂ O₃) C, H; N: Calcd. 7.64; Found 7.13. X-ray Crystallography. A clear colorless crystal of dimensions 0.30 x 0.23 x

0.12 mm² was mounted on glass fiber using a small amount of Epoxy. Data were collected on a Bruker three-circle platform diffractometer equipped with a SMART 6000 CCD detector. The crystals were irradiated using a rotating anode CuK^source (D= 1.54178) with incident beam Gδbel mirrors. Data collection was performed and the unit cell was initially refined using SMART [v5.625] .^74(a) Data Reduction was performed using SAINT [v6.36A]^74(b) mάXPREP [v6.12].^74(c) Corrections were applied for Lorentz, polarization, and absorption effects using SADABS [v2.03].^74(d) The structure was solved and refined with the aid of the programs in the SHELXTL-plus [v6.10] system of programs. ^η4^' The full-matrix least-squares refinement on F² included atomic coordinates and anisotropic thermal parameters for all non-H atoms. The H atoms were included using a riding model. The absolute configuration of C3 was established by making reference to unchanging chiral centres (Cl 9, C21, and C24) in the synthetic procedure, with a resulting Flack parameter of -0.3(2).^74(f) The X-ray data of compound 103 also can be found at the Cambridge Crystallo graphic Data Center (CCDC), http://www.ccdc.cam.ac.uk/ The reference number of this crystal is 285734.

Quantitation of anticholinesterase activity. The action of enantiomers 108- 113 and compounds 117b, 118b, 122b and 123b to inhibit the ability of freshly prepared human AChE and BChE to enzymatically degrade their respective specific substrates, acetyl-(β-methyl)thiocholine and s-butyrylthiocholine (0.5 mmol/L) (Sigma Chemical Co., St. Lois, MO), was quantified.^37"41'^42"44' Samples of AChE and BChE were derived from freshly collected human whole red blood cells and plasma, respectively. Compounds were dissolved in Tween 50/EtOH 3:1 (v:v; <150 μL total volume) and were diluted in 0.1 M Na3P04 buffer (pH 8.0) in half-log concentrations to provide a final concentration range that spanned 0.3 nM to 30 uM. Tween 8OfEtOH was diluted to in excess of 1 in 5000. No inhibitory action on either AChE or BChE was detected in separate experiments where the ChEI activity of the known compound 115a was quantified in excess and without Tween 50/EtOH.

For the preparation of BChE, freshly collected blood was centrifuged (10,000 g, 10 min, 4° C) and plasma was removed and diluted 1 : 125 with 0.1 M Na3Pθ4 buffer (pH 7.4). Plasma was carefully checked to insure an absence of haemolysis. For AChE preparation, erythrocytes were washed five times in isotonic saline, lysed in 9 volumes of 0.1 M Na3P04 buffer (pH 7.4) containing 0.5% Triton-X (Sigma) and then were diluted with an additional 19 volumes of buffer to a final dilution of 1 :200.

Analysis of anticholinesterase activity was undertaken by utilizing a 25 μL sample of each enzyme preparation, and was undertaken at their optimal working pH, 8.0, in 0.1 M Na3P04 buffer (0.75 niL total volume). Compounds were preincubated with enzymes (30 min, at room temperature) and then were incubated with their respective substrates and with 5,5'-dithiobis-2-nitrobenzoic acid (25 min, 37° C). The substrate/enzyme interaction was immediately halted by the addition of excess enzyme inhibitor (physostigmine 1 x 10^"5 M) and production of a yellow thionitrobenzoate anion was then measured by spectrophotometer at 412 run λ. To correct for nonspecific substrate hydrolysis, aliquots were co-incubated under conditions of absolute enzyme inhibition (by the addition of 1 xlO^"5 M physostigmine (115a)), and the associated alteration in absorbance was subtracted from that observed through the concentration range of each test compound. Each agent was analyzed on four separate occasions and assayed along-side physostigmine (115a), as a control and external standard whose activity we have previously reported.^37"41' ^42-44 The mean enzyme activity at each concentration of test compound was then expressed as a percent of the activity in the absence of compound. This was transformed into a logit format (where logit = ln(%activity/100 minus %activity)) and then was plotted as a function of its log concentration. Inhibitory activity was calculated as an ICso, defined as the concentration of compound (nM) required to inhibit 50% of enzymatic activity, which was determined from a correlation between log concentration and logit activity. Only results obtained from correlation coefficients of r >0.98 were considered acceptable. Studies that did not obtain this threshold were repeated. Computer Aided Molecular Modeling. Computer aided molecular modeling was undertaken using Sybyl version 7.0 (Tripos Inc., St. Louis, MO). The numbering of the amino acid residues is based on that for TcAChE. Molecular volume calculations were performed by using multiple volume comparison routine. The atomic coordinates of the transition state of TcAChE were obtained from the protein data bank (PDB entry: IAMN)⁶¹, and were used for transition state studies. In undertaking this, i) molecules of water were removed, ii) the small molecule, m- (N, N, N-trimethylammonio)-2,2,2- trifluoroacetophenone (TMTFA), was extracted and, iii), the carbonyl carbon of TMTFA was retained in the tetrahedral conformation, iv) The carbonyl carbon of the inhibitor, (-)-(3aS)-Ν-2'-methylphenyl carbamoyl phenol of furobenzofuran (109a), was modified into the tetrahedral conformation and then superimposed with that of TMTFA, keeping both phenyl groups on the same side and, as much as possible, in a superimposed conformation. Thereafter, compound 109a was merged into the active domain of TcAChE. The covalent bond between carbonyl carbon of compound 109a and γ-0 of Ser₂₀₀ was created. To maintain the hydrogen bonds associated with acylation, the distances between the NH Of GIy₁₁₈, GIy₁₁₉, AIa₂₀₁ and oxygen of the ligand carbonyl, γ-0 of Ser₂oo and a single H, the hydrogen and N of HiS₄₄₀, the NH of HiS₄₄₀ and oxygen Of GIu₃₂₇ were constrained. AU the residues outside a radius of 10 A from the compound 109a were aggregated. The energy of the enzyme-inhibitor complex was minimized with- the conjugate gradient algorithm. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds, compositions and methods described herein. Other aspects of the compounds, compositions and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions and methods disclosed herein. It is intended that the specification and examples be considered as exemplary.

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(8) Data were calculated by using software Pallas 3.0 produced by CompuDrug International Inc., 705 Grandniew Drive, South San Francisco CA 94708.

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Claims

What is claimed:

1. A compound having the formula I

I wherein each R¹ is, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group,

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, and n is from 1 to 4, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula I is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer.

2. The compound of claim 1, wherein the compound has the formula II

II wherein R¹ is hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula II is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

3. The compound of claims 1 or 2, wherein R² is a C₁-C₄ branched- or straight- chain alkyl group.

4. The compound of claims 1 or 2, wherein R² is methyl.

5. The compound of claims 1 or 2, wherein R¹ is hydrogen.

6. The compound of claims 1 or 2, wherein R¹ is hydrogen and R² is methyl.

7. The compound of claims 1 or 2, wherein R¹ is C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group.

8. The compound of claim 4, wherein R¹ comprises C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group.

9. The compound of claim 8, wherein R³ is hydrogen and R⁴ is a branched- or straight-chain alkyl group or substituted aryl group.

10. The compound of claim 8, wherein R³ is hydrogen and R⁴ is ethyl, o-tolyl, oxp- isopropyl phenyl.

11. The compound of claim 2, wherein R¹ is C(O)NR³R⁴, wherein R³ is hydrogen and R⁴ is ethyl, o-tolyl, or/»-isopropyl phenyl, and R² is methyl.

12. The compound in any of claims 1-11, wherein the compound is the substantially pure (+)-enantiomer.

13. The compound in any of claims 1-11, wherein the compound is the substantially pure (-)-enantiomer.

14. The compound in any of claims 1-11, wherein the compound is racemic.

15. A compound having the formula III

III wherein each R¹ is, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group,

R is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, and n is from 1 to 4, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula III is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

16. The compound of claim 15, wherein the compound has the formula IV

rv wherein R¹ is hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula IVis the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

17. The compound of claims 15 or 16, wherein R² is a C₁-C₄ branched- or straight- chain alkyl group.

18. The compound of claims 15 or 16, wherein R² is methyl.

19. The compound of claims 15 or 16, wherein R¹ is hydrogen.

20. The compound of claims 15 or 16, wherein R¹ is hydrogen and R² is methyl.

21. The compound of claims 15 or 16, wherein R¹ is C(O)NR³R⁴, wherein R³ and R⁴ comprises, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group.

22. The compound of claim 18, wherein R¹ is C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group.

23. The compound of claim 22, wherein R³ is hydrogen and R⁴ is a branched- or straight-chain alkyl group or substituted aryl group.

24. The compound of claim 22, wherein R³ is hydrogen and R⁴ is ethyl, o-tolyl, or />-isopropyl phenyl.

25. The compound of claim 16, wherein R¹ is C(O)NR³R⁴, wherein R³ is hydrogen and R⁴ comprises ethyl, o-tolyl, or/>-isopropyl phenyl, and R² is methyl.

26. The compound in any of claims 15-25, wherein the compound is the substantially pure (+)-enantiomer.

27. The compound in any of claims 15-25, wherein the compound is the substantially pure (-)-enantiomer.

28. The compound in any of claims 15-25, wherein the compound is racemic.

29. A pharmaceutical composition comprising one or more compounds in any of claims 1-28 and a pharmaceutically-acceptable carrier.

30. A method for inhibiting cholinesterase activity in a subject, comprising administering an effective amount of the compound in any of claims 1-28.

31. The method of claim 30, wherein the cholinesterase comprises acetylcholinesterase or butyrylcholinesterase.

32. The method in claims 30 or 31, wherein the compound has the formula II

II wherein R¹ comprises C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula II is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

33. The method in claims 30 or 31, wherein the compound has the formula IV

IV wherein R¹ comprises C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula II is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

34. The method of claims 32 or 33, wherein R³ is hydrogen and R⁴ is ethyl, o-tolyl, or/7-isopropyl phenyl, and R² is methyl.

35. A method of screening a compound for the ability to inhibit cholinesterase activity in a subject, comprising:

(a) administering to the subject the compound of claims 1-28; and

(b) detecting a decrease in the cholinesterase activity as compared to the cholinesterase activity in a control subject not administered the compound, whereby decreased cholinesterase activity identifies the compound as having the ability to inhibit cholinesterase activity in a subject.

36. The method in claim 35, wherein the compound has the formula II

II wherein R¹ comprises C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula II is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

37. The method in claim 35, wherein the compound has the formula IV

IV wherein R¹ comprises C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula IV is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

38. The method of claims 36 or 37, wherein R³ is hydrogen and R⁴ is ethyl, o-tolyl, or/?-isopropyl phenyl, and R² is methyl.

39. A method of treating dementia in a subject diagnosed with dementia, comprising administering to the subject an effective amount of the compound in any of claims 1-28, whereby the compound treats dementia in the subject.

40. The method of claim 39, wherein the dementia is Parkinson's disease, vascular dementia, schizophrenia, or Lewy body dementia.

41. The method of claim 39, wherein the dementia is Alzheimer's Disease or mild cognitive impairment.

42. The method in any of claims 39-41, wherein the compound has the formula II

II wherein R¹ comprises C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula II is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

43. The method in any of claims 39-41, wherein the compound has the formula IV

IV wherein R¹ comprises C(O)NR³R⁴, wherein R³ and R⁴ are, independently, hydrogen, a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group, and

R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula IV is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

44. The method of claims 42 or 43, wherein R³ is hydrogen and R⁴ is ethyl, ø-tolyl, or/>-isopropyl phenyl, and R² is methyl.

45. A method of treating Down's syndrome, abdominal or urinary distension, stroke, head trauma, myasthenia gravis, Sjorgrens syndrome, or type 2 diabetes in a subject diagnosed with such, comprising administering to the subject an effective amount of the compound in any of claims 1-28.

46. A method of treating an ophthalmologic disease in a subject diagnosed with such, comprising administering to the subject an effective amount of the compound in any of claims 1-28.

47. The method of claim 46, wherein the ophthalmologic disease is glaucoma, accommodative esotropia, myasthenia gravis confined to the extraocular and eyelid muscles, Adie (or tonic pupil) syndrome, or louse or mite infestation of lashes.

48. The use of a compound in any of claims 1-28 as a prophylactic or pre-treatment for exposure to a nerve gas, an insecticide, an anticholinergic poison, or excessive echothiophate.

49. The use of a compound in any of claims 1-28 as an imaging agent for the diagnosis of dementia in a subject.

50. The use of a compound in any of claims 1-28 as an agent for reducing pain in a subject.

51. A method of inhibiting production of amyloid β-peptide in a cell, comprising contacting the cell with the compound in any of claims 1-28.

52. The method of claim 51, wherein the cell is in vivo.

53. The method of claim 51, wherein the cell is in vitro.

54. The method of claim 51 , wherein the cell is a human cell.

55. A method of inhibiting production of amyloid β-peptide in a subject, comprising administering to the subject an effective amount of the compound of claims 1- 28.

56. The method of claim 55, wherein the subject is a mammal.

57. The method of claim 55, wherein the subject is a human.

58. The method of Claim 55, wherein the subject is a companion animal.

59. A method of inhibiting production of a C-terminal fragment produced from cleavage of amyloid β-peptide from an amyloid precursor protein, comprising administering to the subject an effective amount of the compound of claims 1- 28.

60. A compound having the formula V

V wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, wherein R² is not methyl, or the pharmaceutically-acceptable salt or ether thereof.

61. The compound of claim 60, wherein R² is a C₁-C₄ branched- or straight-chain alkyl group.

62. A method for making a compound having the formula VIII

VIII wherein R is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, comprising reacting 1,4-cyclohexandione with a compound having the formula VI

wherein R is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group.

63. The method of claim 62, wherein R² is a Cj-C₄ branched- or straight-chain alkyl group.

64. The method of claim 62, wherein R² is methyl.

65. The method of claim 62, wherein the reaction is conducted at an elevated temperature.

66. A compound having the formula VII

VII wherein R² and R⁵ are, independently, a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, or the pharmaceutically-acceptable salt or ester thereof, wherein the compound having the formula VII is the substantially pure (-)- enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (- )-enantiomer and (+)-enantiomer.

67. The compound of claim 66, wherein R² and R⁵ are, independently, a Ci-C₄ branched- or straight-chain alkyl group.

68. The compound of claim 66, wherein R² and R⁵ are methyl.

69. The compound in any of claims 66-68, wherein the compound is the substantially pure (+)-enantiomer.

70. The compound in any of claims 66-68, wherein the compound is the substantially pure (-)-enantiomer.

71. A method for making a compound of claim 66, comprising (a) reacting a compound having the formula VIII

VIII wherein R is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, with a base to produce a first product; and

(b) reacting the first product with a compound having the formula XCH₂COOR⁵, wherein R⁵ is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, and X is a leaving group.

72. The method of claim 71, wherein R² and R⁵ are, independently, a C₁-C₄ branched- or straight-chain alkyl group.

73. The method of claim 71 , wherein R² and R⁵ are methyl.

74. The method of claim 71, wherein the base comprises a hydride, a hydroxide, a carbonate, or an amide.

75. The method of claim 71 , wherein the base is sodium hydride.

76. The method in any of claims 71-75, wherein X is a halide or a substituted sulfonate.

77. The method in any of claims 71-75, wherein X is chloride.

78. A method for producing a compound having the formula X or XI,

X or

XI wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, and wherein the compound having the formula X or XI is the substantially pure (- )-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer, comprising

(a) reacting a compound having the formula VII

VII wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group,

R⁵ is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, wherein the compound having the formula VII is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer, with a reducing agent to produce a first product; and (b) reacting the first product with an acid to produce a compound having the formula X, XI, or a mixture thereof, wherein when a mixture of compounds X and XI are present,

(c) separating compound X from compound XL

79. A method for producing a compound having the formula II or IV,

II or

rv wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, wherein the compound having the formula II or IV is the substantially pure (- )-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer, comprising

(a) reacting a compound having the formula VII

VII wherein R² is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group,

R⁵ is a branched- or straight-chain alkyl group, or a substituted or unsubstituted aryl group, wherein the compound having the formula VII is the substantially pure (-)-enantiomer, the substantially pure (+)-enantiomer, or a racemic mixture of the (-)-enantiomer and (+)-enantiomer, with a reducing agent to produce a first product; and

(b) reacting the first product with an acid to produce to produce a second product;

(c) reacting the second product with a base to produce a deprotonated product; and

(d) reacting the deprotonated product with a compound having the formula R¹ -Y, wherein R¹ is a branched- or straight-chain alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, an ether group, a carboxylate group, or an amide group, and Y comprises a group capable of reacting with the deprotonated product to produce a compound having the formula II, IV, or a mixture thereof, wherein when a mixture of compounds II and IV are present,

(e) separating compound II from compound IV.

80. The method of claim 79, wherein the reducing agent comprises a substituted or unsubstituted borohydride and aluminium hydride.

81. The method of claim 79, wherein the reducing agent comprises lithium aluminium hydride, lithium borohydride, or sodium borohydride.

82. The method of claim 79, wherein the reducing agent comprises hydsrogen transfer via catalytic hydrogenation.

83. The method of claim 79, wherein the base comprises a hydride, a hydroxide, a carbonate, or an amide.

84. The method of claim 79, wherein Y is an isocyanate group or a halide.

85. The method in any of claims 79-84, wherein R² and R⁵ are, independently, a Cj- C₄ branched- or straight-chain alkyl group.

86. The method in any of claims 79-84, wherein R² and R⁵ are methyl.

87. The method in any of claims 79-86, wherein R^]-Y is a branched- or straight- chain alkyl isocyanate or a substituted or unsubstituted aryl group isocyanate.

88. The method in any of claims 79-86, wherein R¹ -Y is ethyl isocyanate, o-tolyl isocyanate, or p-isopropyl phenyl isocyanate.