AU2002232100A1 - Compounds co-inducing cholinergic up-regulation and inflammation down-regulation and uses thereof - Google Patents

Description

COMPOUNDS CO-INDUCING CHOLINERGIC UP-REGULATION AND INFLAMMATION DOWN-REGULATION AND USES THEREOF

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to preparation and use of novel

bifunctional chimeric compounds for the treatment of central nervous

system (CNS) disorders and diseases. More particularly, the present

invention relates to novel chimeric compounds which are conjugates of

reversible or irreversible cholinergic up-regulators and non-steroidal

anti-inflammatory drugs (NSAIDs) and their use in the treatment of various

CNS disorders and diseases such as Alzheimer's disease (AD), cerebral

ischemia or stroke and closed head injury.

The development of new drugs for treatment of Alzheimer's disease

(AD) is presently known to take three possible directions: (i) the

development of cholinergic up-regulators, which includes compounds such

as cholinesterase inhibitors (ChEI), cholinergic Ml agonists, M2

antagonists and nicotinic agonists; (ii) the development of compounds

which decrease the level of beta amyloid peptide (β-A4) such as amyloid

precursor protein (APP) releasers, β-A4 processors and anti-aggregation

agents for the reduction of deposited β-A4 level in the brain; and (iii) the

use of anti-inflammatory drugs, free radical scavengers and antioxidants for

the correction of the inflammatory response and free radical formation

which occur during the progress of the disease (1). Nevertheless, the only clinically used drugs, which presently

demonstrate efficacy in AD treatment and the only AD drugs approved by

the FDA are ChEIs (e.g., ARICEPT and EXELON). The main advantage

of these drugs, compared to other known ChEIs, is the reversible or

pseudo-reversible binding thereof to the active site of acetylcholine esterase

(AChE), which significantly reduces their toxicity.

However, it has been proposed to combine the inhibition of

cholinesterase (ChE) with selective protease inhibition function for

reducing the formation of β-A4 from APP (1). During the last decade there

has been a considerable effort to develop selective muscarinic receptor

agonists, such as Ml and M3 receptor agonists. Compounds such as

AF102B (2), Lu 25-109 (3), WAY- 132983 (4) and Milameline

(CI-979/RU35926) (5) have been shown to act as selective Ml (and M3)

receptor agonists or partial agonists, while some have been further tested

clinically in AD patients (6). In addition, certain nicotinic receptor

agonists, which interact with specific subtypes of neuronal nicotinic

receptors, have been developed for AD therapy (7).

A combination of two cholinergic functions in the same molecule,

such as ChEI with either muscarinic receptor agonist (Ml or M3) or M2

receptor antagonist, has been suggested as well in order to produce higher

pharmacological activity (6). The latter has been demonstrated by Amitai et

al., who developed novel lipophilic analogs of pyridostigmine (PYR-X)

which are both ChEI inhibitors and M2 receptor antagonists (8). Furthermore, BChE and AChE are part of the complex comprising the

plaques in AD brain. It was noted by Inestrosa et al. (9) that inhibition by

peripheral anionic-site inhibitors decreases the formation of these

complexes in AD plaques. Some of these lipophilic PYR-X compounds

have been shown to cross the blood-brain barrier (BBB) in rats, and to act

as almost equipotent inhibitors of human butyrylcholinesterase (BChE) and

acetylcholineesterase (AChE). In this respect it should be noted that the

inhibition of brain BChE is as important as AChE inhibition in AD therapy

(1, 10). As mentioned hereinabove, the use of anti-inflammatory drugs is

known in the art as well, as a different approach for the treatment of AD.

Recent studies have suggested that the symptoms of AD are prevented or

attenuated by anti-inflammatory treatment (11). Specifically, non-steroidal

anti-inflammatory drugs (NSAIDs) are known to act as inhibitors of the

synthesis of IL-6, a cytokine that has been consistently detected in the

brains of AD patients, but not in the brains of non-demented elderly persons

(12).

The proposed mechanism of the NSAIDs as anti-inflammatory drugs

suggests that they inhibit the binding of the prpstaglandin substrate,

arachidonic acid, to the active site of cyclooxygenase (COX). The

constitutive isoform of COX, COX-1, has a clear physiological function in

the prostaglandin biosynthesis. The inducible form, COX-2, is formed by

pro-inflammatory stimuli in migratory cells and inflamed tissues. Thus, the range of activities of NSAIDs against COX-1 compared to COX-2

influences the variations in the side effects of NSAIDs at their

anti-inflammatory effective doses. In other words, the use of NSAIDs for

AD treatment is limited by its possible side effects and toxicity. Moreover,

most NSAIDs are hydrophihc compounds, and therefore have limited

permeability to the brain.

It is therefore a growing need for improved NSAIDs which are more

selective COX-2 inhibitors and are further able to cross the BBB, in order to

achieve potent anti-inflammatory activity with fewer side effects (13).

Some NSAIDs which are more selective toward COX-2 inhibition

are presently known in the art (e.g., IBUPROFEN). However, these

NSAIDs hardly cross the BBB, and the use thereof induces side effects,

such as gastrointestinal effects. Furthermore, a new generation of NSAIDs,

which act as more selective COX-2 inhibitors, has been recently introduced

for clinical use [e.g., Searl's Celebrex (celecoxib) and Merck & Co.'s Vioxx

(rofecoxib)]. Nevertheless, these new drugs still carry the warning about

gastrointestinal effects on their labels, similar to generic NSAIDs (14), and

furthermore, it was recently noted that celecoxib may elevate cardiovascular

risk in humans (15).

In addition, recent studies showed that both ChEI and

anti-inflammatory drugs can be useful for the treatment of acute cerebral

ischemia as well (16). In analogy to AD patients, elevated levels of IL-6

have been detected in cerebral spinal fluid (CSF) of patients with acute stroke (17). Furthermore, it has been found that levels of leukotriene LTC4

and prostaglandin E₂ (PGE₂) were higher in cerebral spinal fluid of stroke

patients than in age-matched controls (18). Therefore, it is presumed that

the use of selective antagonists of LT or inhibitors of its biosynthesis could

be helpful in reducing the ischemic penumbra during acute cerebral

ischemia, by controlling the vasogenic edema. Furthermore, it was

suggested that corticosterone and dexamethasone protection against

hypoxic-ischemic damage is a glucocorticoid receptor-mediated effect (19).

As is reviewed hereinabove, NSAIDs have been shown to act as inhibitors

of prostaglandin biosynthesis, and thus can be useful for treatment of

cerebral ischemia and hypoxia.

Moreover, recent studies have demonstrated the use of the ChEI

ENA-713 (EXELON) for either protection or treatment of neuronal damage

induced by transient brain ischemia in gerbils (20, 21). Post-ischemic

administration of EXELON ameliorated the ischemia- induced pyramidal

cell loss and reduced significantly the number of glial fibrillary acidic

protein-positive astrocytes in the CA1 region of gerbils hippocampus, 14

days post recirculation (21). These findings suggest that ChEIs, such as

EXELON, could be useful for treatment of senile dementia such as

cerebrovascular dementia, and for reducing the neuronal damage caused by

either acute cerebral ischemia or closed head injury.

It is therefore presumed, based on these studies, that the development

of new cholinergic compounds such as reversible or irreversible ChEIs, as well as new NSAIDs, can be useful for the treatment and prevention of

various CNS disorders and diseases, amongst which are AD, cerebral

ischemia, stroke, hypoxia and closed head injury.

Thus, the prior art teaches that cholinergic up-regulators (e.g.,

ChEIs) and NSAIDs are each independently and via different

pharmacotherapy pathways useful in the treatment of CNS disorders and

diseases, as well as head injuries and stroke. However, certain cholinergic

up-regulators and most NSAIDs, especially those comprising a free

carboxylic acid group, are hydrophihc by nature, as they interact with

binding sites of enzymes and/or receptors. As such, the brain

pharmacopenetration of these compounds is limited.

There is thus a widely recognized need for, and it would be highly

advantageous to have novel bifunctional chimeric compounds, covalently

coupling a reversible or irreversible cholinergic up-regulator with a NSAID,

so as to render the chimera sufficiently hydrophobic so as to freely pass the

blood brain barrier, and to exert synergistic copharmacotherapy in the

damaged brain.

SUMMARY OF THE LNVENTION

According to the present invention there are provided: (i) chimeric

compounds which are conjugates of reversible or irreversible cholinergic

up-regulators and non-steroidal anti-inflammatory drugs (NSAIDs); (ii)

reversible cholinesterase inhibitors; (iii) methods for their synthesis; and (iv) use thereof for treatment and/or prevention of central nervous system

(CNS) disorders and diseases.

It is shown herein that such chimeric compounds have a synergistic

effect in several pharmacological aspects including (i) blood brain barrier

permeability; (ii) simultaneous and prolonged pharmacokinetics; (iii)

colocalized pharmacology; (iv) reversible AChE inhibition; and (v) reduced

side effects. Thus, by co-exerting both brain neuronal cholinergic activity,

preferably reversible activity, and brain anti-inflammatory activity, the

compounds of the present invention have higher pharmacological activity

and Therapeutic Index than commonly known ChEIs and prolonged

pharmacokinetics as is compared to presently known drugs; they exert

preventive (prophylactic) therapy for CNS disorders and diseases; they are

sufficiently lipophilic so as to efficiently cross the blood brain barrier; they

are pharmacologically active either as a chimeric compound and/or its

hydrolytic derivatives; due to the cholinergic up-regulator moiety the

compounds of the present invention are targeted (directed) to cholinergic

sites where they exert both cholinergic up-regulation and inflammation

down-regulation; due to the reversible binding to the cholinergic sites of

some of the compounds of the present invention their toxicity is

substantially reduced; gastrointestinal side effects, along with other

systemic side effects associated with the use of NSAIDs are attenuated due

to esterification of the carboxylic acid moiety thereof. According to one aspect of the present invention there is provided a

chimeric compound comprising a cholinergic up-regulator moiety and a

non-steroidal anti-inflammatory moiety being covalently linked thereto.

According to another aspect of the present invention there is

provided a chimeric compound of a general formula:

A-S-B

wherein:

A is a cholinergic up-regulator moiety selected from the group consisting of

a cholinesterase inhibitor residue, a nicotinic receptor agonist residue and a

muscarinic receptor agonist residue; B is a non-steroidal anti-inflammatory

moiety characterized by a functional group selected from the group

consisting of a free carboxylic acid group and a free amine group; and S is a

hydrocarbon spacer being covalently linked to B via a -C(=X)Y- bond,

where X is a non-substituted or substituted oxygen, sulfur or nitrogen atom

and Y is a substituted or non-substituted carbon, oxygen, nitrogen, sulfur,

silicon or phosphor atom linked to the C atom of the bond via a single

covalent bond.

According to yet another aspect of the present invention there is

provided a pharmaceutical composition, comprising, as an active ingredient

any one or more of the chimeric compounds of the present invention.

According to further features in preferred embodiments of the

invention described below, the pharmaceutical composition is formulated for transdermal delivery, nasal administration, administration by inhalation

or administration by injection.

According to still another aspect of the present invention there is

provided a method of treating, ameliorating or preventing a central nervous

system disorder or disease in an organism, the method comprising the step

of administering to the organism a therapeutically effective amount of the

compound per se or as an active ingredient of a pharmaceutical

composition.

According to further features in preferred embodiments of the

invention described below, the central nervous system disorder or disease is

selected from the group consisting of Alzheimer's disease, cerebrovascular

dementia, Parkinson's disease, basal ganglia degenerative diseases,

motoneuron diseases, Scrapie, spongyform encephalopathy and

Creutzfeldt-Jacob's disease.

According to still further features in the described preferred

embodiments the central nervous system disorder or disease is selected

from the group consisting of cerebral ischemia, transient hypoxia and

stroke.

According to still further features in the described preferred

embodiments the central nervous system disorder or disease is a result of a

head injury. According to still further features in the described preferred

embodiments the central nervous system disorder or disease is accompanied

by an inflammatory process.

According to still further features in the described preferred

embodiments the inflammatory process is selected from the group

consisting of an inflammatory process induced by infection, an

inflammatory process induced by a tumor and an inflammatory process

induced by post-operative brain edema.

According to still further features in the described preferred

embodiments the infection is selected from the group consisting of viral

infection and bacterial infection.

According to still further features in the described preferred

embodiments the organism is a mammal.

According to still further features in the described preferred

embodiments the mammal is a human being.

According to an additional aspect of the present invention there is

provided a method of synthesizing the chimeric compound of the present

invention, the method comprising the steps of (a) converting a non-steroidal

anti-inflammatory drug into a non-steroidal anti-inflammatory-ester,

including a hydrocarbon chain terminating with a reactive halide group; and

(b) reacting the non-steroidal anti-infϊammatory-ester including the

hydrocarbon chain terminating with the reactive halide group with a

cholinergic up-regulator, so as to obtain the chimeric compound having the cholinergic up-regulator moiety covalently linked to the non-steroidal

anti-inflammatory moiety via the hydrocarbon spacer.

According to yet an additional aspect of the present invention there is

provided a method of synthesizing the chimeric compound of the present

invention, the method comprising the steps of (a) converting a non-steroidal

anti-inflammatory drug into a non-steroidal anti-inflammatory-amide, the

amide including a hydrocarbon chain terminating with a reactive halide

group; and (b) reacting the non-steroidal anti-inflammatory-amide including

the hydrocarbon chain terminating with the reactive halide group with a

cholinergic up-regulator, so as to obtain the chimeric compound having said

cholinergic up-regulator moiety covalently linked to said non-steroidal

anti-inflammatory moiety via said hydrocarbon spacer.

According to still an additional aspect of the present invention there

is provided a method of synthesizing the chimeric compound of the present

invention, the method comprising the steps of (a) converting a cholinergic

up-regulator into its N(ring)-substituted derivative, the derivative including

a hydrocarbon chain terminating with a reactive hydroxyl group; and (b)

reacting the N(ring)-substituted derivative including the hydrocarbon chain

terminating with the reactive hydroxyl group with a reactive derivative of a

non-steroidal anti-inflammatory drug, so as to obtain the chimeric

compound having the cholinergic up-regulator moiety covalently linked to

the non-steroidal anti-inflammatory moiety. Optionally, the method further

comprising the step of converting the N(ring)-substituted derivative including the hydrocarbon chain terminating with the reactive hydroxyl

group into a tertiary amine N(ring)-substituted derivative including the

hydrocarbon chain terminating with the reactive hydroxyl group, prior to

the step (b).

According to further features in preferred embodiments of the

invention described below, the cholinergic up-regulator moiety and the

non-steroidal anti-inflammatory moiety are covalently linked via a

hydrocarbon spacer.

According to still further features in the described preferred

embodiments the non-steroidal anti-inflammatory moiety is covalently

attached to the spacer via a -C(=X)Y- bond, where X is a non-substituted or

substituted oxygen, sulfur or nitrogen atom and Y is a substituted or

non-substituted carbon, oxygen, nitrogen, sulfur, silicon or phosphor atom

linked to the C atom of the bond via a single covalent bond.

According to still further features in the described preferred

embodiments the -C(=X)Y- bond is selected from the group consisting of

an ester bond and an amide bond.

According to still further features in the described preferred

embodiments the ester bond is selected from the group consisting of a

carboxylic ester bond and a glycol amide ester bond.

According to still further features in the described preferred

embodiments the -C(=X)Y- bond is hydrolizable by brain derived esterases

or amidases. According to still further features in the described preferred

embodiments the hydrocarbon spacer comprises at least one hydrocarbon

selected from the group consisting of an alkyl having 2-20 carbon atoms, a

cycloalkyl having 3-20 carbon atoms and an aryl having 6-20 carbon atoms.

According to still further features in the described preferred

embodiments the cholinergic up-regulator moiety is selected from the group

consisting of a reversible cholinesterase inhibitor and an irreversible

cholinesterase inhibitor.

According to still further features in the described preferred

embodiments the cholinergic up-regulator moiety is selected from the group

consisting of a cholinesterase inhibitor residue, a nicotinic receptor agonist

residue and a muscarinic receptor agonist residue.

According to still further features in the described preferred

embodiments the cholinesterase inhibitor residue is a pyridostigmine

residue.

According to still further features in the described preferred

embodiments the pyridostigmine residue is a 3-N,N-dimethylcarbamoyl

pyridinium bromide residue.

According to still further features in the described preferred

embodiments the nicotinic agonist residue is selected from the group

consisting of a nicotine residue and a cytisine residue. According to still further features in the described preferred

embodiments the muscarinic receptor agonist residue is selected from the

group consisting of an arecoline residue and a pilocarpine residue.

According to still further features in the described preferred

embodiments the non-steroidal anti-inflammatory moiety comprises a

residue of a non-steroidal anti-inflammatory drug characterized by a

functional group selected from the group consisting of a free carboxylic

acid group and a free amine group.

According to still further features in the described preferred

embodiments the non-steroidal anti-inflammatory moiety is selected from

the group consisting of an ibuprofen residue, an indomethacin residue, a

naproxen residue, a diclofenac residue and an aspirin residue.

According to still further features in the described preferred

embodiments the ibuprofen residue is selected from the group consisting of

an (+)-ibuprofen residue, an S-(+)-ibuprofen residue and an R-(-)-ibuprofen

residue.

According to still further features in the described preferred

embodiments the chimeric compound is characterized by lipophilicity

sufficient for permitting the compound to cross a blood brain barrier of an

organism.

According to still further features in the described preferred

embodiments the chimeric compound is characterized by cholinergic

up-regulation activity and inflammation down-regulation activity exerted by the chimeric compound and/or hydrolytic derivatives thereof, and hence

may be defined as a drug and/or a prodrug.

According to a further aspect of the present invention there is

provided a reversible cholinesterase inhibitor having a general formula A:

wherein:

R is C(=Q)-Z-R₃; R₂ is selected from the group consisting of hydrogen, an

alkyl, a hydroxyalkyl, a haloalkyl, an alkylamine, a cycloalkyl and an aryl;

X is a halide; Q and Z are each independently selected from the group

consisting of oxygen and sulfur; and R₃ is selected from the group

consisting of an alkyl, a cycloalkyl and an aryl.

According to yet a further aspect of the present invention there is

provided a method of synthesizing the reversible cholinesterase inhibitor,

the method comprising reacting a pyridine ring substituted at position 3 by

the R_\ with a R₂ residue terminating with the halide group X, so as to

produce a quaternary pyridinium halide substituted at the N(ring) position

by the R₂ residue and at position 3 by the R residue.

According to further features in preferred embodiments of the

invention described below, Q and Z are each oxygen, R₃ is methyl, R₂ is an

alkyl and X is selected from the group consisting of bromide and iodide. According to still a further aspect of the present invention there is

provided a reversible cholinesterase inhibitor having a general formula B:

wherein:

Ri is C(=Q)-Z-R₃; R₂ is selected from the group consisting of hydrogen, an

alkyl, a hydroxyalkyl, a haloalkyl, an alkylamine, a cycloalkyl and an aryl;

Q and Z are each independently selected from the group consisting of

oxygen and sulfur; and R₃ is selected from the group consisting of an alkyl,

a cycloalkyl and an aryl.

According to another aspect of the present invention, there is

provided a method of synthesizing the reversible cholinesterase inhibitor,

comprising: (a) reacting a pyridine ring substituted at position 3 by the R_!

with an organic halide and/or a reactive inorganic halide, so as to produce a

quaternary pyridinium halide substituted by the R] at position 3; and (b)

reducing the quaternary pyridinium halide, so as to produce a tertiary

tetrahydropyridine substituted by the R_! at position 3.

According to further features in preferred embodiments of the

invention described below, Q and Z are each oxygen, R₃ is methyl and R₂ is

an alkyl.

According to still further features in the described preferred

embodiments the reactive inorganic halide is potassium iodide. According to still further features in the described preferred

embodiments the organic halide is the R₂ residue terminating with the

halide group X and the quaternary pyridinium halide is further substituted at

the N(ring) position by the R₂ residue.

According to yet another aspect of the present invention there is

provided a method of treating, ameliorating or preventing a central nervous

system disorder or disease, as described hereinabove, in an organism, the

method comprising the step of administering to the organism a

therapeutically effective amount of the reversible cholinesterase inhibitor

per se or as an active ingredient of a pharmaceutical composition.

The present invention successfully addresses the shortcomings of the

presently known configurations by providing new and potent chimeric

compounds for the treatment and prevention of central nervous system

disorders and diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with

reference to the accompanying drawings. With specific reference now to

the drawings in detail, it is stressed that the particulars shown are by way of

example and for purposes of illustrative discussion of the preferred

embodiments of the present invention only, and are presented in the cause

of providing what is believed to be the most useful and readily understood

description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in

more detail than is necessary for a fundamental understanding of the

invention, the description taken with the drawings making apparent to those

skilled in the art how the several forms of the invention may be embodied in

practice.

In the drawings:

FIGs. la-b are plots demonstrating a time-course of HuAChE

(Figure la) and FBS-AChE (Figure lb) inhibition in vitro by varying

concentrations of DICLO-PO (at 25 °C, in phosphate buffer, 50 Mm, pH

7.4);

FIG. 2 is a plot demonstrating a time-course of whole blood ChE

activity following intramuscular administration of PYR, PO and IBU-PO in

mice;

FIG. 3 shows photographs of rat stomach mucosal tissue following

intraperitoneal administration of 10 mg/kg IBU-PO and DICLO-PO, note

the absence of erosions or ulcers;

FIG. 4 is a bar graph demonstrating a decrease in

carrageenan-induced rat paw edema following intraperitoneal injection of 5

mg/kg NSAIDs and NSAID-PYR-X compounds, compared with injection

of a vehicle only;

FIG. 5 is a comparative bar graph demonstrating an effect of IBU

and IBU-PO on carrageenan-induced brain edema in rats; FIG. 6 is a bar graph demonstrating the effect of intraperitoneal

injection of 10 mg/kg IBU-PO on carrageenan-induced brain edema in

mice;

FIG. 7 shows comparative plots demonstrating the effect of

pretreatment with 5 mg/kg atropine and 2 mg/kg mecamylamine on

IBU-PO-induced (by intraperitoneal injection of 2.5 mg/kg IBU-PO)

hypothermia in mice;

FIG. 8 is a bar graph demonstrating the effect of varying doses of

PO, IBU-PO and NAPRO-PO on edema level induced by a closed head

injury in mice;

FIG. 9 is a bar graph demonstrating the effect of treatment with

varying doses of IBU-PO on survival time in male mice following

hypobaric hypoxia [n=4 per group], compared with a control group treated

with a vehicle only;

FIG. 10 is a bar graph demonstrating the effect of IBU-PO and

known ChE inhibitors on survival time in mice during hypobaric hypoxia

[n=4 per group];

FIG. 11 shows plots demonstrating the competition binding curves

for the displacement of [³H]NMS from rat brain by NSAID-PYR-X

compounds; and

FIG. 12 shows plots demonstrating the competition binding curves

for IBU-PO with various radioactive ligands in rat brain. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of chimeric compounds which are

conjugates of reversible or irreversible cholinergic up-regulators and

non-steroidal anti-inflammatory drugs (NSAIDs) and of novel reversible

cholinesterase inhibitors, pharmaceutical compositions containing same,

methods of their preparation and their use for the treatment of central

nervous system (CNS) disorders and diseases, such as, but not limited to,

Alzheimer's disease, cerebrovascular dementia, cerebral ischemia, transient

hypoxia and stroke, as well as CNS diseases or disorders induced by closed

head injury or accompanied by inflammatory processes.

The principles and operation of the chimeric compounds

according to the present invention may be better understood with reference

to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail,

it is to be understood that the invention is not limited in its application to

the details set forth in the following description or exemplified by the

Examples. The invention is capable of other embodiments or of being

practiced or carried out in various ways. Also, it is to be understood that

the phraseology and terminology employed herein is for the purpose of

description and should not be regarded as limiting.

While conceiving the present invention, it was hypothesized that a

chimeric compound covalently coupling a cholinergic up-regulator moiety

and a NSAID moiety could exert synergistic pharmacological activity toward CNS disorders and diseases. The underlying concepts of this

hypothesis are as follows:

CNS disorders and diseases are in many cases characterized by

reduced cholinergic activity and inflammation. It is indeed known for many

years that CNS disorders and diseases are treatable via hydrophobic

derivatives of cholinergic up-regulators and potentially treatable by

anti-inflammatory drugs. However, certain cholinergic up-regulators and

some non-steroidal anti-inflammatory drugs, especially those comprising a

free carboxylic acid group, are hydrophihc and therefore fail to efficiently

cross the blood brain barrier, where they are to exert their therapeutic

activity.

It was, therefore, hypothesized that covalently coupling a cholinergic

up-regulator and a non-steroidal anti-inflammatory drug via a bond

hydrolizable by brain enzymes, one would achieve a synergistic effect in

several aspects including (i) blood brain barrier permeability; (ii)

simultaneous and prolonged pharmacokinetics; (iii) co-localized

pharmacology; (iv) reduced side effects.

While reducing the present invention to practice, as is further

exemplified in the Examples section that follows, it was found that

covalently coupling a cholinergic up-regulator and a non-steroidal

anti-inflammatory drug via a bond hydrolizable by brain enzymes results in

(i) a combined action of cholinergic up-regulation and inflammation down

regulation of both the chimeric compound and its hydrolytic derivatives, characterized by unitary pharmacokinetics; (ii) sufficient lipophilicity to

cross the BBB; (iii) high affinity to brain cholinergic receptors; (iv) lower

toxicity; (v) larger Therapeutic Index; and (vi) longer duration of action in

the brain compared with other known drugs used for treating CNS disorders

and diseases.

Thus, the chimeric compounds are used according to the present

invention to treat CNS disorders and diseases. Each of the compounds

which are used to treat CNS disorders and diseases according to the present

invention includes a cholinergic up-regulator moiety and a non-steroidal

anti-inflammatory moiety which are covalently coupled.

As used herein in the specification and in the claims section that

follows, the term "cholinergic up-regulator moiety" refers to a residue

derived from a cholinergic compound which retains its cholinergic activity.

As is well accepted in the art, the term "residue" refers herein to a major

portion of a molecule which is covalently linked to another molecule.

Acetylcholine (ACh) is a neurotransmitter that is constantly

produced by a synthetic pathway involving choline acetyltransferase

(ChAT), decomposed by cholinesterase (ChE) and exerts its cholinergic

function via acetylcholine receptors. Thus, cholinergic up regulation can be

achieved by (i) activating its synthetic pathway; (ii) blocking its degradation

by inhibition of ChEs; and/or (iii) mimicking its action via agonists directed

towards cholinergic receptors. Thus, cholinergic compounds according to the present invention

include, for example, cholinesterase inhibitors (ChEI) such as, but not

limited to, a pyridostigmine; nicotinic receptor agonists such as, but not

limited to, nicotine and cytisine, or muscarinic receptor agonists such as,

but not limited to, arecoline and pilocarpine.

The term "non-steroidal anti-inflammatory (NSAID) moiety" as used

herein refers to a residue, as this term is defined hereinabove, of a

non-steroidal anti-inflammatory drug characterized by a functional group

such as, but not limited to, a free carboxylic acid group or a free amine

group. NSAIDs according to the present invention include, for example,

ibuprofen, indomethacin, naproxen, diclofenac and aspirin.

According to a preferred embodiment of the present invention, the

non-steroidal anti-inflammatory moiety is an (+)-ibuprofen residue, an

S-(+)-ibuprofen residue or an R-(-)-ibuprofen residue. The pure optical

isomer S-(+)-ibuprofen, also referred to in the art as dexibuprofen, is known

to exert enhanced anti-inflammatory activity compared to the ibuprofen

racemic mixture and is thus used as an efficacious drug for rheumatoid

arthritis and osteoarthritis (35, 36). However, the other optical isomer,

R-(-)-ibuprofen, which is known to act as an effective anti-inflammatory

drug as well, has been recently found to further act as an anticancer drug

(37). It was therefore expected that a chimeric compound comprising an

S-(+)-ibuprofen residue or an R-(-)-ibuprofen residue will exert higher

pharmacological activity. Indeed, as is shown hereinbelow in the Examples section, the chimeric compound obtained from the optical isomer

S-(+)-ibuprofen was found to exert a pharmacological activity that is about

10 times higher than the chimeric compound obtained from the racemic

ibuprofen.

According to another preferred embodiment of the present invention,

the cholinergic up-regulator moiety and the NSAID moiety are covalently

linked via a hydrocarbon spacer.

As used herein in the specification and in the claims section that

follows, the term "hydrocarbon" refers to a compound that includes

hydrogen atoms and carbon atoms which are covalently attached. The

hydrocarbon can be saturated, unsaturated, branched or unbranched.

The term "hydrocarbon spacer" refers to a hydrocarbon moiety

comprised of at least one hydrocarbon, such as, but not limited to, alkyl,

cycloalkyl and/or aryl.

As used herein in the specification and in the claims section that

follows, the term "alkyl" refers to a saturated aliphatic hydrocarbon

including straight chain and branched chain groups. Preferably, the alkyl

group, has 2 to 20 carbon atoms. Whenever a numerical range; e.g.,

"2-20", is used herein, it means that the group, in this case the alkyl group,

may contain 2 carbon atoms, 3 carbon atoms, etc., up to and including 20

carbon atoms. More preferably, it is a medium size alkyl having 4 to 16

carbon atoms. Most preferably, it is an alkyl having 8 to 14 carbon atoms. A "cycloalkyl" group refers to an all-carbon monocyclic or fused

ring groups (i.e., rings which share an adjacent pair of carbon atoms),

wherein one or more of the rings does not have a completely conjugated

pi-electron system. Examples, without limitation, of cycloalkyl groups

are cyclopropane, cyclobutane, cyclopentane, cyclohexane,

cyclohexadiene, cycloheptane, cycloheptatriene and adamantane.

An "aryl" group refers to an all-carbon monocyclic or fused-ring

polycyclic group (i.e., rings which share adjacent pairs of carbon atoms)

having a completely conjugated pi-electron system. Examples, without

limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl.

According to a preferred embodiment of the present invention, the

NSAID moiety is covalently attached to the hydrocarbon spacer via a

"-C(=X)Y-" bond, where X is, without limitation, a non-substituted or

substituted oxygen, sulfur or nitrogen atom and Y is, without limitation, a

substituted or non-substituted carbon, oxygen, nitrogen, sulfur, silicon or

phosphor atom linked to the C atom of the bond via a single covalent bond.

According to another preferred embodiment of the present invention,

the "-C(=X)Y-" bond is an ester bond or an amide bond.

As used herein in the specification and in the claims section that

follows, the term "ester bond" refers to a "-C(=X)Y-" bond, where X is

without limitation, oxygen or sulfur, and Y is, without limitation, oxygen,

sulfur, glycol amine, glycol ester, O-carbamyl, or O-thiocarbamyl. A "glycol amine" group refers to an -0-CH₂-C(=0)-NR'- group,

where R' is hydrogen, alkyl, cycloalkyl or aryl.

A "glycol ester" group refers to an -0-CH₂-C(=0)-OR'- group,

where R' is as defined above.

An "O-carbamyl" group refers to an -0-C(=0)-NR'- group, where

R' is as defined above.

An "O-thiocarbamyl" group refers to an -0-C(=S)-NR'- group,

where R' is as defined above.

The term "amide bond" as used herein refers to a "-C(=X)Y-" bond,

where X is, without limitation, oxygen or sulfur, and Y is, without

limitation, amine, N-carbamyl or N-thiocarbamyl.

An "amine" group refers to a -NR'- group, where R' is hydrogen,

alkyl, cycloalkyl or aryl.

A "N-carbamyl" group refers to a -NR'-C(=0)-0-R'- group, where

R' is as defined above.

A "N-thiocarbamyl" group refers to a -NR'-C(=S)-0-R'- group,

where R' is as defined above.

According to a preferred embodiment of the present invention, the

ester bond is a carboxylic ester bond or a glycol amide ester bond.

As used herein in the specification and in the claims section that

follows, the term "carboxylic ester bond" refers to a "-C(=0)0-" bond.

The term "glycol amide ester bond" as used herein refers to a

"C(=0)0-CH₂-C(=0)-NR"-" bond, where R" is hydrogen or alkyl. According to another preferred embodiment of the present invention,

the "-C(=Y)X-" bond is hydrolizable by one or more brain derived esterases

or amidases. Such hydrolysis provides for slow release of the NSAID

moiety from the chimeric compound in brain, and its rate is determined by

the nature of the ester bond employed (22). The outcome of such controlled

release of the NSAID moiety is a longer duration of action for the

compound in the brain, following one administration.

However, according to another preferred embodiment of the present

invention, the chimeric compound is characterized by cholinergic

up-regulation activity and inflammation down-regulation activity exerted by

the chimeric compound and/or its hydrolytic derivatives.

The term "hydrolytic derivatives", according to the present

invention, refers to the products formed in vivo by the enzymatic hydrolysis

described hereinabove.

Thus, the pharmacological activity of the chimeric compound of the

present invention is exerted by both a prodrug, which is the parent chimeric

compound itself, and by one or more drugs derived therefrom, which are the

hydrolytic derivatives of the chimeric compound. This dual activity of both

a prodrug and drugs derived therefrom is a novel pharmacological concept.

According to a preferred embodiment of the present invention, the

chimeric compound is a NSAID-PYR-X compound whose chemical

structure is described in Scheme I below: Scheme I

A — CH₂CH₂CH₂(CH₂)_nCH₂CH₂CH₂

0

II Ra

A= RC O-, _RCH N

\ where: n == 2, 4, 6; Ra = H or alkyl and X = halide

where R is one of:

Naproxen residue Diclofenac residue

Indomethacin residue Ibuprofen residue

or

Aspirin residue As used herein in the specification and in the claims section that

follows, the term "NSAID-PYR-X" refers to a chimeric compound

comprised of a 3-N,N-dimethylcarbamoyl pyridinium bromide residue

which is covalently linked through an ester bond or an amide bond to a

NSAID residue, via a hydrocarbon spacer.

The term "3-N,N-dimethylcarbamoyl pyridinium bromide" as used

herein refers to a pyridinium bromide moiety substituted at position 3

thereof with an -0-C(=0)-N(CH₃)₂- group.

According to another preferred embodiment of the present invention,

the chimeric compound is an IBU-4H-PO compound whose chemical

structure is described in Scheme II below:

Scheme II

IBU-4H-PO

As used herein in the specification and in the claims section that

follows, the term "IBU-4H-PO" refers to 2-(4-isobutyl phenyl)-propionic

acid 8-(3-N,N-dimethylcarbaboyl-3,6-dihydro-2H-pyridine- 1 -yl)-octyl

ester.

According to still another preferred embodiment of the present

invention, the chimeric compound is an IBU-OCT-cytisine compound (nicotinic receptor agonist) whose chemical structure is described in

Scheme III below:

Scheme III

IBU-OCT-cytisine

The term "IBU-OCT-cytisine" as used herein refers to ibuprofen

N-octyl-cytisine ester.

Reversible cholinesterase inhibitors:

While continuing to explore the chimeric compounds of the present

invention, the compounds IBU-OCT-arecoline (a muscarinic receptor

agonist), also referred to hereinafter as IOA, and

IBU-OCT-methylnicotinate, also referred to hereinafter as IOMN, were

prepared. Their chemical structures are described in Scheme IV below:

Scheme IV

IBU-OCT-arecoline (IOA)IBU-OCT-methylnicotinate (IOMN)

As used herein in the specification and in the claims section that

follows, the term "IBU-OCT-arecoline" (IOA) refers to

1 - { 8- [2-(4-isobuty l-phenyl)-propionyl]-octyl } - 1 ,2, 5 ,6-tetrahydropyridine-3 -

carboxylic acid methyl ester.

The term "IBU-OCT-methyl nicotinate" (IOMN) refers to l-{8-[2-

(4-isobutyl-phenyl)-propionyl]-octyl}-3-methoxycarbonyl pyridinium

iodide.

However, while evaluating the pharmacological activity of the IOA

and IOMN compounds, it was surprisingly found, as is further described

and exemplified in the Examples section, that these compounds act as

reversible cholinesterase inhibitors.

Thus, contrary to the NSAID-PYR-X compounds of the present

invention and other known ChEIs, which inhibit the cholinesterase by

covalently (and therefore irreversibly) carbamylating the serine residue at

the active site of the enzyme, the IOMN and IOA compounds interact with

the enzyme via electrostatic and hydrophobic interactions which are

completely reversible. This reversible inhibition activity is highly

advantageous since it substantially reduces the toxicity of the compounds.

At this point, it is pertinent to note that the presently known Alzheimer's

disease drugs which are approved by the FDA are ARICEPT and EXELON,

which are reversible and pseudo-reversible AChE inhibitors, respectively. In a search for new reversible AChE inhibitors, it was found that the

known compounds arecoline and methyl nicotinate (MN) are by themselves

reversible AChE inhibitors.

Thus, according to another aspect of the present invention there is

provided a reversible cholinesterase inhibitor having a general formula A:

wherein:

R] is C(=Q)-Z-R₃; R₂ is selected from the group consisting of hydrogen, an

alkyl, a hydroxyalkyl, a haloalkyl, an alkylamine, a cycloalkyl and an aryl;

X is a halide; Q and Z are each independently selected from the group

consisting of oxygen and sulfur; and R₃ is selected from the group

consisting of an alkyl, a cycloalkyl and an aryl.

Further according to the present invention, there is provided another

reversible cholinesterase inhibitor having a general formula B:

wherein:

R_! is C(=Q)-Z-R₃; R₂ is selected from the group consisting of hydrogen, an

alkyl, a hydroxyalkyl, a haloalkyl, an alkylamine, a cycloalkyl and an aryl;

Q and Z are each independently selected from the group consisting of oxygen and sulfur; and R₃ is selected from the group consisting of an alkyl,

a cycloalkyl and an aryl.

The term "haloalkyl", as used herein in the specification and in the

claims section that follows, refers to an alkyl group as defined hereinabove,

which include at least one carbon atom that is substituted by a halogen.

The term "hydroxyalkyl" as used herein, refers to an alkyl group as

defined hereinabove, which includes at least one carbon atom that is

substituted by an -OH group.

The term "alkylamine" as used herein, refers to an alkyl group as

defined hereinabove, which includes at least one carbon atom that is

substituted by an amine group as defined hereinabove.

Chemical synthesis:

Further according to the present invention, there are provided

methods for synthesizing the chimeric compounds of the present invention.

A first method according to the present invention is effected by

converting a non-steroidal anti-inflammatory drug into a non-steroidal

anti-inflammatory-ester which includes a hydrocarbon chain terminating

with a reactive halide group, and thereafter reacting the non-steroidal

anti-inflammatory-ester with a cholinergic up-regulator, thereby obtaining a

chimeric compound having a cholinergic up-regulator moiety and a

non-steroidal anti-inflammatory moiety being covalently linked thereto via

a hydrocarbon spacer and an ester bond. The term "derivative" as used herein refers to the result of a

chemically altering, modifying or changing a molecule or a portion thereof,

such that it maintains its original functionality in at least one respect.

The reactive halide group can be fluoride, chloride, bromide, or

iodide.

In one particular, the non-steroidal anti-inflammatory drug which

includes a free carboxylic acid group is converted into its acetyl chloride

derivative via interaction with an active nucleophilic chloride, such as

oxalyl chloride. Then, the anti-inflammatory drug acetyl chloride derivative

is esterified by a hydrocarbon terminated at a first end thereof with a

hydroxyl and at the opposing end thereof with a halide such as bromide.

Then, the esterified anti-inflammatory drug is reacted with a cholinergic

up-regulator which includes a pyridine ring to form a chimeric compound

having a cholinergic up-regulator moiety and a non-steroidal

anti-inflammatory moiety being covalently linked thereto via a hydrocarbon

spacer and a carboxylic ester bond and being characterized by a quaternary

ammonium halide residue.

A second method according to the present invention is effected by

converting a non-steroidal anti-inflammatory drug into a non-steroidal

anti-inflammatory-amide which includes a hydrocarbon chain terminating

with a reactive halide group, and thereafter reacting the non-steroidal

anti-inflammatory-amide with a cholinergic up-regulator, thereby obtaining

a chimeric compound having a cholinergic up-regulator moiety and a non-steroidal anti-inflammatory moiety being covalently linked thereto via

a hydrocarbon spacer and an amide bond.

In one particular, the non-steroidal anti-inflammatory drug which

includes a free carboxylic acid group is converted into its acetyl chloride

derivative via interaction with an active nucleophilic chloride, such as

oxalyl chloride. Then, the anti-inflammatory drug acetyl chloride derivative

is reacted with a hydrocarbon terminated at a first end thereof with an amine

and at the opposing end thereof with a halide such as bromide to form an

anti-inflammatory drug amide derivative. Then, the anti-inflammatory drug

amide derivative is reacted with a cholinergic up-regulator which includes a

pyridine ring to form a chimeric compound having a cholinergic

up-regulator moiety and a non-steroidal anti-inflammatory moiety being

covalently linked thereto via a hydrocarbon spacer and an amide bond and

being characterized by a quaternary ammonium halide residue.

A third method according to the present invention is effected by

converting a cholinergic up-regulator into its N(ring)-substituted derivative,

where the derivative includes a hydrocarbon chain terminating with a

reactive hydroxyl group, and thereafter reacting the N(ring)-substituted

derivative with a derivative of a non-steroidal anti-inflammatory drug.

Optionally, this method further includes the step of converting the

N(ring)-substituted derivative into its tertiary amine N(ring)-substituted

derivative, prior to the reaction with the derivative of the non-steroidal

anti-inflammatory drug. In one particular, a cholinergic up-regulator which includes a ring

moiety that contains a nitrogen atom, such as pyridine or cytisine, is reacted

with a hydrocarbon terminated at a first end thereof with a hydroxyl and at

the opposing end thereof with a halide such as bromide, to form a

quaternary ammonium halide derivative of the cholinergic up-regulator,

substituted at its N-ring with a hydrocarbon terminated with a hydroxyl.

Then, an acetyl chloride derivative of a non-steroidal anti-inflammatory

drug is esterified by the hydroxyl of the quaternary ammonium derivative,

to form a chimeric compound having a cholinergic up-regulator moiety and

a non-steroidal anti-inflammatory moiety being covalently linked thereto

via a hydrocarbon spacer and an ester bond, and being characterized by a

quaternary ammonium halide residue.

In another particular, the quaternary ammonium derivative of the

cholinergic up-regulator, which is substituted at its N-ring with a

hydrocarbon terminated with a hydroxyl, is reduced into a tertiary amine

derivative. The acetyl chloride derivative of a non-steroidal

anti-inflammatory drug is then esterified by the hydroxyl of the tertiary

amine derivative, to form a chimeric compound having a cholinergic

up-regulator moiety and a non-steroidal anti-inflammatory moiety being

covalently linked thereto via a hydrocarbon spacer and an ester bond and

being characterized by a reduced tertiary amine residue. Further according to the present invention, there are provided

methods of synthesizing the reversible cholinesterase inhibitors of the

present invention.

A first method according to the present invention is effected by

reacting a pyridine ring that is substituted at position 3 by a carboxylate

group with a substituted or non-substituted residue terminating with a halide

group, to form a quaternary pyridinium ring substituted by the substituted or

non-substituted residue at the N(ring) position and by the carboxylate group

at position 3.

The term "carboxylate group" as used herein refers to a

"-C(=Q)Z-R" '-"group, where Q and Z are each independently oxygen or

sulfur and R'" is, without limitation, alkyl, cycloalkyl or aryl, as defined

hereinabove. Representative examples of a carboxylate group are methyl

acetate, methyl thioacetate and ethyl acetate.

Representative examples of a substituted or non-substituted residue

are alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, alkylamine and aryl, as

defined hereinabove.

Representative examples of a halide group are bromide and iodide.

Thus, in one particular, a pyridine ring that is substituted at position

3 by a carboxylate group, such as methyl nicotinate, is reacted with a

substituted or non-substituted alkyl terminating with a halide, such as

bromide or iodide, to form a quaternary pyridinium ring that is substituted by a carboxylate group at position 3 and by the substituted or

non-substituted alkyl at the N(ring) position.

A second method according to the present invention is effected by

reacting a pyridine ring that is substituted at position 3 by a carboxylate

group with an organic halide and/or a reactive inorganic halide, to form a

quaternary pyridinium halide that is substituted at position 3 by carboxylate

group, and reducing thereafter the formed quaternary pyridinium halide, to

form a tertiary tetrahydropyridine ring that is substituted at position 3 by a

carboxylate group.

Representative examples of a reactive inorganic halide are potassium

fluoride, potassium iodide, sodium iodide and sodium bromide.

The term "organic halide" as used herein refers to a substituted or

non-substituted residue, as defined hereinabove, that includes a halide group

at its end.

In one particular, a pyridine ring that is substituted at position 3 by a

carboxylate group, such as methyl nicotinate, is reacted with a substituted or

non-substituted alkyl terminating with a halide group, such as bromide or

iodide, and with a reactive inorganic halide, such as potassium iodide, to

form a quaternary pyridinium ring that is substituted by a carboxylate at

position 3 and by the substituted or non-substituted alkyl at the N(ring)

position. The substituted quaternary pyridinium ring is then reduced, to

form a tertiary tetrahydropyridine ring that is substituted by a carboxylate group at position 3 and by the substituted or non-substituted alkyl at the

N(ring) position.

Pharmaceutical composition:

Further according to the present invention there is provided a

pharmaceutical composition including the chimeric compound of the

invention as an active ingredient.

As used herein a "pharmaceutical composition" refers to a

preparation of one or more of the chimeric compounds described herein,

with other chemical components such as pharmaceutically suitable carriers

and excipients. The purpose of a pharmaceutical composition is to

facilitate administration of a compound to an organism.

Hereinafter, the term "pharmaceutically acceptable carrier" refers to

a carrier or a diluent that does not cause significant irritation to an

organism and does not abrogate the biological activity and properties of

the administered compound. Examples, without limitations, of carriers

are: propylene glycol, saline, emulsions and mixtures of organic solvents

with water. Herein the term "excipient" refers to an inert substance

added to a pharmaceutical composition to further facilitate administration

of a compound. Examples, without limitation, of excipients include

calcium carbonate, calcium phosphate, various sugars and types of starch,

cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be

found in "Remington's Pharmaceutical Sciences," Mack Publishing Co.,

Easton, PA, latest edition, which is incorporated herein by reference.

Routes of administration'. Suitable routes of administration may,

for example, include oral, rectal, transmucosal, transdermal, intestinal or

parenteral delivery, including intramuscular, subcutaneous and

intramedullary injections as well as intrathecal, direct intraventricular,

intravenous, intraperitoneal, intranasal, or intraocular injections.

Composition/formulation: Pharmaceutical compositions of the

present invention may be manufactured by processes well known in the

art, e.g., by means of conventional mixing, dissolving, granulating,

dragee-making, levigating, emulsifying, encapsulating, entrapping or

lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present

invention thus may be formulated in conventional manner using one or

more pharmaceutically acceptable carriers comprising excipients and

auxiliaries, which facilitate processing of the active compounds into

preparations which, can be used pharmaceutically. Proper formulation is

dependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated in

aqueous solutions, preferably in physiologically compatible buffers such

as Hank's solution, Ringer's solution, or physiological saline buffer with

or without organic solvents such as propylene glycol, polyethylene glycol. For transmucosal administration, penetrants are used in the formulation.

Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily

by combining the active compounds with pharmaceutically acceptable

carriers well known in the art. Such carriers enable the compounds of the

invention to be formulated as tablets, pills, dragees, capsules, liquids, gels,

syrups, slurries, suspensions, and the like, for oral ingestion by a patient.

Pharmacological preparations for oral use can be made using a solid

excipient, optionally grinding the resulting mixture, and processing the

mixture of granules, after adding suitable auxiliaries if desired, to obtain

tablets or dragee cores. Suitable excipients are, in particular, fillers such

as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose

preparations such as, for example, maize starch, wheat starch, rice starch,

potato starch, gelatin, gum tragacanth, methyl cellulose,

hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or

physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).

If desired, disintegrating agents may be added, such as cross-linked

polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as

sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,

concentrated sugar solutions may be used which may optionally contain

gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,

titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee

coatings for identification or to characterize different combinations of

active compound doses.

Pharmaceutical compositions, which can be used orally, include

push-fit capsules made of gelatin as well as soft, sealed capsules made of

gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit

capsules may contain the active ingredients in admixture with filler such

as lactose, binders such as starches, lubricants such as talc or magnesium

stearate and, optionally, stabilizers. In soft capsules, the active

compounds may be dissolved or suspended in suitable liquids, such as

fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition,

stabilizers may be added. All formulations for oral administration should

be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of

tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according

to the present invention are conveniently delivered in the form of an

aerosol spray presentation from a pressurized pack or a nebulizer with the

use of a suitable propellant, e.g., dichlorodifluoromethane,

trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In

the case of a pressurized aerosol, the dosage unit may be determined by

providing a valve to deliver a metered amount. Capsules and cartridges of,

e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base

such as lactose or starch.

The chimeric compounds described herein may be formulated for

parenteral administration, e.g., by bolus injection or continuos infusion.

Formulations for injection may be presented in unit dosage form, e.g., in

ampoules or in multidose containers with optionally, an added

preservative. The compositions may be suspensions, solutions or

emulsions in oily or aqueous vehicles, and may contain formulatory agents

such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include

aqueous solutions of the active preparation in water-soluble form.

Additionally, suspensions of the active compounds may be prepared as

appropriate oily injection suspensions. Suitable lipophilic solvents or

vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters

such as ethyl oleate, triglycerides or liposomes. Aqueous injection

suspensions may contain substances, which increase the viscosity of the

suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents

which increase the solubility of the compounds to allow for the

preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for

constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before

use. The chimeric compounds of the present invention may also be

formulated in rectal compositions such as suppositories or retention

enemas, using, e.g., conventional suppository bases such as cocoa butter or

other glycerides.

The pharmaceutical compositions herein described may also

comprise suitable solid of gel phase carriers or excipients. Examples of

such carriers or excipients include, but are not limited to, calcium

carbonate, calcium phosphate, various sugars, starches, cellulose

derivatives, gelatin and polymers such as polyethylene glycols.

Dosage: Pharmaceutical compositions suitable for use in context

of the present invention include compositions wherein the active

ingredients are contained in an amount effective to achieve the intended

purpose. More specifically, a therapeutically effective amount means an

amount of chimeric compound effective to prevent, alleviate or ameliorate

symptoms of disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within

the capability of those skilled in the art, especially in light of the detailed

disclosure provided herein.

For any chimeric compound used in the methods of the invention,

the therapeutically effective amount or dose can be estimated initially

from activity assays in animals. For example, a dose can be formulated in

animal models to achieve a circulating concentration range that includes

the IC₅₀ as determined by activity assays (e.g., the concentration of the test compound, which achieves a half-maximal inhibition of the ChE or COX

activity). Such information can be used to more accurately determine

useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described

herein can be determined by standard pharmaceutical procedures in

experimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (lethal

dose causing death in 50 % of the tested animals) for a subject compound.

The data obtained from these activity assays and animal studies can be

used in formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed

and the route of administration utilized. The exact formulation, route of

administration and dosage can be chosen by the individual physician in

view of the patient's condition. (See e.g., Fingl et al., 1975, in "The

Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to

provide plasma levels of the active moiety which are sufficient to maintain

the cholinesterase (ChE) modulating effects, termed the minimal effective

concentration (MEC). The MEC will vary for each preparation, but can be

estimated from in vitro data; e.g., the concentration necessary to achieve

50-90 % inhibition of a ChE may be ascertained using the assays

described herein. Dosages necessary to achieve the MEC will depend on

individual characteristics and route of administration. HPLC assays or

bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using the MEC value.

Preparations should be administered using a regimen, which maintains

plasma levels above the MEC for 10-90 % of the time, preferable between

30-90 % and most preferably 50-90 %.

Depending on the severity and responsiveness of the condition to

be treated, dosing can also be a single administration of a slow release

composition described hereinabove, with course of treatment lasting from

several days to several weeks or until cure is effected or diminution of the

disease state is achieved.

The amount of a composition to be administered will, of course, be

dependent on the subject being treated, the severity of the affliction, the

manner of administration, the judgment of the prescribing physician, etc.

Packaging: Compositions of the present invention may, if desired,

be presented in a pack or dispenser device, such as an FDA approved kit,

which may contain one or more unit dosage forms containing the active

ingredient. The pack may, for example, comprise metal or plastic foil,

such as a blister pack. The pack or dispenser device may be accompanied

by instructions for administration. The pack or dispenser may also be

accompanied by a notice associated with the container in a form

prescribed by a governmental agency regulating the manufacture, use or

sale of pharmaceuticals, which notice is reflective of approval by the

agency of the form of the compositions or human or veterinary

administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an

approved product insert. Compositions comprising a chimeric compound

of the invention formulated in a compatible pharmaceutical carrier may

also be prepared, placed in an appropriate container, and labeled for

treatment of an indicated condition. Suitable conditions indicated on the

label may include treatment of an Alzheimer's disease, cerebral ischemia,

stroke and a closed head injury.

Ph armacology:

Further according to the present invention, there is provided a

method for treating, ameliorating or preventing a central nervous system

disorder or disease in an organism (e.g., a human being). The method is

effected by administering a therapeutically effective amount of one or

more of the chimeric compounds of the invention to a treated subject.

As used herein, the term "method" refers to manners, means,

techniques and procedures for accomplishing a given task including, but

not limited to, those manners, means, techniques and procedures either

known to, or readily developed from known manners, means, techniques

and procedures by practitioners of the chemical, pharmacological,

biological, biochemical and medical arts.

Herein, the term "treating" includes abrogating, substantially

inhibiting, slowing or reversing the progression of a disease, substantially

ameliorating clinical symptoms of a disease or substantially preventing the

appearance of clinical symptoms of a disease. As used herein, the term "CNS disorder or disease" refers to a

disorder or disease characterized by an impairment of the CNS. The

impairment can be affected by both down-regulation of acetylcholine and

an inflammation process.

The term "administering" as used herein refers to a method for

bringing a chimeric compound of the present invention into an area or a

site in the brain that is impaired by the CNS disorder or disease.

The term "organism" refers to animals, typically mammals having a

blood brain barrier, including human.

The term "therapeutically effective amount" refers to that amount

of the compound being administered which will relieve to some extent one

or more of the symptoms of the disorder or disease being treated.

The present invention is thus directed to chimeric compounds

which are capable of crossing the blood brain barrier, so as to approach the

impaired site or area in the brain, and cause cholinergic up-regulation

together with down-regulation of the inflammation process.

Examples of diseases associated with CNS impairment that are

treatable using the chimeric compounds of the invention, include, without

limitation, Alzheimer's disease, cerebrovascular dementia, Parkinson's

disease, basal ganglia degenerative diseases, motoneuron diseases, Scrapie,

spongyform encephalopathy and Creutzfeldt-Jacob's disease,

Examples of disorders associated with CNS impairment that are

treatable using the chimeric compounds of the invention, include, without limitation, cerebral ischemia, transient hypoxia, and stroke. Further

disorders can be induced by closed head injury, infection, tumor and

post-operative brain edema.

Further according to the present invention, there is provided a

method for treating, ameliorating or preventing a central nervous system

disorder or disease in an organism (e.g., a human being). The method is

effected by administering a therapeutically effective amount of one or

more of the reversible cholinesterase inhibitors of the invention to a

treated subject, either per se or as an active ingredient in a pharmaceutical

composition.

The reversible cholinesterase inhibitors of the invention cause

reversible cholinergic up-regulation at the impaired site or area in the brain,

and may therefore be used in association with CNS disorders and diseases

as defined hereinabove.

Additional objects, advantages, and novel features of the present

invention will become apparent to one ordinarily skilled in the art upon

examination of the following examples, which are not intended to be

limiting. Additionally, each of the various embodiments and aspects of the

present invention as delineated hereinabove and as claimed in the claims

section below finds experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together

with the above descriptions, illustrate the invention in a non limiting

fashion.

MA TERIALS AND EXPERIMENTAL METHODS

Chemical Syntheses and Analyses

The following procedures describe the syntheses and analyses of the

bifunctional chimeric compounds of the invention, and the intermediates

thereof.

Synthesis of indomethacin acid chloride: Indomethacin (2.3 grams,

6.4 mmol) was placed in a dried, nitrogen-purged, 3 -necked 100 ml round

bottom flask. Oxalyl chloride (5.7 grams, 45 mmol) was then added

dropwise, and the reaction was allowed to proceed at room temperature

until evolution of gases ceased. Evaporation (herein and below, under

reduced pressure) of unreacted oxalyl chloride resulted in the formation of

the product as a pale yellow solid (1.6 grams, 66 % yield).

1H-NMR (CDC1₃): δ = 2.41 (s, CH₃, 3H), 3.81 (s, OCH₃, 3H), 4.17

(s, CH₂C(0), 2H), 6.6 (d, Indo-H₇), 6.85 (d, indo-H₆), 6.9 (s, indo-H₄), 7.46

(d, Hα, 2H), 7.6 (d, Hβ, 2H) ppm.

Synthesis of indomethacin bromooctyl ester: Indomethacin acid

chloride (1.6 grams, 4.25 mmol) and l-bromo-8-octanol (0.83 gram, 4

mmol), in dichloromethane (50 ml), were refluxed for 3 days. Evaporation

of the solvent, followed by purification of the resulting crude oil by silica gel column chromatography, using a mixture of 70 %/30 % ether/hexane,

gave 1.2 grams of the product (50.5 % yield).

1H-NMR (CDC1₃): δ = 1.27 (m, CH₂, 8H), 1.61 (quin, CH₂CH₂Br,

2H), 1.82 (quin, OCH₂CH₂, 2Η), 2.38 (s, CH₃, 3H), 3.39 (t, CH₂Br, 2H),

3.65 (s, CH₂C(0), 2H), 3.83 (s, OCH₃, 3H), 4.1 (s, CH₂0, 2H), 6.6 (d,

indo-H₇), 6.85 (d, indo-H₆), 6.9 (s, indo-H₄), 7.46 (d, Hα, 2H), 7.6 (d, Hβ,

2H) ppm.

Synthesis of N-(Indomethacin octanoate)-3-N,N-

dimethylcarbamoyl pyridinium bromide (INDO-PO): Indomethacin

bromooctyl ester (1 gram, 1.8 mmol) and 3-dimethylcarbamoyl pyridine

(0.7 gram, 4.2 mmol), in methoxy ethanol (50ml), were refluxed for 2 days.

Concentration (herein and below, under vacuum) and purification of the

resulting crude oil by silica gel column chromatography, using a mixture of

17:3 chloroform/methanol, gave 0.53 gram product (41% yield).

1H-NMR (CDCI₃): δ = 1.25 (m, CH₂, 8H), 1.58 (quin, OCH₂CH₂,

2Η), 2.01 (quin, CH₂CH₂N, 2H), 2.36 (s, CH₃, 3H), 3.04 (s, NCH₃, 3H),

3.16 (s, NCH₃, 3H), 3.65 (s, CH₂C(0), 2H), 3.82 (s, OCH₃, 3H), 4.1 (s,

CH₂0, 2H), 4.92 (t, CH₂N, 2H), 6.6 (d, indo-H₇), 6.85 (d, indo-H₆), 6.9 (s,

indo-H₄), 7.46 (d, Hα, 2H), 7.6 (d, Hβ, 2H), 8.1 (t, pyr-H₅), 8.26 (d,

pyr-H₄), 9.23 (s, pyr-H₂), 9.33 (d, pyr-H₆) ppm.

MS: m/z = 635 [M+1-Br]⁺.

Synthesis of Ibuprofen acid chloride: (+)Ibuprofen (2.95 grams, 14

mmol) or one of its optical isomers either R(-)-ibuprofen or S-(+)-ibuprofen, were placed in a dried, nitrogen-purged, 3 -necked 100 ml

flask. Oxalyl chloride (11 grams, 86 mmol) was then added dropwise, and

the reaction was allowed to proceed at room temperature until evolution of

gases ceased. Evaporation of the unreacted oxalyl chloride resulted in the

formation of 3.2 grams of the product as a pale yellow solid (100 % yield).

1H-NMR (CDC1₃): δ = 0.93 (d, (CH₃)₂, 6H), 1.61 (s, CH₃CH, 3H),

1.9 (m, CHCH₂, 1H), 2.5 (d, CHCH₂, 2Η), 4.11 (q, CH₃CH, 1H), 7.19 (2d,

Hα+Hβ, 4H) ppm.

Synthesis of Ibuprofen bromooctyl ester: Ibuprofen acid chloride (3

grams, 14 mmol) and l-bromo-8-octanol (2.85 grams, 14 mmol), ^"in

acetonitrile (50 ml), were refluxed for 3 days. Evaporation of the solvent,

followed by purification of the resulting crude oil by silica gel column

chromatography, using a mixture of 1:1 ether/hexane, gave 4.4 grams of the

product (83 % yield).

1H-NMR (CDCI₃): δ = 0.89 (d, (CH₃)₂, 6H), 1.25 (m, CH₂, 10H),

1.48 (d, CH₃CH, 3H), 1.56 (quin, CH₂CH₂Br, 2H), 1.83 (m, CHCH₂, 1H),

2.45 (d, CHCH₂, 2Η), 3.39 (t, CH₂Br, 2H), 3.68 (q, CH₃CH, 1Η), 4.05 (t,

OCΗ₂, 2H), 7.08 (d, Hα, 2H) 7.2 (d, Hβ, 2H) ppm.

Synthesis of N-(Ibuprofen octanoate)-3-N,N-dimethylcarbamoyl

pyridinium bromide (IBU-PO): Ibuprofen bromooctyl ester (2.2 grams,

5.5 mmol) and 3-N,N-dimethylcarbamoyl pyridine (1.2 grams, 7.2 mmol),

in methoxy ethanol (50 ml), were refluxed for 4 days. Evaporation of the

solvent, followed by purification of the resulting crude oil by silica gel column chromatography, using gradient mixture of chloroform/methanol,

gave 1.47 grams of the product (47 % yield).

Η-NMR (CDC1₃): δ = 0.83 (d, (CH₃)₂, 6H), 1.25 (m, CH2, 12H),

1.42 (d, CH₃CH, 3H), 1.83 (m, CHCH₂, IH), 2.38 (d, CHCH₂, 2Η), 2.99 (s,

(CH₃)₂N, 3H), 3.12 (s, (CH₃)₂N, 3H), 3.68 (t, CH₃CH, 1Η), 3.97 (t, OCΗ₂,

2H), 4.91 (t, CH₂N, 2H), 7.03 (d, Hα, 2H), 7.14 (d, Hβ, 2H), 8.12 (t,

pyr~H₅), 8.28 (d, pyr-H₄), 9.23 (s, pyr-H₂), 9.33 (d, pyr-H₆) ppm.

MS: m/z = 483 [M-Br]⁺.

Synthesis of the optical isomer IBU-PO N-(S)-Ibuprofen

(octanoate)-3-N,N-dimethylcarbamoyl pyridinium bromide

(S-(+)-IBU-PO):

To a solid (S)-(+)-4-isopropyl-α-methyl-phenylacetic acid (0.540

gram, 2.62 mmol), oxalyl chloride (1.33 grams, 10.48 mmol) was gradually

added, while stirring, at room temperature under nitrogen atmosphere. The

obtained solution was stirred at room temperature until no evolution of gas

was observed. The excess of oxalyl chloride was then removed at reduced

pressure.

The obtained acid chloride was dissolved in dry dichloromethane

(8.0 ml) and cooled (ice-water bath). A solution of

3-dimethylcarbamoyl- l-(8-hydroxy-octyl)-pyridinium bromide (0.82 gram,

2.19 mmol) and triethylamine (0.530 gram, 5.25mmol) in dry

dichloromethane (8.0 ml) was then added and the reaction mixture was

stirred at room temperature overnight. Addition of ether, filtration of the obtained solid precipitate, washing the solid with ether and evaporation of

the ether remnants gave a crude oil. Purification by silica gel column

chromatography using a mixture of 5 %/95 % methanol/dichloromethane

gave 0.573 gram of the product as an oil (46.6 % yield).

1H-NMR (CDC1₃): δ - 0.89 (d(j=6.6Hz), (CH₃)₂C), 1.11-1.44 (m,

(CH₂)₄), 1.48 (dG=7.2Hz), CH₃CHC0₂), 1.55 (m, 2H), 1.83 (m, (CH₃)₂CH),

2.03 (m, 2Η), 2.44 (dG=7.1Hz), (CH₃)₂CHCH₂), 3.06 (s, (CH₃)₂NCO), 3.18

(s, (CH₃)₂NCO), 3.68 (q(j=7.2Hz), CH₃CHC0₂), 4.03 (t(}=6.7Ηz),

C0₂CH₂), 5.00 (t(j=7.5Hz), CH₂N⁴ , 7.08 (d(j=8.0Hz), 2 Ar-H), 7.19

(d(j=8.0Hz), 2 Ar-H), 8.08 (ddG=6.0,8.6Hz), 1 pyr-H), 8.27 (dG=8.6Hz), 1

pyr-H), 9.27 (s, 1 pyr-H), 9.41 (d(j=6.0Hz), 1 pyr-H) ppm.

Synthesis of Naproxen acid chloride: Naproxen (2.7 grams, 12

mmol) was placed in a dried, nitrogen-purged, 3 -necked 100 ml round

bottom flask. Oxalyl chloride (10.4 grams, 82 mmol) was then added

dropwise, and the reaction was allowed to proceed at room temperature

until evolution of gases ceased. Evaporation of the unreacted oxalyl

chloride resulted in the formation of 2.9 grams of the product as a pale

yellow solid (100% yield).

1H-NMR (CDC1₃): δ = 1.59 (d, CHCH₃, 3Η), 3.91 (s, CH₃0, 3H),

4.15 (q, CHCH₃, IH), 7.12 (m, nap-Hj+Hs, 2H), 7.4 (d, nap-H₄), 7.68 (m,

nap-H₅+H₇+H₈, 3H) ppm.

Synthesis of Naproxen bromooctyl ester: Naproxen acid chloride (3

grams, 12 mmol) and l-bromo-8-octanol (2.3 grams, 11 mmol), in acetonitrile (50 ml), were refluxed for 2 days. Evaporation of the solvent

gave a white precipitate to which ether (50 ml) was added. The resulting

ether solution was evaporated. Purification of the residual oil by silica gel

column chromatography, using a mixture of 1:1 ether/hexane, gave 2.81

grams of the product (57% yield).

1H-NMR (CDC1₃): δ = 1.17 (m, CH₂, 8H), 1.29 (m, OCH₂CH₂, 2Η),

1.59 (d, CHCH₃, 3Η), 1.79 (m, CH₂CH₂Br, 2H), 3.35 (t, CH₂Br, 2H), 3.83

(q, CHCH₃, IH), 3.89 (s, CH₃0, 3H), 4.05 (t, OCH₂, 2H), 7.1 (m,

nap-Hj+H₃, 2H), 7.6 (m, nap-H₅+H₇+H₈, 3H) ppm.

Synthesis of N-(Naproxen octanoate)-3-(N,N-dimethylcarbamoyl)

pyridinium bromide (NAPRO-PO): Naproxen bromooctyl ester (1.2

grams, 2.8 mmol) and 3-N,N-dimethylcarbamoyl pyridine (1 gram, 6

mmol), in methoxy ethanol (50 ml), were refluxed for 4 days. Evaporation

of the solvent, followed by purification of the resulting crude oil by silica

gel column chromatography, using a mixture of 17:3 chloroform/methanol,

gave 0.87 gram of the product (52 % yield).

1H-NMR (CDCI₃): δ = 1.22 (m, CH₂, 8H), 1.25 (m, OCH₂CH₂, 2Η),

1.5 (d, CHCH₃, 3Η), 1.9 (m, CH₂CH₂Br, 2H), 3.0 (s, CH₃N, 3H), 3.13 (s,

CH₃N, 3H), 3.8 (q, CHCH₃, IH), 3.87 (s, CH₃0, 3H), 4.01 (t, OCH₂, 2H),

4.9 (t, CH₂N, 2H), 7.1 (m, nap-H₇+H₃, 2H), 7.4 (d, nap-H₄), 7.6 (m,

nap-H₅+H₇+H₈, 3H), 8.16 (t, pyr-H₅), 8.25 (d, pyr-H₄), 9.15 (s, pyr-H₂),

9.24 (d, pyr-H₆) ppm. Synthesis of Diclofenac bromooctyl ester: Diclofenac (0.97 gram,

3.27 mmol) and l-bromo-8-octanol (2.9 grams, 13.8 mmol) were placed in

a 50 ml flask. 37 % Hydrochloric acid (2 ml) was added thereafter and the

mixture was refluxed for 1 hour. The reaction was monitored by TLC and

1H-NMR analysis. Chloroform (150 ml) was then added to the resulting

yellow oil and the solution was dried (over anhydrous Na₂S0₄) and

concentrated. Purification of the crude oil by silica gel column

chromatography, using chloroform, gave 1.2 grams of the product (75%

yield).

1H-NMR (CDC1₃): δ = 1.34 (m, CH₂ ,8H), 1.65 (quin, CH₂CH₂Br,

2H), 1.84 (quin, OCH₂CH₂, 2Η), 3.4 (t, CH₂Br, 2H), 3.81 (s, CH₂C(0), 2H),

4.14 (t, OCH₂, 2H), 6.57 (d, H₈), 6.98 (t, H₉+H₁₀, 2H), 7.13 (t, H„), 7.25 (d,

H₄), 7.34 (d, H₃+H₅, 2H) ppm.

MS (El): m/z = 487 |M^f], 277, 242, 214.

Synthesis of N-(Diclofenac octanoate)-3-(N,N-dimethylcarbamoyl)

pyridinium bromide (DICLO-PO): Diclofenac bromooctyl ester (0.460

gram, 0.94 mmol) and 3-dimethylcarbamoyl pyridine (0.5 gram, 3 mmol),

in methoxy ethanol (10 ml), were refluxed for 4 days. Evaporation of the

solvent, followed by purification of the crude oil by silica gel column

chromatography using a mixture of 17:3 chloroform/methanol, gave 0.58

gram of the product, as a dark green oil (94% yield).

1H-NMR (CDCI₃): δ = 1.28 (m, CH₂, 8H), 1.6 (quin, OCH₂CH₂,

2H), 2.01 (quin, CH₂CH₂N, 2H), 3.04 (s, CH₃N, 3H), 3.16 (s, CH₃N, 3H), 3.79 (s, CH₂C(0)0, 2H), 4.01 (t, OCH₂, 2H), 4.98 (t, CH₂N, 2H), 6.52 (d,

H₈), 6.97 (t, H₉+H₁₀, 2H), 7.11 (t, H„), 7.23 (d, H₄), 7.33 (d, H₃+H₅, 2H),

8.1 (t, pyr-H₅), 8.26 (d, pyr-H₄), 9.27 (s, pyr-H₂), 9.41 (d, pyr-H₆) ppm.

MS: m/z = 572 [M-Br]⁺.

Synthesis of 3-(N,N-dimethylcarbamoyl)-l-(8-hydroxy-octyl)

pyridinium bromide: 3-N,N-dimethylcarbamoyl pyridine (0.88 gram, 5.3

mmol) and 1-bromo 8-octanol (1.55 grams, 7.4 mmol), in methoxyethanol

(80 ml), were refluxed for 2 days. Evaporation of the solvent followed by

purification of the resulting crude oil by silica gel column chromatography,

using a mixture of 1:4 methanol/chloroform, gave 0.62 gram of the product

(31% yield).

1H-NMR (CDC1₃): δ = 1.29 (m, CH₂, 8H), 1.49 (m, HOCH₂CH₂,

2Η), 2.05 (m, CH₂CH₂N, 2H), 3.03 (s, NCH₃, 3H), 3.16 (s, NCH₃, 3H), 3.57

(s, CH₂OH, 2H), 4.92 (t, CH₂N, 2H), 8.15 (t, pyr-H₅), 8.31 (d, pyr-H₄), 9.32

(s, pyr-H₂), 9.45 (d, ρyr-H₆) ppm.

Synthesis of 3-N,N-Dimethylcarbamic acid l-(8-hydroxy-octyl)-

l,2,5,6-tetrahydro-pyridine-3-yl ester: Solid sodium borohydride (3.1

grams, 81.9 mmol) was added in portions during a period of 10 minutes into

a cooled (ice-water bath) stirred solution of

3-N,N-dimethylcarbamoyl-l-(8-hydroxy-octyl)-pyridinium bromide (6.2

grams, 16.5 mmol) and methanol (150 ml). The cooled reaction mixture

was stirred for 30 minutes followed by 1 hour stirring at room temperature.

Evaporation of the solvent, dissolving the formed residue in water (35ml), Extraction with 3x70 ml dichloromethane, drying over anhydrous MgS0₄

and concentration gave 4.5 grams of yellow oil. Purification by

chromatography on a silica gel column, using a mixture of 95 %/5 %

chloroform/methanol, gave 2.45 grams of the product, as a colorless oil

(49.7 % yield).

1H-NMR (CDC1₃): δ = 1.31 (s, (CH₂)₄, 8H), 1.54 (m, 4H), 2.25 (m,

2H), 2.44 (m, 2H), 2.59 (m, 2H), 2.93 (s, NCH₃, 3H), 2.95 (s, NCH₃, 3H),

3.00 (s, 2H), 3.62 (t, 2H), 5.44 (s, C=CH, 1Η) ppm.

Synthesis of 2-(4-Isobutyl phenyl)-propionic acid 8-(5-dimethy

lcarbamoyl-3,6-dihydro-2H-pyridine-l-yl)octyl ester (IBU-4H-PO):

2-(4-Isobutyl-phenyl)-propionyl chloride, prepared by reacting ibuprofen

(0.560 gram, 2.72 mmol) with oxalyl chloride, and 3-N,N-dimethylcarbamic

acid l-(8-hydroxy-octyl)-l,2,5,6-tetrahydro-pyridine -3-yl ester (0.6 gram,

2.01 mmol), in dichloromethane (10 ml), were stirred at room temperature

overnight. Dichloromethane (90 ml) was added thereafter to the reaction

mixture. Washing with 6 % aqueous sodium carbonate, drying the organic

layer (over anhydrous MgS0 ) and evaporation of the solvent, gave crude

yellow oil (0.98 grams). Purification by column chromatography on a silica gel

(68 grams), using a mixture of 20 %/80 % methanol/chloroform, gave 0.382

gram of the pure product, as a colorless oil (39 % yield).

1H-NMR (CDCI₃): δ = 0.89 (dG=6.6Ηz), (CH₃)₂CH, 6H), 1.24 (s,

(CH₂)₄, 8H), 1.48 (d(j=7.2Hz), CH₃CHC0₂, 3H), 1.55 (m, 4H), 1.83 (m,

(CH₃)₂CH, 1Η), 2.25 (m, 2Η), 2.43 (2H), 2.44, (d, (CH₃)₂CHCH₂, 2Η), 2.57 (tQ=5.7Hz), CH₂N, 2H), 2.93 (s, N(CH₃), 3H), 2.94 (s, N(CH₃), 3H), 3.04 (s

2H), 3.68 (q, CH₃CHC0₂, 1Η), 4.04 (t ^'=6.7Ηz), C0₂CH₂, 2H), 5.43 (bs,

C=C ), 7.08 (d(j=8.0Hz), Ar-2H), 7.20(d(j=8.0Hz), Ar-2H) ppm.

MS: m/z = 486 pvf], 414[M⁺-(CH₃)₂NCO, 100%].

Synthesis of 2-Acetoxy-benzoic acid 8-bromo-octyl ester (Aspirin

bromooctyl ester): (a) 2-Acetoxybenzoyl chloride was synthesized as

described by Liebeskind et al. (23). (b) l-Bromo-8-octanol (3 grams, 14.3

mmol) and pyridine (2.94 grams, 37.2 mmol), in dichloromethane (45ml),

were stirred in a cooled ice-water bath, under N₂ atmosphere. A solution of

2-acetoxybenzoyl chloride (3.7 grams, 18.6 mmol) in dichloromethane

(5ml) was added gradually, and the reaction mixture was stirred at room

temperature for 48 hours. After evaporation of the solvent, 300 ml ether

were added, and washed with 2 x 50 ml water, 3 x 70 ml 0.3N dilute

hydrochloric acid, 3 x 70 ml 5 % aqueous sodium bicarbonate and 3 x 70 ml

water. Drying the ether phase, concentration, extraction of the residue with

4 x 40 ml hexane and evaporation of the hexane extracts gave 4.7 grams of

a viscous oil (88.3 % yield).

1H-NMR (CDC1₃): δ = 1.30-1.50 (m, (CH₂)₄, 8H), 1.74 (m, 2H),

1.85 (m, 2H), 2.35 (s, OCOCH₃,3H), 3.41 (t(j=6.8Hz), CH₂Br, 2H), 4.27

(t(j=6.7Hz), C0₂CH₂, 2H), 7.10 (dd, Ar-IH), 7.31 (dt, Ar-IH), 7.55 (m,

Ar-IH), 8.02 (dd, Ar-IH) ppm. Synthesis of l-[8-(2-Acetoxy-benzoyl)-octyl]-3-N,N-

dimethylcarbamoyl pyridinium bromide (ASP-PO): 2-Acetoxy-benzoic

acid bromooctyl ester (1.01 grams, 2.73 mmol) and

3-N,N-dimethylcarbamoyl pyridine (0.56 gram, 3.37 mmol), in

2-methoxyethanol (25 ml), were refluxed overnight. Evaporation of the

solvent, followed by ether extractions to remove residual solvent and

non-polar impurities, gave 1.4 grams of insoluble oil. Purification by

chromatography on a silica-gel column with a mixture of 95 %/5 %

chloroform-methanol gave 0.84 gram of a viscous oil (57.5% yield).

^- MR (CDC1₃): δ = 1.24-1.50 (m, (CH₂)₄, 8H), 1.71 (m, 2H),

2.04 (m, 2H), 2.35 (s, OCOCH₃, 3H), 3.04 (s, CONCH₃, 3H), 3.17 (s,

CONCH₃, 3H), 4.25 (t(j=6.7Hz), C0₂CH₂, 2H), 4.99 (t(j=7.4Hz), CΑ_$f,

2H) 7.10 (dd, Ar-IH), 7.33 (dt, Ar-IH), 7.56 (m, Ar-IH), 8.01 (dd, Ar-IH),

8.10 (dd, Pyr-IH), 8.30 (m, Pyr-IH), 9.30 (s, Pyr-IH), 9.42 (d, Pyr-IH)

ppm.

Synthesis of N-(8-hydroxyoctyl)-cytisine: l-Bromo-8-octanol

(0.198 gram, 0.947 mmol), cytisine (0.150 gram, 0.789 mmol), and

potassium carbonate (0.120 gram, 0.868 mmol), in ethanol (4.0 ml) were

refluxed overnight. Water (0.25 ml) and potassium carbonate (0.100 gram)

were then added and the reaction mixture was shortly stirred. The solvents

were thereafter removed under reduced pressure, and the residue was

extracted with dichloromethane. Drying the organic extract over MgS0

and evaporation of the solvent gave a crude residue (0.290 grams)). Purification by silica gel column chromatography, using ethyl acetate, gave

0.189 gram of pure N-(8-hydroxyoctyl)-cytisine (75.3 % yield).

1H-NMR (CDC1₃): δ = 0.95-1.40 (m, 10H), 1.43-1.60 (m, 2H),

1.70-1.94 (m, 2H), 2.10-2.31 (m, 4H), 2.41 (brs, IH), 2.80-3.00 (m, 4H),

3.63 (t(j=6.3Hz), CH₂OH), 3.89 (m(j=l.lHz), 6.4 (d(j=15.4Hz), IH), 4.04

(d(j=15.4Hz), IH), 6.00 (dd(j=l.1,6.9Hz), C=CH, IH), 6.44

(dd(j=1.4,9.0Hz), C=CH, IH), 7.28 (ddG=6.9,9.0Hz), C=CH, lH) ppm.

¹³C-NMR (CDCI3): δ = 25.6 (-CH₂-), 26.1 (-CH₂-), 26.4 (-CH₂-),

28.2 (-CH-), 29.1 (-CH₂-), 29.5 (-CH₂-), 33.1 (-CH₂-), 35.7 (-CH-), 50.3

(-CH₂-), 56.9 (-CH₂-), 60.3 (-CH₂-), 60.5 (-CH₂-), 62.7 (-CH₂-), 104.9

(-CH=), 116.4 (-CH=), 138.8 (-CH=), 152 (-C-), 163.8 (-CO) ppm.

MS (El): m/z = 318 [M"], 288, 203 [M⁺-(CH₂)₇OH, 77 %], 172, 160,

146, 117, 102, 84, 69, 58 (100 %).

Synthesis of ibuprofen N-octyl-cytisine ester

(IBU-OCT-CYTISINE): N-(8-hydroxyoctyl)-cytisine (0.150 gram, 0.472

mmol) and triethylamine (0.130 mg, 1.287 mmol), in dry dichloromethane

(3.0 ml), were stirred in a cooled ice- water bath, under nitrogen atmosphere.

A solution of 2-(4-isobutyl-phenyl)-propionyl chloride (the acid chloride of

ibuprofen, 0.143 gram, 0.637 mmol) in dry dichloromethane (1 ml) was

added gradually and the reaction mixture was stirred at room temperature

overnight. The solvent was then removed under reduced pressure and water

(5.0 ml) was added to the residue. A solution of concentrated potassium

carbonate was added to adjust the pH to a value of 9-10, and the mixture was extracted with ether (3 x 20 ml). Drying the combined organic extracts

over MgS0 and evaporation of the solvent gave 0.248 gram of crude oil.

Purification by silica gel column chromatography, using a mixture of ethyl

acetate/ether (1:5 v/v ratio) gave 0.187 gram of pure ibuprofen

N-octyl-cytisine ester as a colorless oil (78.3 % yield).

1H-NMR (CDC1₃): δ = 0.89 (d(j=6.6Hz), (CH₃)₂C, 6H), 0.94-1.43

(m, 10H), 1.48 (d(j=7.1Hz), CH₃CHC0₂, 3H), 1.40-1.64 (m, 2H), 1.70-1.95

(m, 2H), 2.10-2.32 (m, 4H), 2.40 (brs, IH), 2.44 (d,G=7.1Hz),

(CH₃)₂CHCH₂), 2.80-3.05 (m, 4Η), 3.68 (q(p7.1Hz), CH₃CHC0₂, 1Η),

3.87 (ddG=6.6,15.4Ηz), IH), 4.02 (dG=15Hz), IH), 4.02 (t, C0₂CH₂, 2H),

5.96 (dG=6.7Hz), C=CH, IH), 6.41 (dG=9.0Hz), C=CH, IH), 7.08

(dG=8.0Hz), 2 Ar-H), 7.19 (dG=8.0Hz), 2 Ar-H), 7.25 (ddG=6.8,9.0Hz),

C=CH, lH) ppm.

¹³C-NMR (CDC1₃): δ - 18.6 (CH₃CH), 22.5 (-(CH₃)₂CH-), 25.7

(-CH₂-), 26.1 (-CH₂-), 26.5 (-CH₂-), 26.8 (-CH₂-), 28.2 (-CH-), 28.5

(-CH2-), 29.1 (-CH₂-), 29.2 (-CH₂-), 30.2 (-(CH₃)₂CH-), 35.7

(-CH-cytisine), 45.1 (-(CH₃)₂CHCH₂-), 45.3 (-CH₃CTΪ-), 50.2 (-CH₂-), 57.5

(-CH₂-), 60.3 (-CH₂-), 60.5 (-CH₂-), 64.8 (-CH₂-), 104.5 (-CH=), 116.5

(=CH-), 127.2 (2 Ar-CH=), 129.3 (2 Ar-CH=), 138.0 (Ar-C-), 138.6

(-CH=), 140.4 (Ar-C-), 151.8 (-C-), 163.7 (-C=0), 174.9 (-C0₂-) ppm.

MS (El): m/z = 506 [M^], 491, 433, 398, 360, 318, 301, 204, 203

(100 %), 161, 149. Synthesis of l-(8-Hydroxy-octyl)-3-methoxycarbonyl pyridinium

bromide (or iodide): l-Bromo-8-octanol (2.02 grams, 9.66 mmol), methyl

nicotinate (5.13 grams, 37.45 mmol) and potassium iodide (1.62 grams,

9.76 mmol), in methanol (25 ml), were refluxed, under nitrogen

atmosphere, overnight. Evaporation of the solvent, followed by two ether

extractions to remove the residual solvent and starting materials, gave 4.2

grams of ether insoluble oil. Purification by silica gel column

chromatography, using a mixture of 10 %/90 % methanol/chloroform, gave

2.7 grams of the product as a viscous yellow oil (71 % yield).

1H-NMR (CDC1₃): 6 = 1.2-1.6 (m, (CH₂)₅, 1 OH), 2.10 (m, 2H), 3.62

(tG=6.4Hz), CH₂OH), 4.08 (s, C0₂CH₃), 5.06 (tQ=7.6Hz), CΑ^ ), 8.38

(dd, Pyr-H), 8.97 (d(j=8.1Hz), pyr-H), 9.43 (s, pyr-H), 9.94 (d(j=6.1Hz),

pyr-H) ppm.

Synthesis of l-(8-Hydroxy-octyl)-l,2,5,6-tetrahydro-pyridine-

3-carboxylic acid methyl ester (N-hydroxyoctyl-arecoline): Solid sodium

borohydride (0.520 gram, 13.74 mmol) was added in portions into a cold

(ice- water bath) stirred solution of l-(8-hydroxy-octyl)-3-methoxycarbonyl

pyridinium iodide (1.36 grams, 3.46 mmol) in methanol (40 ml), and the

reaction mixture was stirred for 30 minutes. The solvent was then

evaporated and the formed residue was dissolved in 30 ml water.

Extraction with 200 ml ether, washing the organic layer with water (30 ml),

potassium carbonate solution (2 x 30 ml) and water (2 x 30 ml), drying over

MgS0₄ and concentration gave a crude yellow oil (1.10 grams). Purification by silica gel column chromatography using a mixture of 2 %

methanol in chloroform, gave 0.384 gram of the product (41.2 % yield).

1H-NMR (CDC1₃): δ = 1.2-1.9 (m, (CH₂)₆), 2.38 (m, 2H), 2.47 (m,

2H), 2.55 (tO=5.6Hz), 2H), 3.20 (bs, NCH₂C=C), 3.66 (tG=6.6Ηz),

CH₂OH), 3.76 (s, C0₂CH₃), 7.02 (bs, CH=C) ppm.

Synthesis of l-{8-[2-(4-Isobutyl-phenyl)-propionyl]-octyl}-

l,2,5,6-tetrahydro-pyridine-3-carboxylic acid methyl ester

(IBU-OCT-Arecoline, IOA): 2-(4-Isobutyl-phenyl)-propionyl chloride,

formed by reacting ibuprofen (0.560 gram, 2.72 mmol) with oxalyl

chloride, was added to l-(8-Ηydroxyoctyl)-l,2,5,6-tetrahydro-pyridine-

3 -carboxylic acid methyl ester (0.316 gram, 1.17 mmol) in dry

dichloromethane (10 ml), and the reaction mixture was stirred at room

temperature, under nitrogen atmosphere, overnight. Chloroform (70 ml)

was added thereafter. Washing with 10 % potassium carbonate solution (3

x 20 ml), drying the organic layer over MgS0 and evaporation gave a

crude oil. Purification by a silica gel column chromatography, using a

mixture of 0.8 % methanol in chloroform, gave 0.37 gram of the pure

product as a viscous oil (69 % yield).

Optionally, the product can be obtained by sodium borohydride

reduction of l-{8-[2-(4-isobutyl-phenyl)-propionyl]-octyl}-

3 -methoxycarbonyl-pyridinium iodide.

1H-NMR (CDCI3): δ = 0.89 (dG=6.6Hz), (CH₃)₂CH), 1.25 (m,

(CH₂)₄), 1.48 (dG=7.2 Hz), CH₃CHC0₂), 1.55 (m, 4H), 1.84 (m, (CH₃)₂CH), 2.35 (m, 2Η), 2.44 (dG=7.1Hz), (CH₃)₂CHCH₂), 2.45 (m, 2Η),

2.53 (t, 2H), 3.18 (bs, NCH₂-C=C), 3.68 (q(j=7.1Ηz), CH₃CHC0₂), 3.73 (s,

C0₂CH₃), 4.04 (tQ=6.6Ηz), CO₂CH₂), 7.00 (bs, CH=C), 7.08 (dO=8.1Ηz),

Ar-2H), 7.20 (d(j=8.1Hz), Ar-2H) ppm.

Synthesis of l-{8-[2-(4-Isobutyl-phenyl)-propionyl]-octyl}-3-

methoxycarbonyl-pyridinium iodide (IB U-OCT-methylnicotinate,

IOMN): Ibuprofen- 8-bromooctyl ester (0.547 grams, 1.38 mmol), methyl

nicotinate (0.797 gram, 5.82 mmol) and potassium iodide (0.250 gram, 1.51

mmol), in methanol (10 ml), were refluxed, under nitrogen atmosphere, for

26 hours. Evaporation of the solvent and trituration of the residue with

ether, gave an ether-insoluble residue which was dissolved in chloroform.

Filtration to remove inorganic salts and evaporation gave 0.53 gram of

crude yellow oil. Purification by chromatography on a silica gel column,

using a mixture of 1 :24 methanol/chloroform, gave 0.4 gram of the product

(50 % yield).

1H-NMR (CDCI₃): δ = 0.89 (dG=6.6Hz), (CH₃)₂CH), 1.17-1.45 (m,

(CH₂)₅), 1-48 (dG=7.1Hz), CH₃CHC0₂), 1.55 (m, 2H), 1.84 (m, (CH₃)₂CH),

2.06 (m, 2Η), 2.44 (dG=7.2Hz), (CH₃)₂CHCH₂), 3.68 (q(j=7.1Ηz),

CH3CHCO2), 4.03 (tG=6.7Ηz), CO₂CH₂), 4.08 (s, C0₂CΗ₃), 5.03

(t(j=7.6Hz), CHzN"^"), 7.08 (dG=8.0Hz), Ar-2H), 7.19 (dG=8.0Hz), Ar-2H),

8.37 (ddG=6.1&8.0Hz), pyr-H₅), 8.97 (dG=8.0Hz), pyr-H₄), 9.37 (bs,

pyr-H₂), 9.93 (dG=6.1Hz), pyr-H₆) ppm. Synthesis of 7-Bromo-heptylamine: Bromo-heptanenitrile (1.50

gram, 7.89 mmol) in dry THF (40 ml) were cooled (ice-water bath) and

stirred under nitrogen atmosphere. A borane solution in THF (1 M BH₃.

THF, 20ml) was gradually added and the reaction mixture was stirred at

room temperature overnight. After cooling the reaction mixture (ice- water

bath), a solution of IN HC1 was added to achieve a pH value of 1-2 and

cease the hydrogen evolution. A solution of 10 % concentrated potassium

carbonate solution was thereafter added to the reaction mixture (to achieve

an alkaline Ph). Extraction with ether (3 x 35 ml), washing the combined

ether extracts with concentrated potassium carbonate solution, drying over

K₂C0₃ and evaporation of the solvent gave crude 7-bromo-heptylamine.

1H-NMR (CDC1₃): δ = 1.20-1.76 (m, 8H), 1.86 (m, CH₂CH₂Br),

2.72 (tG=7.1Hz), -CH₂NH₂)₅ 3.41 (tQ=6.8Hz), CH₂Br) ppm.

Synthesis of Ibuprofen 7-bromohepty amide:

7-Bromo-heptylamine was prepared as described hereinabove above and

was immediately thereafter dissolved in dry dichloromethane (50 ml).

Triethylamine (2.1 gram, 20.79 mmol) was added and the reaction mixture

was cooled (water-ice bath) and stirred. 2-(4-Isobutyl-phenyl)-propionyl

chloride (the acid chloride of ibuprofen) (2.5 gram, 11.13 mmol) was then

added and the reaction mixture was stirred at room temperature for one day.

Addition of ether (200 ml), washing with concentrated potassium carbonate

solution, IN HC1 and water, drying the organic phase over MgS0₄ and

evaporation of the solvent gave a crude oil. Purification by chromatography on a silica gel column, using mixtures of ether/hexane with

increased ether concentrations followed by a mixture of 30 %/70 %

ether/hexane gave 1.50 gram (49.7 % yield) of pure

ibuprofen-7-bromoheptyl amide as an oil, which crystallized upon standing

(m.p. = 39-40.5 °C.

1H-NMR (CDC1₃): δ = 0.91 (dG=6.6Hz), (CH₃)₂CH-], 1.16-1.31 (m,

2(-CH₂-), 4H), 1.34-1.47 (m, 2(-CH₂-), 4H), 1.52 (dG=7.2Hz), CH₃CHC0₂,

3H), 1.82 (m, CH₂CH₂Br, 2H), 1.87 (m, (CH₃)₂CH, 1Η), 2.47 (dG=7.2Ηz),

(CH₃)₂CHCH₂, 2Η), 3.18 (m, CONHCH₂), 3.39 (tG=6.8Ηz), CH₂Br, 2H),

3.53 (qG=7.2Hz), CH₃CHCON), 5.28 (brs, CONΗ), 7.13 (dG=8.1Ηz), 2

Ar-H), 7.20 (dG=8.1Hz), 2 Ar-H) ppm.

MS(EI) m/z 381 and 383 (M^^" very small); 302(M -Br, 100%).

Synth esis of 3-Dimethylcarbamoyl-l-{7-[2-(4~isob utyl-ph enyl)

-propionylaminoj-heptylj-pyridinium bromide) (IBU-Heptylamide-PYR,

IBU-Am-PH): A solution of ibuprofen-7-bromoheptyl amide (0.216 gram,

0.565 mmol) and 3-(N,N-dimethylcarbamoyl)pyridine (0.306 gram, 1.843

mmol), in 2-methoxy ethanol (5.0 ml), was refluxed overnight. Evaporation

of the solvent and purification by silica gel column chromatography, using a

mixture of 9 %/91 % methanol/ chloroform gave 0.190 gram of the amide

as an oil (61.4 % yield).

1H-NMR (CDC1₃): δ = 0.89 (dG=6.6Hz), (CH₃)₂C, 6H), 1.15-1.53

(m, (CH₂)₄), 1.49 (dG=7.2Hz), CH3CHCON), 1.83 (sep, (CH₃)₂CH), 2.04

(m, 2Η), 2.43 (d(j=7.1Hz), (CH₃)₂CHCH₂), 3.05 (s, (CΗ₃)₂NCO), 3.18 (s, (CH₃)₂NCO), 3.09-3.30 (m, CONCH₂), 3.63 (qG=7.1Hz), CH₃CHC0₂),

4.97 (tG=7.6Ηz), CHzN^1"), 6.00 (t, CONH), 7.08 (dG=8.0Hz), 2 Ar-H), 7.24

(dG=8.0Hz), 2 Ar-H), 8.06 (ddG=6.0,8.7Hz), 1 pyr-H), 8.28 (dG=8.7Hz), 1

pyr-H), 9.38 (s, 1 pyr-H), 9.49 (dG=6.0Hz), 1 pyr-H) ppm.

MS m/z 468.

The following equations (Scheme V) further illustrate the syntheses

of the final products INDO-PO, IBU-PO, IBU-4H-PO, (S)-IBU-PO,

NAPRO-PO, DICLO-PO, ASP-PO, IBU-OCT-Cytisine,

IBU-OCT-Arecoline (IOA), IBU-OCT-methylnicotinate (IOMN),

IBU-Heptylamide-Pyr (IBU-am-PH), and the various intermediates thereof:

Scheme V

Indomethacin acid chloride

Indomethacin bromooctyl ester

N-(Indomethacin octanoate) 3-(N,N-dimethylcarbamoyl) pyridinium bromide

(INDO-PO)

Ibuprofen acid chloride

Ibuprofen bromooctyl ester

N-(Ibuprofen octanoate) 3-N,N-dimethylcarbamoyl pyridinium bromide

(IBU-PO)

o

(S)-IBU-PO

Naproxen acid chloride

Naproxen bromooctyl ester

N-(Naproxen octanoate) 3-(N,N-dimethylcarbamoyl) pyridinium bromide

(NAPRO-PO)

O

Diclofenac bromooctyl ester

N-(Diclofenac octanoate) 3-(N,N-dimethylcarbamoyl) pyridinium bromide

(DICLO-PO)

3-(N,N-Dimethylcarbamoyl)-l-(8-hydroxy-octyl) pyridinium bromide

N,N-Dimethylcarbamic acid l-(8-hydroxy-octyl)-l,2, 5, 6-tetrahydro

-pyridine-3-yl ester

-(4-Isobutyl-phenyl)-propionic acid 8-(3-N,N-dimethylcarbamoyl

-3,6-dihydro-2H-pyridine-l-yl)octyl ester (IBU-4H-PO)

l-[8-(2-Acetoxy-benzoyl)-octyl]-3-N,N-dimethylcarbamoyl

pyridinium bromide (ASP-PO)

- HCI

Synthesis of N-(8-Hydroxyoctyl)-cytisine

Ibuprofen N-octyl-cytisine ester (IBU-OCT-CYTISINE)

l-(8-Hydroxy-octyl)-3-methoxycarbamoyl-pyridinium bromide (or iodide)

l-(8-Hydroxy-octyl)-l,2,5,6-tetrahydro-pyridine~3-carboxylic acid methyl

ester

l-{8-[2-(4-Isobutyl-phenyl)-propionyl]-octyl}-l,2,5,6-tetrahydro-pyridine-3-

carboxylic acid methyl ester (IBU-OCT-Arecoline, IOA)

l-{8-[2-(4-isobutyl-phenyl)-propionyl]-octyl}-3-methoxycarbonyl-pyridinium

iodide (IBU-N-octyl-methylnicotinate, IOMN)

7-Bromo-heptylamine

Br(CH₂)₆CN + BH₃- THF ^THF >. Br(CH₂)₆CH₂NH₂

Ibuprofen-7-bromoheptyl amide

3-Dimethylcarbamoyl-l-{7-[2-(4-isobutyl-phenyl)-propionylamino]- heptylj-pyridinium bromide (IBU-Am-PH)

Activity Assays:

Inhibition of ChEs in vitro and in vivo: Purified recombinant

human acetylcholineesterase (rHuAChE, 20 μl of 1.5 U/ml), fetal bovine

serum- AChE (FBS-AChE 20μl of 5U/ml)) or purified human plasma

butyrylcholinesterase (BChE) (HuBChE, 5U/ml) was incubated in the

presence of fixed concentrations (ranging from 0.01 μM to 0.6 μM) of

NSAID-PYR-X compounds, IOA and IOMN at 25 °C. Aliquots of such

enzyme/inhibitor solution (20 μl) were transferred at specified time

intervals into a cuvette containing 1.5 mM acetylthiocholine (ATCh, Sigma,

USA) and 1.5 mM DTNB (Ellman reagent 29, Sigma, USA) in 1 ml

phosphate buffer (50 mM, pH 7.4). The residual activity of AChE was

measured by following the rate of increase in OD at 412 nm using a GARY

3 double-beam spectrophotometer (Varian, USA) (24).

In vivo inhibition of whole blood ChE in mice was measured by

injecting the tested compounds to the animals and then sampling a 10 μl

blood with a glass capillary from the eye orbit vein. The blood sample was

transferred immediately thereafter into a cold water solution (0.09 ml, kept

at 2-4 °C). The blood ChE activity was measured by transferring a 20 μl

diluted blood sample into a 1 ml cuvette containing DTNB and ATCh in 1

ml phosphate buffer (50mM, pH 7.4).

In vivo toxicity and Therapeutic Index: NSAID-PYR-X

compounds were dissolved in 1:3 propylene glycohwater solution and were injected intramuscularly (0.1 ml per 25 grams) or intraperitoneally (0.2 ml

per 25 grams) to mice. Toxic signs and mortality rates were observed

during 24 hours. The LD₅₀ values were calculated according to Spearman

Kerber method (25).

The Therapeutic Index (TI) for ChE inhibitors was calculated

according to the following equation:

TI = LD₅o/ΕD₅o

Wherein:

LD₅₀ is the dose causing lethality to 50 % of the animals: and

ED₅₀ is defined as the dose causing 50 % ChE inhibition in blood.

Lipophilicity: A solution of 0.1 mM PYR-X, NSAID-PYR-X

compound, IOA or IOMN in 1 ml n-octanol was prepared and placed in a

plastic test tube. 1 ml phosphate buffer (50 mM, pH 7.4) was thereafter

added and the mixture was stirred extensively by vortex for 2-3 minutes at

25 °C. Phase separation at 2000 RPM for 10 minutes, was followed by

transferring the upper n-octanol solution into a fused silica cuvettes and

reading its OD at 272 or 333 nm. The selected wavelength was the λ_max of

absorption according to the UV-visible spectra of a certain compound in

n-octanol. The compound's concentration in n-octanol was determined by

interpolation of the OD at λ_max, using a calibration curve for the specific

compound in n-octanol. The partition coefficient (k) was calculated

according to the following equation:

k = C₀/C_b, wherein,

C₀ is the concentration of the compound in n-octanol

C_b is the concentration of the compound in phosphate buffer which

was obtained by subtracting the observed C₀ from the initial known

concentration of the compound in n-octanol.

Inhibition of COX activity in vitro: The activity of cyclooxygenase

I and II (COX I and COX II, Cayman Chemicals (MI, USA)) and the

inhibition thereof by NSAIDs and NSAID-PYR-X compounds were

measured by an immunoassay measuring prostaglandin E₂ (PGE₂)

displacement. PGE₂ was produced in vitro from arachidonic acid (AA) by

purified COX I or II. The assay was based on a competition binding

technique in which PGE₂ competes with a fixed amount of alkaline

phosphatase-labeled PGE₂ for sites of a mouse monoclonal antibody.

During incubation, the mouse monoclonal antibody becomes bound to the

goat anti-mouse antibody coated onto a microplate (R&D Systems, MN,

USA). Following wash to remove excess conjugate and unbound sample,

an alkaline phosphatase substrate solution was added to the wells to

determine bound enzyme activity. Immediately following color

development, absorbance at 405 nm was determined. The intensity of the

color is inversely proportional to the concentration of PGE₂. The

production of PGE₂ levels at the specified incubation time with COX I or

COX II served as a quantitative assay for COX activity. Inhibition of COX activity was determined in the presence of various NSAID and

NSAID-PYR-X compounds.

Peripheral anti-inflammatory activity: The anti-inflammatory

activity of NSAID-PYR-X and NSAID compounds was evaluated by using

the rat paw edema experimental model (26). Carrageenan (CAR, Sigma)

solution (1 % in saline) was intramuscularly injected into the rat hind paw,

and the edema volume was measured quantitatively by using a

plethysmometer (model 7140, Ugo Basile, Italy) attached to an electronic

block reader (model 7141). A plethysmometer is a volume meter designed

for accurate measurement of rat paw swelling. It consists of a water filled

Perspex cell (2.5 cm in diameter) into which the rat paw is dipped up to a

marked sign. The small difference in water level caused by volume

displacement is recorded by a transducer coupled to a LCD read-out which

shows the exact paw volume (control or treated). The maximal edema

volume was obtained 2 hours after CAR injection. The efficacy of

NSAID-PYR-X compounds against CAR-induced edema was measured by

intraperitoneal injection of a NSAID-PYR-X compound 30 minutes prior to

the CAR injection, followed by measurement of the edema volume 2 hours

after the CAR injection. The level of edema (% EDEMA) was calculated

according to the following equation:

% EDEMA = (V N₀ - 1) x 100, wherein:

V₀ = Pretreated rat paw volume (ml) (measurements which were

taken before CAR injection); and V_t = Treated rat paw volume (ml) (measurements which were taken 2

hours after CAR injection).

Anti-inflammatory activity in the brain:

Studies in mice: Male albino mice (25-30 grams) were injected with

either 5 or 10 μl solution of either 1 % CAR or saline solution, through the

skull, into the left lateral ventricle, using a syringe equipped with a stainless

steel needle that is 3 mm longer than a plastic spacer. The exact location

for the injection into the left lateral ventricle was determined by measuring

the specific coordinates using anatomical atlas of mouse brain and validated

histologically by injection of 1 % solution of Evans Blue dye in saline.

Intraperitoneal NSAID-PYR-X treatment (5 or 10 mg/kg at 5 ml/kg) was

applied 30 minutes before CAR injection. The mice were sacrificed 4 hours

after the injection and their brains were isolated, dissected into four parts

hemispheres and weighed immediately. The brain tissue was placed in a

drying oven (140 °C) for 24 hours and weighed thereafter. The increase in

brain water content due to CAR-induced inflammation, with and without

treatment, was obtained by the following equation:

% Water Content = [(W₀ - W₂₄)/W₀ ] x 100, wherein:

Wo = the weight before drying; and

W₂ = the weight after drying for 24 hours.

These values served as a quantitative measure for brain edema

induced by CAR injection. The decrease in the brain edema level after systemic intraperitoneal injection of an NSAID-PYR-X compound reflected

its efficacy as an anti-inflammatory agent (27).

Studies in rats: Male Sprague-Dawley rats (300-420 grams) were

anesthetized with equithisine solution and placed in a small animal

stereotaxic instrument (Schuleler Co. Inc., NY, USA). The skull bone was

exposed and a fixed cannule (Guide and Internal Cannule 6 mm, Bilaney,

USA) was implanted into the left lateral ventricle. Solutions of either 1- %

CAR or saline were injected through an internal cannule at a rate of

lμl/minute using an electric pump (CMA 100 Microiηjection Pump -

Carnegie Medicin Stockholm, Sweden). NSAID-PYR-X treatment was

applied intraperitoneally prior to the injection of CAR or saline, and the

measurements were proceeded as described hereinabove.

Hypothermic effect: Male albino mice (25-35 grams) were placed in

a constant temperature environment (22±1 °C). The mice were

intraperitoneally injected either with 1:3 propylenglycol/water (PG:DDW)

which served as a vehicle (carrier) or with a solution of IBU-PO dissolved

in the 1:3 propylenglycol/water vehicle. The animals' rectal temperature

was measured at specified time intervals after administration, using a

bimetal rod thermometer coupled to Physitemp model BAT- 12 reader

(Physitemp Instruments, NJ, USA). In order to delineate the

pharmacological mechanism of IBU-PO-induced hypothermia, either

atropine (5mg/kg) or mecamylamine (2 mg/kg) were injected subcotenously 30 minutes before the IBU-PO injection, and the rectal temperature was

monitored during the same specified time intervals.

Protection against closed head injury: Mice were subjected to a

head injury which was caused by a stainless steel rod weighing 333 grams

dropped from a height of 2 or 3 cm, and were intraperitoneally injected with

various doses of PO, IBU-PO and NAPRO-PO (5, 7.5 and 10 mg/kg), 15

minutes thereafter. Clinical assessment of the animals was performed 1

hour post the head trauma using a neurological severity score (NSS) (28).

Animals were examined by using specified motor tests wherein each failure

in a particular test adds one point, while untreated animal has a score of 0.

The NSS was determined again after 24 hours and the difference in NSS

(ΔNSS) was calculated according to the following equation:

ΔNSS = NSS(t=lh) - NSS(t=24h)

Following the neurological score assessment, the mice were

sacrificed and the affected hemispheres were weighed immediately

thereafter and further after drying in oven for 24 hours at 140 °C. The

percentage of brain water content was calculated according to:

% Water Content = [(W₀ - W₂₄)/W₀] x 100

wherein,

W₀ = the weight before drying; and

W₂₄ = the weight after drying for 24 hours.

Protection against hypobaric hypoxia: Male albino mice (25-35

grams, n = 4) were measured for their rectal temperature and were intraperitoneally injected thereafter either with 1 :3 PG:DDW (vehicle) or

with an IBU-PO dissolved in 1:3 PG:DDW. 30 minutes after the injection,

rectal temperatures were measured for the indication of the drug effect, and

the mice were placed in a glass dessicator at a pressure of 200 mm Hg for

15 minutes (29, 30) during which time of mice mortality was determined.

The protection ratio (PR) of IBU-PO against hypobaric hypoxia was

calculated using the following equation:

PR = t_D/t_v

wherein,

t_D is the time of death following drug treatment; and

t_v is time of death following injection with the vehicle.

Binding to rat brain muscarinic receptors: Rat brain homogenates

were prepared according to a known procedure (31) and were stored at -70

°C until use. Competition binding of ligands to brain receptor was

performed with specified concentrations of NSAID-PYR-X ligands in the

presence of a fixed concentration of the following radioactive muscarinic

antagonists: [³H]N-methylscopolamine (NMS) (for all muscarinic receptor

subtypes), [ H]pirenzepine (selective for Ml receptors) and [ H]AFDX

(selective for M2 receptors). The NSAID-PYR-X Ligands were incubated

in the presence of the radioactive agonists and rat brain homogenate

suspension (0.1 mg/ml protein in 50 mM Na/K phosphate buffer, pH 7.4)

for 2 hours at 25 °C. Cholinergic muscarinic physiological response: Isolated guinea pig

ileum was placed in a 5 ml glass bath filled with physiological Ringer

solution (pH 7.4) kept at 37 °C by Haake thermostat. A constant stream of

95 %/5 % 0₂/C0₂ was bubbled into the solution during the physiological

measurement. Muscle contraction was induced by 0.5 μM carbamylcholine

(CCh) and specified concentrations of NSAID-PYR-X were added

thereafter. The antagonist effect of the tested compounds was determined

by the decrease in CCh-induced muscle contraction which was caused by

the addition of the tested compounds into the bath (32).

Stability of compounds in human plasma in vitro: NSAID-PYR-X

compound IBU-PO (0.1 mM) was dissolved in 0.75 ml human plasma and

incubated for specified time intervals at 37 °C. The plasma solutions were

separated thereafter by centrifugation (SA600 head, refrigerated Sorval

centrifuge) at 15,000 rpm for 3 hours, using a Fugisep filtration membrane

tubes (4 ml) with a cutoff number of 10 kDa. Water was then evaporated to

dryness using a SpeedVac heated centrifuge under vacuum, and the residual

oily material was treated with 1 :1 acetonitrile: water solution and filtered

through a membrane filter (Whatman, PURADISC 25AS 0.45 μ filters). 50

μl of the filtered solutions were injected into HPLC column (RP-18 Merck,

125 x 4 mm) using isocratic carrier mixture of 75 %/25 %

acetonitrile/phosphate buffer (5 mM, pH 7.4) containing 0.35 mM

tetramethyl ammonium chloride. Quantitative analysis of the degradation

product of IBU-PO, PO-OH, was performed using pyridostigmine as an internal standard. Electrospray Mass Spectrometric (ESMS) analysis was

performed on parallel samples that were prepared similarly to the

HPLC-injected samples.

EXPERIMENTAL RESULTS

Inhibition kinetics of AChE and BChE in vitro: The kinetic

parameters of the inhibition of purified rHuAChE, FBS-AChE and

HuBChE by various NSAID-PYR-X compounds were measured and were

further compared with the measured kinetic parameters of ChEs' inhibition

by the known acetylcholine up regulators pyridostigmine (PYR) and

Octyl-pyridostigmine (PO), and by the plasma hydrolysis product of

IBU-PO (PO-OH) which was identified by ESMS analysis.

Table 1 below presents the inhibition kinetic parameters measured

for IBU-PO, S-(+)-IBU-PO, IBU-am-PH, INDO-PO, NAPRO-PO, ASP-PO

and DICLO-PO, as well as for PO-OH, PYR and PO. All NSAID-PYR-X

compounds demonstrated inhibition of AChE and BChE with bimolecular

rate constants (k_;) ranging from 5.4 x 10 to 2.8 x 10⁶ M^{"1 '} minute^"1. These

rate constants are comparable with the values obtained with known ChEIs

(e.g. PYR and PO). Moreover, the values obtained for the dissociation

constants (K_τ) indicate that the effective concentrations of most

NSAID-PYR-X compounds range from 1.6 x 10^"7 to 1.6 x 10^"5 M and are

thus similar to the values obtained for PYR. The value obtained for the

optical isomer S-(+)-IBU-PO was, as predicted, significantly higher (2.2 x

10^"4), while the value obtained for the chimeric compound that include an amide bond, IBU-am-PH, was somewhat lower. Nevertheless, these results

suggest that the introduction of the NSAID moiety into the chimeric

compound did not affect the potency of the pyridostigmine moiety toward

the inhibition of AChE and/or BChE. Furthermore, the rate constants of the

ChE's inhibition obtained by the plasma hydrolysis product PO-OH are

comparable to those obtained by its parent chimer IBU-PO. These data

demonstrate an inhibitory activity of both the prodrug (IBU-PO) and the

released drug form (PO-OH).

Table 1

Table 1 Continued.

Figure 1 demonstrates the time-course of inhibition of FBS-AChE

and HuAChE by various concentrations of DICLO-PO. Inhibition kinetics

is similar to that obtained with other known carbamate derivatives,

approaching a steady-state at time periods that depend on the carbamate

derivative concentration.

The kinetic measurements performed with arecoline, methyl

nicotinate (MN), and the chimeric compounds IOA and IOMN, surprisingly

showed that these compounds demonstrate reversible inhibition of AChE.

Table 2 below presents the IC₅₀ values obtained for AChE inhibition

in the presence of these compounds. The data show that the chimeric

compound IOMN demonstrates reversible inhibition of AChE with an IC₅₀

value that is comparable to the Ki value obtained with pyridostigmine (see

Table 1 hereinabove), whereas both methyl nicotinate (MN) and Arecoline

demonstrate much lower activity as AChE inhibitors. However, the IC₅₀

value obtained with the chimeric compound IOA shows that the reversible

AChE inhibition activity thereof is one order of magnitude lower than that

obtained with IOMN. Table 2

Inhibition kinetics of AChE and BChE in vivo: Figure 2 shows the

time-course of whole blood ChE inhibition following the injection of PYR,

PO or IBU-PO in mice. Using doses of 4 or 10 mg/kg of PO or IBU-PO

resulted in 30-50 % ChE inhibition, for a period of at least 5 hours. In

particular, intraperitoneal injection of 4 mg/kg IBU-PO resulted in 50 %

inhibition of blood ChE within 30 minutes, with a duration time of several

hours. In contrast, using 0.13 mg/kg PYR resulted in less than 20 % ChE

inhibition, with a much shorter duration time (1-1.5 hour) than that

observed for IBU-PO. These data indicate that the chimeric compound

IBU-PO demonstrates both longer duration of action in vivo and higher

Therapeutic Index (as is described herein below), as is compared with PYR.

In vivo toxicity and therapeutic index: Table 3 below presents the

LD₅₀ values obtained for PYR, PO, NSAIDs, NSAID-PYR-X compounds

and IOMN in mice. All the NSAID-PYR-X compounds are 10-50 fold less

toxic than PYR, having LD₅₀ values that range from 22.5 to 57.4 mg/kg of

intramuscular injection. The least toxic NSAID-PYR-X compounds are

IBU-PO and DICLO-PO, having LD₅₀ values of 57.4 and 53.4 mg/kg (intramuscular) and 61.2 and 53.4 mg/kg (intraperitoneal), respectively.

Thus, further studies were pursued mainly with IBU-PO, due to its lower

toxicity and outstanding activity in some of the pharmacological models

described below. However, the chimeric compound of IBU and methyl

nicotinate, IOMN, was found to be even less toxic than these

NSAID-PYR-X compounds, having a LD₅₀ value greater than 100 mg/Kg

(intraperitoneal).

Table 3

Table 3 Continued.

Furthermore, as is shown in Figure 3, intraperitoneal injection of 10

mg/kg of IBU-PO and DICLO-PO did not cause any detrimental effect on

the mucosal tissue of rat stomach. It is well established in the prior art that

NSAID such as ibuprofen and indomethacin cause erosions and bleeding

ulcers in the gastrointestinal system. Thus, these observations are

consistent with the notion that the esterification of the carboxylic acid group

in NSAID's markedly reduces the gastrointestinal side effects thereof (33).

The Therapeutic Index (TI) is defined by the following equation:

The TI calculated for IBU-PO in mice is 15.3 (=61.2/4). This value

is significantly higher than TIs calculated for the known ChE inhibitors

physostigmine, TACRINE and EXELON, which are 3.4, 3.5 and 12.5,

respectively. Lipophilicity: The lipophilicity of the compounds of the present

invention was measured by phase partition experiments (n-octanol and

phosphate buffer). Table 4 below presents the partition coefficient (k_p)

values between n-octanol and phosphate buffer of PO, PO-OH,

NSAID-PYR-X compounds, IOMN and IOA. The k_p values obtained for

IBU-PO, IBU-4H-PO, NAPRO-PO and INDO-PO are significantly higher

than those obtained for PO and PO-OH. Furthermore, the K_p value obtained

for the tertiary amine form IBU-4H-PO is much higher than its quaternary

congener IBU-PO. However, it was unexpectedly found that the K_p value

obtained for the quaternary ammonium IOMN (K_p = 28.4, see Table 4) is

much higher than the value obtained for its tertiary amine derivative IOA

(K_p = 4).

Table 4

Inhibition of cyclooxygenase (COX) isoenzymes in vitro: The

inhibition of constitutive cyclooxygenase (COX I) and its inflammation

inducible isoenzyme COX II by NSAIDs and NSAID-PYR-X chimeric

compounds was measured using a quantitative competitive PGE₂ immunoassay. Purified COX I (bovine seminal vesicles) and COX II

(sheep placenta) were used for the assay. Table 5 below presents the

inhibition level of COX I and COX II obtained with 1 μM of NSAIDs and

NSAID-PYR-X compounds. The data are consistent with the prior art as to

the selectivity of ibuprofen, diclofenac and, to some extent, indomethacin

toward COX II. The chimeric compounds IBU-PO and INDO-PO further

demonstrate some selectivity toward COX II as compared to COX I. The

data further show that NSAID-PYR-X chimera are effective COX I and

COX II inhibitors at concentrations that are equivalent to those of NSAIDs

(about 1 μM). Moreover, these concentrations are similar to the Ki values

of AChE and BChE inhibition accepted for the chimeric compounds as is

presented in Table 1 hereinabove.

Table 5

Peripheral anti-inflammatory activity: The effect of NSAIDs and

NSAID-PYR-X compounds on CAR-induced rat paw edema was evaluated

by intraperitoneally injecting the compounds 30 minutes before CAR and

measuring the change in paw volume 2 hours after CAR injection.

Figure 4 shows the effect of the NSAIDs IBU and INDO, and the

chimeric compounds IBU-PO and INDO-PO on carrageenan

(CAR)-induced rat paw edema, as well as the effect of injecting the vehicle

(1:3 PG/DDW) only. The results demonstrate that injecting 5 mg/kg of all

compounds resulted in the same significant decrease in edema level (to

about 30 %) as compared to injecting the vehicle only, thus indicating that

the new NSAID-PYR-X chimeric compounds display peripheral

anti-inflammatory activity that is comparable to that of clinically used

NSAIDs. Furthermore, it should be noted that the chimeric compounds

were injected together with a carrier which by itself induce larger edema

then CAR without any treatment (73 vs. 59 %, respectively). It is therefore

concluded that these chimeric compounds display higher anti-inflammatory

activity than their corresponding NSAID compounds.

Anti-inflammatory activity in brain: The effect of IBU and IBU-PO

on CAR-induced brain edema in rats, was measured following the injections

thereof 10 minutes prior to intra cerebral ventricle (icv) (left lateral

ventricle) injection of CAR (1 % solution in saline).

Figure 5 demonstrates the effect of IBU and IBU-PO on the water

content in rats' brain. While pretreatment with IBU resulted in no change in the edema water content elicited by CAR (83 %), the pretreatment with

the chimeric compound IBU-PO reduces the % water content to 82 %

which is similar to that obtained after saline icv injection, indicating its

capability to cross the BBB and act as an anti-inflammatory drug.

Figure 6 demonstrates the effect of IBU-PO on CAR-induced brain

edema in mice. Intraperitoneal injection of 10 mg/kg IBU-PO significantly

reduces the brain water content in mice, thus indicating its efficacy as a

central anti-inflammatory drug in both mice and rats.

Hypothermic effect: Figure 7 shows the time-course of hypothermia

induced by IBU-PO in mice. Intraperitoneal injection of 2.5 mg/kg IBU-PO

resulted in an IBU-PO-induced hypothermic effect which is maximal at 30

minutes and persists up to at least 6 hours following injection. Pretreatment

with 5 mg/kg atropine applied subcotenously partially reverses the

hypothermia, whereas pretreatment with 2 mg/kg of the nicotinic antagonist

mecamylamine applied subcotenously cause a significant delay in the

maximal hypothermia induced by IBU-PO from 30 to 60 minutes.

Protection against closed head injury: The neuroprotective activity

of the novel chimeric compounds was evaluated using a closed head injury

model in mice (28), which were injected with various doses of PO, IBU-PO

and NAPRO-PO 15 minutes after subjection to a head injury.

Table 6 below presents the percent water content and difference in

neurological severity score (ΔNSS) of the animals with and without

treatment, compared with sham animals with no head injury. As a rule, larger ΔNSS value represents higher drug efficacy against the damage

induced by the closed head injury.

Figure 8 demonstrates the effect of NSAID-PYR-X compounds on

the edema level caused by a head injury.

The values presented in Table 6 and Figure 8 show the high efficacy

demonstrated by IBU-PO (water (%): 80.94, 81.45 and 80.91 and ΔNSS:

1.25, 2.0 and 2.5) as compared to the known ChEI derivative PO (water

(%): 81.17, 82.77 and 83.43 and ΔNSS: 1.25, 1.5 and 1.4) at doses of 5, 7.5

and 10 mg/kg, respectively. The water content and ΔNSS values obtained

for IBU-PO are comparable to those obtained for the most efficacious

neuroprotective compounds tested so far in this model (e.g., TEMPOL and

HU-211). NAPRO-PO demonstrates a moderate protective activity against

head trauma (water (%): 81.38, 82.09 and 81.38 and ΔNSS: 2.0, 1.52 and

2.25) at the same respective doses as mentioned above. Nevertheless, its

efficacy is still higher then PO.

Table 6

Table 6 Continued

p<0.05 vs vehicle treated (PG:DDW, 1 :3); ^# p=0.058 vs vehicle treated

Protection against hypobaric hypoxia: The neuroprotective,

anti-ischemic and anxiolytic activity of the chimeric compounds of the

invention was evaluated using the hypobaric hypoxia model in mice (29,

30), by measuring their effect on survival time of mice during hypobaric

hypoxia.

Figure 9 demonstrates the effect of injecting various doses of

IBU-PO to mice, prior to induction of hypobaric hypoxia, compared with a

control group injected with a vehicle only. The protection of IBU-PO

against hypoxia is 7.5-9 fold larger than that of the vehicle control (100

seconds). Moreover, some of the mice treated with IBU-PO survived even

the maximal hypoxia period of 15 minutes.

Figure 10 shows, in comparison, the effect of other known ChE

inhibitors, e.g., physostigmine (0.15 mg/kg injected intramuscularly),

huperzine-A (0.2 mg/kg injected intramuscularly) PO (5 mg/kg injected intraperitoneally) and ibuprofen (0.10 mg/kg injected intraperitoneally) on

hypoxia in mice. The only ChEI that provides good protection against hypoxia

is physostigmine, suggesting that ChE inhibition by itself does not necessarily

provide protection against hypoxia. Hypothermic effect of 2-3 °C was

observed after injection of IBU-PO and physostigmine. However, it was

previously observed that the anti-hypoxia effect induced by ChE inhibitors and

cholinergic agonists is not attributed solely to hypothermia but rather to direct

neuronal effect (34).

Binding to brain cholinergic receptors: The novel NSAID-PYR-X

chimeric compounds were tested for their interaction with cholinergic

muscarinic receptor subtypes by measuring their competitive displacement

of radioactive muscarinic ligands from rat brain in vitro.

Figure 11 demonstrates the binding curves for the displacement of

[³H]NMS from rat brain by NSAID-PYR-X compounds.

Table 7 below presents the IC₅₀ and Ki values obtained for IBU-PO,

INDO-PO, DICLO-PO and NAPRO-PO. The Ki values range from 1.0 x

10^"6 to 6.9 x 10^"6 M, whereas the effective concentrations obtained for the

interaction of the NSAID-PYR-X chimeric compounds with the muscarinic

receptors are similar to those received for ChE and COX inhibition, as

described hereinabove.

Table 8 below presents the binding parameters of IBU-PO to Ml and

M2 muscarinic receptors, using [ H pirenzepine and [ HJAFDX-346 as the

specific labeled ligands, as well as [³H]MK-801 for NMDA receptors. The Ki values obtained for IBU-PO from the displacement of [³H]pirenzepine

and [³H]AFDX-346 are 5.1xl0^"7 and 4.4xl0^"7 M, respectively, indicating

that IBU-PO binds at the same affinity to both Ml and M2 muscarinic

receptor subtypes. IBU-PO demonstrates lower affinity toward NMDA

receptors as evidenced from the competition with tritiated MK-801 (Ki =

4.3xl0^"5 M).

Figure 12 shows all respective binding curves for IBU-PO with the

various radioactive receptor ligands in rat brain.

Table 7

K_D [ H]NMS = 0.2 nM, dissociation constant of radioactive ligand.

L [³H]NMS = 0.48 nM, concentration of radioactive ligand.

Table 8

Stability in human plasma in vitro: The rate of hydrolysis of

NSAID-PYR-X chimeric compounds in human plasma was evaluated by

incubating IBU-PO in human plasma at 37 °C, in vitro, for various time

intervals, in the presence of pyridostigmine as an internal standard.

The estimated half-life time (t]_/2) for the hydrolysis of IBU-PO in

plasma at 37 °C, based on HPLC analysis, is 4.5-5 hours.

The degradation products of the hydrolysis of IBU-PO, at various

time intervals in plasma, are PO-OH and ibuprofen, as was determined by

quantitative electrospray mass spectrometry (ESMS) analysis, using

pyridostigmine as an internal standard.

The ESMS spectra obtained for the degradation products show

unequivocally that the dimethylcarbamoyl moiety in the ChEI derivative

remains attached to the pyridine ring during hydrolysis of the chimeric

compound in plasma.

It is appreciated that certain features of the invention, which

are, for clarity, described in the context of separate embodiments, may also

be provided in combination in a single embodiment. Conversely, various

features of the invention, which are, for brevity, described in the context of

a single embodiment, may also be provided separately or in any suitable

subcombination. Although the invention has been described in conjunction with

specific embodiments thereof, it is evident that many alternatives,

modifications and variations will be apparent to those skilled in the art.

Accordingly, it is intended to embrace all such alternatives, modifications

and variations that fall within the spirit and broad scope of the appended

claims. All publications, patents and patent applications mentioned in this

specification are herein incorporated in their entirety by reference into the

specification, to the same extent as if each individual publication, patent or

patent application was specifically and individually indicated to be

incorporated herein by reference. In addition, citation or identification of

any reference in this application shall not be construed as an admission that

such reference is available as prior art to the present invention.

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Claims (133)

WHAT IS CLAIMED IS:

1. A chimeric compound comprising a cholinergic up-regulator

moiety and a non-steroidal anti-inflammatory moiety being covalently

linked thereto.

2. The chimeric compound of claim 1, wherein said cholinergic

up-regulator moiety and said non-steroidal anti-inflammatory moiety are

covalently linked via a hydrocarbon spacer.

3. The chimeric compound of claim 2, wherein said non-steroidal

anti-inflammatory moiety is covalently attached to said spacer via a

-C(=X)Y- bond, where X is a non-substituted or substituted oxygen, sulfur

or nitrogen atom and Y is a substituted or non-substituted carbon, oxygen,

nitrogen, sulfur, silicon or phosphor atom linked to said C via a single

covalent bond.

4. The chimeric compound of claim 3, wherein said bond is

selected from the group consisting of an ester bond and an amide bond.

5. The chimeric compound of claim 4, wherein said ester bond is

selected from the group consisting of a carboxylic ester bond and a glycol

amide ester bond.

6. The chimeric compound of claim 3, wherein said bond is

hydrolizable by a brain derived esterase.

7. The chimeric compound of claim 3, wherein said bond is

hydrolizable by a brain derived amidase.

8. The chimeric compound of claim 2, wherein said hydrocarbon

spacer comprises at least one hydrocarbon selected from the group

consisting of an alkyl having 2-20 carbon atoms, a cycloalkyl having 3-20

carbon atoms and an aryl having 6-20 carbon atoms.

9. The chimeric compound of claim 1, wherein said cholinergic

up-regulator moiety is selected from the group consisting of a

cholinesterase inhibitor residue, a nicotinic receptor agonist residue and a

muscarinic receptor agonist residue.

10. The chimeric compound of claim 9, wherein said

cholinesterase inhibitor residue is a pyridostigmine residue.

11. The chimeric compound of claim 10, wherein said

pyridostigmine residue is a 3-N,N-dimethylcarbamoyl pyridinium bromide

residue.

12. The chimeric compound of claim 9, wherein said nicotinic

agonist residue is selected from the group consisting of a nicotine residue

and a cytisine residue.

13. The chimeric compound of claim 9, wherein said muscarinic

receptor agonist residue is selected from the group consisting of an

arecoline residue and a pilocarpine residue.

14. The chimeric compound of claim 1, wherein said

non-steroidal anti-inflammatory moiety comprises a residue of a

non-steroidal anti-inflammatory drug characterized by a functional group

selected from the group consisting of a free carboxylic acid group and a free

amine group.

15. The chimeric compound of claim 14, wherein said

non-steroidal anti-inflammatory moiety is selected from the group

consisting of an ibuprofen residue, an indomethacin residue, a naproxen

residue, a diclofenac residue and an aspirin residue.

16. The chimeric compound of claim 15, wherein said ibuprofen

residue is selected from the group consisting of an (±)-ibuprofen residue,

S-(+)-ibuprofen residue and R-(-)-ibuprofen residue.

17. The chimeric compound of claim 1, characterized by

lipophilicity sufficient for permitting the compound to cross a blood brain

barrier of an organism.

18. A chimeric compound of a general formula:

A-S-B

wherein:

A is a cholinergic up-regulator moiety selected from the group

consisting of a cholinesterase inhibitor residue, a nicotinic receptor agonist

residue and a muscarinic receptor agonist residue;

B is a non-steroidal anti-inflammatory moiety characterized by a

functional group selected from the group consisting of a free carboxylic

acid group and a free amine group; and

S is a hydrocarbon spacer being covalently linked to B via a

-C(=X)Y- bond, where X is a non-substituted or substituted oxygen, sulfur

or nitrogen atom and Y is a substituted or non-substituted carbon, oxygen,

nitrogen, sulfur, silicon or phosphor atom linked to said C via a single

covalent bond.

19. The chimeric compound of claim 18, wherein said

cholinesterase inhibitor residue is a pyridostigmine residue.

20. The chimeric compound of claim 19, wherein said

pyridostigmine residue is a 3-N,N-dimethylcarbamoyl pyridinium bromide

residue.

21. The chimeric compound of claim 18, wherein said nicotinic

agonist residue is selected from the group consisting of a nicotine residue

and a cytisine residue.

22. The chimeric compound of claim 18, wherein said muscarinic

receptor agonist residue is selected from the group consisting of an

arecoline residue and a pilocarpine residue.

23. The chimeric compound of claim 18, wherein said

non-steroidal anti-inflammatory moiety is selected from the group

consisting of an ibuprofen residue, an indomethacin residue, a naproxen

residue, a diclofenac residue and an aspirin residue.

24. The chimeric compound of claim 23, wherein said ibuprofen

residue is selected from the group consisting of an (±)-ibuprofen residue, a

S-(+)-ibuprofen residue and a R-(-)-ibuprofen.

25. The chimeric compound of claim 18, wherein said bond is

selected from the group consisting of an ester bond and an amide bond.

26. The chimeric compound of claim 25, wherein said ester bond

is selected from the group consisting of a carboxylic ester bond and a glycol

amide ester bond.

27. The chimeric compound of claim 18, wherein said

hydrocarbon spacer comprises at least one hydrocarbon selected from the

group consisting of an alkyl having 2 -20 carbon atoms, a cycloalkyl having

3-20 carbon atoms and aryl having 6-20 carbon atoms.

28. A pharmaceutical composition comprising, as an active

ingredient, the compound of claim 1, and a pharmaceutically acceptable

carrier.

29. The pharmaceutical composition of claim 28, wherein said

cholinergic up-regulator moiety and said non-steroidal anti-inflammatory

moiety are covalently linked via a hydrocarbon spacer.

30. The pharmaceutical composition of claim 29, wherein said

non-steroidal anti-inflammatory moiety is covalently attached to said spacer

via a -C(=X)Y- bond, where X is a non-substituted or substituted oxygen,

sulfur or nitrogen atom and Y is a substituted or non-substituted carbon,

oxygen, nitrogen, sulfur, silicon or phosphor atom linked to said C via a

single covalent bond.

31. The pharmaceutical composition of claim 30, wherein said

bond is selected from the group consisting of an ester bond and an amide

bond.

32. The pharmaceutical composition of claim 31, wherein said

ester bond is selected from the group consisting of a carboxylic ester bond

and a glycol amide ester bond.

33. The pharmaceutical composition of claim 30, wherein said

bond is hydrolizable by a brain derived esterase.

34. The pharmaceutical composition of claim 30, wherein said

bond is hydrolizable by a brain derived amidase.

35. The pharmaceutical composition of claim 29, wherein said

hydrocarbon spacer comprises at least one hydrocarbon selected from the

group consisting of an alkyl having 2-20 carbon atoms, a cycloalkyl having

3-20 carbon atoms and an aryl having 6-20 carbon atoms.

36. The pharmaceutical composition of claim 28, wherein said

cholinergic up-regulator moiety is selected from the group consisting of a

cholinesterase inhibitor residue, a nicotinic receptor agonist residue and a

muscarinic receptor agonist residue.

37. The pharmaceutical composition of claim 36, wherein said

cholinesterase inhibitor residue is a pyridostigmine residue.

38. The pharmaceutical composition of claim 37, wherein said

pyridostigmine residue is a 3-N,N-dimethylcarbamoyl pyridinium bromide

residue.

39. The pharmaceutical composition of claim "36, wherein said

nicotinic agonist residue is selected from the group consisting of a nicotine

residue and a cytisine residue.

40. The pharmaceutical composition of claim 36, wherein said

muscarinic receptor agonist residue is selected from the group consisting of

an arecoline residue and a pilocarpine residue.

41. The pharmaceutical composition of claim 28, wherein said

non-steroidal anti-inflammatory moiety comprises a residue of a

non-steroidal anti-inflammatory drug characterized by a functional group

selected from the group consisting of a free carboxylic acid group and a free

amine group.

42. The pharmaceutical composition of claim 41, wherein said

non-steroidal anti-inflammatory moiety is selected from the group consisting of an ibuprofen residue, an indomethacin residue, a naproxen

residue, a diclofenac residue and an aspirin residue.

43. The pharmaceutical composition of claim 42, wherein said

ibuprofen residue is selected from the group consisting of an (+)-ibuprofen

residue, S-(+)-ibuprofen residue and R-(-)-ibuprofen residue.

44. The pharmaceutical composition of claim 28, characterized by

lipophilicity sufficient for permitting the compound to cross a blood brain

barrier of an organism.

45. The pharmaceutical composition of claim 28, formulated for

transdermal delivery.

46. The pharmaceutical composition of claim 28, formulated for

nasal administration.

47. The pharmaceutical composition of claim 28, formulated for

administration by inhalation.

48. The pharmaceutical composition of claim 28, formulated for

administration by injection.

49. A pharmaceutical composition comprising, as an active

ingredient, the compound of claim 18, and a pharmaceutically acceptable

carrier.

50. The pharmaceutical composition of claim 49, wherein said

cholinergic up-regulator moiety and said non-steroidal anti-inflammatory

moiety are covalently linked via a hydrocarbon spacer.

51. The pharmaceutical composition of claim 50, wherein said

non-steroidal anti-inflammatory moiety is covalently attached to said spacer

via a -C(=X)Y- bond, where X is a non-substituted or substituted oxygen,

sulfur or nitrogen atom and Y is a substituted or non-substituted carbon,

oxygen, nitrogen, sulfur, silicon or phosphor atom linked to said C via a

single covalent bond.

52. The pharmaceutical composition of claim 51, wherein said

bond is selected from the group consisting of an ester bond and an amide

bond.

53. The pharmaceutical composition of claim 52, wherein said

ester bond is selected from the group consisting of a carboxylic ester bond

and a glycol amide ester bond.

54. The pharmaceutical composition of claim 51, wherein said

bond is hydrolizable by a brain derived esterase.

55. The pharmaceutical composition of claim 51, wherein said

bond is hydrolizable by a brain derived amidase.

56. The pharmaceutical composition of claim 50, wherein said

hydrocarbon spacer comprises at least one hydrocarbon selected from the

group consisting of an alkyl having 2-20 carbon atoms, a cycloalkyl having

3-20 carbon atoms and an aryl having 6-20 carbon atoms.

57. The pharmaceutical composition of claim 49, wherein said

cholinergic up-regulator moiety is selected from the group consisting of a

cholinesterase inhibitor residue, a nicotinic receptor agonist residue and a

muscarinic receptor agonist residue.

58. The pharmaceutical composition of claim 57, wherein said

cholinesterase inhibitor residue is a pyridostigmine residue.

59. The pharmaceutical composition of claim 58, wherein said

pyridostigmine residue is a 3-N,N-dimethylcarbamoyl pyridinium bromide

residue.

60. The pharmaceutical composition of claim 57, wherein said

nicotinic agonist residue is selected from the group consisting of a nicotine

residue and a cytisine residue.

61. The pharmaceutical composition of claim 57, wherein said

muscarinic receptor agonist residue is selected from the group consisting of

an arecoline residue and a pilocarpine residue.

62. The pharmaceutical composition of claim 49, wherein said

non-steroidal anti-inflammatory moiety comprises a residue of a

non-steroidal anti-inflammatory drug characterized by a functional group

selected from the group consisting of a free carboxylic acid group and a free

amine group.

63. The pharmaceutical composition of claim 62, wherein said

non-steroidal anti-inflammatory moiety is selected from the group

consisting of an ibuprofen residue, an indomethacin residue, a naproxen

residue, a diclofenac residue and an aspirin residue.

64. The pharmaceutical composition of claim 63, wherein said

ibuprofen residue is selected from the group consisting of an (±)-ibuprofen

residue, S-(+)-ibuprofen residue and R-(-)-ibuprofen residue.

65. The pharmaceutical composition of claim 49, characterized by

lipophilicity sufficient for permitting the compound to cross a blood brain

barrier of an organism.

66. The pharmaceutical composition of claim 49, formulated for

transdermal delivery.

67. The pharmaceutical composition of claim 49, formulated for

nasal administration.

68. The pharmaceutical composition of claim 49, fonnulated for

administration by inhalation.

69. The pharmaceutical composition of claim 49, formulated for

administration by injection.

70. A method of synthesizing the chimeric compound of claim 1,

the method comprising the steps of:

(a) converting a non-steroidal anti-inflammatory drug into a

non-steroidal anti-inflammatory-ester, including a

hydrocarbon chain terminating with a reactive halide group;

and (b) reacting said non-steroidal anti-inflammatory-ester including

said hydrocarbon chain terminating with said reactive halide

group with a cholinergic up-regulator, so as to obtain the

chimeric compound having said cholinergic up-regulator

moiety covalently linked to said non-steroidal

anti-inflammatory moiety via said hydrocarbon spacer.

71. The method of claim 70, wherein said hydrocarbon chain has

at least one hydrocarbon selected from the group consisting of an alkyl

having 2-20 carbon atoms, a cycloalkyl having 3-20 carbon atoms and an

aryl having 6-20 carbon atoms.

72. The method of claim 70, wherein said non-steroidal

anti-inflammatory drug is characterized by a functional group selected from

the group consisting of a free carboxylic acid group and free amine group.

73. The method of claim 72, wherein said non-steroidal

anti-inflammatory drug is selected from the group consisting of ibuprofen,

indomethacin, naproxen, diclofenac and aspirin.

74. The method of claim 73, wherein said ibuprofen is selected

from the group consisting of (+)-ibuprofen, S-(+)-ibuprofen and

R-(-)-ibuprofen.

75. The method of claim 70, wherein said cholinergic

up-regulator is selected from the group consisting of a cholinesterase

inhibitor, a nicotinic agonist and a muscarinic agonist.

76. The method of claim 75, wherein said cholinesterase inhibitor

is a pyridostigmine.

77. The method of claim 76, wherein said pyridostigmine is

3-N,N.-dimethylcarbamoyl pyridinium bromide.

78. The method of claim 75, wherein said nicotinic agonist is

selected from the group consisting of nicotine and cytisine.

79. The method of claim 75, wherein said muscarinic agonist is

selected from the group consisting of arecoline and pilocarpine.

80. A method of synthesizing the chimeric compound of claim 1,

the method comprising the steps of:

(a) converting a non-steroidal anti-inflammatory drug into a

non-steroidal anti-inflammatory-amide, said amide including

a hydrocarbon chain terminating with a reactive halide

group; and (b) reacting said non-steroidal anti-inflammatory-amide

including said hydrocarbon chain terminating with said

reactive halide group with a cholinergic up-regulator, so as

to obtain the chimeric compound having said cholinergic

up-regulator moiety covalently linked to said non-steroidal

anti-inflammatory moiety via said hydrocarbon spacer.

81. The method of claim 80, wherein said hydrocarbon chain has

at least one hydrocarbon selected from the group consisting of an alkyl

having 2-20 carbon atoms, a cycloalkyl having 3-20 carbon atoms and an

aryl having 6-20 carbon atoms.

82. The method of claim 80, wherein said non-steroidal

anti-inflammatory drug is characterized by a functional group selected from

the group consisting of a free carboxylic acid group and free amine group.

83. The method of claim 82, wherein said non-steroidal

anti-inflammatory drug is selected from the group consisting of ibuprofen,

indomethacin, naproxen, diclofenac and aspirin.

84. The method of claim 83, wherein said ibuprofen is selected

from the group consisting of (+)-ibuprofen, S-(+)-ibuprofen and

R-(-)-ibuprofen.

85. The method of claim 80, wherein said cholinergic

up-regulator is selected from the group consisting of a cholinesterase

inhibitor, a nicotinic agonist and a muscarinic agonist.

86. The method of claim 85, wherein said cholinesterase inhibitor

is a pyridostigmine.

87. The method of claim 86, wherein said pyridostigmine is

3-N,N-dimethylcarbamoyl pyridinium bromide.

88. The method of claim 85, wherein said nicotinic agonist is

selected from the group consisting of nicotine and cytisine.

89. The method of claim 85, wherein said muscarinic agonist is

selected from the group consisting of arecoline and pilocarpine.

90. A method of synthesizing the chimeric compound of claim 1,

the method comprising the steps of:

(a) converting a cholinergic up-regulator into its

N(ring)-substituted derivative, said derivative including a

hydrocarbon chain terminating with a reactive hydroxyl

group; and (b) reacting said N(ring)-substituted derivative including said

hydrocarbon chain terminating with said reactive hydroxyl

group with a reactive derivative of a non-steroidal

anti-inflammatory drug, so as to obtain the chimeric

compound having said cholinergic up-regulator moiety

covalently linked to said non-steroidal anti-inflammatory

moiety.

91. The method of claim 90, wherein said hydrocarbon chain has

at least one hydrocarbon selected from the group consisting of an alkyl

having 2-20 carbon atoms, a cycloalkyl having 3-20 carbon atoms and an

aryl having 6-20 carbon atoms.

92. The method of claim 90, wherein said non-steroidal

anti-inflammatory drug is characterized by a functional group selected from

the group consisting of a free carboxylic acid group and a free amine group.

93. The method of claim 92, wherein said non-steroidal

anti-inflammatory drug is selected from the group consisting of ibuprofen,

indomethacin, naproxen, diclofenac and aspirin.

94. The method of claim 93, wherein said ibuprofen is selected

from the group consisting of (±)-ibuprofen, S-(+)-ibuprofen and

R-(-)-ibuprofen.

95. The method of claim 90, wherein said cholinergic

up-regulator is selected from the group consisting of a cholinesterase

inhibitor, a nicotinic agonist and a muscarinic agonist.

96. The method of claim 95, wherein said cholinesterase inhibitor

is a pyridostigmine.

97. The method of claim 96, wherein said pyridostigmine is

3-N,N-dimethylcarbamoyl pyridinium bromide.

98. The method of claim 95, wherein said nicotinic agonist is

selected from the group consisting of nicotine and cytisine.

99. The method of claim 95, wherein said muscarinic antagonist

is selected from the group consisting of arecoline and pilocarpine.

100. The method of claim 90, further comprising the step of

converting said N(ring)-substituted derivative including said hydrocarbon

chain terminating with said reactive hydroxyl group into a tertiary amine N(ring)-substituted derivative including said hydrocarbon chain terminating « with said reactive hydroxyl group, prior to said step (b).

101. A method of treating, ameliorating or preventing a central

nervous system disorder or disease in an organism, the method comprising

the step of administering to said organism a therapeutically effective

amount of the compound of claim 1.

102. The method of claim 101, wherein said central nervous

system disorder or disease is selected from the group consisting of

Alzheimer's disease, cerebrovascular dementia, Parkinson's disease, basal

ganglia degenerative diseases, motoneuron diseases, Scrapie, spongyform

encephalopathy and Creutzfeldt-Jacob's disease.

103. The method of claim 101, wherein said central nervous

system disorder or disease is selected from the group consisting of cerebral

ischemia, transient hypoxia and stroke.

104. The method of claim 101, wherein said central nervous

system disorder or disease is a result of a head injury.

105. The method of claim 101, wherein said central nervous

system disorder or disease is accompanied by an inflammatory process.

106. The method of claim 105, wherein said inflammatory process

is selected from the group consisting of an inflammatory process induced by

infection, an inflammatory process induced by a tumor and an inflammatory

process induced by post-operative brain edema.

107. The method of claim 106, wherein said infection is selected

from the group consisting of viral infection and bacterial infection.

108. The method of claim 101, wherein said organism is a

mammal.

109. The method of claim 101, wherein said mammal is a human

being.

110. The chimeric compound of claim 1, wherein said cholinergic

up-regulator moiety is selected from the group consisting of a reversible

cholinesterase inhibitor residue and an irreversible cholinesterase inhibitor

residue.

111. The chimeric compound of claim 18, wherein said cholinergic

up-regulator moiety is selected from the group consisting of a reversible

cholinesterase inhibitor residue and an irreversible cholinesterase inhibitor

residue.

112. The pharmaceutical composition of claim 28, wherein said

cholinergic up-regulator moiety is selected from the group consisting of a

reversible cholinesterase inhibitor residue and an irreversible cholinesterase

inhibitor residue.

113. The pharmaceutical composition of claim 49, wherein said

cholinergic up-regulator moiety is selected from the group consisting of a

reversible cholinesterase inhibitor residue and an irreversible cholinesterase

inhibitor residue.

114. The method of claim 70, wherein said cholinergic

up-regulator moiety is selected from the group consisting of a reversible

cholinesterase inhibitor residue and an irreversible cholinesterase inhibitor

residue.

115. The method of claim 80, wherein said cholinergic

up-regulator moiety is selected from the group consisting of a reversible

cholinesterase inhibitor residue and an irreversible cholinesterase inhibitor

residue.

116. The method of claim 90, wherein said cholinergic

up-regulator moiety is selected from the group consisting of a reversible cholinesterase inhibitor residue and an irreversible cholinesterase inhibitor

residue.

117. A reversible cholinesterase inhibitor having a general formula

A:

wherein,

R₂ is selected from the group consisting of hydrogen, an alkyl, a

hydroxyalkyl, a haloalkyl, an alkylamine, a cycloalkyl and an aryl;

X is a halide.

Q and Z are each independently selected from the group consisting of

oxygen and sulfur; and

R₃ is selected from the group consisting of an alkyl, a cycloalkyl and an

aryl.

118. The reversible cholinesterase inhibitor of claim 117, wherein Q

and Z are each oxygen, R₃ is methyl, R₂ is alkyl and X is selected from the

group consisting of bromide and iodide.

119. A method of synthesizing the reversible cholinesterase inhibitor

of claim 117, comprising reacting a pyridine ring substituted at position 3 by

said R] with a R₂ residue terminating with said X, so as to produce a quaternary

pyridinium halide substituted at the N(ring) position by said R₂ and at position

3 by said R

120. A method of treating, ameliorating or preventing a central

nervous system disorder or disease in an organism, the method comprising

the step of administering to said organism a therapeutically effective

amount of the reversible cholinesterase inhibitor of claim 117.

121. The method of claim 120, wherein said central nervous

system disorder or disease is selected from the group consisting of

Alzheimer's disease, cerebrovascular dementia, Parkinson's disease, basal

ganglia degenerative diseases, motoneuron diseases, Scrapie, spongyform

encephalopathy and Creutzfeldt-Jacob's disease.

122. The method of claim 120, wherein said central nervous

system disorder or disease is selected from the group consisting of cerebral

ischemia, transient hypoxia and stroke.

123. The method of claim 120, wherein said central nervous

system disorder or disease is a result of a head injury.

124. A reversible cholinesterase inhibitor having a general formula B:

wherein,

R_ is C(=Q)Z-R₃;

R₂ is selected from the group consisting of hydrogen, an alkyl, a

hydroxyalkyl, a haloalkyl, an alkylamine, a cycloalkyl and an aryl;

Q and Z are each independently selected from the group consisting

of oxygen or sulfur; and

R₃ is selected from the group consisting of an alkyl, a cycloalkyl and an

aryl.

125. The reversible cholinesterase inhibitor of claim 124, wherein Q

and Z are each oxygen, R₃ is methyl and R₂ is alkyl.

126. A method of synthesizing the reversible cholinesterase inhibitor

of claim 124, comprising:

(a) reacting a pyridine ring substituted at position 3 by said R_Ϊ with

an organic halide and/or a reactive inorganic halide, so as to

produce a quaternary pyridinium halide substituted by said Ri at

position 3; and (b) reducing said quaternary pyridinium halide, so as to produce a

tertiary tetrahydropyridine ring, substituted by said R] group at

position 3.

127. The method of claim 126, wherein said reactive inorganic halide

is potassium iodide.

128. The method of claim 126, wherein said organic halide is a R₂

residue terminating with a halide group and said quaternary pyridinium halide

is further substituted at the N(ring) position by said R₂.

129. A method of treating, ameliorating or preventing a central

nervous system disorder or disease in an organism, the method comprising

the step of administering to said organism a therapeutically effective

amount of the reversible cholinesterase inhibitor of claim 124.

130. The method of claim 129, wherein said central nervous

system disorder or disease is selected from the group consisting of

Alzheimer's disease, cerebrovascular dementia, Parkinson's disease, basal

ganglia degenerative diseases, motoneuron diseases, Scrapie, spongyform

encephalopathy and Creutzfeldt-Jacob's disease.

131. The method of claim 129, wherein said central nervous

system disorder or disease is selected from the group consisting of cerebral

ischemia, transient hypoxia and stroke.

132. The method of claim 129, wherein said central nervous

system disorder or disease is a result^'of a head injury.

133. The chimeric compound of claim 1, characterized by

cholinergic up-regulation activity and inflammation down-regulation

activity exerted by said chimeric compound and by hydrolytic derivatives

thereof.