WO2008118326A1

WO2008118326A1 - Muscarinic agonists and methods of use thereof

Info

Publication number: WO2008118326A1
Application number: PCT/US2008/003637
Authority: WO
Inventors: William S. Messer; Frederick Tejada
Original assignee: University Of Toledo
Priority date: 2007-03-23
Filing date: 2008-03-20
Publication date: 2008-10-02

Abstract

A method of forming analogs of CDD- 0304, i.e., tetra(ethyleneglycol) (4- methoxy-1,2,5-thiadiazol-3-yl)[3-(1-methyl-1,2,4,5-tetrahydropyrid-3-yl)-1,2,5- thiadiazo1-4-yl] ether hydrochloride includes replacing the 1,2,5-thiadiazole moiety with a terminal nitrogen moiety or an OH group; and b) varying the length of the linking group by replacing the tetra (ethylene) glycol moiety with one of: ethylene glycol, di(ethylene) glycol, penta(ethylene) glycol, or diether diol. Also, such analog compounds and their uses are disclosed.

Description

TITLE

MUSCARINIC AGONISTS AND METHODS OF USE THEREOF

INVENTORS: William S. Messer, Jr., Frederick R. Tejada

BACKGROUND OF THE INVENTION

[0001] Schizophrenia is a psychiatric disorder that afflicts approximately two million Americans. The yearly cost to society for patient care is estimated to be $23 billion per year, in the United States alone. Approximately $2.3 billion was spent on antipsychotic medications in 1999, and the antipsychotic drug market is expected to grow to more than $6 billion by 2006.

[0002] The underlying cause of schizophrenia is unknown, although an imbalance of activity at dopamine synapses has been proposed to play a role in the positive symptoms such as hallucinations, delusions and disordered thought patterns.¹'² For many years, dopamine antagonists such as haloperidol and chlorpromazine have been widely used in the treatment of schizophrenia. Unfortunately, many of the classical antipsychotic compounds produce unwanted side effects such as Parkinsonism, acute dystonia and akathisia. The adverse effects are mainly due to a blockade of dopamine (D₂) receptors, although many antipsychotic compounds interact with other G protein-coupled receptors. The classical antipsychotics are effective in treating the positive symptoms of schizophrenia, but are much less useful in treating negative symptoms such as social withdrawal and blunted affect and exacerbate the cognitive deficits associated with schizophrenia. [0003] In the 1980s, the atypical antipsychotic clozapine was found to be effective in treating both positive and negative symptoms of schizophrenia with a relatively lower incidence of extrapyramidal side effects. Despite its effectiveness in treating schizophrenia patients, the clinical utility of clozapine is limited by the development of agranulocytosis in a small subset (0.6 %) of patients. Other atypical antipsychotics, including olanzapine, risperidone and quetiapine, may be as effective as clozapine in treating the positive symptoms of schizophrenia, yet do not produce agranulocytosis. Over the past decade, therapeutic approaches toward the treatment of schizophrenia have focused on developing more effective antipsychotic compounds with reduced side effect profiles.

[0004] Although a number of treatments are available to treat the positive and negative symptoms, relatively few efforts have focused on developing compounds that can improve cognitive disturbances associated with schizophrenia.³ Cognitive deficits, including impaired working memory, attention and executive functions, are strongly linked to the long-term disabilities found in patients with schizophrenia. The cognitive deficits associated with schizophrenia represent an important clinical target for drug development that has not been addressed by the pharmaceutical industry.

[0005] The dopamine hypothesis of schizophrenia has had a pronounced effect on the strategies for developing antipsychotics.⁵ However, even recent atypical antipsychotics do not completely relieve all schizophrenic symptoms.⁶ [0006] While recent advances have been made in the understanding of the cholinergic nervous system and the receptors therein, there is still a need to develop compositions which do not have the adverse affects of the currently available treatment regimes. One avenue being pursued involves cholinergic receptors which are proteins embedded in the cell membrane that respond to the chemical acetylcholine. Cholinergic receptors are subdivided into the nicotinic and muscarinic receptor families, and muscarinic receptors represent a family of five subtypes. [0007] Muscarinic receptors mediate a variety of physiological responses to the neurotransmitter acetylcholine in the central and peripheral nervous systems. M₁ receptors are found in high abundance within the cerebral cortex and hippocampus brain regions implicated in memory and cognitive function.⁷ Post-mortem and genetic study suggest that activating the Mi receptor might be critical in reversing the cognitive deficits in schizophrenia.⁸ Similar to Mj receptors, M₂ receptors, also are expressed in the hippocampus and most other brain regions implicated in learning and memory processes. Since M₁ and M₂ receptors play a role in cognitive and memory function,^9"11 agonists with M₁ and M₂ activity might be particularly useful in treating memory and cognitive deficits associated with schizophrenia/'¹² Acetylcholine stimulates smooth muscle contraction in a variety of tissues and promotes secretion from exocrine glands. These effects are mediated by M₃ receptors.

[0008] Though less well characterized pharmacologically, M₄ receptors appear to play a role in the perception of pain, and M₅ receptors may regulate dopaminergic activity in the brain. On the other hand, M₄ receptors regulate the release of dopamine and have been implicated in schizophrenia.¹²'¹³ A recent study indicates that loss of midbrain M₄ receptors in knockout mice causes a state of dopaminergic hyperexcitability.¹⁴ This in turn may be responsible for the pathological mechanism for affective and cognitive disorders and psychoses, in which dysregulated dopaminergic transmission plays a key role.

[0009] Taken together, an M₁, M₂, and M₄ agonist could provide efficacy in a broad range of symptomatic domains of schizophrenia, including enhancement of cognitive function. However, the development of such selective muscarinic agonists has been hindered by the high degree of homology among the five receptor subtypes. [00010] Several lines of evidence suggest that selective muscarinic agonists might be useful in the treatment of schizophrenia. Anticholinergic drugs, including the glycolate esters, produce psychotomimetic effects in humans.¹⁵ Muscarinic antagonists have been used in schizophrenic patients to control the parkinsonism associated with administration of antipsychotics with dopamine antagonist activity, yet at higher doses, muscarinic antagonists exacerbate the symptoms of schizophrenia, producing confusion and hallucinations. In contrast, Alzheimer's disease patients treated with cholinesterase inhibitors, which elevate levels of acetylcholine, exhibit improvements in neuropsychiatric symptoms such as agitation, hallucinations and psychosis. ^" In addition, the selective M₁ZM₄ muscarinic agonist xanomeline significantly improved psychiatric symptoms such as hallucinations in phase II clinical trials in Alzheimer's patients.¹⁹ Unfortunately, xanomeline produced unwanted side effects associated with activation of M₃ receptors, including salivation, diarrhea and profuse sweating, that limited patient compliance.²⁰ The side effects seem to be associated with rapid metabolism of the alkyloxy side chain, or the N-methyl tetrahydropyridyl group, following oral administration, resulting in a nonselective, yet active compound with limited therapeutic utility. Despite a second phase II clinical trial with a patch formulation, the liabilities of xanomeline still outweigh its benefits.

[00011] Follow-up preclinical studies with structurally related compounds identified strong antipsychotic activity in (5R,6R)-6-(3-butylthio-l,2,5-thiadiazol-4- yl)-l-azabicyclo[3.2.1]octane (BuTAC), which displays partial agonist activity at M₂ and M₄ receptors.²¹ Muscarinic agonists such as xanomeline and BuTAC may exert an antipsychotic action by regulating the release of dopamine in the frontal cortex. 2⁴ Xanomeline and BuTAC produce very few of the adverse side effects (e.g., catalepsy) associated with classical antipsychotics such as haloperidol, suggesting that selective muscarinic agonists might provide a useful alternative therapeutic approach to treating the symptoms of schizophrenia. Moreover, muscarinic agonists might be particularly useful in improving cognitive function (including memory function, language use and constructional praxis) in schizophrenic patients.

[00012] Behavioral studies of muscarinic receptor knockout mice also suggest the utility Of M₁ and M₄ agonists in the treatment of psychosis.²⁴ For example, M₄ receptors modulate locomotor activity produced by the stimulation OfD₁ dopamine receptors. M₄ knockout mice also show enhanced sensitivity to the effects of PCP on the pre-pulse inhibition model of psychosis. Since M₁ and M₂ receptors play a role in cognitive and memory function,⁹'¹⁰ agonists with M₁ and M₂ activity might be particularly useful in treating memory and cognitive deficits associated with schizophrenia/ ²⁴

[00013] Several of the co-inventors herein have discovered muscarinic agonists, which are claimed in US Patent Nos. 6,096,767; 6,211,204 Bl ; 6,376,675 B2; 6,369,081 Bl ; and 6,602,891 B2, which are expressly incorporated herein by reference.

[00014] Efforts to develop selective muscarinic agonists have been hampered by the high degree of amino acid homology within the binding pocket of muscarinic receptors. Compounds with larger size and functional groups that interact with the extracellular loops of muscarinic receptors may interact with unique amino acid residues and selectively activate Mj and M₄ receptors.

[00015] In particular, the clinical utility of muscarinic agonists for the treatment of schizophrenia has not been adequately assessed due to the lack of compounds exhibiting an appropriate combination of agonist activity and selectivity for M] , M₂ and M₄ receptors. Previously, a bivalent derivative of xanomeline, CDD-0273, was developed with strong agonist activity at Mj and M₄ receptors and very low activity at M₃ and M₅ receptors. While bivalent ligands (i.e., CDD-0273) are unsuitable as drug candidates due to their limited membrane permeability and bioavailability, they have helped define accessory binding sites for muscarinic ligands with improved receptor subtype selectivity.

[00016] In view of the foregoing, it would be desirable to provide muscarinic agonists that result in the selective activation of muscarinic receptors, particularly so side effects are minimized during treatment of the conditions noted above.

[00017] Thus, there is a need for muscarinic agonists with activity at M₁, M₂ and M₄ receptors which then would be useful in the treatment of Alzheimer's disease and schizophrenia, and other cognitive impairment disorders.

SUMMARY OF THE INVENTION

[00018] There is provided herein relates to analogs of the potent muscarinic receptor agonist, CDD-0304, tetra(ethylene glycol)(4-methoxy-l,2,5-thiadiazol-3- yl) [3 -methyl- 1 ,2,5,6-tetrahyrdopyrid-3-yl)-l ,2,5,thiadiazol-4-yl] ether hydrochloride. [00019] In another aspect, the present invention relates to a method of forming analogs of CDD-0304. i.e., tetra(ethyleneglycol) (4-methoxy-l,2,5-thiadiazol-3-yl)[3- (1 -methyl- 1 ,2,4,5-tetrahydropyrid-3-yl)- 1 ,2,5-thiadiazol-4-yl]ether hydrochloride comprising at least one of the following steps: [00020] replacing the 1,2,5-thiadiazole moiety with a terminal nitrogen moiety, including primary amines or amides; and [00021] varying the length of the linking group by replacing the tetra (ethylene) glycol moiety with one of: ethylene glycol, di(ethylene) glycol, penta(ethylene) glycol, or diether diol. [00022] In another aspect, the present invention relates to a method for an analog oftetra(ethyleneglycol) (4-methoxy-l,2,5-thiadiazol-3-yl)[3-(l-methyl-l,2,4,5- tetrahydropyrid-3-yl)- 1 ,2,5-thiadiazol-4-yl]ether hydrochloride comprising: [00023] replacing the 1,2,5-thiadiazole moiety with OH; and [00024] varying the length of the linking group by replacing the tetra (ethylene) glycol moiety with one of: ethylene glycol, di(ethylene) glycol, penta(ethylene) glycol, or diether diol. [00025] Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[00026] Fig 1. - Table 1 shows the inhibition of [³I I]-(R)-QNB binding to wild- type and chimeric receptors expressed as pK, values. Data represent the mean (± s.e.m,) from three assays, each performed in triplicate.

[00027] Fig. 2 - Table 2 shows the stimulation of phosphoinositide metabolism by muscarinic agonists at wild-type and chimeric muscarinic receptors. Data represent the mean (± s.e.m.) from three assays, each performed in triplicate.

[00028] Fig. 3 - Table 3 shows the stimulation of phosphoinositide metabolism by selected muscarinic agonists at wild type and mutant muscarinic receptors. Data represent the mean (± s.e.m.) from three experiments, each performed in triplicate.

[00029] Fig. 4 is an illustration of Scheme 1, showing the synthesis of analogues CDD-0320, CDD-0317, CDD-0319, CDD-0325, CDD-0322 and CDD-0324 of the compound CCD-0304, namely, tetra(ethyleneglycol) (4-methoxy-l,2,5- thiadiazol-3-yl)[3-(l -methyl- 1 ,2,4,5-tetrahydropyrid-3-yl)- 1 ,2,5-thiadiazol-4-yl]ether hydrochloride (CCD-0304).

[00030] Fig. 5 is an illustration of Scheme 2 showing the synthesis of analogues CDD-0300 and CDD-0323 of the compound CCD-0304, namely, tetra(ethyleneglycol) (4-methoxy- 1 ,2,5-thiadiazol-3-yl)[3-( 1 -methyl- 1 ,2,4,5- tetrahydropyrid-3-yl)- 1 ,2,5-thiadiazol-4-yl]ether hydrochloride (CCD-0304).

[00031] Figs. 6A and 6B - Table 4 shows the optimized O_α ...O_ω, and corresponding O_α...N distances (A) for bound bivalent ligands and the hydrogen

bonds farmed with the M₁ wild-type as well as o3 and o2 chimeric receptor.

[00032] Fig. 7 - Table 5 shows the effect of basic terminal group on the inhibition of [3H]-(R)-QNB binding to human muscarinic receptor subtypes expressed in A9 cells. pKi values were obtained by nonlinear least squares curve-fitting of data, as described herein. Data represent the mean + SEM from at least three independent experiments, each performed in triplicate.

[00033] Fig. 8 - Table 6 shows the binding Properties for wild-type Ml and M5 expressed as pKi ± s.e.m.

[00034] Figs. 9A and 9B - Table 7 shows the stimulation of PI metabolism through activation OfM₁, M₃ and M₅ receptors and inhibition of adenylyl cyclase activity through activation of M₂and M₄.

[00035] Fig. 10 is a graph comparing cAMP release (%), for the M₂ receptor for the Ligand and Ligand+HSM (10 μM) in the following: Carbachol (CCh), CDD- 0300, CDD-0320, CDD-0317, and CDD-0319, thus showing the reversal of the effects of hyoscy amine at M₂ and M₄ receptors.

[00036] Fig. 11 is a graph comparing cAMP release (%), for the M4 receptor for the Ligand and Ligand+HSM(10 μM) in the following: CCh, CDD-0300, CDD-0320, CDD-0317, CDD-0319 and CDD-0322, thus showing the reversal of the effects of hyoscy amine at M₂ and M₄ receptors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

[00037] In one aspect, there is provided herein compounds that activate M] and M₄ receptors, which enhance memory function and modulate dopamine function, respectively.

[00038] In another aspect, there is provided herein compounds having improved muscarinic receptor selectivity profiles (specifically at Mi and M₄ receptors).

[00039] These compounds are muscarinic agonists that are useful in the treatment of a variety of neurological disorders including Alzheimer's disease and schizophrenia. Of particular interest are compounds that activate selectively Mi and M₄ receptors, which enhance memory function and modulate dopamine function, respectively. Efforts to develop selective muscarinic agonists have been hampered by the high degree of amino acid homology within the binding pocket of muscarinic receptors. Compounds with larger size and functional groups that interact with the extracellular loops of muscarinic receptors may interact with unique amino acid residues and selectively activate Mi and M₄ receptors. A series of novel muscarinic agonists is described possessing a unique set of features that results in the selective activation of muscarinic receptors.

[00040] In a continuing effort to design and develop Mi, M₂and M₄ selective muscarinic agonists, a second generation derivative containing a terminal thiadiazole moiety was synthesized and pharmacologically evaluated. Tetra(ethylene glycol) (3- methoxy-l,2,5-thiadiazol-4-yl)[3(l-methyl-l,2,5,6-tetrahydropyrid-3-yl)-l,2,5- thiadiazol-4-I]ether, CDD-0304, displayed functional activity at Mi and M₂ receptors, partial agonist activity at M₄ receptors, and was essentially inactive at M₃ and M₅ receptors. Compound CDD-0304 thus represents a lead compound for the development of selective muscarinic agonists with potential utility in the treatment of schizophrenia. Preliminary chimeric receptor data indicates that amino acids found in the second and third extracellular loops of the Mj receptor are critical for agonist binding and activity, respectively.

[00041] The interaction of muscarinic agonists with transmembrane domain amino acid residues was evaluated by examining receptor binding properties at wild-type and mutant (Thrl92Ala) M₁ receptors. Previous studies had identified Thrl92 as an important amino acid residue involved in the binding of small muscarinic agonists such as acetylcholine and carbachol. The binding affinities of carbachol, xanomeline and compound CDD-0273 were examined at wild-type and mutant (Thrl92Ala) M₁ receptors. Carbachol exhibited a lower affinity for (Thri92Ala) Mj receptors (pK; of 2.7 ± 0.16) than for wild-type M] receptors (pK; of 5.7 ± 0.06). Xanomeline also displayed a lower affinity for (Thrl92Ala) M j receptors (pK; of 5.4 + 0.07) than for wild-type M₁ receptors (pK_; of 7.9 ± 0.29). In a similar fashion, CDD-0273 displayed lower affinity for (Thrl 92Al a) M₁ receptors (pKj of 7.0 ± 0.10) than for wild-type M₁ receptors (pK_; of 9.9 + 0.28). The data indicate that xanomeline and CDD-0273 interact with transmembrane domains Of M₁ receptors in a similar fashion to smaller molecules. Their relatively high affinity for M] receptors may also depend on interaction with residues outside of the common binding site for agonists located within the transmembrane domain.

[00042] In order to assess this possibility, chimeric receptors were developed. Exchange of the second (o2) extracellular loop of the M₁ receptor with the corresponding sequence from M₅ receptors lowered the binding affinity and activity of both compounds CDD0304 and CDD-0273 (Table 1 and 2).

[00043] The activities of carbachol and xanomeline were relatively unchanged, although the potency of xanomeline was decreased (Table 2). The o2 loop may play a role in the binding and activity of relatively large muscarinic agonists such as compounds CDD-0304 and CDD-0273.

[00044] Exchange of the third (o3) extracellular loop of the Mi receptor with the corresponding sequence from Ms receptors dramatically lowered the activity of xanomeline, CDD-0304 and CDD-0273. The third extracellular loop of Mj receptors therefore may interact with functional groups common to xanomeline, CDD-0304 and CDD-0273. Replacement of both the o2 and o3 loops of the M₁ receptor with the corresponding sequences from M₅ receptors virtually eliminated agonist activity for CDD-0304 and CDD-0273. The data strongly indicate that compounds CDD-0304 and CDD-0273 interact with the second and third extracellular domains of Mi receptors and that these interactions are important for conferring agonist activity and selectivity.

[00045] Based on the data from chimeric Mj/M₅ receptors, the inventors discovered that mutation of individual amino acid residues in the second and/or third extracellular loops would result in a loss of binding affinity or agonist activity at residues that are involved in the binding of CDD-0304 and CDD-0273.

[00046] In contrast, smaller agonists such as carbachol should not be dramatically affected by such mutations, since it interacts primarily with highly conserved residues within the transmembrane domains. Thus carbachol served as a useful control for comparing the effects of individual mutations. Xanomeline also served as a helpful control since it shares many structural features with CDD-0304 and CDD-0273.

[00047] The first set of site-directed mutagenesis studies examined the effects of replacing the four nonconserved residues located in the third extracellular loop OfM₁ receptors with the corresponding residues of M₅ receptors. No significant differences were noted in the binding or activity of the mutant receptors (data not shown). A second set of studies focused on replacing residues found in the second extracellular loop of Mi receptors with the corresponding residues of M₅ receptors. Two mutant Mj receptors were created and characterized Mj (E 17 OK) and

[00048] As summarized in Table 3, the data indicate that while carbachol activity remained unaffected, mutation of Glul70 and Glnl85 to the corresponding M₅ residues lowered the potency and activity of CDD0304. Therefore, both residues appear to contribute to the potency and activity of CDD0304 at Mi receptors as compared with M₅ receptors.

[00049] The data indicate that derivatives of compound CDD-0304 containing hydrogen bond donors (e.g., primary amines or amides) will exhibit even better functional selectivity for M₁ vs. M₅ receptors. This can be tested by synthesizing and characterizing new ligands incorporating hydrogen bond donating groups in place of the terminal 1,2,5-thiadiazole moiety found in CDD-0304, with either a tetraethylene glycol or pentaethylene glycol spacer.

[00050] In one aspect, the present invention relates to compounds which have an improved M] /M₄ agonist activity and selectivity and CNS penetration. The compounds are designed and synthesized based on structural modifications of the compound CDD-0304.

[00051] CDD-0304 consists of the agonist pharmacophore, i.e., xanomeline, which is linked via an ethylene glycol spacer to a terminal 3 -methoxy- 1,2,5- thiadiazole ring. Xanomeline can undergo N-oxidation and N-demethylation at the tetrahydropyridine ring which affects its muscarinic agonist properties. In order to prevent this N-oxidation and N-demethylation, the N-methyl group is incorporated in a ring, thus giving various azabicyclic and tricyclic systems.

[00052] In one aspect, the present invention relates to a method for forming a first set of compounds which have a suitable terminal nitrogen-containing moiety that binds in its protonated form to negatively charged residues found in the M₁-WiId Type receptor in order to improve agonist activity and selectivity, and to the compounds formed thereby.

[00053] In another aspect, the present invention relates to a method for forming a second set of compounds which have different linking groups, and the compounds formed thereby. The linking groups are both varied in the nature and length of the linking group.

[00054] In still another aspect, the present invention relates to CDD-0304 analogs which optimize M₁, M₂ and M₄ agonist activity and selectivity and CNS penetration for the treatment of various neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia.

[00055] In one aspect, disclosed herein is a method of increasing the activity of a muscarinic receptor comprising contacting the receptor with an effective amount of at least one CCD-0304 analog compound.

[00056] In another aspect, disclosed herein is a method of treating a subject suffering from a muscarinic receptor related disorder comprising identifying a subject in need thereof and administering to the subject a therapeutically effective amount of at least one CCD-0304 analog compound. By "muscarinic related disorder," it is meant a disorder whose symptoms are ameliorated by activating a muscarinic receptor.

[00057] In another aspect, disclosed herein is a method of treating schizophrenia or psychosis of any origin in a subject, comprising identifying a subject in need thereof and administering to the subject a therapeutically effective amount of at least one CDD-0304 analog compound. In some embodiments, the method comprises treating a subject with a pharmacologically active dose of at least one CDD-0304 analog compound, for the purpose of controlling the positive (hallucinations and delusion), negative (apathy, social withdrawal, anhedonia) and cognitive symptoms of schizophrenia or related psychosis.

[00058] In another aspect, the present invention relates to a method of ameliorating at least one symptom in a subject of a condition where it is beneficial to increase the level of activity of at least one of an M] , M₂ and/or M₄ muscarinic receptor comprising: determining that the subject would benefit from an increased level of activity of at least one of an M₁ M₂ and/or M₄ muscarinic receptor; and administering an amount of at least one analog of the 1,2,5,6-tetrahydropyridine compound CDD-0304 which is therapeutically effective to increase the level of activity of said at least one of an Mi , M₂ and/or M₄ muscarinic receptor and to ameliorate said at least one symptom to the subject.

[00059] In certain embodiments, it is within the scope of the contemplated invention that the CDD-0304 analog compounds may be administered in a single daily dose, or the total daily dosage may be administered as a plurality of doses, (e.g., divided doses two, three or four times daily). Furthermore, compounds for the present invention may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, or via topical use of ocular formulations, or using those forms of transdermal skin patches well known to persons skilled in the art.

[00060] It is to be further understood that the dosage regimen can be selected in accordance with a variety of factors. These include type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the kidney and liver functions of the patient; and the particular compounds employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the disease or disorder that is being treated. For example, the daily dosage of the products may be varied over a wide range from 0.01 to 1000 mg per adult human per day. An effective amount of the drug is ordinarily supplied at a dosage level of about 0.0001 mg/kg to about 25 mg/kg body weight per day. Preferably, the range is from about 0.001 to 10 mg/kg of body weight per day, and especially from about 0.001 mg/kg to 1 mg/kg of body weight per day. The compounds may be administered on a regimen of 1 to 4 times per day.

[00061] It is also within the contemplated scope of the present invention that the CDD-0304 analog compounds may be used alone at appropriate dosages defined by routine testing in order to obtain optimal pharmacological effect, while minimizing any potential toxic or otherwise unwanted effects. In addition, the CDD-0304 analog compounds may be used as adjunctive therapy with known drugs to reduce the dosage required of these traditional drugs, and thereby reduce their side effects.

[00062] The term "therapeutically effective amount" is used to indicate an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. This response may occur in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, and includes alleviation of the symptoms of the disease being treated.

[00063] The term "pharmaceutically acceptable addition salts" refers to salts known in the art to be acceptable in pharmaceutical practice, for example acid addition salts such as hydrochloric acid salts, maleic acid salts, and citric acid salts. Pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorus, and the like, as well as the salts derived from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Also contemplated are the salts of amino acids such as arginate, gluconate, galacturonate, and the like.

[00064] The term "metabolite" refers to a form of a compound obtained in a human or animal body by action of the body on the administered form of the compound, for example a de-methylated analogue of a compound bearing a methyl group on the tetrahydropyridyl moiety. This is a result of action by the body on the methylated compound after its administration. Metabolites may themselves have biological activity.

[00065] The term "prodrug" refers to a form of a compound which after administration to a human or animal body is converted chemically or biochemically to a different compound in said body having biological activity. A prodrug form of a compound may itself have biological activity.

[00066] The novel compounds of embodiments of the present invention, and compounds which may be used in accordance with embodiments of the present invention may have at least one chiral center, and may accordingly exist as enantiomers or as mixtures of enantiomers (e.g., racemic mixtures). Where the compounds possess two or more chiral centers, they may additionally exist as diastereoisomers .

[00067] In some embodiments of the present invention, there are provided pharmaceutical compositions and the use of certain compounds in the manufacture of pharmaceutical compositions. Such compositions may be in a form suitable for oral (e.g., in the form of capsules, tablets, granules, powders or beads), rectal, parenteral, intravenous, intradermal, subcutaneous, transdermal or topical administration, or for administration by insufflation or nasal spray, iontophoretic, buccal, or sublingual lingual administration. Such compositions may be in unit dosage form. Certain of the compounds in some embodiments of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention.

[00068] Examples

[00069] The following is a detailed example of a preferred process to prepare compounds described herein. It will be understood that the following examples are not intended to limit the scope of the invention.

[00070] The compounds described herein can be prepared as described in the schemes shown in the Figs. 4 and 5. For the Scheme in shown n Fig. 4, the general procedure is: (i) TsCl, Pyridine, 0⁰C, (ii) CH₃I, acetone/CHCl₂, (iii) NaBH₄, MeOH/CHCl₃, (iv) R, MeCN or DMF. Heater or r.t., (v) ethereal HCl. For the Scheme II shown in Fig. 5, the general procedure is: (NaH/THF, polyethylene glycol, reflux, (ii) CH₃I, acetone, r.t., (iii) NaBH₄/MeOH.CHCl₃ 0-5 ⁰C, (iv) HCl.

[00071] For example, the 1,2,5,6-tetrahydropyridine compounds were prepared by the Williamson ether formation method to incorporate the thiadiazoles with the corresponding n-(ethylene glycol) linkers, where n is the number of ethylene glycol unit. The intermediate 3-(3-chloro-l,2,5-thiadiazol-4-yl)pyridine was synthesized using the published procedure.²⁵The intermediate 3-chloro-4-methoxy- 1,2,5- thiadiazole was prepared using previous procedure.²⁶

[00072] Example 1 - Analogues of CDD-304

[00073] A series of analogs were synthesized with terminal nitrogen-containing moiety that can bind in its protonated form to negatively charged residues found in the M₁-WT receptor and its o2 and o3 chimers when these loops are replaced by those from M₅-WT. The general schemes for the synthesis of the CDD- analogs are shown in Figs. 4 and 5.

[00074] In Fig 1. - Table 1 shows the inhibition of [³11 ]-(R)-QNB binding to wild-type and chimeric receptors expressed as pK; values. Data represent the mean (± s.e.m,) from three assays, each performed in triplicate. Compound 1 is CDD-273 and compound 2 is CDD-304.

[00075] In Fig. 2 - Table 2 shows the stimulation of phosphoinositide metabolism by muscarinic agonists at wild-type and chimeric muscarinic receptors. Data represent the mean (± s.e.m.) from three assays, each performed in triplicate. Compound 1 is CDD-273 and compound 2 is CDD-304.

[00076] In Fig. 3 - Table 3 shows the stimulation of phosphoinositide metabolism by selected muscarinic agonists at wild type and mutant muscarinic receptors. Data represent the mean (± s.e.m.) from three experiments, each performed in triplicate. Compound 1 is CDD-273 and compound 2 is CDD-304.

[00077] In Figs. 6A and 6B - Table 4 shows the optimized O_α...O_ω, and corresponding O_α...N distances (A) for bound bivalent ligands and the hydrogen bonds formed with the Mj wild- type as well as o3 and o2 chimeric receptor.

[00078] In Fig. 7 - Table 5 shows the effect of a basic terminal group on the inhibition of [3H]-(R)-QNB binding to human muscarinic receptor subtypes expressed in A9 cells. pKi values were obtained by nonlinear least squares curve-fitting of data. Data represent the mean + SEM from at least three independent experiments, each performed in triplicate.

[00079] In Fig. 8 - Table 6 shows the binding properties of wild-type M₁ and M₃ express as pKi ± SEM.

[00080] In Figs. 9A and 9B - Table 7 shows the stimulation of PI metabolism through activation of Mi, M₃ and M₅ receptors and inhibition of adenylyl cyclase activity through activation of M₂ and M₄. The data represent the mean ±SEM from at least three independent experiments, each performed in triplicate.

[00081] In Figs. 10 and 11 are graphs showing the reversal of the effects of hyoscyamine at M₂ and M₄ receptors.

[00082] The skilled artisan will appreciate that many factors influence the selection of any compound for application in clinical therapy, e.g., effectiveness for the intended purpose, safety, possible side-effects and therapeutic index. The skilled artisan will thus appreciate how to interpret the expression "pharmaceutically acceptable quaternary compounds" which are structurally derived from the inventive compounds having a tertiary nitrogen atom, as this expression is used in the present specification and claims.

[00083] While the invention as been described with reference to a preferred embodiment, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof.

[00084] Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.

[00085] Each of the following references is incorporated by reference herein in its entirety, including any drawings.

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Claims

CLAIMS What is claimed is:

1. A method for forming an analog of tetra(ethyleneglycol) (4-methoxy- l,2,5-thiadiazol-3-yl)[3-(l -methyl- l,2,4,5-tetrahydropyrid-3-yl)-l,2,5-thiadiazol-4- yl]ether hydrochloride comprising at least one of the following steps: a) replacing the 1,2,5-thiadiazole moiety with a terminal nitrogen moiety; and b) varying the length of the linking group by replacing the tetra (ethylene) glycol moiety with one of: ethylene glycol, di(ethylene) glycol, penta(ethylene) glycol, or diether diol.

2. A method for forming an analog of tetra(ethyleneglycol) (4-methoxy- l,2,5-thiadiazol-3-yl)[3-(l-methyl-l,2,4,5-tetrahydropyrid-3-yl)-l,2,5-thiadiazol-4- yl] ether hydrochloride comprising at least one of the following steps: a) replacing the 1,2,5-thiadiazole moiety with OH; and b) varying the length of the linking group by replacing the tetra (ethylene) glycol moiety with one of: ethylene glycol, di(ethylene) glycol, penta(ethylene) glycol, or diether diol.

3. The method of claim 1 , wherein the nitrogen-containing moiety includes one or more primary amines or primary amides.

4. An analog of tetra(ethyleneglycol) (4-methoxy-l,2,5-thiadiazol-3-yl)[3- ( 1 -methyl- 1 ,2,4,5-tetrahydropyrid-3-yl)- 1 ,2,5-thiadiazol-4-yl]ether hydrochloride wherein the 1,2,5-thiadiazole moiety is replaced with a terminal nitrogen moiety; and the linking group, the tetra (ethylene) glycol moiety, is replaced with one of: ethylene glycol, di(ethylene) glycol, penta(ethylene) glycol, or diether diol.

5. An analog of tetra(ethyleneglycol) (4-methoxy-1.2,5-thiadiazol-3-yl)[3- (l-methyl-l,2,4,5-tetrahydropyrid-3-yl)-l,2.5-thiadiazol-4-yl]ether hydrochloride wherein the 1,2,5-thiadiazole moiety is replaced with OH; and the tetra (ethylene) glycol moiety is replaced with one of: ethylene glycol, di(ethylene) glycol, penta(ethylene) glycol, or diether diol.

6. The analog of claim 4, wherein the nitrogen-containing moiety includes one or more primary amines or primary amides.

7. A method of activating at least one of an M₁ , M₂ and/or M₄ muscarinic receptor in a subject comprising contacting said receptor with at least one analog of claim 4.

8. The method of claim 7, wherein the subject is human.

9. The method of claim 7, further comprising contacting said subject with an additional therapeutic agent.

10. A method of activating at least one of an M₁ , M₂ and/or M₄ muscarinic receptor in a subject comprising contacting said receptor with at least one analog of claim 5.

11. The method of claim 10, wherein the subject is human.

12. The method of claim 10, further comprising contacting said subject with an additional therapeutic agent.

13. A method of activating at least one of an M₁ , M₂ and/or M₄ muscarinic receptor in a subject comprising contacting said receptor with at least one analog of claim 6.

14. The method of claim 13, wherein the subject is human.

15. The method of claim 13, further comprising contacting said subject with an additional therapeutic agent.

16. A method of ameliorating at least one symptom in a subject of a condition where it is beneficial to increase the level of activity of at least one of an M₁ and/or M₄ muscarinic receptor comprising: determining that the subject would benefit from an increased level of activity of at least one of an M], M₂ and/or M₄ muscarinic receptor; and administering an amount of at least one analog of claim 4 which is therapeutically effective to increase the level of activity of said at least one of an M₁, M₂ and/or M₄ muscarinic receptor and to ameliorate said at least one symptom to the subject.

17. The method of claim 16, wherein the subject is human.

18. The method of claim 16, further comprising contacting said subject with an additional therapeutic agent.

19. A method of ameliorating at least one symptom in a subject of a condition where it is beneficial to increase the level of activity of at least one of an M₁ and/or M₄ muscarinic receptor comprising: determining that the subject would benefit from an increased level of activity of at least one of an M₁, M₂ and/or M₄ muscarinic receptor; and administering an amount of at least one analog of claim 5 which is therapeutically effective to increase the level of activity of said at least one of an M₁, M₂ and/or M₄ muscarinic receptor and to ameliorate said at least one symptom to the subject.

20. The method of claim 19, wherein the subject is human.

21. The method of claim 19, further comprising contacting said subject with an additional therapeutic agent.

22. A method of ameliorating at least one symptom in a subject of a condition where it is beneficial to increase the level of activity of at least one of an M] and/or M₄ muscarinic receptor comprising: determining that the subject would benefit from an increased level of activity of at least one of an M₁, M₂ and/or M₄ muscarinic receptor; and administering an amount of at least one analog of claim 6 which is therapeutically effective to increase the level of activity of said at least one of an M₁, M₂ and/or M₄ muscarinic receptor and to ameliorate said at least one symptom to the subject.

23. The method of claim 22, wherein the subject is human.

24. The method of claim 22, further comprising contacting said subject with an additional therapeutic agent.

25. The method of claim 24, wherein the subject suffers from a condition selected from the group consisting of hallucinations, delusions, disordered thought, behavioral disturbance, aggression, suicidality, mania, impaired cognitive function, schizophrenia, and two or more any of the foregoing conditions.

26. A pharmaceutical composition for treating schizophrenia comprising an efficacious amount of at least one analog of claim 4 and at least one pharmaceutically acceptable carrier, diluent or excipient therefore.

27. A pharmaceutical composition for treating schizophrenia comprising an efficacious amount of at least one analog of claim 5 and at least one pharmaceutically acceptable carrier, diluent or excipient therefore.

28. A pharmaceutical composition for treating schizophrenia comprising an efficacious amount of at least one analog of claim 6 and at least one pharmaceutically acceptable carrier, diluent or excipient therefore.

29. A method of treating schizophrenia, comprising administering to a patient in need thereof an efficacious amount of an analog of claim 4 and pharmaceutically acceptable salts thereof and mixtures or salts thereof.

30. A method of treating schizophrenia, comprising administering to a patient in need thereof an efficacious amount of an analog of claim 5 and pharmaceutically acceptable salts thereof and mixtures or salts thereof.

31. A method of treating schizophrenia, comprising administering to a patient in need thereof an efficacious amount of an analog of claim 6 and pharmaceutically acceptable salts thereof and mixtures or salts thereof.

32. A method of ameliorating symptoms of schizophrenia, comprising administering to a patient in need thereof an efficacious amount of an analog of claim 4 and pharmaceutically acceptable salts thereof and mixtures or pharmaceutically acceptable salts thereof.

33. The method of claim 32, wherein the subject is human.

34. The method of claim 32. further comprising contacting said subject with an additional therapeutic agent.

35. A method of ameliorating symptoms of schizophrenia, comprising administering to a patient in need thereof an efficacious amount of an analog of claim

5 and pharmaceutically acceptable salts thereof and mixtures or pharmaceutically acceptable salts thereof.

36. The method of claim 35, wherein the subject is human.

37. The method of claim 35, further comprising contacting said subject with an additional therapeutic agent.

38. A method of ameliorating symptoms of schizophrenia, comprising administering to a patient in need thereof an efficacious amount of an analog of claim

6 and pharmaceutically acceptable salts thereof and mixtures or pharmaceutically acceptable salts thereof.

39. The method of claim 38, wherein the subject is human.

40. The method of claim 38, further comprising contacting said subject with an additional therapeutic agent.

41. A compound of claim 4, or an acid addition salt, solvate or hydrate thereof.

42. A compound of claim 5, or an acid addition salt, solvate or hydrate thereof.

43. A compound of claim 6, or an acid addition salt, solvate or hydrate thereof.

44. A pharmaceutical composition, comprising a therapeutically effective amount of a compound according to claim 4, and a pharmaceutically acceptable carrier.

45. A pharmaceutical composition, comprising a therapeutically effective amount of a compound according to claim 5, and a pharmaceutically acceptable carrier.

46. A pharmaceutical composition, comprising a therapeutically effective amount of a compound according to claim 6, and a pharmaceutically acceptable carrier.