WO2006050148A2 - Propargyl trifluoromethoxy aminobenzimidazole derivatives - Google Patents

Abstract

Disclosed are compounds having the structure: wherein X is a) where R1 is H, C1-C6 alkyl, C3-C6 alkenyl, or C3-C6 alkynyl; R2 is H or C1-C4 alkyl; and R3 is H or C1-C4 alkyl, or b) -OH, halide, SR5, SO3H or SO2R5

Description

PROPARGYL TRIFLUOROMETHOXY AMINOBENZIMIDAZOLE DERIVATIVES

This application claims benefit of U.S. Provisional Application No. 60/623,734, filed October 29, 2004, the contents of which are hereby incorporated by reference.

Throughout this application various publications, published patent applications, and published patents are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Background of the Invention

N-Propargyl- (R) -1-aminoindan (rasagiline) mesylate, a highly selective MAO-B inhibitor, is currently being developed as an anti-Parkinsonian drug. In addition to its anti-Parkinsonian activity, this compound has been shown to be neuroprotective both in vitro, in various neuronal cell culture models, and in vivo (M. B.H. Youdim et al . , Cell. MoI. Neurobiol. (2002) 21:555-73, and Akao et al . , J. Neurochem. (2002) 82:913-23) . It has also been reported to enhance cognition, increase survival, and prevent stroke in SH rats (S. Eliash et al., J. Neural. Transm. (2001) 108:909-23); to enhance SOD and catalase activities in the dopaminergic system in the rat (K. Kitani et al., J. Neural Transm. (2000) Suppl. 60:139-56); and to regulate amyloid precursor protein (M. Yogev-Falach et al., Neural Plasticity (2002) 9(2) :124) . This activity profile of rasagiline has been attributed to the propargylamine pharmacophore attached to the bicyclic indan ring system.

Propargyl trifluoromethoxy aminobenzothiazole derivatives have been shown to be effective in several experimental allergic encephalomyelitis ("EAE") models, see, for example, PCT International Publication No. WO 2004/047756, published June 10, 2004. EAE is an accepted animal model of autoimmune disorder (Tisch et al. Proc. Natl. Acad. Sci. USA (1994) 91:437-438) .

2-Amino derivatives of benzimidazoles (no propargyl, no OCF₃) have been known as antiallergics (emedastine, astemizole, mizolastine) , anthelmintic (albendazole, cyclobendazole, flubendazole, mebendazole) , antiemetic (lerisetron) . 2- Propargylamino benzimidazole derivatives (no trifluoromethoxy group) were reported as antiarrhythmics (PCT International Application Publication No. WO 91/18904), herbicides (U.S. Patent No. 3,399,987), photographic stabilizers (U.S. Patent No. 4,451,557) .

5 (6) -Trifluoromethoxy-lH-benzimidazol-2-yl-amine has been disclosed in U.S. Patent No. 5,049,574 as an excitatory amino acid neurotransmitter antagonist for the treatment of psychotic diseases including schizophrenia, epilepsy, and neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and cerebral ischemia.

Summary of the Invention

Heterocycles containing an imidazole ring instead of the thiazole ring have now been found to retain their efficacy in EAE models. Moreover, the presence of a basic nitrogen in the five membered ring enables the preparation of acid addition salts having good water solubility.

Trifluoromethoxy substituted benzimidazoles containing a propargylamino moiety at the 2 position are novel .

The subject invention provides a compound having the structure:

wherein X is

a)

where

Ri is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl;

R₂ is H or C_x-C₄ alkyl; and R₃ is H or Ci-C₄ alkyl ,

or

b) -OH, halide, SR₅, SO₃H or SO₂Rs

where R₅ is Ci-C₄ alkyl, and

wherein R₄ is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl,

or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

Brief Description of the Figures

Figure 1: Daily mean scores of compound 6 in the CSJL study.

Figure 2: Daily mean scores of compound 6 in the Biozzi study.

Detailed Description of the Invention

The subject invention provides a compound having the structure:

wherein X is

a)

where Ri is H, Ci-C₆ alkyl, C3-C6 alkenyl, or C₃-C₆ alkynyl; R₂is H or C₁-C₄ alkyl; and R₃ is H or Ci-C₄ alkyl, or

b) -OH, halide, SR₅, SO₃H or SO₂R₅

where R₅ is Ci-C₄ alkyl, and wherein R₄ is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl,

or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In an embodiment, the compound has the structure:

wherein Ri is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl; R₂ is H or C₁-C₄ alkyl; R₃ is H or C₁-C₄ alkyl; and R₄ is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl,

or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In a further embodiment, the compound has the structure:

wherein Ri is H or Ci-C₄ alkyl or C₃-C₆ alkynyl; R₂ is H or Ci-C₄ alkyl; R₃ is H or Ci-C₄ alkyl; and R₄ is H or Ci-C₄ alkyl or C₃- C₆ alkynyl,

or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound has the structure:

In a further embodiment, the compound has the structure:

In a further embodiment, the compound has the structure:

In these embodiments, R₄ may be Ci-C₄ alkyl or C3-C₆ alkynyl .

The chiral carbon in the disclosed compounds may be in the R configuration when R₂ is Ci-C₄ alkyl. Alternatively, the chiral carbon may be in the S configuration when R₂ is Ci-C₄ alkyl.

Alternatively, in the disclosed embodiments, Ri may be H or methyl; R₂ may be H or methyl; R₃ may be H or methyl; and R₄ may be H, methyl, or propargyl . Pharmaceutically acceptable salts of the disclosed embodiments are also provided and may be mesylate, maleate, fumarate, tartarate, hydrochloride, hydrobromide, esylate, p- toluenesulfonate, benzoate, acetate, phosphate or sulfate salt.

In another embodiment, the compound has the structure:

or

In an alternative embodiment, the compound has the structure:

In yet another embodiment, the compound has the structure:

In a further embodiment, the compound has the structure:

In a further embodiment, the compound has the structure:

or

In a further embodiment, the compound has the structure:

Any one of the disclosed compounds, individually, may be a hydrochloride salt. Alternatively, any one of the disclosed compounds, individually, may be a mesylate salt.

The subject invention also provides a method for treating a subject afflicted with a neurologic disorder comprising administering to the subject a therapeutically effective amount of any one of the compounds disclosed herein so as to thereby treat the neurologic disorder in the subject. The neurologic disorder may be Parkinson's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis, stroke, a neuromuscular disorder, schizophrenia, cerebral infarction, head trauma, glaucoma, facialis or Huntington's Disease.

The subject invention also provides a method for treating a subject afflicted with multiple sclerosis comprising administering to the subject a therapeutically effective amount of a compound of any one of the compounds disclosed herein so as to thereby treat multiple sclerosis in the subject. Such a method may further comprise also administering to the subject a therapeutically effective amount of levodopa, glatiramer acetate, interferon beta-lb, interferon beta-la, steroids or Mitoxantrone. In the disclosed methods, the therapeutically effective amount may be from about 1 to about 1000 mg/day, e.g. 2 to 800 mg/day, 5 to 500 mg/day, lOto 300 mg/day or 20 to 100 mg/day.

In the disclosed methods, the therapeutically effective amount of the compound may be administered by injection, systemically, orally or nasally.

The subject invention also provides a pharmaceutical composition comprising any one of the compounds disclosed herein and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the subject invention can further comprise a therapeutically effective amount of levodopa, glatiramer acetate, interferon beta-lb, interferon beta-la, steroids or Mitoxantrone.

In a specific embodiment, the pharmaceutical composition comprises a therapeutically effective amount of glatiramer acetate.

The subject invention also provides a process for the manufacture of a pharmaceutical composition comprising admixing any one of the compounds disclosed herein with a pharmaceutically acceptable carrier.

The subject invention also provides a packaged pharmaceutical composition for treating a neurologic disorder in a subject comprising:

(a) the pharmaceutical composition of the subject invention; and

(b) instructions for using the composition for treating the neurologic disorder in the subject. The subject invention also provides a compound having the structure:

wherein X is -OH, halide, SR₅, SO₃H or SO₂Rs

where R₅ is Ci-C₄ alkyl, and

wherein R₄ is H, C_x-C₆ alkyl, C3-C6 alkenyl or C3-C6 alkynyl,

or an enantiomer thereof, or a tautomer thereof.

In an embodiment, the compound has the structure:

or

In another embodiment, the compound has the structure:

or

In a further embodiment, the compound has the structure:

or

In a further embodiment, the compound has the structure:

In yet a further embodiment, the compound has the structure;

or

In yet another embodiment, the compound has the structure:

or

The subject invention also provides a process of manufacturing a compound having the structure:

wherein R₁Is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl; R₂ is H or Ci-C₄ alkyl; R₃ is H or Ci-C₄ alkyl; and R₄ is H, C_x-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl,

or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

reacting

with diphosgene or triphosgene to provide:

(b) treating A with a chlorinating agent to provide

(c) reacting B with

to provide the compound.

In an embodiment of the process, the product of step (c) may be reacted with BrR4, IR₄, or CIR₄ in a polar solvent in the presence of a base.

In another embodiment of the process, the chlorinating agent in step (b) may be POCl₃.

In a further embodiment of the process, the polar organic solvent may be acetonitrile and the base may be potassium carbonate.

The subject invention also provides a process of manufacturing a compound having the structure:

wherein R₁Is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl; R₂ is H or C₁-C₄ alkyl; R₃ is H or Ci-C₄ alkyl; and R₄ is H, C₁-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl, or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

(a) reacting

with an isothiocyanate of the formula R₃C≡C-CH(R₂)NCS in the presence of a solvent to provide:

b) reacting C with methyl iodide to provide the compound.

In an embodiment of the process, the product of step (b) may be reacted with BrR₄, IR₄, ClR₄, BrR₅, IR₅, or ClR₅ in a polar solvent in the presence of a base.

In another embodiment of the process, the solvent in step (a) may be CCl₄.

The subject invention also provides a process of manufacturing compounds having the structure:

comprising the steps of:

(a) reacting

with an isothiocyanate of the formula R₃C≡C-CH(R₂)NCS in the presence of a solvent to provide a reaction product; and

(b) reacting the product of step (a) with methyl iodide to manufacture the compound.

In an embodiment of the process, the product of step (b) may be purified. In another embodiment of the process, the solvent in step (a) may be CCI4.

The subject invention also provides a process of manufacturing compounds having the structure:

or

comprising the steps of:

with oxone to manufacture the compound.

In another embodiment of the process, the solvent in step (a) may be CCI₄.

The subject invention also provides a process of manufacturing compounds having the structure:

or

comprising the steps of:

(a) reacting

with potassium ethyl xanthogenate to provide:

(b) reacting D with sodium percarbonate to provide the compound.

The subject invention also provides the use of any one of the compounds disclosed herein for manufacturing a medicament useful- for treating a neurologic disorder in a subject. The neurologic disorder may be Parkinson's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis, stroke, a neuromuscular disorder, schizophrenia, cerebral infarction, head trauma, glaucoma, facialis or Huntington's Disease.

The subject invention also provides the use of any one of the compounds disclosed herein for manufacturing a medicament useful for treating multiple sclerosis in a subject. The medicament may further comprise levodopa, glatiramer acetate, interferon beta-lb, interferon beta-la, steroids or Mitoxantrone.

The subject invention also provides the use of any one of the compounds disclosed herein for manufacturing a medicament in a package having instructions for administration of the medicament to treat a neurologic disorder in a subject.

As used herein, "Ci-C_x", e.g. "Ci-C_x alkyl", is defined to include any one of the groups having 1, 2, ... x-1, or x carbons in a linear or branched arrangement. Similarly, "C₃-C_x alkenyl" is defined to include any one of the groups having 3, 4, ... x-1 or x carbons in a linear or branched arrangement.^' Similarly, "C₃~C_X alkynyl" is defined to include any one of the groups having 3, 4, ... x-1 or x carbons in a linear or branched arrangement.

Those skilled in the art will be familiar with the fact that some compounds of this invention can exist as tautomers. The compounds of this invention are therefore also to be understood as meaning, hereinabove and hereinbelow, the relevant tautomers, even when the latter are not mentioned specifically in each individual case. This invention also relates to the use of all such tautomers and mixtures thereof.

The invention further contemplates the use of prodrugs which are converted in vivo to the therapeutic compounds of the invention (see, e.g., R.B. Silverman, 1992, "The Organic Chemistry of Drug Design and Drug Action", Academic Press, Chapter 8, the entire contents of which are hereby incorporated by reference) . Such prodrugs can be used to alter the biodistribution (e.g., to allow compounds which would not typically enter the reactive site of the protease) or the pharmacokinetics of the therapeutic compound.

As set out above, certain embodiments of the present compounds can contain a basic functional group, such as amino or alkylamino, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19) . The term "pharmaceutically acceptable salts" as used herein also includes a quaternary ammonium salt.

It will be noted that the structure of some of the compounds of this invention includes asymmetric carbon atoms and thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. All such isomeric forms of these compounds are expressly included in this invention. Each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemical^ controlled synthesis.

When the compounds of the present invention are administered as pharmaceuticals, to humans and mammals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can performs its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; pplyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations .

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA) , butylated hydroxytoluene (BHT) , lecithin, propyl gallate, alpha- tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA) , sorbitol, tartaric acid, phosphoric acid, and the like. Formulations of the present invention include those suitable for oral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, pills, tablets, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. In solid dosage forms of the invention for oral administration

(capsules, tablets, pills, dragees, powders, granules and the like) , the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose) , lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose) , surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes . The active ingredient can also be in micro¬ encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert dilutents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils) , glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert dilutents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration, " "administered systematically, " "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

It is noted that the compounds of the invention can prevent neuronal death and improve the outcome in various models resembling human degenerative disorders.

The phrase "neurologic disorder" as used herein refers to a disorder whose adverse affects are localized in the nervous system.

The term "neurotrauma" as used herein, refers to damage to the central or peripheral nervous system caused by a traumatic event, such as head trauma, spinal trauma, neurotoxic injury, stroke, ischemia, hypoxia, or anoxia.

The term "stroke" or "ischemic stroke" as used herein means a brain infarct manifested by neurologic deficits. "Stroke" may refer to a "stroke in evolution" in which the infarction is still enlarging or a "completed stroke" in which the infarction size is no longer growing. (THE MERCK MANUAL, 17th EDITION, 1999 MERCK & CO.)

The phrase "treatment of stroke" as used herein is meant to include the treatment of the brain infarction per se or the treatment of the symptoms caused by the brain infarction. Such symptoms may include neurological deficits, cognitive disturbances, brain edema, decreased cerebral blood flow, catecholamine fluctuations, or neurological or motor disabilities. The invention is further illustrated by the following examples which in no way should be construed as being further limiting. It should be understood that the models used throughout the examples are accepted models and that the demonstration of efficacy in these models is predictive of efficacy in humans.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

Synthesis

Prop-2-ynyl- [5 (6) -trifluoromethoxy-lH-benzoimidazol-2-yl] - amines 6 and 15 were prepared via a 3-step procedure from 4- trifluoromethoxy-benzene-1,2-diamine 2 {Scheme I₁ route I) as follows: The latter was reacted with diphosgene or triphosgene (Clark et al, J. Am. Chem. Soc. (1958) 80:1957-62) to give 5 (6) -trifluoromethoxy-lH-benzoimidazol-2-ol 13 (Clark et al, J. Am. Chem. Soc. (1958) 80:1957-62), which was then converted to 2-chloro-5 (6) -trifluoromethoxy-lH-benzoimidazole 14 (PCT International Application Publication No. WO 91/189042). Reaction of the latter with N-propargylamines at elevated temperature, either neat in a sealed tube or in a suitable solvent, e.g., n-butanol, eventually afforded the corresponding 2-propargylamino derivatives (PCT International Application Publication No. WO 91/189042) . The procedure described above afforded compound 15 and compound 6. In an alternative embodiment, compound 6 was prepared via route II (PCT International Application Publication No. WO 91/189042). (Scheme I)₁ as follows: a regioisomeric mixture of l-(2-amino- 5-trifluoromethoxy-phenyl)3-prop-2-ynyl-thiourea 5 and l-(2- amino-4-trifluoromethoxy-phenyl) 3-prop-2-ynyl-thiourea 4 was reacted with methyl iodide in an appropriate solvent, such as ethanol. The thioureas 4 and 5 were prepared (PCT International Application Publication No. WO 91/189042; PCT International Application Publication No. WO 02/0769603) by the reaction of 4-trifluoromethoxy-benzene-1, 2-diamine 2 with propargyl isothiocyanate 3 in a suitable solvent such as carbon tetrachloride. The diamine 2 was prepared by reduction (Yagupolskii et al. , J. Gen. Chem. (1961) 31:8454; Mandel et al., J. Med. Chem. (1970) 13(6) :10435) of 2-nitro-4- trifluoromethoxy-phenylamine 1, a suitable reducing agent being, for instance, either stannous chloride and HCl or catalytic hydrogenation in the presence or absence of a mineral acid.

Ring substituted alkyl and alkynyl prop-2-ynyl- [5 (6) - trifluoromethoxy-lH-benzoimidazol-2-yl] -amines 7-10 were prepared (Sch. 2) by reacting 6 with alkyl and alkynyl halides, such as methyl iodide (PCT International Application Publication No. WO 91/189042) or propargyl bromide (Popov et al., Khim. Geterotsikl. Soedin (1973) 4:5516) in a polar organic solvent, such as acetonitrile, in the presence of a base, such as potassium carbonate. The compounds were prepared and used as their acid addition salts .

5 (6) -Trifluoromethoxy-2-substituted benzimidazoles were prepared from 2 as shown in Sch. 3. Compounds 11 and 12 were obtained in the conversion of the thioureas 4 and 5 to compound 6.

A synthesis scheme for the preparation of a group of propargylamino benzimidazoles is outlined below in Scheme 1.

Scheme 1

rou dι- or tn-phosgene

SMe

CF₃O_V^,N

\ 1 />-SMe

12

A synthesis scheme for the preparation of a group of ring substituted propargylamino benzimidazoles is outlined below in Scheme 2.

Scheme 2

A synthesis scheme for the preparation of a group of benzimidazoles is outlined below in Scheme 3.

Scheme 3

11 17 Synthetic Examples

Example 1.

Prop-2-ynyl- [5(6) -trifluoromethoxy-lH-benzoimidazol-2-yl] - amine HCl (6) Via thioureas 4 and 5:

1.1 4-Trifluoromethoxy-benzene-1,2-diamine (2)

1.1.1 by stannous chloride

A solution of 2-nitro-4-trifluoromethoxy-phenylamine 1 (25.0 g, 0.112 mol) in EtOH (200 ml) was added to solution of stannous chloride dihydrate (82.0 g, 0.363 mol) in cone HCl (330 ml) . The resulting suspension was stirred for 0.5 h at 70-80°C, cooled at 0-5°C, and basified to pH=ll-12 by 40% NaOH solution, and the product extracted with ether (2^x200 ml) . The organic solution was washed with water, dried over Na₂Sθ₄ and evaporated to dryness under reduced pressure to give the title compound as a brown oil (21.1 g, 98%).

1.1.2 by catalytic hydrogenation

2-Nitro-4-trifluoromethoxy-phenylamine 1 (25.0 g, 0.112 mol) was dissolved in EtOH (300 ml), and cone HCl (29 ml) and 10% Pd/C (2.5 g) was added, and the mixture was reacted for 1 h with H₂ at 2-3 atm at 30-35⁰C. The suspension was filtered and the filtrate was evaporated to dryness under reduced pressure. The solid was collected, washed with cold EtOH (20-30 ml) and dried to give 25.8 g (80 %) of the HCl salt of the title compound, mp 255-26O⁰C (decomp.).

1.2 1- (2-amino-5-trifluoromethoxy-phenyl)3-prop-2-ynyl- thiourea (5) and 1- (2-amino-4- trifluoromethoxy-phenyl) 3- prop-2-ynyl-thiourea (4) To a solution of 4-trifluoromethoxy-benzene-1, 2-diamine 2 (21.1 g, 0.11 mol) in CCl₄ (200 ml) was added dropwise (over 45-60 min) a solution of propargyl isothiocyanate (11.6 g, 0.12 mol) in CCl₄ (100 ml ). The suspension was stirred at rt for 2.5-3 h, and the solid was collected, washed with CCl₄ and dried to give two regioisomeric thioureas 4 and 5 (29.2 g, 92%) .

1.3 Prop-2-ynyl- [5(6) -trifluoromethoxy-lH-benzoimidazol-2- yl]-amine HCl (6)

The thioureas mixture (55 g, 0.19 mol) was dissolved in ethanol (1 lit) and methyl iodide (114.0 g, 50 ml, 0.80 mol) was added. The clear solution was heated at reflux for 3 h under N₂, cooled to rt and evaporated to dryness under reduced pressure. The residue was dissolved in CH₂Cl₂ (300 ml) and the solution washed with 1% ammonia solution at pH=10-ll. The organic layer was separated, dried over sodium sulfate and evaporated to dryness. The residue was dissolved in CH₂Cl₂ and cooled at -20⁰C overnight. The resulting white solid was collected by filtration, washed with CH₂Cl₂ and dried to give 12.6 g. (26%) of 6 as a free base The yield was further increased (7.5 g, 15.5%) by flash column chromatography (30:70 EtOAc:CHC1₃) of the filtrate, to give a total of 20.1 g (41.5%) . This crude product was crystallized from acetone- chloroform (1:9) to afford 18.4 g (38%) of the free base mp 161-162°C.

The latter was dissolved in ether (500 ml) and 20% HCl in EtOH was added to pH=l-2, and the white solid was collected by filtration and dried to give 19.2 g (91%) of the HCl salt, mp 171-2°C. Anal calcd for CnH₈F₃N₃O.HCl : C, 45.30; H, 3.11; N, 14.41; Cl, 12.15. Found: C, 45.40; H, 3.21; N, 14.43; Cl, 11.75.

¹H NMR (DMSO-d₆, 300 MHz) δ: 9.81 (m, IH, -MfCH₂C≡CH) , 7.51 (d,

IH, C4-H), 7.43 (d, IH, C7-H) , 7.25 (dd, IH, C5-H) , 4.34 (dd, 2H, -CH_∑C≡CH) , 3.47 (t, IH, -CH₂C≡CH) . MS: 256 (MH⁺, 100) , 217 (24) .

The mesylate salt was prepared by dissolving the free base

(40.0 g) in methyl t-butyl ether (600 ml) at 50-55^aC, and adding dropwise over a period of 30 min a solution of methanesulfonic acid (16.6 g) in methyl t-butyl ether (100 ml) , stirring the resulting suspension for 30 min at rt, and ice-cooling for another 30 min. The solid was collected by filtration and dried to give the mesylate salt as a white solid, mp: 123-5²C.

Anal calcd for CnH₈F₃N₃O-CH₄O₃S : C, 41.03; H, 3.44; N, 11.96; S, 9.13. Found: C, 41.29; H, 3.54; N, 11.68; S, 9.31.

¹H NMR (DMSO-d₆, 300 MHz) δ: 9.56 (m, IH, NH), 7.52 (d, IH, C4- H) , 7.46 (d, IH, C7-H) , 7.27 (dd, IH, C5-H) , 4.28 (dd, 2H, - CH^C≡CH), 3.49 (t, IH, -CH₂C≡CH) , 2.39 (s, 3H, Me) . MS: 256 (MH⁺, 100) , 217 (24) .

Example 2.

Prop-2-ynyl- [5(6) -trifluoromethoxy-lH-benzoimidazol-2-yl] - amine HCl (6) Via compound 14:

A mixture of 2-chloro-5 (6) -trifluoromethoxybenzimidazole (14, 2.0 g, 8.5 mmol) and N-propynyl amine (8.0 g, 0.145 mol) was heated in sealed tube for 1 hr at 140°C (oil bath temperature) . Excess N-propynylamine was removed under reduced pressure and the residue was purified by flash column chromatography (20:80 EtOAc:hexane) to give 0.32 g (15 %) of the free base, mp: 160- 161°C, which was converted to the HCl salt as described above.

Example 3.

(1-Methyl- 6-trifluoromethoxy-lH-benzoimidazol-2-yl) -prop-2- ynylamine HCl (7) and (1-Methyl- 5-trifIuoromethoxy-1H- benzoimidazol-2-yl) -prop-2-ynylamine HCl (8)

A mixture of prop-2-ynyl- [5 (6) -trifIuoromethoxy-1H- benzimidazol-yl] -amine 6 (0.9 g, 3.5 mmol) , methyl iodide (2.0 g, 14.1 mmol) and K₂CO₃ (0.5 g, 3.6 mmol) in acetonitrile (10 ml) was heated at reflux for 3 h, cooled to rt and diluted with water (50 ml) . This aqueous solution was extracted with ether (50 ml), the organic phase was separated, dried over Na₂SO,! and evaporated to dryness under reduced pressure. The crude product was purified by flash column chromatography (1:1 EtOAc rhexane) to give 0.5 g (53%) of a 1:1 mixture of 5- and 6-trifluoromethoxy (l-methyl-lH-benzimidazol-2-yl) -prop-2- ynylamine.

This mixture was dissolved in ether (20 ml) and 20% HCl in EtOH was added to pH=l-2. The white solid was collected by filtration and dried to give 0.5 g (47%) .

¹H NMR of 7 HCl salt (DMS0-d₆, 300 MHz) δ: 9.96 (br s, IH,

HN₃ ⁺), 7.65 (d, IH, C4-H) , 7.45 (d, IH, C7-H) , 7.33 (dd, IH,

C5-H) , 4.39 (br s, 2H, CH_∑C≡CH) , 3.71 (s, 3H, Me), 3.48 (t, IH, -CH₂C=CH) .

¹H NMR of 8 HCl salt (DMSO-d₆, 300 MHz) δ: 9.96 (br s, IH,

HN₃ ⁺), 7.73 (d, IH, C4-H) , 7.56 (d, IH, C7-H) , 7.30 (dd, IH, C6-H) , 4.40 (br s, 2H, Cff^C≡CH) , 3.71 (s, 3H, Me), 3.47 (t, IH, -CH₂C≡CH) . Example 4 .

Prop-2-ynyl- (l-prop-2-ynyl-6-trifIuoromethoxy-1H- benzoimidazol-2-yl) -amine HCl (9) and Prop-2-ynyl- (l-prop-2- ynyl-5-trifluoromethoxy-lH-benzoimidazol-2-yl) -amine HCl (10)

A mixture of prop-2-ynyl- [5 (6) -trifIuoromethoxy-1H- benzimidazol-yl] -amine 6 (1.3 g, 5.1 mmol) , propargyl bromide

(0.86 ml, 7.6 mmol, 80 % in toluene) and K₂CO₃ (1.38 g, 10.0 mmol) in acetonitrile (15 ml) was heated at reflux for 2.5 h.

The solvent was evaporated under reduced pressure to dryness to give a viscous brown oil (1.8 g) , which was purified by flash column chromatography (5:95 EtOACrCHCl₃) to give prop-2- ynyl- (l-prop-2-ynyl-5-trifluoromethoxy-lH-benzoimidazole-2- yl) -amine (0.6 g, 40%, eluting first) and prop-2-ynyl- (1-prop- 2-ynyl-6-trifluoromethoxy-lH-benzoimidazole-2-yl) -amine (0.58 g, 39%) . The two free bases were converted to the respective HCl salts. Total yield: 75%.

¹H NMR of 10 free base (DMSO-d_6/ 300 MHz) δ: 7.52 (t, IH, - MJCH₂C=CH) , 7.32 (d, IH, C7-H) , 7.27 (d, IH, C4-H) , 6.95 (dd, IH, C6-H) , 4.95 (d, 2H, N₁-CHsCsCH) , 4.22 (dd, 2H, CH₂C=CH) , 3.37 (t, IH, N₁-CH₂C=CH), 3.15 (t, IH, -CH₂C=CH) . MS: 294 (MH⁺, 57), 254 (100), 227 (31), 208 (67) .

¹H NMR of 9 free base (DMSO-d₆, 300 MHz) δ: 7.51 (t, IH, - i\7HCH₂C≡CH) , 7.32 (d, IH, C7-H) , 7.29 (d, IH, C4-H) , 7.0 (dd, IH, C5-H) , 4.98 (d, 2H, N₁-CHzC=CH), 4.18 (d, 2H, CH_jC=CH) , 3.37 (t, IH, N₁-CH₂C=CH), 3.16 (t, IH, -CH₂C=CH) . MS: 294 (MH⁺, 96), 254 (100), 227 (41), 208 (88) .

Example 5.

Methyl-prop-2-ynyl- [5(6) -trifluoromethoxy-lH-benzoimidazol-2- yl] -amine HCl (15) 5.1 5 (6) -Trifluoromethoxy-lH-benzoimidazol-2-ol (13)

To a solution of 4-trifluoromethoxy-benzene-1, 2-diamine 2 (4.5 g, 23.4 itimol) and triethylamine (5.0 g, 49.5 iranol) in methylene chloride (100 ml) was added dropwise a solution of diphosgene (2.4 g, 12.1 mmol) in methylene chloride (10 ml) at 0-5°C. The resulting suspension was stirred for 1 h at rt and filtered. The collected white solid was washed with water and dried to give 3.85 g (75%), mp 260-262⁰C.

5.2 2-Chloro-5 (6) -trifluoromethoxy-lH-benzoimidazol (14) from 5 (6) -trifluoromethoxy-lH-benzoimidazol-2-ol (13) :

5(6)-Trifluoromethoxy-lH-benzoimidazol-2-ol (13) (3.8 g, 17 iranol) was heated under reflux in POCl₃ (30 ml) for 2 h, cooled to rt, and POCl₃ was removed under reduced pressure. The residual oil was poured onto ice-water, and 25% aqueous ammonia was added to pH 8-9. The resulting white solid was collected by filtration and dried to give 2.6 g (65%), mp 163- 165°C.

¹H NMR (DMSO-d₆, 300 MHz) δ: 7.64 (d, IH, C4-H) , 7.57 (d, IH, C7-H) , 7.25 (dd, IH, C5-H) . MS: 239, 237 (MH⁺, 100, 35) .

From 5 (6) -trifluoromethoxy-lH-benzoimidazol-2-sulfonic acid (16) :

A mixture of 5 (6) -trifluoromethoxy-lH-benzoimidazol-2-sulfonic acid (16, 2.82 g, 10 mmol), PCl₅ (6.24 g) and POCl₃ (5 ml) was refluxed for 6 h, cooled to rt and added carefully to ice- water. Aqueous ammonia was added to the mixture (pH 5-6), and the precipitated solid collected by filtration, washed with water and dried to give 1.3 g (5.5 mmol, 55 %) of the title compound. 5.3 Methyl-prop-2-ynyl- [5(6) -trifIuoromethoxy-1H- benzoimidazol-2-yl] -amine HCl (15)

In n-butanol:

A mixture of 2-chloro-5 (6) -trifluoromethoxy-lH-benzoimidazol (14) (0.5 g, 2.1 mmol) and N-methyl-N-propynylamine (1 g, 14.5 mmol) in n-butanol (5 ml) was heated under reflux for 4 h under a nitrogen atmosphere. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (20:80 EtOAc:hexane) to give 0.14 g (25%) of the title free base, which was then converted to the HCl salt

(0.1 g) .

Neat (sealed tube) :

A mixture of compound 14 (8.0 g, 34 mmol) and Ν-methyl-Ν- propynylamine (20.0 g, 0.29 mol) was heated in sealed tube for 1 hr at 130-140°C (oil bath temperature) . Excess Ν-methyl-Ν- propynylamine was removed under reduced pressure and the residue was purified by flash column chromatography (20:80 EtOAc:hexane) to give 2.7 g (30%) of the free base, mp: 163- 164°C. This free base was converted to the hydrochloride by dissolving it in ether (200 ml) and adding dropwise ethanolic HCl to pH 1-2. The salt was collected by filtration and dried to give 2.7 g of a white solid, mp: 207-8²C.

Anal. Calcd. For C_I2H_IOF₃N₃O-HCIJC, 47.15; H, 3.63; N, 13.75; Cl, 11.60.

Found: C, 47.17; H, 3.67; N, 13.83; Cl, 11.62.

¹H NMR (DMSO-d_6/ 300 MHz) δ: 7.55 (d, IH, C4-H) , 7.43 (d, IH, C7-H) , 7.28 (dd, IH, C5-H) , 4.64 (d, 2H, -CT₂C≡CH) , 3.59 (t, IH, -CH₂C≡CH) , 3.33 (s, 3H, Me) . MS: 270 (MH⁺, 100), 231 (14) . The mesylate salt was prepared by dissolving the free base (1.16 g, 4.3 mmol) in ether (85 ml) and adding methanesulfonic acid (0.35 ml) . The resulting suspension was stirred at room temperature for 2 h, filtered and the collected solid washed well with ether followed by hexane, to give 1.4 g of an off- white solid, mp: 182-4°C.

¹H NMR (DMSO-d₆ 600 MH_z)δ:760 (d, IH, C4-H) , 7.54 (br s, IH, C7-H) , 7.30 (dd, IH, C5-H) , 4.51 (d, 2H, -CH₂C=CH)₁ 3.60 (br t, IH, -CH₂C≡CH) , 3.30 (s, 3H, N-Me), 2.49 (s, 3H, Me) .

¹³C NMR (DMSO-d₆, 150 MH₂) δ: 151.36 (C2, imidazole), 144.11, 130.85, 129.29 (Ar-C3_a, C6, C7_a) , 120.0 (CF₃O), 116.77, 112.65, 105.45 (3^xArCH), 77.11, 76.87 (triple bond), 40.72 (-CH₂C≡CH) , 39.73 (MeSO₃H), 36.67 (NMe) .

MS: 270 (MH⁺, 100), (MH⁺ - CH₂C≡CH, 15) .

Example 6.

5 (6) -Trifluoromethoxy-lH-benzoimidazol-2-sulfonic acid (16)

5 (6) -Trifluoromethoxy-lH-benzoimidazol-2-thiol (18, 3.27 g, 13.97 mmol, prepared from 2 and potassium ethyl xanthogenate as described in US 4555518) was added portionwise over 1 h to a solution of sodium percarbonate (6.0 g) in water (65 ml) . The reaction mixture was refluxed for 4 h, cooled to room temperature, treated with charcoal and filtered. The filtrate was acidified to pH 1 (cone HCl) , and the resulting suspension was filtered. The collected solid was dried to give 2.9 g (10.28 mmol, 73.5 %) of the title compound. Example 7 .

2-Methylsulfanyl-5 (6) -trifluoromethoxy-lH-benzoimidazole (11) and l-methyl-2-methylsulfanyl-5 (6) -trifIuoromethoxy-1H- benzoimidazole (12) .

Compounds 11 (solid, R_f=0.7, 23.6 g, mp: 142-3°C) and 12 (oil, R_f=O.75) were obtained as side products during the chromatographic separation of 6 (Example 1.3, EtOAc:CHCl₃ 30:70) .

¹H NMR (DMSO-d₆, 200 MHz) δ:

Compound 11: 7.53 (d, IH, Ar-H), 7.49 (s, IH, Ar-H), 7.10 (d, IH, Ar-H), 2.77 (s, 3H, S-Me) .

Compound 12: 7.60 (d, IH, Ar-H), 7.55 (s, IH, Ar-H), 7.15 (d, IH, Ar-H), 3.71 (s, 3H, N-Me), 2.77 (s, 3H, S-Me) .

Example 8.

2-Methylsulfonyl-5 (6) -trifluoromethoxy-lH-benzoimidazole (17)

A solution of 2-methylsulfanyl-5 (6) -trifIuoromethoxy-1H- benzoimidazole (11, 4.9 g, 19.7 mmol) in MeOH (100 ml) was cooled to 0-5⁰C, and a solution of potassium peroxymonosulfate (18.3 g, 29.7 mmol) in water (100 ml) was added dropwise. The reaction mixture was heated to room temperature, stirred overnight and concentrated under reduced pressure. The suspension was filtered to give 5.5 g (99%) of the title compound as a white solid, mp: 115-117°C.

¹H NMR (CDCl₃, 300 MHz) δ: 7.83 (br s, 2H, Ar), 7.42 (d, IH, Ar), 3.52 (s, 3H, Me) ppm. MS : 281 (MH⁺, 100), 233 (12), 218 98) . Biological Activity

1. EAE studies

1.1 General

Preparation of test solutions of compound 6:

A 8.0 mg/mL stock solution of the test article was prepared daily in purified water for dose level of 80.0 mg/kg. The sample was crushed, weighed and dissolved in purified water. The 8.0 mg/mL solution was vortexed and diluted to 0.5, 1.0, 2.0 and 4.0 mg/mL solution in purified water for dose levels of 5, 10, 20 and 40 mg/kg x 2.

Test article administration:

The mice were administered with the respective dose levels of compound 6 at volume dose level of 200 μL/mouse. The test formulations were vortexed before dispension in syringe.

These test articles were administered to the respective groups by oral gavage daily for 30 consecutive days, starting from the day of induction until the termination of the study.

The test formulations of compound 6 were administered to the treatment groups twice a day except on weekends. On weekends the same concentrations of the test articles were administered once a day.

The vehicle was administered to the control group in a similar manner. Quantitation of Experimental Observations:

Morbidity and mortality:

All animals were examined once daily to detect if any was dead or moribund.

a. Clinical signs:

Scoring of EAE clinical signs was initiated from day 10 (CSJL) and Day 15 (Biozzi) post-EAE induction and was continued daily for the duration of the study. The clinical signs were recorded on observation cards according to a grading system described in Table 1.

Table 1 Evaluation of the EAE clinical signs

All mice having scores of 1 and above were considered sick. All animals having a score of 4 were sacrificed on humane grounds.

For calculation purposes, the score of animals that were sacrificed (4) or died (5) was carried forward.

b. Interpretation of results:

Calculation of the incidence of disease (Disease ratio) :

^■ The number of sick animals in each group was summed

^■ The incidence of disease was calculated as

No. sick mice (treated grp)

Incidence of disease =

No. sick mice (control grp)

Calculation of mean delay in onset of disease:

The mean delay in onset of disease expressed in days was calculated by subtracting the mean onset of disease in the control group from the test group.

For calculation purposes, the onset period for a mouse that did not develop EAE during the observation period was considered as 31 days.

Calculation of the mean maximal score and percent inhibition:

The mean maximal score (MMS) of each group was calculated as:

Σ maximal score of each mouse/number of mice in the group.

The percent inhibition (%I) was calculated as %I = 1- MMS of treated group x 100 MMS of control group

Calculation of the mean group score and percent inhibition: The daily scores of each mouse in the test group was summed and the individual mean daily score (IMS) was calculated as

Σ daily score of mouse/observation period (days).

The mean group score (GMS) was calculated as

Σ IMS of each mouse/number of mice in the group.

The percent inhibition (%I) was calculated as

(%I)= 1 - [GMS of treated group] x 100

GMS of control group

1.2 EAE in CSJL mice

Healthy, nulliparous, non-pregnant female mice of the CSJL/FI strain were obtained from Harlan Breeding Center, Jerusalem, Israel. The animals weighed about 17-20 g on arrival, and were approximately 7 weeks of age. The body weights of the animals were recorded on the day of delivery. Overtly healthy animals were assigned to study groups arbitrarily before treatment commenced. The mice were individually identified by markings on the body. Animal housing and care conditions were maintained according to SOP' s # 33.22.01 and 33.2.02.

EAE was induced by injecting the encephalitogenic mixture (emulsion) consisting of MSCH and commercial CFA containing 1 mg/ml Mycobacterium tuberculosis to the foot pads of the animals. Pertussis toxin was injected iv on the day of induction and 48 hrs later. The mortality rates, incidence, MMS and GMS values and the mean duration of the disease are given in Table 2. The daily- mean score values are depicted in Figure 1.

Table 2.

1.3 EAE in Biozzi mice (Effect of oral treatment with compound 6 on chronic relapsing EAE in Biozzi mice) Relapsing remitting (RR) EAE was induced in Biozzi mice by the subcutaneous injection of encephalitogenic agent (mouse spinal cord homogenate + incomplete freund's adjuvant enriched with mycobacterium tuberculosis) into one flank of the Biozzi mice. The injection was repeated one week later on the other flank.

Healthy, specific pathogen free, female Biozzi mice, obtained from Harlan Animal Breeding center, Jerusalem, Israel, were used in the study.

The mice were 7-9 weeks old on receiving day, weighing 2Og +_ 15%.

Animals housing and care conditions will be maintained according to Sops # 33.22.01 and 33.22.02.

On the day of first injection of the encephalitogenic agent, the mice were allocated to treatment groups and the treatment was initiated (preventive model) . The groups were treated orally, twice a day, daily for 59 consecutive days with 10.0, 20.0 or 40.0 mg/kg of compound 6 each and the control group treated with the vehicle (water) . On weekends the mice were treated once.

All the test articles were administered as a solution in water.

The mice were examined and scored for EAE clinical signs from Day 15 after disease induction, until the termination of the study (60 days after initiation of treatment) .

The activity of the groups was calculated by comparing the incidence of disease, duration of disease, group mean score and mean maximal score to the values obtained for the control group. The results are shown in Table 3. The daily mean scores are shown in Figure 2.

Table 3: Treatment with compound 6, Incidence, Mortality, Mean maximal score, Group mean score and duration during first and second relapse.

2. Neuroprotection studies (H₂O₂ model)

This assay, developed in primary cultures of embryonic cortical neurons, allows screening of compounds on neuronal cells that have undergone oxidative stress through exposure to H₂O₂.

Preparation of Cells: Cortices are removed from E17-18 rat embryos (Sprague-Dawley) into Hank's balanced salt solution (HBSS) and membranes and blood vessels are removed. The cortices are then chopped up into l-2mm squares and chemically dissociated in papain, followed by mechanical dissociation through tituration.

Cells are plated in 6-well plates pre-coated with Collagen and Poly-L-lysine at IxIO⁶ viable cells/well diluted in 2 ml of medium. Cells are raised in Minimum Essential Medium-Eagle (MEM; Biological Industries) , supplemented by 10% Foetal Calf Serum (FCS) and 5% Horse Serum (HS) , 2mM Glutamax (Gibco) , 6g/l glucose and Penicillin/Streptomycin. At the time of application media is further supplemented by B27 without antioxidants (Gibco) , a neuronal supplement, and the cultures are incubated in a 5% CO₂ incubator at 37⁰C.

After 4 days in vitro (DIV) half the medium was exchanged with the appropriate medium as described above, but further supplemented with lOμM Cytosine-Arabinofuranoside (to give a final concentration of 5μM) , to inhibit proliferation of glial cells. Cell counts showed that the percentage of neurons in these cultures is approximately 90%.

Toxicity:

At 6 DIV the medium is completely replaced with fresh NB medium supplemented with B27 w/o AO in preparation for toxicity tests. The cells are allowed to acclimatize to the fresh medium for 2-3 hours. For cells undergoing neuroprotective treatment, the test compound or positive control was added 45 minutes prior to the 250 μM H₂O₂ insult. The positive control used was 4-Hydroxy-TEMPO (TEMPOL; Sigma) at I mM. Cell death assay:

Cell death was measured by ascertaining levels of lactate dehydrogenase (LDH; Promega kit) . LDH is a cytosolic enzyme that is released into the medium in cell cultures when the cells die and the cell membrane is ruptured. It is a widely accepted method for measuring cell death and allows for on¬ line monitoring of cell death, if necessary, by removal of samples of the medium.

LDH in the samples catalyzes a chemical reaction with the solution provided in the kit, resulting in the formation of formazan (red) . The amount of cell death is determined by measuring the formation of formazan using visible wavelengths

(490nm) in a standard ELISA plate reader. Total cell death is determined by freezing all the cells at -80⁰C for 30 minutes followed by thawing at 37°C for 15 minutes.

The percentage of cell death is calculated as follows:

% Toxicity = [ (ODtest compound " 0D_controi ) / ( OD_totai-OD_control ) ] X 100

OD=optical density at 490nm

Percentage of cell death in wells exposed only to the H2O2 insult should be between 30-60%.

The extent of neuroprotection provided by test compounds or positive control is measured relative to the percentage of cell death in wells only exposed to the insult and not to the treatment and is calculated as follows:

^Neuroprotection =100- [(% death_test compound / % death_toXi_City) x 100] The results of the compounds of the invention (each treatment was allocated 1x6 well plate) are given below in Table 4.

Table 4.

Claims

What is claimed is:

1. A compound having the structure:

wherein X is

a)

where

Ri is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl ;

R₂ is H or Ci-C₄ alkyl; and

R₃ is H or Ci-C₄ alkyl,

or

b) -OH, halide, SR₅, SO₃H or SO₂R₅

where R₅ is Ci-C₄ alkyl, and

wherein R₄ is H, C_x-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl, or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 having the structure:

wherein

Ri is H, C₁-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl;

R₂ is H or Ci-C₄ alkyl;

R₃ is H or Ci-C₄ alkyl; and

R₄ is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl,

or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

3. The compound of claim 2, having the structure:

wherein Ri is H or Ci-C₄ alkyl or C₃-C₆ alkynyl;

R₂ is H or C1-C₄ alkyl;

R₃ is H or Ci-C₄ alkyl; and

R₄ is H or Ci-C₄ alkyl or C₃-Cε alkynyl,

or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof.

The compound of claim 3, having the structure:

The compound of claim 3, having the structure:

6. The compound of claim 3, having the structure:

7. The compound of claim 3, 4, 5 or 6 wherein R₄ is Ci-C₄ alkyl or C₃-C6 alkynyl.

8. The compound of claim 3, 4, 5 or 6, wherein when R₂ is Ci- C₄ alkyl, the chiral carbon is in the R configuration.

9. The compound of claim 3, 4, 5 or 6, wherein when R₂ is Ci- C₄ alkyl, the chiral carbon is in the S configuration.

10. The compound of claim 5 or 6, wherein

R₁ is H or methyl;

R₂ is H or methyl;

R₃ is H or methyl; and

R₄ is H, methyl, or propargyl.

11. The pharmaceutically acceptable salt of the compound of any one of claims 2-10, wherein the salt is the mesylate, maleate, fumarate, tartarate, hydrochloride, hydrobromide, esylate, p-toluenesulfonate, benzoate, acetate, phosphate or sulfate salt.

12. The compound of claim 5 or 6 having the structure:

or

13. The compound of claim 6 having the structure:

14. The compound of claim 6 having the structure:

15. The compound of claim 5 having the structure:

16. The compound of claim 5 or 6 having the structure:

or

17. The compound of claim 5 having the structure:

18. The hydrochloride salt of the compound of any one of claims 2-10 or 12-20.

19. The mesylate salt of the compound of any one of claims 2- 10 or 12-20.

20. A method for treating a subject afflicted with a neurologic disorder comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 2-19 so as to thereby treat the neurologic disorder in the subject. 21. The method of claim 20, wherein the neurologic disorder is Parkinson's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis, stroke, a neuromuscular disorder, schizophrenia, cerebral infarction, head trauma, glaucoma, facialis or Huntington's Disease.

22. The method of claim 20 or 21, wherein the therapeutically effective amount is from about 1 to about 1000 mg/day.

23. A method for treating a subject afflicted with multiple sclerosis comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 2-19 so as to thereby treat multiple sclerosis in the subject.

24. The method of claim 23, further comprising administering to the subject a therapeutically effective amount of levodopa, glatiramer acetate, interferon beta-lb, interferon beta-la, steroids or Mitoxantrone.

25. The method of claim 23, wherein the therapeutically effective amount is from about 1 to about 1000 mg/day.

26. The method of any one of claims 20-25 wherein the therapeutically effective amount of the compound is administered by injection, systemically, orally or nasally.

27. A pharmaceutical composition comprising the compound of any one of claims 2-19 and a pharmaceutically acceptable carrier.

28. The pharmaceutical composition of claim 27, further comprising a therapeutically effective amount of levodopa, glatiramer acetate, interferon beta-lb, interferon beta-la, steroids or Mitoxantrone.

29. The pharmaceutical composition of claim 27, further comprising a therapeutically effective amount of glatiramer acetate.

30. A process for the manufacture of a pharmaceutical composition comprising admixing the compound of any one of claims 2-19 with a pharmaceutically acceptable carrier.

31. A packaged pharmaceutical composition for treating a neurologic disorder in a subject comprising:

(a) the pharmaceutical composition of claim 27; and

(b) instructions for using the composition for treating the neurologic disorder in the subject.

32. The compound of claim 1 having the structure:

wherein X is -OH , halide , SR₅ , SO₃H or SO₂Rs

where R₅ is Ci -C₄ alkyl , and

wherein R₄ is H , Ci-Cε alkyl , C₃ -C₆ alkenyl or C₃ -Cε alkynyl ,

or an enantiomer thereof , or a tautomer thereof . 33. The compound of claim 32 having the structure:

or

34. The compound of claim 32 having the structure:

or

35. The compound of claim 32 having the structure:

or

36. The compound of claim 32 having the structure:

37 . The compound of claim 32 having the structure :

or

38. The compound of claim 32 having the structure:

or

39. A process of manufacturing the compound of claim 2 comprising the steps of:

(a) reacting

with diphosgene or triphosgene to provide:

(b) treating A with a chlorinating agent to provide

(c) reacting B with

to provide the compound.

40. The process of claim 39, further comprising reacting the product of step (c) with BrR₄, IR₄, or ClR₄ in a polar solvent in the presence of a base.

41. The process of claim 39, wherein the chlorinating agent in step (b) is POCl₃.

42. The process of claim 40, wherein the polar organic solvent is acetonitrile and the base is potassium carbonate.

43. A process of manufacturing the compound of claim 2 comprising the steps of:

(a) reacting

with an isothiocyanate of the formula R₃C=C-CH(R₂)NCS in the presence of a solvent to provide:

b) reacting C with methyl iodide to provide the compound.

44. The process of claim 43, further comprising reacting the product of step (b) with BrR₄, IR₄, ClR₄, BrR₅, IR₅, or ClR₅ in a polar solvent in the presence of a base.

45. A process of manufacturing the compound of claim 35 or 36 comprising the steps of:

(a) reacting

with an isothiocyanate of the formula R₃C≡C-CH(R₂)NCS in the presence of a solvent to provide a reaction product; and

(b) reacting the product of step (a) with methyl iodide to manufacture the compound.

46. The process of claim 45, further comprising purifying the compound.

47. A process of manufacturing the compound of claim 38 comprising the steps of:

(a) reacting

with potassium peroxymonosulfate to manufacture the compound.

48. The process of any one of claims 43-47, wherein the solvent in step (a) is CCl₄.

49. A process of manufacturing the compound of claim 37 comprising the steps of:

(a) reacting

with potassium ethyl xanthogenate to

provide :

D; and

(b) reacting D with sodium percarbonate to provide the compound.

50. Use of the compound of any one of claims 2-19 for manufacturing a medicament useful for treating a neurologic disorder in a subject.

51. The use of claim 50, wherein the neurologic disorder is Parkinson's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis, stroke, a neuromuscular disorder, schizophrenia, cerebral infarction, head trauma, glaucoma, facialis or Huntington' s Disease.

52. Use of the compound of any one of claims 2-19 for manufacturing a medicament useful for treating multiple sclerosis in a subject.

53. The use of claim 52, wherein the medicament further comprises levodopa, glatiramer acetate, interferon beta- Ib, interferon beta-la, steroids or Mitoxantrone.

54. Use of the compound of any one of claims 2-19 for manufacturing a medicament in a package having instructions for administration of the medicament to treat a neurologic disorder in a subject.

where R₅ is C₁-C₄ alkyl, and

wherein R₄ is H, Ci-C₆ alkyl, C₃-C₆ alkenyl, or C₃-C₆ alkynyl, or an enantiomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof. Also disclosed are uses and processes for preparation of the compounds .