Abstract:
This invention features a method for treating a neurodegenerative disease. The method includes administrating to a subject in need thereof a compound of the formula (I):  
                         
 
     A is H, R, or  
                         
 
     each of Ar 1 , Ar 2 , and Ar 3 , independently, is phenyl, thienyl, furyl, or pyrrolyl; each of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 , independently, is H, halogen, R, C(O)OH, C(O)OR, C(O)SH, C(O)SR, C(O)NH 2 , C(O)NHR, C(O)NRR′, ROH, ROR′, RSH, RSR′, NHR, NRR′, RNHR′, or RNR′R″; or R 1  and R 2  together, R 3  and R 4  together, or R 5  and R 6  together are ORO; wherein each of R, R′, and R″, independently is C 1 ˜C 6  alkyl; and n is 1, 2, or 3.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    This application is a continuation-in-part of, and claims priority to U.S. application Ser. No. 10/242,826, filed on Sep. 13, 2002, the contents of which are incorporated herein by reference. 
     
    
     
       BACKGROUND  
         [0002]    Neurodegenerative diseases, which represent a large group of neurological disorders, include Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington disease, glaucoma neurodegeneration and amyotrophic lateral sclerosis. These diseases are characterized by a progressive and specific loss of neurons. Patients with neurodegenerative diseases are likely to show localized to generalized atrophy of central and/or peripheral neurons, leading to compromises in both mental and physical functions. Mentally, patients exhibit forgetfulness, poor memory, reduced mental capacity, emotional disturbance, or unclear speech. Physically, patients exhibit partial to complete incontinence, aspiration of food particles, tremor, poor balance, muscle rigidity, or muscle paralysis.  
         SUMMARY OF THE INVENTION  
         [0003]    In one respect, this invention features a method for treating a neurodegenerative disease. The method includes administrating to a subject in need thereof a fused pyrazolyl compound of the formula (I):  
                         
 
           [0004]    A is H, R, or  
                         
 
           [0005]    (referred to as “(CH 2 ) n Ar 3 (R 5 )(R 6 )” hereinafter); each of Ar 1 , Ar 2 , and Ar 3 , independently, is phenyl, thienyl, furyl, or pyrrolyl; each of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 , independently, is H, halogen, R, C(O)OH, C(O)OR, C(O)SH, C(O)SR, C(O)NH 2 , C(O)NHR, C(O)NRR′, ROH, ROR′, RSH, RSR′, NHR, NRR′, RNHR′; or RNR′R″, or R 1  and R 2  together, R 3  and R 4  together, or R 5  and R 6  together are ORO. Each of R, R′, and R″, independently is C 1 ˜C 6  alkyl; and n is 1, 2, or 3. The compound is in an effective amount for treating a neurodegenerative disease.  
           [0006]    Formula (I) includes a pyrazolyl core and at least two aryl groups, e.g., Ar 1 , or Ar 2 . A subset of the compounds of formula (I) are featured by that A is (CH 2 ) n Ar 3 (R 5 )(R 6 ). In these compounds, Ar 1  is phenyl, and R 1  and R 2  are substituted at positions 4 and 5 of phenyl, respectively. Alternatively, Ar 2  is 5′-furyl, and one of R 3  and R 4  is substituted at position 2 of 5′-furyl. In some embodiments, Ar 3  is phenyl.  
           [0007]    Another subset of the compounds of formula (I) are featured by that A is H. In these compounds, Ar 1  is phenyl, and R 1  and R 2  are substituted at positions 4 and 5 of phenyl, respectively. Alternatively, Ar 2  is 5′-furyl, and one of R 3  and R 4  is substituted at position 2 of 5′-furyl.  
           [0008]    The term “aryl” as used herein includes phenyl, thienyl, furyl, or pyrrolyl, each of which optionally includes one or more substituted moieties. The substituted moieties may be the same as or different from R 1 , R 2 , R 3 , R 4 , R 5 , or R 6 . They can be halogen, amino, hydroxyl, mercapto, cyano, C 1 ˜C 6  alkyl, C 1 ˜C 6  alkenyl, C 1 ˜C 6  alkoxy, aryl, heteroaryl, or heterocycloalkyl, wherein alkyl, alkenyl, alkoxy, aryl, heteroaryl, and heterocycloalkyl are optionally substituted with C 1 ˜C 6  alkyl, halogen, amino, hydroxyl, mercapto, cyano. The term “alkyl” includes both linear and branched alkyl, which optionally includes one or more just-described substituted moieties.  
           [0009]    Set forth below is an example of a fused pyrazolyl compound that can be used to practice the method of this invention:  
                         
 
           [0010]    1-benzyl-3-(5′-methoxymethyl-2′-furyl)-indazole  
           [0011]    The fused pyrazolyl compounds described above include the compounds themselves, as well as their salts and their prodrugs, if applicable. Such salts, for example, can be formed between a negatively charged substituent (e.g., carboxylate) on a fused pyrazolyl compound and a cation. Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as teteramethylammonium ion. Likewise, a positively charged substituent (e.g., amino) can form a salt with a negatively charged counterion. Suitable counterions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, or acetate. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing fused pyrazolyl compounds described above.  
           [0012]    As used herein, the term “neurodegenerative disease” refers to a disorder associated with a progressive and specific loss of neurons. Examples include, but are not limited to, Parkinson&#39;s disease, Huntington&#39;s disease, inflammatory brain disease, Alzheimer&#39;s disease, amyotrophic lateral sclerosis, HIV-associated dementia, multiple sclerosis, glaucoma neurodegeneration and excitoneurotoxicity.  
           [0013]    It was unexpected that the method of this invention protects neurons from cytotoxicity induced by a variety of neurotoxicants such as 1-methyl-4-phenylpyridinium (MPP + ), kainic acid, 3-nitropropionic acid (3-NP), and lipopolysaccharide (LPS). Other features, objects, and advantages of the invention will be apparent from the description and from the claims.  
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0014]    A fused pyrazolyl compound used to practice the method of this invention can be prepared by procedures well known to a skilled person in the art (see, e.g., U.S. Pat. No. 5,574,168). They include the synthetic route that follows: An aryl aryl ketone is first prepared by coupling an arylcarbonyl chloride with another aryl compound. Either aryl compound is optionally mono- or multi-substituted. The ketone then reacts with an arylalkylhydrazine, the aryl group of which is also optionally mono- or multi-substituted, to form a hydrazone containing three aryl groups. The hydrazone group is transformed into a fused pyrazolyl core via an alkylene linker, another aryl group is fused at 4-C and 5-C of the pyrazolyl core, and the third aryl group is directly connected to 3-C of the pyrazolyl core. Derivatives of the fused pyrazolyl compound may be obtained by modifying the substituents on any of the aryl groups.  
         [0015]    The chemicals used in the above-described synthetic route may include, for example, solvents, reagents, catalysts, protecting group and deprotecting group reagents. The methods described above may also additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the fused pyrazolyl compound. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing applicable fused pyrazolyl compounds are known in the art and include, for example, those described in R. Larock,  Comprehensive Organic Transformations,  VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,  Protective Groups in Organic Synthesis,  2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser,  Fieser and Fieser&#39;s Reagents for Organic Synthesis,  John Wiley and Sons (1994); and L. Paquette, ed.,  Encyclopedia of Reagents for Organic Synthesis,  John Wiley and Sons (1995) and subsequent editions thereof.  
         [0016]    A fused pyrazolyl compound thus synthesized can be further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization.  
         [0017]    One aspect of this invention is a method for treating a neurodegenerative disease, such as Parkinson&#39;s disease, Huntington&#39;s disease, inflammatory brain disease, Alzheimer&#39;s disease, amyotrophic lateral sclerosis, HIV-associated dementia, multiple sclerosis, or excitoneurotoxicity. The method includes administering to a subject in need thereof an effective amount of one or more fused pyrazolyl compounds and a pharmaceutically acceptable carrier. As used herein, the term “treating” refers to curing, healing, alleviating, relieving, altering, remedying, ameliorating, or preventing neurodegenerative diseases. “An effective amount” is defined as the amount of the fused pyrazolyl compound which, upon administration to a subject in need thereof, is required to confer therapeutic effect on the subject. An effective amount of the fused pyrazolyl compound can range from about 0.01 mg/kg to about 300 mg/kg. Effective doses will also vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other agents for treating neurodegenerative diseases.  
         [0018]    To practice the method of the present invention, a fused pyrazolyl compound can be administered orally, parenterally, by inhalation spray or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.  
         [0019]    A composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added. An inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.  
         [0020]    A sterile injectable composition, for example, a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer&#39;s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.  
         [0021]    A carrier in a pharmaceutical composition must be “acceptable” in the sense of being compatible with the active ingredient of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the fused pyrazolyl compound, or one or more solubilizing agents, can be utilized as pharmaceutical excipients for delivery of the fused pyrazolyl compound. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&amp;C Yellow #10.  
         [0022]    In vivo screening can be performed by following procedures well known in the art. See the specific examples below.  
         [0023]    Without further elaboration, it is believed that the above description has adequately enabled the present invention. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All of the publications, including patents, cited herein are hereby incorporated by reference in their entirety.  
       Chemical Synthesis  
       [0024]    Calcium borohydride was first prepared by stirring anhydrous calcium chloride (88.8 mg, 0.8 mmole) with sodium borohydride (60 mg, 1.6 mmole) in anhydrous THF (20 mL) for 4 hrs. A 30 mL THF solution containing 88.0 mg 1-benzyl-3-(5′-methoxycarbonyl-2′-furyl)-indazole (0.27 mmole) was then added dropwise to the calcium borohydride solution at 30±2° C. The mixture was heated under reflux for 6 hrs, cooled, quenched into crushed ice, placed at a reduced pressure to remove THF, and filtered to obtain a solid product. The solid was extracted with dichloromethane. The extract was concentrated to 50 mL and a solid precipitated after petroleum ether was added. The precipitate was collected and purified by column chromatography (silica gel-benzene) to obtain 70.0 mg 1-benzyl-3-(5′-hydroxymethyl-2′-furyl)-indazole at a yield of 87%. This compound is referred to as “Indazole 1” below.  
         [0025]    mp: 108-109° C. MS (%), m/z: 304 (M + ). IR (KBr)λ max : 3350 cm −1  (—OH).  1 H-NMR (DMSO-d 6 , 200 MHz) δ:4.51 (2H, d, J=5.5 Hz, —CH 2 O—), 5.31 (1H, t, J=5.5 Hz, —OH), 5.70 (2H, s, ═NCH 2 —), 6.48 (1H, d, J=3.4 Hz, H-4′), 6.97 (1H, d, J=3.4 Hz, H-3′) 7.21-7.31 (6H, m, H-5, phenyl), 7.45 (1H, t, J=8.2 Hz, H-6), 7.75 (1H, dd, J=8.2, 1.8 Hz, H-7), 8.12 (1H. dd, J=8.2. 1.0 Hz. C4-H).  
       Biological Assays  
     Methods  
       [0026]    Animals: Sprague-Dawley (SD) rats weighing 200˜300 g were used. These animals were housed in a room with controlled temperature (24±1° C.) and humidity (55±5%) under a 12:12 h light-dark cycle. They were allowed free access to food and water. When surgery was performed, the rats were under anesthesia using trichloroacetaldehyde (400 mg/kg).  
         [0027]    Model of Parkinson&#39;s disease: SD rats received unilateral intranigral injection of MPP +  (9 μg) or LPS (25 μg) using a Hamilton syringe at the coordinates 5.3 mm posterior to Bregma, 2 mm right to the midline, and 7.8 mm ventral to the dura. The rats were tested for circling behavior using apomorphine (i.p., 5 mg/kg) 7 days after the injection of MPP + . The rats were then deeply anesthetized with tricholoacetaldehyde and perfused with saline followed by cold phosphate-buffered 4% paraformaldehyde solution (pH 7.4). Their brains were then removed rapidly, postfixed in 4% paraformaldehyde solution for 24 h for immunohistochemical study.  
         [0028]    Model of Huntington&#39;s disease: Rats were injected with 3-nitropropionic acid (15 mg/kg/day, i.p.) for 5 successive days. In addition to examining the learning behavior using rotarod test, their brains were removed for staining of neuronal loss via cresyl violet staining, GABAergic neuron, NOS interneuron and gliosis immunostaining.  
         [0029]    Model of Alzheimer&#39;s disease: Rats received a single i.p. injection of 3-NP (20 mg/kg) dissolved in phosphate buffer and 4 hr later they received an intrahippocampal administration of amyloid β (Aβ) fragment 25-35 (2 μg/μl) in the dentate gyrus (coordinates: AP −3.6, ML −2.0, DV −2.7). Day-3 after drug administration, the hippocampal region was isolated for immunohistochemical staining of a neuronal marker.  
         [0030]    Model of kainic acid-induced neurotoxicity: Kainic acid (KA) (0.2 μg) was unilaterally injected into hippocampus of rats (coordinates: AP −4.5; ML −2; DV −2.8). Following the injection, the rats were left to recover for one week. The hippocampal region was isolated for immunohistochemical staining of a neuronal marker.  
         [0031]    Immunohistochemistry: Brain sections of various regions were isolated for the examination of dopamine (DA), glutamate decarboxylase (GAD), and a neuronal marker, e.g., glial fibrillary acidic protein (GFAP) or CD11b. The brain sections (30 μm) were incubated with the primary antibody including anti-tyrosine hydroxylase (TH, a marker of dopamine neuron), anti-neuroal-nuclei, anti-glutamate decarboxylases, anti-GFAP or anti-CD11b for 48 hr at 4° C. The brain sections were then incubated in biotinylated goat anti-rabbit IgG or anti-mouse IgG. Staining was revealed by the ABC method (Vector Laboratories, Burlingame, Calif.) and developed using 0.04% (w/v) diaminobenzidine to produce a brown reaction product.  
         [0032]    Cresyl violet staining: Frozen sections were obtained from cryostat and mounted onto the gelatin-coated slides, dried overnight. After washing in water, the sections were stained using a 1% solution of cresyl violet acetate (Sigma) for 3 min, differentiated in 1% glacial acetic acid solution, washed with running water, and covered with Glycerin-gelatin.  
         [0033]    Staining of NADPH/d+(NOS) interneurons: NADPH diaphorase staining was performed on free-floating cryostat sections by incubating for 60 min in the dark at 37° C. in a reaction mixture containing 0.1 M Tris-HCl buffer (pH 8.0), 5 mM magnesium chloride, 2 mg/ml NADPH (reduced form; Sigma), and 1 mg/ml nitroblue tetrazolium (Sigma). The reaction was stopped with Tris-HCl buffer and then the sections were mounted on glass slides, dried overnight, cleared with xylene and cover-slipped with Permount.  
         [0034]    Rotarod Test: Three trials were performed for each experiment in a rotarod test. For each trial, a rat was placed on a rod and timed until it fell off the rod. The test interval was 10 min. The rod was turned by an electric motor at 12 rpm.  
       Results  
       [0035]    Prevention of MPP + -induced neurodegeneration: MPP +  (9 μg) in the absence or presence of Indazole 1 (1.2 nmole) was unilaterally injected into either substantria nigra or striatum. One week after local single injection of MPP +  into substantia nigra (SN), marked cell losses of dopaminergic neurons and dopaminergic terminals in striatum were observed. In contrast, co-injection of MPP +  and Indazole 1 into SN significantly protected dopaminergic neurons against the toxic insult caused by MPP +  and preserved the axonal projection. 80.1±2.6 (n=6) of the nigral dopaminergic neurons survived in Indazole 1-treated rats, while only 32.1±5.9% (n=10) survived in the controls. In addition, systemic injection of Indazole 1 (i.p., 1 mg/kg/day; for 3-5 days) also exerted profound neuroprotection against local single injection of MPP + -induced neurotoxicity in SN (survived neuron: 80.0±2.2%, n=9).  
         [0036]    Local injection of MPP +  into striatum also caused DA neuronal loss in SN (survived neuron: 61.2±6.5%, n=4). Co-injection of Indazole 1 with MPP +  into striatum increased the survival of DA neuron in SN (survived neuron: 85.2±2.9%, n=6).  
         [0037]    Prevention of LPS-induced neuronal death: LPS-induced DA neuronal loss in SN is used as an animal model for the study of Parkinson&#39;s or inflammatory CNS diseases. The brain sections were obtained 7 days after unilateral intranigral injection of LPS (25 μg). 54.5±4.1% (n=11) TH-positive neurons survived in response to single injection of LPS in comparison with contralateral un-injected side. Co-injection of LPS with Indazole 1 (1.2 nmole) antagonized the neurotoxicity of LPS (survived neuron: 74.5±3.4%, n=10).  
         [0038]    Prevention of kainic acid-induced hippocampal neuronal death: Kainic acid (0.2 μg) was unilaterally injected into hippocampus in the absence or presence of Indazole 1 (1.2 nmole). After 7 days, injection of kainic acid alone caused almost 60% of pyramidal neuron loss, in comparison with contralateral side. On the other hand, single intra-hippocampal injection (1.2 nmole) or i.p. injection (1 mg/kg/day, 7 days) of Indazole 1 significantly protected neurons from kainic acid-induced neuronal death. It was observed that 85.6±4.9% (n=10) neurons survived with intra-hippocampal single injection of Indazole 1, 88.0±3.1% (n=10) neurons with i.p. injection of Indazole 1 (1 mg/kg), and 66.4±3.2% (n=3) neurons with i.p. injection of Indazole 1 (0.1 mg/kg), while only 37.8±6.5% (n=11) neurons survived in controls.  
         [0039]    Prevention of 3-NP-induced neuronal death in striatum: Rats were injected intraperitoneally with 3-NP (15 mg/kg/day) for 5 successive days. Other rats were injected concomitantly with 3-NP (15 mg/kg/day) and Indazole 1 (1 mg/kg) for 5 successive days. The brain sections of all of the rats were stained with cresyl violet. Systemic 3-NP administration produced a localized neuronal loss in the striatum. However, Indazole 1 treatment markedly reduced 3-NP-induced neuronal lesion.  
         [0040]    To examine protection of Indazole 1 against 3-NP-induced GABAergic neurotoxicity in striatum, the brain sections were then stained with antibody against GAD. 3-NP administration caused the loss of GABAergic neuronal loss, whereas, Indazole 1 treatment protected neurons from 3-NP intoxication.  
         [0041]    To examine protection of Indazole 1 against 3-NP-induced NADPH-d/nNOS neuronal death in striatum, the brain sections were stained by NADPH-d histochemistry to illustrate nitric oxide producing neurons. The number of survived neurons was counted from 4 brain slices per rat. It read 226±1/slice in control, 116±10/slice with injection of 3-NP, and 204±4/slice with injection of 3-NP and Indazole 1 (n=6 for each). Thus, 3-NP intoxication caused neuronal death of NADPH-d/NOS interneurons. On the other hand, Indazole 1 prevented the damage of NADPH-d/NOS interneurons.  
         [0042]    To examine prevention of Indazole 1 against 3-NP-induced gliosis, the brain sections were then stained with antibody against GFAP, a marker of astrocyte. The result showed that chronic treatment with 3-NP caused gliosis much more severely than the concomitant treatment with 3-NP and Indazole 1.  
         [0043]    To examine the effect of Indazole 1 on 3-NP-induced motor damage, the motor function was tested using rotarod on day 5. The rotarod was turned by an electric motor at 12 rpm. Each rat was tested thrice at an interval of 10 min and the time staying on rotarod was recorded after chronic systemic administration of 3-NP. The performance of the control rats improved with increasing numbers of trial. However, 3-NP administration severely impaired the motor learning ability. Treatment with Indazole 1 significantly improved the motor function. Time on rotarod at trial 3 was measured as 50.8±20.1 sec (n=8) in control, 2.9±0.4 sec (n=9) with injection of 3-NP, and 100.8±19.4 sec (n=13) with injection of 3-NP and Indazole1.  
         [0044]    Prevention of Aβ-induced neuronal death in hippocampus: Rats received a single i.p. injection of 3-NP (20 mg/kg) dissolved in phosphate buffer and 4 hr later they received an intrahippocampal administration of Aβ fragment 25-35 (2 μg/μl) in the dentate gyrus. Day-3 after drug administration, the hippocampal region was isolated for immunohistochemical staining of a neuronal marker. Injection of Aβ in combination with single dose of 3-NP caused neuronal loss in dentate gyrus, in comparison with contralateral side. On the other hand, single intra-hippocampal injection of Indazole 1 (1.2 nmole) markedly protected neurons from Aβ-induced neuronal death.  
         [0045]    In conclusion, Indazole 1 protects neurons from cytotoxicity induced by a variety of neurotoxicants, i.e., MPP + , kainic acid, 3-NP, LPS, and Aβ. Indazole 1-related compounds are thus good candidate drugs for treating of many neurodegenerative diseases including Parkinson&#39;s disease, Huntington&#39;s disease, inflammatory brain disease, Alzheimer&#39;s disease, amyotrophic lateral sclerosis, HIV-associated dementia, multiple sclerosis, glaucoma neurodegeneration and excitoneurotoxicity.