TWI668007B - Compounds for use in prevention and treatment of neurodegenerative diseases and pain - Google Patents

Abstract

The present invention discloses compounds for the prevention and treatment of neurodegenerative diseases and pain. In a particular embodiment of the invention, the compound is selected from the group consisting of N ⁶ -[(3-halothien-2-yl)methyl]adenosine, N ⁶ -[(4-halothen-2-yl) a group consisting of methyl]adenosine and N ⁶ -[(5-halothiphenyl-2-yl)methyl]adenosine. In another embodiment of the invention, the compound is selected from the group consisting of N ⁶ -[(2-bromothien-3-yl)methyl]adenosine, N ⁶ -[(4-bromothiophen-3-yl) )methyl]adenosine, N ⁶ -[(5-bromothien-3-yl)methyl]adenosine, N ⁶ -[(2-chlorothiophen-3-yl)methyl]adenosine, N ⁶ - a group consisting of [(4-chlorothiophen-3-yl)methyl]adenosine and N ⁶ -[(5-chlorothiophen-3-yl)methyl]adenosine. The invention also discloses methods of making and using same.

Description

Compound for preventing and treating neurodegenerative diseases and pain

The present invention relates to compounds for the prevention and/or treatment of neurodegenerative diseases or pain or both.

U.S. Patent Publication No. US20120295863 discloses bifunctional compounds that bind to adenosine A2A receptors and adenosine transporters to prevent and treat neurodegenerative diseases. A selective A2A adenosine receptor (A2AR) agonist, named CGS21680 (CGS for short), has been shown to attenuate Huntington's disease (HD) symptoms in a gene-transferred mouse model and enhance ubiquitin The ubiquitin-proteasome system activity is insufficient to rescue the urea cycle of HD disease (Chiang et al., 2009 Hum Mol Genet. 18:2929-2942; Chou et al., 2005 J Neurochem. 93:310-320) . However, conventional CGS has strong immunosuppressive effects and other side effects and is therefore not suitable for clinical use.

N6-(4-hydroxybenzyl)adenosine, such as T1-11 of US20120295863, is also an A2AR agonist, showing potential for the treatment of neurodegenerative diseases. However, the development of T1-11 into oral drugs is still difficult due to poor bioavailability (F < 5%). Oral bioavailability is an important property of drug development as it represents the percentage of systemic circulation that is achieved after absorption and metabolism.

In one aspect, the invention relates to a compound of formula (I) or (IA): ; Or a pharmaceutically acceptable salt thereof, wherein X is a halogen.

The halogen (F, Cl, Br, or I) may be at the 3-, 4-, or 5-position of formula (I) or at the 2-, 4-, or 5-position of formula (IA).

In a particular embodiment of the invention, the compound is selected from the group consisting of N6-[(3-halothien-2-yl)methyl]adenosine (N6-[(3-halothien-2-yl)methyl]adenosine ), N6-[(4-halothien-2-yl)methyl]adenosine (N6-[(4-halothien-2-yl)methyl]adenosine), and N6-[(5-halothiphenyl-2- Group of methyl]adenosine (N6-[(5-halothien-2-yl)methyl]adenosine).

In another embodiment of the invention, the compound is selected from the group consisting of N6-[(2-halothen-3-yl)methyl]adenosine (N6-[(2-halothien-3-yl)methyl] Adenosine), N6-[(4-halothien-3-yl)methyl]adenosine (N6-[(4-halothien-3-yl)methyl]adenosine), and N6-[(5-halothiophene-3) a group consisting of -methyl]adenosine (N6-[(5-halothien-3-yl)methyl]adenosine).

Further in another embodiment of the invention, the compound is selected from the group consisting of N6-[(5-bromothien-2-yl)methyl]adenosine (N6-[(5-bromothien-2-yl)methyl) ]adenosine), N6-[(4-bromothien-2-yl)methyl]adenosine (N6-[(4-bromothien-2-yl)methyl]adenosine), N6-[(3-bromothiophene-2) -yl)methyl]adenosine (N6-[(3-bromothien-2-yl)methyl]adenosine), N6-[(5-chlorothiophen-2-yl)methyl]adenosine (N6-[(5 -chlorothien-2-yl)methyl]adenosine), N6-[(4-chlorothien-2-yl)methyl]adenosine (N6-[(4-chlorothien-2-yl)methyl]adenosine), and N6 a group consisting of -[(3-chlorothien-2-yl)methyl]adenosine (N6-[(3-chlorothien-2-yl)methyl]adenosine).

In another embodiment of the invention, the compound is selected from the group consisting of N6-[(2-bromothien-3-yl)methyl]adenosine (N6-[(2-bromothien-3-yl)methyl] Adenosine), N6-[(4-bromothien-3-yl)methyl]adenosine, N6-[(5-bromothiophene-3-) Amino]adenosine (N6-[(5-bromothien-3-yl)methyl]adenosine), N6-[(2-chlorothiophen-3-yl)methyl]adenosine (N6-[(2- Chlorothien-3-yl)methyl]adenosine), N6-[(4-chlorothien-3-yl)methyl]adenosine), and N6- A group consisting of [(5-chlorothien-3-yl)methyl]adenosine).

In another aspect, the invention relates to a process for the preparation of a compound of formula (I) or (IA) above, which comprises step (I) or (II): (I): (a) in the presence of a base The next reaction is 6-chloropurine ribofuranoside and (thienyl)methylamine, which is substituted with fluorine, chlorine, bromine, or iodine and has the formula 3 or 3A: (3); (3A), to obtain a compound of formula (I) or (IA); or (II): (a1) to react (2',3'-O-isopropylidene) adenosine in the presence of a base ((2') , 3'-O-isopropylidene)adenosine) and a hydroxy protecting agent to obtain a (2',3'-O-isopropylidene) adenosine derivative having a hydroxy protecting group; (b) reacting with a hydroxyl group Protecting the (2',3'-O-isopropylidene) adenosine derivative with an amine-based protecting agent to obtain a (2',3'-O-isopropylidene) adenosine derivative , having a hydroxy protecting group and an amine protecting group; (c) performing a coupling reaction by reacting the (2',3'-O-isopropylidene) adenine derivative having a hydroxyl group and an amine protecting group with Substituted (thienyl)methyl compound, such as Formula 7 or 7A: (7); (7A), wherein X is F, Cl, Br, or I, and Y is X, OH, methanesulfonate (OSO2CH3, OMs), p-toluenesulfonate (OSO2C6H4-p) -CH3, OTs), or trifluoromethanesulfonate (OSO2CF3, OTf) to obtain a product containing a protecting group and a substituted (thienyl)methyl group; and (d) under acidic conditions The product of step (c) removes the protecting group to give a compound of formula (I) or (IA).

In a particular embodiment of the invention, the base of step (a) can be diisopropylethylamine. The hydroxy protecting agent may be tert-butyldimethylsilyl chloride. The amine based protecting agent can be Di-tert-butyl dicarbonate. The base of step (a1) may be imidazole.

In another embodiment of the present invention, in the step (I) (a), the base is diisopropylethylamine, and the substituted (thienyl)methylamine is: (i) (5-bromothiophene) -2-yl)methanamine (5-bromothien-2-yl)methanamine to obtain N6-[(5-bromothien-2-yl)methyl]adenosine; or (ii) (5-chlorothiophene) 2-ylthion-2-ylmethanamine to obtain N6-[(5-chlorothien-2-yl)methyl]adenosine.

Further in another embodiment of the invention, the coupling reaction is carried out in the presence of triphenylphosphine and diisopropylazodicarboxylate. The compound containing a substituted (thienyl)methyl group is (5-bromothien-2-yl)methanol.

In a further aspect, the invention relates to a composition comprising: (a) a therapeutically effective amount of a compound as described above or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier, Excipient, or carrier.

In yet another aspect, the invention relates to the use of a compound as described above for the manufacture of a medicament for the treatment of a neurodegenerative disease and/or pain in a subject in need thereof. Alternatively, the invention relates to a compound as described above for use in the treatment of a neurodegenerative disease and/or pain in a subject in need thereof. A neurodegenerative disease can be a protein-misfolding disease.

In a specific embodiment of the invention, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Prion disease. ), Huntington's disease, and the group consisting of spinal cerebellar ataxias. Spinocerebellar ataxia may be selected from the group consisting of a second type of spinal cerebellar ataxia, a third type of spinal cerebellar ataxia, and a seventh type of spinal cerebellar ataxia. Pain can be acid-induced pain. Acid-induced pain can be acid-induced muscle pain. Acid-induced muscle pain can be acid-induced chronic muscle pain.

In a specific embodiment of the invention, the pain is selected from the group consisting of inflammatory pain, cancer pain, chest pain, back pain, neck pain, shoulder pain, migraine, headache, myofascial pain, joints Groups of pain, muscle pain syndrome, neuropathic pain, peripheral pain, sympathetic pain, postoperative pain, post-traumatic pain, and multiple sclerosis pain.

In another embodiment of the invention, the pain may be dysfunctional pain. The dysfunctional pain can be selected from the group consisting of fibromyalgia, myofascial pain, bladder pain syndrome, and pain caused by intestinal syndrome.

The invention and other aspects of the invention will be apparent from the following description of the preferred embodiments of the invention.

The drawings illustrate one or more embodiments of the invention, and in the written description The same or similar elements of a particular embodiment are denoted by the same reference numerals throughout the drawings.

[Definitions] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, unless otherwise defined. In the event of a conflict, the definition will be based on this document.

The words "treatment" or "treatment" mean the administration of an effective amount of a therapeutic agent to an individual in need thereof, having neurodegenerative diseases and/or pain, or symptoms or predispositions of the disease and/or pain, for the purpose of healing Relieving, soothing, treating, ameliorating, or preventing the disease and/or pain, its symptoms, or its propensity. The individual can be defined by a health care professional based on results from any applicable diagnostic method.

By "an effective amount" is meant an amount of the active compound that is to be administered to the individual to be treated. The effective dose will vary, as will be understood by those skilled in the art, depending on the route of administration, the use of the excipient, and the likelihood of being used in conjunction with other treatments.

"Guidance for Industry and Reviewers Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers" published by the Food and Drug Administration of the US Department of Health and Services reveals that "therapeutically effective amount" can be calculated from the following formula: HED = Animal dose mg/kg ́ (animal weight kg/human weight kg) 0.33.

JMF 1907 refers to the compound N6-[2-(indol-3-yl)ethyl]adenosine and is the compound 6 disclosed in U.S. Patent Publication No. 20120295863 A1.

Chemically synthesized in a method, 6-chloropurine nucleofuranoside (2) is treated in the presence of a base, and optionally substituted (thienyl)methylamine (3) to obtain the desired compound of formula (I) .

For example, diisopropylethylamine is used as a base, and the substitution reaction of 6-chloropurine nucleofuranoside with (5-bromothien-2-yl)methylamine is heated under a 1-propanol solvent. This was carried out to obtain N6-[(5-bromothien-2-yl)methyl]adenosine (JMF3464, structure 1). First diagram of the process Second diagram of the process

In another method, (2',3'-O-isopropylidene) adenosine (4) is treated with tert-butyl dimethyl fluorenyl chloride (TBDMSCl) in the presence of a base imidazole to obtain Mercaptoether derivative (5). The 6-amino group is protected as carbamate 6 which contains a tertiary tert-butoxycarbonyl (Boc) substituent. The desired compound of formula (I) is obtained by introducing a selectively substituted (thienyl)methyl group and removing all protecting groups under acidic conditions.

For example, the coupling reaction of compound 6 with (5-bromothien-2-yl)methanol (compound 7, wherein X = 5-Br, Y = OH) is carried out by using triphenylphosphine and diisopropyl azo. Promoted by diisopropyl azodicarboxylate (DIAD) to obtain Compound 8. The overall deprotection of the sulfhydryl group, the acetonide, and the Boc group of the compound 8 is carried out under acidic conditions to obtain the compound 1.

The compound (IA) was obtained by the same chemical method. Third process

The oral bioavailability rate measures the oral bioavailability of the test compound, and male ICR mice (6 weeks old; 20-25 g) are administered orally (10 mg/kg) or intravenously (1 mg/kg). Blood samples were taken after testing the compounds. Blood samples collected at the indicated time were extracted with methanol containing 0.1% formic acid, and 10 μL of each extracted sample was injected into UPLC-MSMS for quantification. The results (first A-first B) showed that the oral bioavailability of T1-11 and JMF3464 in ICR mice was 2.8% and 17.4%, respectively, indicating that the oral absorption rate of JMF3464 was higher than that of T1-11. More than double.

JMF3464 binds to A2AR and ENT1 and protects neuronal apoptosis. The inventors first confirmed the pharmacological properties of JMF3464 by radioligand binding assay. Table 1 shows the pharmacological properties of T1-11, JMF1907, and JMF3464. The binding of the designated compound to the A ^2A adenosine receptor (A ^2A R) and the aequon nucleoside transporter 1 (ENT1) was confirmed by standard binding procedures. As shown in Table 1, JMF3464 binds to A ^2A R and adenosine transporter - equilibrative nucleoside transporter 1 (adenosine transporter - equilibrative nucleoside transporter 1). The affinity of JMF3464 for A2AR is similar to that of T1-11 and JMF1907 (T1-11 analog), and its affinity for ENT1 is better than that of T1-11 and JMF1907. The inventors' study also indicated that N6-[(5-chlorothien-2-yl)methyl]adenosine (JMF3818) also inhibited apoptosis of PC12 cells caused by serum removal. The inhibition of A ^2A receptor and adenosine transporter (ENT1) by 10 μM of JMF3818 was 54% and 96%, respectively (second panel). Table 1

JMF3464 has a beneficial effect on the main symptoms of HD in Huntington's disease (HD) gene-transferred mouse model. Since A ^2A R and ENT1 are located in the striatum and involve striatal function, the inventors hypothesized Chronic treatment of JMF3464 will regulate the progression of HD. The inventors tested the utility of JMF3464 in the HD gene transfer mouse model (R6/2), in which the A ^2A R agonist has a benefit. Mice were administered JMF 3464 (0.11 mg/kg/day) at 7 weeks of age to resist progressive deterioration of motor coordination and were assessed by roller performance tests (Figure 3A). The shortened lifespan of R6/2 mice was also improved by continuous (long-term) treatment of JMF3464.

The beneficial effect of JMF3464 on type 2 spinocerebellar ataxia (SCA2) may be a mechanism of A _2A R receptor signaling pathway by activating proteasome activity to prevent mutation Htt aggregation or behavior in R6/2 gene-transferred mice. Performance (Huang et al., 2011 PLoS One . 6:e20934; Lee et al., 2012 PLoS One . 7:e38865), A _2A R stimulants may be effective in alleviating other polyglutamate (polyQ) diseases, such as SCA2 is also beneficial. Indeed, the inventors' data show that the T1-11 line is effective in preventing mutations in the SCA2 gene-transforming mouse ( ATAXN2 ^Q127 ), ATXN2 aggregation (Fig. 4A) and behavioral performance (Fig. 4B), which supports the above hypothesis. In view of the fact that the bioavailability of JMF3464 is significantly better than that of T1-11, the inventors concluded that JMF3464 also has beneficial effects on SCA2.

The beneficial effects of T1-11 and its analogues on the third type of spinal cerebellar ataxia (SCA3) compared with wild-type mice, the carrier-treated performance of polyglutamate amplification, dysregulated -3- Q79 SCA3 gene-transferred mice exhibited various ataxia symptoms, including impaired roller performance (figure A and sixth A), with an onset age of approximately 3-4 months. As shown in Figures 5A and 6A, the 4-month-old SCA3 gene-transferred mice were treated with T1-11 (1 mg per day) or JMF1907 (1 mg per day) orally, and the roller performance was significantly improved. . Similar to SCA3 patients, the neurons of the SCA3 gene-transferred mice found significant neuronal death (figure B and sixth B). According to the results of the roller test, daily oral treatment of T1-11 (fifth B and fifth C) or JMF1907 (sixth and sixth C) significantly alleviated death of the cerebral neurons of SCA3 gene-transferred mice (p. Five B, fifth C, sixth B, and sixth C). The results provide evidence that continuous oral administration of T1-11 or JMF1907 improves the neurological and neuropathological phenotype of SCA3 gene-transferred mice.

JMF3464 has a high bioavailability. Therefore, JMF3464 is also expected to have beneficial effects on mutational deregulation-3-Q79-induced ataxia and neurodegeneration in SCA3 gene-transferred mice. As expected, daily application of JMF3464 (0.3 mg per day) alleviated the ataxia of SCA3 gene-transferred mice (Figure 7A) and cerebral neuronal death (Figures 7B and 7C).

The beneficial effects of JMF1907 and JMF3464 on amyotrophic lateral sclerosis (ALS) are shown in Figure 8A - Figure 8B. Gene-transferred mice are given 4 different doses of JMF1907 (3.7, 1.25, 0.5, And 0.1 mg/kg/day), the roller and grip strength test performance was significantly better than the control group. A dose of 1.25 mg/kg showed the greatest test benefit. These data indicate a significant improvement in motor function. JMF3464 is an analog of T1-11 and JMF1907, which has a high bioavailability. Therefore, JMF3464 is expected to play a beneficial role in ALS mice. Treatment of NSC34 cells with JMF3464 normalizes the mislocalization of TDP-43 caused by AMPK activation (Fig. 9), which supports the notion that JMF3464 can prevent the initial steps of ALS pathogenicity. These findings support that the JMF3464 is useful for ALS.

The beneficial effect of JMF3464 on pain Although T1-11 (ip) showed excellent analgesic effect on two fibromyalgia mouse models (acid-induced chronic widespread pain and intermittent cold compression mode), its bioavailability Very low. Oral administration of T1-11 (up to 2 mg/kg) showed no analgesic effect on acid-induced chronic widespread pain patterns, in which mice developed fibromyalgia after treatment with creatine and genistein treatment (p. Ten A map). JMF3464 is an analog of T1-11 and exhibits analgesic effects in a fibromyalgia mouse model in which the mice develop chronic extensive pain after 2 days of intermittent cold compression (Fig. B). Oral administration of JMF3464 (1 mg/kg) was consistent with its good bioavailability and showed analgesic effect in intermittent cold compression mode (Fig. 10C).

EXAMPLE 1 All reagents and solvents were reagent grades and their use did not require further purification unless otherwise indicated. Tetrahydrofuran and diethyl ether were distilled from Na/benzophenone, and CH ₂ Cl ₂ was distilled from CaH ₂ . All air or moisture sensitivity experiments were performed under argon. All glasses were oven dried for more than 2 hours and cooled to room temperature on desiccators. The microwave reaction was carried out in a focused single mode microwave unit (CEM Discover). The apparatus is provided with a continuously focused microwave delivery device which is provided with a selective power output unit.

The melting point was recorded by a Yanaco micro apparatus. The optical rotation was measured by a digital polarimeter (Japan JASCO Co. DIP-1000). The unit of [a]D is 10 ^{– 1} deg cm ² g ^{– 1} . Infrared spectroscopy (IR) was measured by an infrared spectrometer (Nicolet Magna 550-II). The NMR spectrum was measured by a nuclear magnetic resonance apparatus (Varian Unity Plus-400) (400 MHz). In addition, the chemical shifts (δ) corresponded to the δH 7.24/ δC 77.0 of CHCl ₃ /CDCl ₃ (the central spectrum of t) δH 2.05/ δC 29.92 of the line (CH ₃ ) ₂ CO/(CD ₃ ) ₂ CO, δH 3.31/ δC 49.0 of CH ₃ OH/CD ₃ OD and (CH ₃ ) ₂ SO/(CD ₃ ) ₂ SO δH 2.49 (m) / δC 39.5 (m) and expressed in ppm. The splitting patterns are single-single (s), doublet (dt), triplet (triplet, t), quadruple (quartet, q), multiplet (multiplet, m) And broad peak (broad, br). Coupling constants (J) are in Hz. Electrospray Mass Spectrometry (ESI-MS) experiments were performed on a high resolution mass spectrometer (Bruker Daltonics BioTOF III). Analytical thin-layer chromatography (TLC) was performed on thin-layer chromatography plates (E. Merck 60 F254 plates, 0.25 mm), while the compounds were UV-ray, p-methoxybenzaldehyde ( Anisaldehyde) spray or ninhydrin spray display. Column chromatography was performed with a column packed with 70-230 mesh silicone.

The purity of the compound was determined by HPLC to be ≥95% at a wavelength of 280 nm (Agilent HP-1100).

2',3'-O-isopropylidene-5'-O-(tri-butyldimethylmethyl) adenosine (5) (2',3'-O-Isopropylidene-5'-O- (tert-butyldimethylsilyl) adenosine (5 )) in a nitrogen atmosphere at 0 ^o C, the silicon based the three-butyldimethylsilyl chloride (tert -butyldimethylsilyl chloride) (TBDMSCl, 452 mg, 3 mmol) was added to an ice-bath the cooling containing 2 ', 3' - (O - isopropylidene-yl) adenosine (2 ', 3' - ( O-isopropylidene) adenosine) (4, 614 mg, 2.00 mmol) and imidazole (imidazole) (408 mg , 6 mmol) of anhydrous CH ₂ Cl ₂ (12 mL). The ice bath was removed and the mixture was stirred at room temperature for 12 h. Methanol (4 mL) was added and the mixture was stirred for additional 15 min then concentrated over EtOAc. The solid residue was dissolved in CH ₂ Cl ₂ and washed sequentially with 1 M HCl, DI water, and brine. The organic layer was collected, dried on MgSO _4, and filtered. The filtrate was concentrated under reduced pressure to give compound 5 ( s s, s,

6-N-tertiary butoxycarbonyl-2',3'-O-isopropylidene-5'-O-(tri-butyldimethylmethyl) adenosine (6) (6-N- tert-Butoxycarbonyl-2',3'-O-isopropylidene-5'-O-(tert-butyldimethylsilyl)adenosine (6)) will contain compound 5 (819 mg, 1.57 mmol) and 4-(dimethyl) under nitrogen A solution of 4-(dimethylamino)pyridine (DMAP, catalytic amount) in dry THF (10 mL) was stirred for 2 min. The solution was cooled in an ice bath and di-tert-butyl butyl carbonate ((Boc) ₂ O, 1.08 mL, 4.71 mmol). The ice bath was removed and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was concentrated by rotary evaporation under reduced pressure. The crude product was dissolved in CH ₂ Cl _2, and then click to 1 M HCl, deionized water, and brine wash. The organic layer was collected, dried on MgSO _4, and filtered. The filtrate was concentrated under reduced pressure to give EtOAc (EtOAc: EtOAc:EtOAc:

Methylamine (0.25 mL of a 40% solution in methanol, 2.54 mmol) was added to a MeOH solution (8 mL). The mixture was stirred at room temperature for 20 hours until TLC analysis showed the starting material was completely consumed. The mixture was concentrated by rotary evaporation under reduced pressure. The crude product was purified by EtOAc EtOAc EtOAc EtOAc (EtOAc (EtOAc) Yellow oil.

6-N-(5-bromothien-2-yl)methyl-6-N-tertiary butoxycarbonyl-2',3'-O-isopropylidene-5'-O- (tertiary Aminoglycine adenosine (8,X = 5-Br)(6-N-(5-Bromothien-2-yl)methyl-6-N-tert-butoxycarbonyl-2', 3'-O- Isopropylidene-5'-O- (tert-butyldimethylsilyl)adenosine (8, X = 5-Br)) will contain compound 6 (300 mg, 0.57 mmol), (5-bromothien-2-yl)methanol (164 mg, 0.85 mmol), and triphenylphosphine (226 mg, 0.85 mmol) of anhydrous THF (9 mL) was stirred at 45 ^o based nitrogen C of 2 minutes. Diisopropyl azodicarboxylate (DIAD, 0.168 mL, 0.85 mmol) was added dropwise. The mixture was stirred for an additional 45 minutes until TLC analysis showed compound 6 disappeared. The mixture was concentrated by rotary evaporation under reduced pressure. To flash chromatography (EtOAc / hexane = 1: 9) The crude product was purified to obtain Compound 8 (X = 5-Br) , as a pale yellow oil.

N6-[(5-Bromothien-2-yl)methyl]adenosine (JMF3464) Method A: Deionized water (5 mL) containing Compound 8 (X = 5-Br, 138 mg, 0.19 mmol) The suspension of THF (1 mL) was stirred and cooled in an ice bath. At 0 ^o C was added dropwise trifluoroacetic acid (Trifluoroactetic acid) (5 mL) . The mixture was stirred for 10 minutes, the ice bath was removed, and the mixture was stirred for additional 30 min. The mixture was concentrated by rotary evaporation under reduced pressure. The crude product was purified flash chromatography (MeOH / EtOAc = 1:49), to obtain JMF3464 (compound 1), as a white powder.

Method B: will contain (5-bromothien-2-yl)methylamine (768 mg, 4 mmol), 6-chloropurine nucleofuranoside (143 mg, 0.5 mmol), and diisopropylethylamine (2 mL, 12 mmol) of 1-propanol (20 mL) mixture was heated at line 70 ^o C 7 hours. A mixture containing the desired product and unreacted (5-bromothien-2-yl)methylamine at room temperature with THF containing di-tertiary butyl dicarbonate (0.92 mL, 4 mmol). mL) was treated with NaHCO3 (672 mg, 8 mmol) for 2 hours. The mixture was concentrated by rotary evaporation and purified by flash chromatography (EtOAc,EtOAcEtOAc Recrystallization from MeOH gave the desired product JMF3464 (59 mg, 27% yield). The product purity was 96% as indicated by HPLC on an HC-C18 column (Agilent, 4.6 x 250 mm, 5 μm) with a gradient of 50% aqueous MeOH.

Method C: will contain (5-bromothien-2-yl)methanamine (384 mg, 2 mmol), 6-chloropurine nucleofuranoside (287 mg, 1 mmol), and diisopropylethylamine (3 mL, 17 mmol) of EtOH (8 mL) was added to a sealed tube (15 mL). The sealed tube was placed in a single focusing mode microwave reactor (CEM Discover) Cabin, and at 80 ^o C to 100 W irradiation for 20 minutes. The solvent was removed by rotary evaporation. The residue was purified by flash chromatography eluting EtOAc EtOAc EtOAc

C ₁₅ H ₁₆ BrN ₅ O ₄ S; yellow powder; mp 141.4-141.7 ^o C; [α] ²⁵ _D = -43.0 (DMSO, c = 1.0); TLC (2-propanol/hexane (2:3) R _f = 0.38; ¹ H NMR (DMSO- d ₆ , 400 MHz) δ 8.55 (1 H, br s), 8.40 (1 H, s), 8.29 (1 H, br s), 7.03 (1 H, d, J = 3.6 Hz), 6.78 (1 H, d, J = 4.0 Hz), 5.90 (1 H, d, J = 6.0 Hz), 5.45 (1 H, d, J = 6.0 Hz), 5.34–5.32 (1 H, m), 5.19(1 H, d, J = 4.4 Hz), 4.76(2 H, s), 4.63–4.59(1 H, m), 4.15–4.14(1 H, m), 3.96– 3.95 (1 H, m), 3.69–3.65 (1 H, m), 3.57–3.52 (1 H, m), ¹³ C NMR (DMSO- d ₆ , 100 MHz) δ 153.9, 152.2, 148.5, 145.0, 140.2 , 129.6, 126.3, 120.0, 109.7, 87.9, 85.8, 73.5, 70.6, 61.6, 42.9, ESI-MS calcd for C ₁₅ H ₁₇ BrN ₅ O ₄ S: 442.0185, observed: m/z 442.0189 [M + H] ⁺ .

N6-(thiophen-3-yl-methyl)adenosine (JMF3461) will contain 3-(aminomethyl)thiophene (0.25 mL, 2.5 mmol), 6-chloropurine nucleofuranoside (143 mg, 0.5 mmol) and diisopropylethyl amine (2 mL, 12 mmol) of 1-propanol (25 mL) the mixture was heated at 80 ^o C for 6 hours. The mixture was concentrated by rotary evaporation and recrystallised from MeOH to afford the desired product JMF 3461 (136 mg, 75% yield). The product purity was 99% as indicated by HPLC on an HC-C18 column (Agilent, 4.6 x 250 mm, 5 μm) with a gradient of 50% aqueous MeOH. C ₁₅ H ₁₇ N ₅ O ₄ S; yellow powder; mp 134.3–135.1 ^o C; [α] ²⁴ _D = –58.6 (DMSO, c = 1.0); TLC (2-propanol/hexane, (2:3) )) R _f = 0.33; ¹ H NMR (DMSO- d ₆ , 400 MHz) δ 8.36 (2 H, s s), 8.22 (1 H, s), 7.43 (1 H, dd, J = 3, 5 Hz ), 7.28(1 H, d, J = 1.6 Hz), 7.09(1 H, d, J = 4.8 Hz), 5.88(1 H, d, J = 6.4 Hz), 5.43(1 H, d, J = 6.0 Hz), 5.38 (1 H, q, J = 4.6 Hz), 5.17 (1 H, d, J = 4.4 Hz), 4.69 (2 H, s), 4.63–4.16 (1 H, m), 4.14– 4.13(1 H, m), 3.97–3.95 (1 H, m), 3.69–3.64 (1 H, m), 3.57–3.52 (1 H, m), ¹³ C NMR (DMSO- d ₆ , 100 MHz) δ 154.4, 152.3, 148.5, 140.8, 139.9, 127.9, 125.1, 120.0, 119.8, 87.9, 85.9, 73.5, 70.7, 61.7, 42.9; ESI-MS calculated C ₁₅ H ₁₈ N ₅ O ₄ S: 364.1080, observed : m/z 364.1079 [M + H] ⁺ .

N6-(thiophen-2-yl-methyl)adenosine (JMF3462) will contain 2-(aminomethyl)thiophene (0.25 mL, 2.5 mmol), 6-chloropurine nucleofuran glucoside (143 mg, 0.5 mmol), and diisopropylethyl amine (2 mL, 12 mmol) of a mixture of 1-propanol (25 mL) was heated at 80 ^o C 7 hours. The mixture was concentrated by rotary evaporation and recrystallised from MeOH to afford the desired product JMF 3462 (154 mg, 85% yield). The product purity was 99% as indicated by HPLC on an HC-C18 column (Agilent, 4.6 x 250 mm, 5 μm) with a gradient of 50% aqueous MeOH. C ₁₅ H ₁₇ N ₅ O ₄ S; white powder; mp 149.2–149.7 ^o C; [α] ²⁵ _D = –68.2 (DMSO, c = 1.0); TLC (2-propanol/hexane, (2:3) )) R _f = 0.35; ¹ H NMR (DMSO- d ₆ , 400 MHz) δ 8.51 (1 H, br s), 8.39 (1 H, s), 8.27 (1 H, br s), 7.32 (1 H) , d, J = 5.2 Hz), 7.28 (1 H, d, J = 3.2 Hz), 6.93 (1 H, dd, J = 1.8, 2.6 Hz), 5.89 (1 H, d, J = 6.0 Hz), 5.46–5.45(1 H, m), 5.36(1 H, q, J = 4.6 Hz), 5.20–5.18(1 H, m), 4.64(2 H, s), 4.16–4.13(1 H, m) , 3.97–3.95 (1 H, m), 3.70–3.65 (1 H, m), 3.58–3.52 (1 H, m), 3.57–3.52 (1 H, m), ¹³ C NMR (DMSO- d ₆ , 100 MHz) δ 154.1, 152.2, 148.5, 142.9, 140.0, 126.5, 125.3, 124.7, 120.0, 87.9, 85.9, 73.5, 70.6, 61.6, 42.9; ESI-MS calculated C ₁₅ H ₁₈ N ₅ O ₄ S: 364.1080 , observed: m/z 364.1081 [M + H] ⁺ .

N6-[(4-bromothien-2-yl)methyl]adenosine will contain (4-bromothiophen-2-yl)methanamine (1152 mg, 6 mmol), 6-chloropurine nucleofuranoside (214 mg) , 0.75 mmol), and diisopropylethyl amine (3 mL, 18 mmol) of 1-propanol mixture (30 mL) was heated at line 70 ^o C 7 hours. The mixture containing the desired product and the unreacted (3-bromothiophen-2-yl)methylamine was THF (8 mL) containing di-tert-butyl butyl carbonate (1.4 mL, 6 mmol) at room temperature. (mL) and NaHCO ₃ (1 g, 1.2 mmol) were treated for 2 hours. The mixture was concentrated by rotary evaporation and purified by flash chromatography (EtOAc, EtOAc/EtOAc = 1:9) to obtain N ⁶ -[(4-bromothiophen-2-yl)methyl]adenosine.

N6-[(3-bromothien-2-yl)methyl]adenosine will contain (3-bromothiophen-2-yl)methylamine (576 mg, 3 mmol), 6-chloropurine nucleofuranoside (430 mg) 1.5 mmol) and diisopropylethylamine (4.5 mL, 15.5 mmol) in EtOH (10 mL) were added to a sealed tube (15 mL). The sealed tube was placed in a single focusing mode microwave reactor (CEM Discover) Cabin, and at 80 ^o C to 100 W irradiation for 20 minutes. The solvent was removed by rotary evaporation. The residue was purified by flash chromatography (EtOAc, EtOAc/EtOAc = EtOAc) to afford N ⁶ -[(3-bromothiophen-2-yl)methyl].

N6-[(2-bromothiophen-3-yl)methyl]adenosine will contain (2-bromothiophen-3-yl)methylamine (384 mg, 2 mmol), 6-chloropurine nucleofuranoside (287 mg) , 1 mmol), and diisopropylethylamine (3 mL, 17 mmol) in EtOH (8 mL) were added to a sealed tube (15 mL). The sealed tube was placed in a single focusing mode microwave reactor (CEM Discover) Cabin, and at 80 ^o C to 100 W irradiation for 20 minutes. The solvent was removed by rotary evaporation. The residue was purified by flash chromatography (EtOAc, EtOAc/EtOAc = EtOAc) to afford N ⁶ -[(2-bromothiophen-3-yl)methyl].

N6-[(4-bromothiophen-3-yl)methyl]adenosine will contain (4-bromothiophen-3-yl)methylamine (768 mg, 4 mmol), 6-chloropurine nucleofuranoside (143 mg) , 0.5 mmol), and diisopropylethyl amine (2 mL, 12 mmol) of a mixture of 1-propanol (20 mL) was heated at line 70 ^o C 7 hours. A mixture containing the desired product and unreacted (4-bromothiophen-3-yl)methylamine at room temperature with THF (6.sup.2 mL, 4 mmol) in THF (6) (mL) and NaHCO ₃ (672 mg, 8 mmol) were treated for 2 hours. The mixture was concentrated by rotary evaporation and purified by flash chromatography (EtOAc, EtOAc/EtOAc = 1:9) to obtain N ⁶ -[(4-bromothiophen-3-yl)methyl]adenosine.

6-N-(5-bromothiophen-3-yl)methyl-6-N-tertiary butoxycarbonyl-2',3'-O-isopropylidene-5'-O- (tertiary Aminoglycine (6-N-(5-Bromothien-3-yl)methyl-6-N-tert-butoxycarbonyl-2', 3'-O-isopropylidene-5'-O-(tert -butyldimethylsilyl)adenosine) will contain anhydrous compound 6 (360 mg, 0.68 mmol), (5-bromothiophen-3-yl)methanol (197 mg, 1.02 mmol), and triphenylphosphine (271 mg, 1.02 mmol) The THF solution (10 mL) was stirred under a nitrogen atmosphere of 45 ^o C for 2 min. Diisopropyl azodicarboxylate (DIAD, 0.20 mL, 1.02 mmol) was added dropwise. The mixture was stirred until TLC analysis showed compound 6 disappeared. The mixture was concentrated by rotary evaporation under reduced pressure. The crude product was purified by flash chromatography (EtOAc/hexane = 1:9) to afford 6- N- (5-bromothiophen-3-yl)methyl-6- N -tris-butoxycarbonyl-2 ',3'- O -isopropylidene-5'- O- (tri-butyldimethylmethyl) adenosine.

N6-[(5-bromothien-3-yl)methyl]adenosine will contain 6- N- (5-bromothien-3-yl)methyl-6- N -tertiarybutoxycarbonyl-2' , 3'- O -isopropylidene-5'- O- (tri-butyldimethylmethyl) adenosine (138 mg, 0.19 mmol) in deionized water (5 mL) with THF (1 mL) The suspension was stirred and cooled in an ice bath. At 0 ^o C was added dropwise trifluoroacetic acid (5 mL). The mixture was stirred for 10 minutes, the ice bath was removed, and the mixture was stirred for an additional 30 minutes. The mixture was concentrated by rotary evaporation under reduced pressure. The crude product (MeOH / EtOAc = 1:49) was purified by flash chromatography to afford N ⁶ -[(5-bromothiophen-3-yl)methyl]adenosine.

(5-Chlorothiophen-2-yl)methylamine will contain 2-(aminomethyl)thiophene (1 mL, 10 mmol), di-tert-butyl butyl carbonate (2.5 mL, 11 mmol), and NaHCO _A mixture of ₃ (840 mg, 10 mmol) in THF (13 mL) was stirred at room temperature for 3 s to afford a suspension of pale yellow solid. The mixture was concentrated under reduced pressure, and with CH ₂ Cl ₂ and H ₂ O and extracted residue. The organic phase was dried MgSO _4, filtered, and purified by flash chromatography, wherein the silica gel column was Eluant EtOAc / hexane (1:20), to obtain three-butyl (thiophen-2-yl) Methyl carbamate (C ₁₀ H ₁₅ NO ₂ S, 1.62 g, 76% yield).

Containing three-butyl (thiophen-2-yl) methyl carbamate (106 mg, 0.5 mmol), N - chlorosuccinimide (PEI) (N-chlorosuccinimide) (73 mg , 0.55 mmol) of a mixture of benzene ( 0.3 mL) system was stirred at 80 ^o C. After 2 hours, acetic acid (0.3 mL, 5 mmol) was added and further reacted for 21 hours. The mixture was extracted with CH ₂ Cl ₂ and H ₂ O. The organic phase was dried MgSO _4, filtered, and purified by flash chromatography, wherein the silica gel column was Eluant EtOAc / hexane (1:20), to obtain three-butyl (5-chloro-thiophene -2 -tert-butyl(5-chlorothien-2-yl)methyl carbamate (C ₁₀ H ₁₄ ClNO ₂ S, 82.6 mg, 67% yield).

Preparation of the compound containing the front (65 mg, 0.26 mmol) was stirred with TFA (1 mL, 13 mmol) of a mixture of CH ₂ Cl ₂ (1 mL) at room temperature for 3 hours. The solution was concentrated under reduced pressure to give (5-chlorothiophen-2-yl)methylamine (~100% yield). C ₅ H ₆ NSCl; pale yellow solid; ¹ H NMR (400 MHz, CD ₃ OD) δ 7.07 (1H, d, J = 4.0 Hz), 6.95 (1 H, d, J = 3.6 Hz), 4.26 ( 2H, s); ¹³ C NMR (100 MHz, CD ₃ OD) δ 134.9, 132.8, 130.7, 128.0, 38.8; ESI-HRMS calc. C ₅ H ₇ ClNS: 147.9988, observed: m/z 147.9995 [M + H] ⁺ .

N6-[(5-chlorothien-2-yl)methyl]adenosine (JMF3818) will contain (5-chlorothiophen-2-yl)methanamine (35.4 mg, 0.24 mmol), 6-chloropurine nucleofuranoside (0.12 mmol), and diisopropylethyl amine (0.36 mL, 2 mmol) the mixture of EtOH (1 mL) in a sealed tube system 80 ^o C the stirring system by which microwave radiation for 30 min. The mixture was cooled to room temperature and concentrated under reduced pressure to obtain a pale yellow oil, which in turn to H ₂ O and washed with MeOH to obtain the title compound JMF3818. _{C 15 H 16 ClN 5 O 4} S; white ^{solid; 1 H NMR (400 MHz,} CD 3 OD) δ 8.29 (1 H, s), 8.27 (1 H, s), 6.88 (1 H, d, J = 3.6 Hz), 6.79 (1 H, d, J = 3.6 Hz), 5.96 (1 H, d, J = 6.8 Hz), 4.74–4.77 (1 H, m), 4.32–4.34 (1 H, m), 4.17 (1 H, d, J = 2.8 Hz), 3.89 (1 H, dd, J = 12.4, 2.0 Hz), 3.75 (1 H, dd, J = 12.4, 2.8 Hz); ¹³ C NMR (100 MHz, CD ₃ OD) δ 156.0, 153.6, 142.8, 142.0, 129.9, 127.0, 126.6, 121.7, 91.5, 88.4, 75.6, 72.9, 63.7, 40.4; ESI–HRMS calculated C ₁₅ H ₁₇ ClN ₅ O ₄ S: 398.0690, Observed: m/z 398.0692 [M + H] ⁺ .

Pharmacokinetic studies. The compound is administered as an aqueous solution of physiological saline. Male ICR mice were purchased from BioLASCO Taiwan Co., Ltd. To measure the bioavailability of oral test compounds (T1-11 and JMF3464), male ICR mice were administered after oral (10 mg/kg) or intravenous (1 mg/kg) administration of the test compound. Zhou Da; 20-25g) Collect blood samples. On the T1-11 side, blood samples were collected 2, 10, 30, 60, 120, 360 minutes after intravenous administration and 15, 30, 45, 60, 120, 360 minutes after oral administration. For JMF3464, blood samples were collected 2, 10, 30, 60, 120, 240, 360, 480 minutes after intravenous administration and 15, 30, 60, 90, 120, 240, 360, 480 minutes after oral administration. The blood sample was extracted with methanol containing 0.1% formic acid, and then 10 μL of the extracted sample was injected into UPLC-MSMS for quantification.

Pharmacokinetic parameters were obtained with a pharmacokinetic program (WinNonlin) that fits the data to a noncompartmental model. Pharmacokinetic parameters were evaluated via regression of time and log concentration, including area under plasma concentration versus time curve (AUC) vs. last sampling time (AUC _0–120 ), for infinite time (AUC _0–∞ ), terminal phase half-life ( T _1/2 ), the maximum concentration of the compound in the plasma (C _max ), the C _max time (T _max ), and a one-order rate constant ( k ) associated with the end portion of the curve. Total plasma clearance (CL) was calculated as dose/AUC _iv . The oral bioavailability ( F ) for oral administration of test compounds was calculated by dividing the oral dose of AUC _{0 - ∞} by the intravenous (iv) dose of AUC _{0 - ∞} .

Example 2 Radiation ligand binding assay . Radiation ligand binding assays were performed with MDS Pharma Services Taiwan (Taipei, Taiwan) using standard binding procedures. For the A _2A R binding assay, membrane proteins collected from HEK293 cells overexpressing human A _2A R were cultured in a reaction buffer containing ³ H-CGS 21680 (50 nM) at 25 ° C [50 mM Tris-HCl (pH 7.4). , 10 mM MgCl ₂ , 1 mM EDTA, and 2 U/mL adenosine deaminase] for 90 minutes. 50 μM in the adenosine -5'- N - Amides the ethyl (adenosine-5'-N-ethylcarboxamide ) exists, evaluate non-specific binding. To measure the binding affinity of T1-11 to A ₃ R, membrane proteins collected from Chinese hamster ovary (CHO)-K1 cells overexpressing human A ₃ R and ³ H-AB-MECA (0.5 nM) were cultured at 25 °C. 25 mM HEPES (pH 7.4), 5 mM MgC ₂ , 1 mM CaCl, and 0.1% bovine serum albumin in a reaction buffer for 60 minutes. Non-specific binding was assessed in the presence of 1 μM IB-MECA (Tocris Bioscience, Ellisville, MO, USA). The adenosine transporter binding assay was carried out as described above. Membrane fraction collected from the cerebral cortex of Duncan Hartley-derived guinea pigs and ³ H-labeled 6-[(4-nitrobenzyl)thio]-9-β-D-ribose ³ H-labeled 6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine (NBTI, 0.5 nM) was cultured in 25 °C containing 50 mM Tris-HCl (pH 7.4). 30 minutes in the buffer. Non-specific binding was assessed in the presence of 5 μM NBTI, a potent inhibitor of balanced nucleoside transporters. It should be noted that NBTI is a high affinity inhibitor of ENT1 and 0.5 nM only inhibits human (h) ENT1. The reaction was terminated by GF/B glass fiber filtration and reaction buffer washing.

Cell Culture and Transient Transfection Rat PC12 cell line purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) was maintained in DMEM supplemented with 10% horse serum and 5% FBS, and cultured at 37 ° C in CO _{2 .} Incubator (5%). In LIPOFECTAMINE ^TM 2000 (Invitrogen) as a carrier, according to the manufacturer & steps plastids transferred to the cell. The quality system was presented from Dr. Pulst (Department of Neurology, University of Utah, USA). Normally, 5 μg of DNA was combined with 5 μl of LIPOFECTAMINETM 2000 to each well of a 6-well culture dish. The number of cells in the disc is (1~1.5) x 10 ⁶ cells/well. After 6 hours of transfection, the cells were additionally treated with the reagent for 24 hours. Images were taken with a Zeiss Axiovert 200M inverted fluorescent microscope (Göttingen, Germany).

MTT test. Survival was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic assay. Briefly, after treatment, MTT was added to the medium (0.5 mg/ml) and incubated at 37 ° C for 2-3 hours. In the 96-well culture plate, the number of cells in the disc was 1 x 104 cells/well. After the medium was removed, DMSO was added to each well to dissolve the formazan crystals, and then measured at a wavelength of 570/630 nm by a micro-enzyme-linked immunosorbent assay (ELISA) reader. The absorbance of a culture well.

Example 3 Animal and drug administration . Male R6/2 mice (Mangiarini et al., 1996 Cell . 87: 493-506) and sibling control were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and with female control mice (B6CBAFI/) J) Mating. The progeny were identified by polymerase chain reaction (PCR) genotyping technique, and the genomic DNA extracted from the tail tissue was identified, and the primer for gene transfer (5'-CCGCTCAGGTTCTGCTTTTA-3'; SEQ ID NO: 1, was used. And 5'-GGCTGAGGAAGCTGAGGAG-3'; SEQ ID NO: 2) to ensure that the number of CAG repeats is maintained at about 150. Animals were maintained at the Institute of Biomedical Sciences Animal Care Facility under the 12/12-h light/dark cycle. Mouse body weight was recorded once a day. Animal experiments were carried out under the accreditation procedure of the Academia Sinica Institutional Animal Care and Utilization Committee (Taipei, Taiwan).

Roller performance . Motion coordination was assessed using a roller device (UGO BASILE, Comerio, Italy), which was performed at constant speed (12 rpm) for 2 minutes (Carter et al., 1999 J Neurosci . 19: 3248-3257). All mice were trained for 2 days at 4 weeks of age to familiarize them with the roller device. Subsequently, the animals were tested three times a week at 4 to 12 weeks of age. At each test, the animals were placed in the device before starting to rotate. Automatically record latency to fall. Each test was administered to each mouse for up to 2 minutes for a total of three times.

Example 4 Animals . C57BL/6 mice were purchased from the National Laboratory Animal Center (Taiwan). The genetically transformed mouse ( ATAXN2 ^Q127 ) was supplied by Dr. Pulst. The mice were housed in a soundproof chamber (22 ± 2 ° C) under a light/dark cycle of 12/12-h and temperature control. Food and water are freely available. Every effort is made to minimize the number of animals used and their pain and discomfort, based on the National Institutes of Health's Guide to the Care and Use of Laboratory Animals. Principles and instructions. These experiments were also reviewed and approved by the Institutional Animal Care and Use Committee at the National Research Institute of Chinese Medicine (approval number: 100-15).

Filter retardation test . This method is followed by the description of Wanker et al. (1999 Methods Enzymol . 309:375-386) with some modifications. Briefly, the harvested cells were resuspended in lysis buffer (50 mM Tris-HCl (pH 8.8), 100 mM NaCl, 5.0 mM MgCl ₂ , 1 mM EDTA, and 0.5% (w/v) IPGEAL, which contained 1x protease inhibitor mixed component (Roche Diagnostics, Indianapolis, IN, USA)) and subjected to ultrasonic shock for 10 seconds (1 pulse/second). Each group of equal concentrations of protein (15-20 μg/well) was filtered through a 2% sodium dodecyl sulfate (SDS) pre-equilibrated cellulose acetate membrane (0.2 μm; Whatman, Maidstone, Kent, UK). A Bio-Dot SF device (Bio-Rad, Hercules, CA, USA) was used. Each well was washed twice with 200 μl of 0.1% SDS during aspiration. The stain was blocked in TBS (100 mM Tris-HC1 and 150 mM NaC1; pH 7.4) containing 3% skim milk powder for 1 hour at room temperature, followed by anti-polyglutamic acid (1:5000; MAB1574) The antibody was cultured in 3% bovine serum albumin (BSA) containing 0.02% NaN ₃ (4 ° C overnight) to detect normal and mutant ATAXN2. The subsequent method is the same as the foregoing.

Roller performance . This study used 5-week-old wild-type and genetically-transferred mice. Motion coordination was assessed with a roller device (UGO BASILE, Comerio, Italy), which increased in 5 minutes (10 to 28 rpm). All mice were trained for 2 days at 5 weeks of age to familiarize them with the roller device. In addition, at this week, some of the genetically-transferred mice began to take up T1-11 dissolved in daily drinking water. Subsequently, the animals were formally tested twice a week at 6 weeks of age. For each test, the animals were placed in the device prior to the start of rotation. Automatically record delayed drop time. Each mouse was given two trials each time. The mice were allowed to rest for 20 to 30 minutes between the two trials.

Example 5 behavioral test . The mouse balance and coordination function was determined by a roller test. The mice were placed on a moving roller of a roller device (Accelerating Model, Ugo Basile Biological Research Apparatus) and then accelerated until the mouse fell from the roller to the platform and the timer was stopped. The delayed fall time was measured in four daily trials over a four day period.

Immunohistochemical staining . Wild-type or SCA3 gene-transferred mice were anesthetized and perfused with PBS containing 4% polyoxymethylene. The brain is equilibrated with the Tissue-Tek embedding medium and frozen in liquid nitrogen. Coronal sections (20 μm) prepared by cryosection were permeabilized with 0.1% Triton X-100 and incubated with 4 ^o C of diluted anti-NeuN monoclonal antiserum (Chemicon) for 48 hours. Subsequently, the sections were washed and cultured with biotinylated horse anti-mouse IgG and cultured with avidin-biotin-horseradish peroxidase complex. . Next, the sections were washed and developed with a diaminobenzidine solution. NeuN-positive neurons were observed and counted on a Leica DM2500 microscope equipped with a Retiga-2000R CCD camera (QImaging) and a 3-axis computer-controlled MAC 600 mobile platform (Ludl Electronics), supplemented with StereoInvestigator software (MBF Bioscience). Each analysis covered 15 brain treatment sections from each mouse.

Example 6 Animal and drug delivery . The gene-transgenic mouse B6SJL-Tg (Prnp-TARDBP) 4Jlel/J line was purchased from the Jackson Laboratory (Bar Harbor, Maine USA) and was bred by the National Laboratory Animal Center. The gene-transferred mouse strain was screened by PCR, wherein the forward primer was 5'-GGT GGT GGG ATG AAC TTT GG-3' (SEQ ID NO: 3) and the reverse primer was 5'-GTG GAT AAC CCC TCC CCC AGC CTA GAC-3' (SEQ ID NO: 4). The wild type is a non-gene-transferred homologous mouse. Six-week old mice underwent surgery to load the ALZET micro-osmotic pump model 1004 (DURECT Corporation, Cupertino, CA, USA) containing DMSO or the designated JMF 1907, which was subcutaneously embedded in the ventrolateral side of the abdomen. Replace the pump every 28 days.

Grip. Grip force was measured with a grip meter (TSE Systems, Inc., MO, USA). In short, the mouse is lifted from the tail and held by the adjustable height grip attached to the force sensor. A pulling force is applied from the tail of the mouse. When the mouse releases the grip, the maximum force is displayed on the digital display panel connected to the control unit. Each mouse was tested 3 times.

Roller performance . The motion coordination of wild-type and genetically-transferred mice was evaluated by a device (UGO BASILE, Comerio, Italy), which was performed at a constant speed (40 rpm) for 120 seconds. All mice were trained for 2 days per week for 2 weeks. Subsequently, the mice were formally tested twice a week at 9 weeks of age. For each test, the animals were placed in the device prior to the start of rotation. Automatically record delayed drop time. Each mouse was given three trials at a time. Mice were allowed to rest for 20 minutes between trials.

Cell culture and transfection. The motor neuron cell line (NSC34) is from Dr. Neil Cashman (Brain Research Centre, The University of British Columbia, Canada) and is housed in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal calf Serum (FCS), 2 mM L-glutamic acid, and 1% penicillin/streptomycin (Invitrogen Gibco BRL, Carlsbad, CA, USA) were cultured at 37 ° C with 5% CO ₂ .

Example 7 mice . Female C57BL6N mice, 8-12 weeks old, were purchased from BioLASCO (Yi-Lan, Taiwan).

Acid-induced chronic widespread pain pattern . The fibromyalgia pattern was modified from the acid-induced chronic pain pattern established by the Sluka team (Sluka et al., 2003 Pain . 106: 229-239). The mice were briefly anesthetized with 2% evaporative isoflurane and intramuscularly injected with 20 mL of genistein (1 mM) in the left gastrocnemius muscle. After 3 minutes, the same position was injected with 20 mL of acidic saline (pH 4.0). Subsequently, the mice developed persistent mechanical hyperalgesia for more than 2 weeks. After 4 days of developing mechanical hyperalgesia in mice, the analgesic effect of T1-11 (administered orally by 0.9-mm/7-cm gastric tube) was tested. Mechanical hyperalgesia was detected by testing the paw-reflexion of 0.2-mN von Frey filament stimulation in the hind paw of the mouse.

Intermittent cold compression mode . The fibromyalgia pattern was established by the Ueda team, in which the mice were treated with intermittent cold compression for 2 days (Nishiyori and Ueda, 2008 Mol Pain . 4:52). The mice developed persistent (>2 weeks) mechanical and temperature hyperalgesia after intermittent cold compression treatment. Intermittent cold compression in 5 days, the plurality of test JMF3464 (oral or intraperitoneal injection) of mice analgesic effect. The detection of mechanical hyperalgesia was performed by testing the hind paw of the mouse for the 0.2-mN von Frey filament stimulation.

The foregoing description of the exemplary embodiments of the present invention is intended to Many modifications and variations are possible in light of the foregoing implementation.

The present invention and its practical applications are intended to be illustrative of the invention and the specific embodiments thereof, and the various embodiments of the invention are intended to be used in a particular application. Alternative embodiments will be apparent to those skilled in the art without departing from the spirit and scope.

All of the references cited and referred to in this specification are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the

no

The first panel shows the concentrations of T1-11 (A) and JMF 3464 (B) in the blood of ICR mice after intravenous (1 mg/kg) or oral (10 mg/kg) T1-11 or JMF3464. The oral bioavailability of T1-11 and JMF3464 was estimated to be 2.8% and 17.4%, respectively. The second panel shows the effect of A _2A R agonists (CGS21680, T1-11, JMF3464, and JMF3818) on cell death induced by deprivation of serum. The PC12 cell line deprived of serum was treated with the indicated reagents or not treated for 24 hours. Cell viability was compared to the mean of the serum-containing control group and expressed as a percentage of the MTT test results. Data points represent mean ± SEM (n = 3 ~ 6). Figures 3A through 3B show the effect of JMF3464 on movement disorders and longevity. JMF3464 (0.11 mg/mouse/day), JMF1907 (0.11 mg/mouse/day), or vehicle (control group) were administered subcutaneously to the designated 7-week old mice, which were administered with an ALZET osmotic minipump. 6 weeks. The roller performance (A) and life (B) of the mice were evaluated. * p < 0.05. *** p < 0.005. Figures 4A to 4B show the effect of T1-11 on preventing protein aggregation and behavioral expression induced by overexpression of the mutant SCA2 gene in SCA2 gene-transferred mice. (A) pSCA2-22Q-EGFP or pSCA2-104Q-EGFP transfected cell lines were treated with 10 μM T1-11 or 10 μM JMF1907 (T1-11-derived A _2A -R agonist) for 24 hours. The cells are harvested and subjected to a filter retardation assay or image capture. (B) Wild type (WT) or gene transfer mice (SCA2) are drinking water with or without T1-11. The roller performance is used to measure the behavioral function of the mouse. Figures 5A through C show that T1-11 improves motor dysfunction and pontine neuronal death in SCA3 gene-transferred mice. (A) SCA3 gene-transferred mice were given drinking water containing vehicle (0.2% DMSO) or T1-11 (0.1 mg/ml) at 4 weeks of age. The roller test showed that the vehicle-treated 4-month-old deregulated-3-Q79 (ataxin-3-Q79) gene-transferred mice exhibited a significantly shorter delayed fall than the wild-type (WT) mice. Time and movement are not coordinated. The roller performance of the 4-week-old SCA3 gene-transferred mice (1 mg per day) treated with T1-11 was significantly better than the carrier-treated deregulated-3-Q79 mice of the same age. Each point shows the mean of the 7-8 mice + SE. (BC) Immunohistochemical staining of the neuronal marker NeuN indicated that daily oral administration of T1-11 (1 mg per day) significantly improved cerebral neuronal death in 4 months old SCA3 gene-transferred mice (SCA3 + T1- 11). The scale is 50 μm. Each set of data shows the mean + SE of 7-8 mice. * P < 0.01 compared to SCA3 gene-transferred mice. Figures 6A to 6C show that JMF1907 alleviates the ataxia symptoms and the death of the pons in the SCA3 gene-transferred mice. (A) The roller test indicated that the roller performance of the 4 week old SCA3 mice (1 mg per day) treated with JMF 1907 was significantly improved compared to the 4 week old SCA3 gene transfected mice treated with vehicle. Each point shows the mean of 6 mice + SE. (BC) Immunocytochemical staining of NeuN showed that oral administration of JMF1907 (1 mg per day) significantly prevented cerebral neuronal death in 4 month old SCA3 mice (SCA3 + JMF1907). The scale is 50 μm. Each set of data represents the mean + SE of 6 mice. *P < 0.01 compared to SCA3 mice. Figures 7A through 7C show that JMF3464 improves ataxia and pons death in the SCA3 gene-transferred mice. (A) SCA3 gene-transferred mice exhibited impaired roller performance. Daily administration of JMF3464 (0.3 mg per day) significantly improved roller performance in 4 week old SCA3 mice. (BC) Immunocytochemical staining of the neuronal marker NeuN confirmed that daily oral administration of JMF3464 (0.3 mg per day) significantly improved the death of the pons in the 4-month-old SCA3 gene-transferred mice. Each set of data represents the mean + SE of 6 mice. # P < 0.01, compared to the SCA3 gene transgenic mice. Figures 8A through 8B show that treatment with JMF1907 improved the motor function of TDP-43 gene-transferred mice. Four different doses of JMF 1907 (3.7, 1.25, 0.5, 0.1 mg/kg) were tested. CTL: Gene-transferred mice were treated with DMSO. WT: wild type mice, treated with DMSO. Statistics were performed by two-way ANOVA. In terms of the roller, statistical significance was achieved in the experimental group of 1.25 & 0.5 mg/kg, the experimental group of 0.1 mg/kg in 12-21 weeks, and the experimental group of 3.7 mg/kg in 10-12 weeks. In terms of grip strength, all test doses were statistically significant in all experimental groups. N = 18 (CTL), 15 (WT), 15 (0.1), 15 (0.5), 15 (1.25), 5 (3.7). Figure 9 shows that JMF3464 treatment reduced TDP-43 mislocalization of NSC34 cells. Cells were pretreated with JMF 3464 (30 mM) for 1 hour and then treated with AICAR (1 mM, Al) for an additional 24 hours in the presence of JMF3464. TDP-43 (red) localization was analyzed by immunostaining. Mark the position of the core with Hochest (blue). Figures 10A through C show the analgesic effects of JMF3464 on fibromyalgia mouse models. (A-1 and A-2) Oral administration of T1-11 showed no analgesic effect on the model of fibromyalgia in mice, in which mice developed chronic muscle pain after treatment with intramuscular injection of acid and genistein. The hollow arrows indicate the time the mice received the acid injection. The black arrow indicates when the mouse received T1-11 (po). (B) JMF 3464 showed an analgesic effect on a model of fibromyalgia in mice in which the mice developed chronic extensive pain after 2 days of intermittent cold stress treatment. The analgesic effect of JMF3464 is dose dependent. The effective dose starts at 100 mg/kg (ip). (C) Oral administration of JMF 3464 (1 mg/kg) showed an analgesic effect on intermittent cold compression mode.

no

Claims (6)

a compound of formula (I) or (IA): ; Or a pharmaceutically acceptable salt thereof, wherein X is a halogen. A compound according to claim 1 which is selected from the group consisting of N ⁶ -[(3-halothien-2-yl)methyl]adenosine, N ⁶ -[(4-halothen-2-yl) a group consisting of methyl]adenosine and N ⁶ -[(5-halothiphenyl-2-yl)methyl]adenosine. A compound according to claim 2, which is selected from the group consisting of N ⁶ -[(5-bromothien-2-yl)methyl]adenosine, N ⁶ -[(4-bromothien-2-yl) Methyl]adenosine, N ⁶ -[(3-bromothien-2-yl)methyl]adenosine, N ⁶ -[(5-chlorothien-2-yl)methyl]adenosine, N ⁶ -[ a group consisting of (4-chlorothien-2-yl)methyl]adenosine and N ⁶ -[(3-chlorothien-2-yl)methyl]adenosine. A compound according to claim 1 which is selected from the group consisting of N ⁶ -[(2-halothen-3-yl)methyl]adenosine, N ⁶ -[(4-halothen-3-yl) a group consisting of methyl]adenosine and N ⁶ -[(5-halothiphenyl-3-yl)methyl]adenosine. A compound according to claim 4, which is selected from the group consisting of N ⁶ -[(2-bromothien-3-yl)methyl]adenosine, N ⁶ -[(4-bromothiophen-3-yl) Methyl]adenosine, N ⁶ -[(5-bromothien-3-yl)methyl]adenosine , N ⁶ -[(2-chlorothiophen-3-yl)methyl]adenosine, N ⁶ -[ a group consisting of (4-chlorothiophen-3-yl)methyl]adenosine and N ⁶ -[(5-chlorothiophen-3-yl)methyl]adenosine. A composition comprising: (a) a therapeutically effective amount of a compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier, Excipient, or carrier.