(S)-ENANTIOMERIC FORM OF A HETEROCYCLIC COMPOUND HAVING MOTIVATION IMPROVING AND/OR REFERENCE MEMORY ENHANCING ACTIVITY
20210380545 · 2021-12-09
Inventors
Cpc classification
A61P25/28
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to the (S)-enantiomer of the chemical compound having the general formula (I):
##STR00001## wherein R.sub.1 and R.sub.2 are, equal or independently, aryl, heteroaryl, fused aryl, fused heteroaryl, mono or multi-substituted aryl, mono or multi-substituted heteroaryl, mono or multi-substituted fused aryl, mono or multi-substituted fused heteroaryl; and R.sub.TA is a 2-1,3-, or 4-1,3-or 5-1,3-thiazole ring with the general Formula (IIa), which can also be substituted.
The invention also relates to said compound according to formula (I) for use in improving motivation and/or cognitive functions and/or for use in improving the reference memory in human individuals.
Claims
1. An (S)-enantiomer (with respect to the sulfoxide group) of the chemical compound having the general formula (I): ##STR00013## wherein R.sub.1 and R.sub.2 are, equal or independently, aryl, heteroaryl, fused aryl, fused heteroaryl, mono or multi-substituted aryl, mono or multi-substituted heteroaryl, mono or multi-substituted fused aryl, mono or multi-substituted fused heteroaryl; R.sub.TA is a 2-1,3-, or 4-1,3- or 5-1,3-thiazole ring with the general Formula (IIa), ##STR00014## R.sub.3 is present on the ring according to Formula (IIa) 1 or 2 times, equal or independently, wherein R.sub.3 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, alkylamino, hydroxyalkylamino, alkoxy, arylalkyl, hydroxyalkyl, thioalkyl, haloalkyl, haloaryl, haloarylalkyl, haloalkoxy, mono or multi-substituted alkyl, mono or multi-substituted cycloalkyl, mono or multi-substituted heterocycloalkyl, mono or multi-substituted alkylamino, mono or multi-substituted arylamino, mono or multi-substituted hydroxyalkylamino, mono or multi-substituted alkoxy, mono or multi-substituted arylalkyl, mono or multi-substituted hydroxyalkyl, mono or multi-substituted thioalkyl, mono or multi-substituted haloalkyl, mono or multi-substituted haloaryl, mono or multi-substituted haloarylalkyl, mono or multi-substituted haloalkoxy and carboxylate ester, wherein substituted means substituted with a residue selected from the group consisting of alkyl, cycloalkyl, heterocycloalkyl, hydroxyalkyl, alkylthio, ether, hydroxyl, fluoride, chloride, bromide and iodide.
2. The compound according to claim 1, wherein one of R.sub.1 and R.sub.2 or both of R.sub.1 and R.sub.2 is/are aryl or substituted aryl, and/or wherein R.sub.3 is hydrogen or alkyl or oxyalkyl or alkyl substituted with at least one residue selected from the group consisting of heterocycloalkyl, carboxylic acid, amide and ester.
3. The compound according to claim 1, wherein R.sub.TA is a thiazole group.
4. The compound according to claim 1, wherein R.sub.1 and R.sub.2 are, equal or independently, aryl or substituted aryl, wherein R.sub.3 is present on R.sub.TA according to Formula (IIa) 2 times and selected from the group consisting of R.sub.4, R.sub.5, R.sub.6 and R.sub.7, wherein R.sub.TA is a 4-1,3-thiazole ring with the general Formula (IIIa) ##STR00015## wherein R.sub.4 is selected from the group consisting of oxirane, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and wherein R.sub.5 is selected from the group consisting of hydrogen, methyl, n-propyl, iso-propyl, fluoro, chloro, bromo and iodo, or wherein R.sub.TA is a 5-1,3-thiazole ring with the general Formula (IIIb) ##STR00016## wherein R.sub.6 is selected from the group consisting of hydrogen, methyl, n-propyl, iso-propyl, fluoro, chloro, bromo, iodo, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, wherein R.sub.7 is selected from the group consisting of hydrogen, methyl, n-propyl, iso-propyl, fluoro, chloro, bromo, iodo, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, and wherein substituted means substituted with a residue selected from the group consisting of alkyl, cycloalkyl, heterocycloalkyl, hydroxyalkyl, alkylthio, ether, hydroxyl, fluoride, chloride, bromide and iodide.
5. The compound according to claim 1, wherein it is selected from the group consisting of (S)-2-(diphenylmethanesulfinylmethyl)-1,3-thiazole, (S)-4-(diphenylmethanesulfinylmethyl)-1,3-thiazole, (S)-2-(diphenylmethanesulfinylmethyl)-4-methyl-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-1,3-thiazole and (S)-4-(diphenylmethanesulfinylmethyl)-2-methyl-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-2-chloro-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-4-methyl-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-4-chloro-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-2-methyl-4-methyl-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-2-methyl-4-chloro-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-2-chloro-4-methyl-1,3-thiazole, (S)-5-(diphenylmethanesulfinylmethyl)-2-cyclopropyl-1,3-thiazole, (S)-4-(diphenylmethanesulfinylmethyl)-2-oxirane-1,3-thiazole, (S)-4-(diphenylmethanesulfinylmethyl)-2-cyclopropyl-5-methyl-1,3-thiazole, (S)-4-(diphenylmethanesulfinylmethyl)-2-cyclopropyl-5-chloro-1,3-thiazole, (S)-4-(diphenylmethanesulfinylmethyl)-2-cyclopropyl-1,3-thiazole.
6. The compound according to claim 1, wherein it is (S)-5-(diphenylmethanesulfinylmethyl)-2-methyl-1,3-thiazole or (S)-5-(diphenylmethanesulfinylmethyl)-1,3-thiazole-.
7. A method for treating age-related cognitive decline in a human individual in need thereof, the method comprising administering the compound of claim 1 to the human individual.
8. A method for improving motivation or cognitive functions, or motivation and cognitive functions in a human individual in need thereof, the method comprising administering the compound of claim 1 to the human individual.
9. A method for improving reference memory, or motivation and reference memory in a human individual in need thereof, the method comprising administering the compound of claim 1 to the human individual.
10. The method according to claim 8, wherein the human individual does not have defects of cognitive functions or reference memory deficits or motivation deficits, or does not have defects of cognitive functions and reference memory deficits and motivation deficits caused by diseases of the brain.
11. The method according to claim 10, wherein the human individual does not have defects of cognitive functions or reference memory deficits or motivation deficits, or does not have defects of cognitive functions and reference memory deficits and motivation deficits.
12. The method according to claim 8, wherein the human individual has defects of cognitive functions or reference memory deficits or motivation deficits, or has defects of cognitive functions and reference memory deficits and motivation deficits caused by diseases of the brain.
13. The method according to claim 12, wherein the human individual has defects of cognitive functions or reference memory deficits and/or motivation deficits, or reference memory deficits and motivation deficits caused by Alzheimer; Down syndrome; vascular cognitive impairment; stroke; frontotemporal dementia; behavioural, semantic or progressive aphasia type dementia; dementia with Lewy bodies; subcortical dementias; Parkinson's disease dementia; alcohol related dementia; dementia caused by traumatic brain injury; Huntington's disease related dementia; AIDS-related dementia; attention deficit disorders; reference memory deficiencies related to ageing; reference memory disorders related to viral infections of the brain; or schizophrenia.
14. The method according to claim 13, wherein the motivation deficits are caused by depression or depressive disorders.
15. The method according to claim 8, wherein the human individual is an aging individual.
16. A method for inhibiting dopamine transporter (DAT)-mediated dopamine reuptake in the brain of a human individual in need thereof, the method comprising administering the compound of claim 1 to the human individual.
17. A method for improving motivation or cognitive functions, or motivation and cognitive functions in a human individuals, who does not have defects of cognitive functions or reference memory deficits or motivation deficits, or who does not have defects of cognitive functions and reference memory deficits and motivation deficits, the method comprising administering the compound of claim 1 to the human individual.
18. The method according to claim 17, wherein the cognitive function to be improved is the learning capability and/or the reference memory, or the learning capability and the reference memory.
19. A pharmaceutical preparation comprising at least one compound according to claim 1 and a pharmaceutically acceptable carrier and/or diluent.
20. A method for treating age-related cognitive decline in a human individual in need thereof, the meth od corn rising administering the pharmaceutical preparation of claim 19 to the human individual.
21. A method for improving motivation or cognitive functions or for improving motivation and reference memory in a human individuals in need thereof, the method comprising administering the pharmaceutical preparation of claim 19 to the human individual.
22. A method for inhibiting DAT-mediated dopamine reuptake in the brain of a human individual in need thereof, the method comprising administering pharmaceutical preparation of claim 19 to the human individual.
23. A method for inhibiting DAT-mediated dopamine reuptake in the brain of a human individual who does not have defects of cognitive functions or reference memory deficits or motivation deficits, the method comprising administering the compound of claim 1 to the human individual.
24. The compound according to claim 2, wherein the thiazole group is a 2-thiazole group or a 4-thiazole group.
25. The compound according to claim 24, wherein the thiazole group is 2-methyl-4-thiazole.
26. The method according to claim 7 wherein the age-related cognitive decline is according to the US version of 2018 ICD-10-CM Diagnosis Code R41.81.
27. The method according to claim 20 wherein the age-related cognitive decline is according to the US version of 2018 ICD-10-CM Diagnosis Code R41.81.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0135]
[0136]
DETAILED DESCRIPTION
Examples
A. Syntheses
1. Synthesis of 5-(chloromethyl)thiazole Hydrochloride
[0137] 5-(Hydroxymethyl)thiazole (0.63 g, 5.5 mmol) was dissolved in 20 mL of dichloromethane in a 100 mL round-bottomed flask, which was subsequently placed in an ice bath set up on a magnetic stirrer. Thionyl chloride (0.40 mL, 5.5 mmol) was carefully dripped in via a syringe and the reaction was allowed to proceed overnight. The reaction mixture was condensed on a rotary evaporator and the dark yellow grainy substance thus obtained was additionally dried in high vacuum to remove lingering thionyl chloride as well as water and afforded 0.93 g (5.5 mmol) of a highly hygroscopic yellow powdery product in quantitative yield.
[0138] 2. Synthesis of [(diphenylmethyl)sulfanyl]methanimideamide Hydrobromide
##STR00010##
[0139] Diphenylmethanol (1.29 g, 7 mmol) was dissolved in 30 mL of methanol in a 100 mL three-necked round-bottomed flask, thiourea (0.61 g, 8 mmol) was added and the reaction mixture was refluxed (external temperature 87 to 90° C.) for half an hour until homogeneity was achieved. Hydrogen bromide (3.64 mL, 32.2 mmol) was added in a dropwise fashion over the course of half an hour, and the reaction mixture was further refluxed for 3 hours.
[0140] The reaction mixture was allowed to cool down to room temperature, it was poured from the three-necked flask into a 100 mL round-bottomed flask and methanol was evaporated. The remaining substance was suspended in 30 mL of dichloromethane, stirred for half an hour (batch extraction of remaining diphenylmethanol) and filtered through a Buechner funnel. The solid residue was washed with additional 30 mL of dichloromethane and suspended afterwards in 30 mL of water, stirred for half an hour (batch extraction of unreacted thiourea) and filtered through a Buechner funnel. The residue was washed with 30 mL of water and dried. 1.62 g (5 mmol) of white, fine powdered product was obtained, amounting to isolated yield of 71%.
[0141] 3. Synthesis of 5-((diphenylmethylthio)-methyl)thiazole
##STR00011##
[0142] Compound A (1.62 g, 5 mmol) was dissolved in 30 mL of methanol in a 100 mL round-bottomed flask mounted of a magnetic stirrer. 5-(Chloromethyl)thiazole hydrochloride (0.93 g, 5.5 mmol) was added and dissolved, potassium carbonate (3.45 g, 25 mmol) was subsequently introduced and the reaction mixture was refluxed for 4 hours (external temperature was 87 to 90° C.). The majority of potassium carbonate was disposed of by filtering of the hot reaction mixture on through a Buechner funnel; methanol was evaporated from the filtrate. The remaining substance was suspended in 50 mL of water, and organic products were extracted with ethyl acetate (75 mL, 3 portions). The extracts were pooled, dried with anhydrous Na.sub.2SO.sub.4, filtered and condensed under reduced pressure to afford an oily mixture which was purified by column chromatography on silica gel (2.5% solution of methanol in dichloromethane was used as eluent). 1.25 g (4.25 mmol) of the desired product was obtained in this manner, which corresponds to isolated yield of 84%.
4. Synthesis of (S)-5((diphenylmethanesulfinyl)-methyl-1,3-thiazole) (Compound C1)
[0143] ##STR00012##
[0144] 5-((Benzhydrylthio)methyl)thiazol (1.25 g, 4.25 mmol) was dissolved in 25 mL of acetone in a 100 mL round-bottomed flask. Titanium isopropoxide (0.37 mL, 1.25 mmol), (S,S)-(−) diethyl tartrate (0.43 mL, 2.5 mmol) and water (11.25 μL, 0.625 mmol) were added to the mixture, which was stirred for 5 minutes to achieve homogeneity, upon which the flask was equipped with reflux condenser and the mixture was stirred for an hour at 65° C. (external temperature). The mixture was given 15 minutes to cool down to room temperature, N,N-Diisopropylethylamine (0.15 mL, 0.85 mmol) was added and, after 10 minutes of stirring, Cumene hydroperoxide (0.78 mL, 4.25 mmol) was added and the reaction was allowed to proceed for 20 hours. Acetone was evaporated from the mixture and the oily orange-brown residue was purified by column chromatography on silica gel (2.5% solution of methanol in dichloromethane was used as eluent). Thus 0.99 g of ((S)-5-((diphenylmethanesulfinyl)-methyl-1,3-thiazole) (Compound C1) was obtained, which corresponds to a 74% yield.
B. Experiments Demonstrating Motivation and Cognitive Enhancing Effects
[0145] The compound of the present disclosure can be used for motivation and cognitive enhancement of reference memory of normal and aged individuals with and without a variety of human diseases.
[0146] The compounds of the present disclosure were demonstrated to have motivation and cognitive enhancing activity and more specifically motivation and reference memory enhancing activity in an animal model.
[0147] It has been suggested that animal models employing tests of effort-based decision making can be used to study functions that are related to aspects of human motivational dysfunction (Salamone et al. 2006, 2016a, b, c). This strategy has been validated by clinical research showing that patients with major depression, Parkinson's disease and other disorders show a low-effort bias when tested on effort-related choice procedures (Treadway et al. 2012; Yang et al. 2014; Chong et al. 2016; Barch et al. 2017).
B-1. Holeboard Test:
Animals and Keeping Conditions:
[0148] Male Sprague-Dawley rats (17 to 18 weeks and 22 to 23 months old), bred and maintained in the Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna were used. The animals were transferred to a separate experimental room one week before the start of the experimental procedure and kept there throughout the experiment individually in standard Makrolon cages filled with autoclaved woodchips temperature: 22±2° C.; humidity: 55±5%; 12 h artificial light/12 h dark cycle: light on at 7:00 am). The study was carried out according to the guidelines of the Ethics committee, Medical University of Vienna, and were approved by the Federal Ministry of Education, Science and Culture, Austria.
Holeboard Apparatus and Training:
[0149] The holeboard (1 m×1 m) was made of black plastic and was surrounded by translucent plexiglass walls. The walls were equipped with spatial cues. Distal cues were provided by room structures outside the board. Four out of sixteen regularly arranged holes (diameter and depth 7 cm) were baited (dustless precision pellets, 45 mg, Bioserv®). The pattern of baited holes remained the same during the entire test. To avoid olfactory orientation pellets were also present in an area below the board. Prior to the experiment for three days the rats were handled for 15 min to get familiarized to the experimenter, followed by two days of habituation to the hole-board during which the animals could explore the maze for 15 min each day with access to food pellets. The weight of each rat was reduced by food restriction to reach 85% of its initial body weight before the start of the experiment. Tap water was given ad libitum. Rats were trained for three days (five trials on day one, four trials on day 2 and a retention trial at day 3). Every trial lasted for 120 s or until all four pellets were found. The apparatus was cleaned with 0.1% Incidin® (Ecolab, Dusseldorf, Germany) between trials to remove odor cues. The interval between two successive trials for an individual was 20 min. A camera mounted on the room ceiling recorded the performance of the rats in the maze. The hole visits and removals of pellets were noted for each trial. To compare rats with similar levels of motivation, rats with less than 40-hole visits in total over the ten trials were excluded from the analysis. Reference memory errors were noted as the number of visits to the unbaited holes. The Reference Memory Index (RMI) was calculated using the formula (first+revisits of baited holes)/total visits of all holes. All behavioral training/testing was performed during the light phase of the light-dark cycle.
Results:
Behaviour in Young Males
[0150] A significant trial effect was found (F.sub.9,324=22.00, p<0.001), but no trial x treatment interaction (F.sub.27,324=0.93, p=0.569) and no overall treatment effect between groups in young males (F.sub.3,33=0.75, p=0.528) in the memory indices.
Behaviour in Old-Males
[0151] And, a significant trial effect was determined (F.sub.9,315=15.18, p<0.001), but no trial x treatment interaction (F.sub.27,315=1.38, p=0.103), however, an overall treatment effect between groups in the memory indices (F.sub.3,35=3.29, p=0.032). Tukey-post hoc tests revealed significantly enhanced RMI in rats treated with compound C1 at a dosage of 10 mg/kg body weight (p=0.021), whereas rats treated with 1 mg (p=0.357) or 5 mg (p=0.171) performed similar as compared to controls.
[0152] Training session-specific analysis between vehicle (DMSO/Tween 80/saline solution) and 10 mg compound C.sub.1 treated rats revealed a significantly enhanced RMI in treated rats at day 1 (F.sub.1,18=5.40, p=0.032), a significant trial effect (F.sub.4,72=9.41, p<0.001) and a trial x treatment interaction (F.sub.4,72=3.95, p=0.006). At day 2 there was no significant trial effect (F.sub.3,54=2.41, p=0.077), no significant trial x treatment interaction (F.sub.3,54=1.42, p=0.248), but a significant treatment effect (F.sub.1,18=18.02, p<0.001). At the retention trial at day 3 a higher RMI was found in treated rats (T=−3.40, df=18, p=0.003) compared to controls.
Differences in Behaviour Between Young and Old Males
[0153] Young animals treated with 10 mg/kg bodyweight showed lower RMI than old males treated with the same dosage (F.sub.1,18=5.01, p=0.038) over the entire training. A significant trial effect (F.sub.9,162=16.86, p<0.001) was found, and no trial x age interaction (F.sub.9,162=0.80, p=0.616).
[0154] Day specific analyses revealed a significant trial effect (F.sub.4,72=15.24, p<0.001), but no trial x age interaction (F.sub.4,72=1.04, p=0.393), and no age effect (F.sub.1,18=2.04, p=0.170) at day 1. A significant trial effect (F.sub.3,54=4.89, p=0.004), no trial x age interaction (F.sub.3,54=0.86, p=0.466), but a significant age effect (F.sub.1,18=6.71, p=0.018) could be determined at day 2. No significant differences in RMI between groups could be observed at the retention trial at day 3 (T=1.87, df=18, p=0.077).
[0155]
B-2. Motivational Studies Showing the Effect of Compound C1:
[0156] Animals were offered a choice between high effort instrumental actions leading to highly valued reinforcers vs. low effort/low reward options. That is, effort-related choice behavior was used to model effort-related motivational dysfunctions in humans.
[0157] Rats were assessed using the fixed ratio 5/chow feeding choice test. Tetrabenazine (TBZ: 9,10-dimethoxy-3-(2-methylpropyl)-1,3,4,6,7, 11b-hexahydrobenzo[a]quinolizin-2-one) was used as the vesicular monoamine transport blocker which inhibits DA storage, depletes brain DA, and reduces post-synaptic DA receptor signalling (Nunes et al. 2013). TBZ is known to shift choice behaviour and induce a low effort bias. And, it is also known that the reallocation of behaviour from high to low effort options that is produced by TBZ is not due to alterations in food preference or hedonic taste reactivity, reduced appetite or impairments in reference memory (Randall et al. 2012, 2014; Nunes et al. 2013; Correa et al. 2015; Pardo et al. 2015; Yohn et al. 2015a).
[0158] Tetrabenazine (1.0 mg/kg) shifted choice behavior, decreased lever pressing and increased chow intake. Compound C1 was co-administered at doses ranging from 3.0 to 24.0 mg/kg. The highest dose partially but significantly reversed the effects of tetrabenazine. Compound C1 was able to reverse the effort-related effects of TBZ. This suggests that compound C1 could be useful as a treatment for effort-related motivational dysfunction in humans.
[0159] The behavioural studies evaluated the motivational effects of compound C1 in rats using a test of effort-based choice behaviour, the concurrent fixed ratio 5 (FR5)/chow feeding choice task. Effort-related choice was studied using procedures that offer high effort options leading to highly valued reinforcers vs. low effort/low reward options (Salamone and Correa 2012; Salamone et al. 2016a,b,c). That is, the ability of compound C1 to attenuate the effort-related effects of TBZ in rats tested on the concurrent FR5/chow feeding choice task was evaluated.
Animals and Keeping Conditions:
[0160] Adult male Sprague-Dawley rats (Envigo Sprague Dawley, Indianapolis, Ind., USA; weights 275-299 g upon arrival) were pair-housed in a colony maintained at 23° C., with a 12-h light/dark cycle (lights on 07:00). Rats were food deprived to 85% of their free-feeding body weight for operant training and allowed modest growth throughout the experiment. Water was available ad libitum in the home cages. Animal protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee, and the studies were conducted according to National Institutes of Health (NIH) guidelines.
Behavioral Procedure: FR5/Chow Feeding Choice Task:
[0161] Behavioural sessions were conducted in operant chambers (28×23×23 cm3; Med Associates, Fairfax, Vt.) with 30 minute sessions 5 days/week. Rats were initially trained to lever press on a continuous reinforcement FR1 schedule (high-carbohydrate 45 mg pellets, Bio-Serv, Frenchtown, N.J.) and then shifted to the FR5 schedule. After 5 weeks of training on the FR5 schedule, chow was introduced. Weighed amounts of laboratory chow (Laboratory Diet, 5P00 Prolab RMH 3000, Purina Mills, St. Louis, Mo.; typically 15-20 g) were concurrently available on the floor of the chamber during the FR5/chow feeding choice task sessions. At the end of each 30 minute session, rats were immediately removed from the chambers, number of lever presses was recorded, and the amount of chow consumed was determined by weighing the remaining food (including spillage). Rats were trained on the FR5/chow feeding choice procedure for 5 weeks, after which drug testing began. On baseline and drug treatment days, rats consumed all of the operant pellets that were delivered during each session.
Drug Treatments and Dose Selection:
[0162] Compound C1 ((S)-5((diphenylmethanesulfinyl)-methyl-1,3-thiazole)) was dissolved in dimethyl sulfoxide (DMSO), Tween 80, and 0.9% saline. The DMSO/Tween 80/saline solution was administered as the vehicle control. TBZ was obtained from Tocris Bioscience (Ellisville, Mo.) and was dissolved in DMSO, 0.9% saline, and titrated with HCl. The DMSO/saline solution was administered as the vehicle control. The TBZ dose was 1.0 mg/kg.
Behavioural Experiments:
[0163] Trained rats (n=8) were administered either TBZ (1.0 mg/kg) or vehicle, and compound C1 (6.0, 12.0, and 24.0 mg/kg) or vehicle, on drug testing days. Rats received TBZ or vehicle 120 minutes before testing and compound C1 or vehicle 30 minutes before testing. The experiment used a within-groups design, with each rat receiving each drug treatment in a randomly varied order (one treatment per week, with none of the treatment sequences repeated across different animals). The following five treatment combinations were given: [0164] TBZ vehicle+compound C1 vehicle; [0165] mg/kg TBZ+compound C1 vehicle; [0166] 1/0 mg/kg TBZ+6.0 mg/kg compound C1; [0167] 1.0 mg/kg TBZ+12.0 mg/kg compound C1; [0168] 1.0 mg/kg TBZ+24.0 mg/kg compound C1.
[0169] Total number of lever presses and gram quantity of chow intake from the 30 minute sessions were analysed using repeated measures ANOVA. A statistical program (SPSS; version 2015) was used to perform all analyses. When there was a significant ANOVA, non-orthogonal planned comparisons were performed, using the overall error term to assess differences between each treatment and the control condition. The number of comparisons was restricted to the number of treatments minus one (Keppel, 1991).
Results of FR5/Chow Feeding Choice Task:
[0170] Repeated measures ANOVA revealed that there was an overall significant effect of treatment with compound C1 on lever pressing [F(4,28)=34.625, p<0.001]. Planned comparisons showed that TBZ significantly decreased lever pressing compared to vehicle treatment (TBZ/vehicle vs. vehicle/vehicle [F(1,28)=105.87, p<0.001]). There also was a significant overall effects of treatment with compound C1 on chow intake [F(4,28)=27.280, p<0.001], and TBZ alone significantly increased chow intake relative to vehicle treatment (TBZ/vehicle vs. vehicle/vehicle [F(1,28)=66.625, p<0.001]). Additional planned comparisons revealed that co-administration of the dose of 24.0 mg/kg compound C1 with TBZ significantly attenuated the effects of TBZ on lever pressing (TBZ plus 24.0 mg/kg vs. TBZ/vehicle [F(1,28)=13.2866, p<0.01]) and chow intake (TBZ plus 24.0 mg/kg vs. TBZ/Veh [F(1,28)=61.014, p<0.001]).
[0171]
[0172] In detail,
Reuptake Inhibition Assays.
[0173] [3H]5-HT (Hydroxytryptamine; 5-[1,2-3H[N]]; 27,8 Ci/mmol), [3H]DA (Dihydroxyphenylethylamine; 3,4-[ring-2,5,6-3[H]]-Dopamine; 36,6 Ci/mmol) and [3H]MPP+ (Methyl-4-phenylpyr-idinium iodide; 1-[methyl-3H]; 80 Ci/mmol) were purchased from Perkin Elmer, Boston, Mass.
[0174] Effects of Compound C1 ((S)-5-(diphenylmethanesulfinylmethyl)-1,3-thiazole), (R)-5-(diphenylmethanesulfinylmethyl)-1,3-thiazole (in the following also referred to as Compound C2) and of racemic 5-(diphenylmethanesulfinylmethyl)-1,3-thiazole (in the following also referred to as Compound C3) on reuptake of their respective substrates were analyzed as described by Y. D. Aher et al. in Front. Behav. Neurosci. 10 (2016) 20 (“A novel heterocyclic compound CE-104 enhances spatial working memory in the radial arm maze in rats and modulates the dopaminergic system”) and S. Sucic et al., in J. Biol. Chem. 285 (2010) 10924-10938 (“The N terminus of monoamine transporters is a lever required for the action of amphetamines”). HEK293 cells stably expressing human isoforms of the dopamine transporter (DAT), the nor-epinephrine transporter (NET) and the serotonin transporter (SERT) were used for reuptake inhibition assays. All cell lines were seeded on 96-well plates pre-coated with poly-D-lysine (PDL) (5×104 cells/well) 24 h prior to the experiment. Each well was washed with 100 μL of Krebs-HEPES buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2.2H2O, 2 mM MgCl2.6H2O, 5 mM D-(+)-Glucose mono-hydrate, pH 7.3). Cells were pre-incubated 5 min in KHB containing different dilutions (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 0.1 mM and 1 mM) of Compound C1, Compound C2 and Compound C3. Compound C1, Compound C2 and Compound C3 were separately dissolved first in 99.9% dimethyl sulfoxide (DMSO) and subsequently diluted in KHB. Afterwards, cells were incubated in KHB containing the same dilutions of Compound C1/Compound C2/Compound C3 with addition of 0.2 μM [3H]-dopamine (for HEK-DAT), 0.05 μM [3H]MPP+(for HEK-NET) and 0.4 μM [3H]5-HT (for HEK-SERT). Incubation times were 1 min for HEK-DAT and HEK-SERT and 3 min for HEK-NET. For determination of non-specific uptake in HEK-DAT and HEK-NET 10 μM mazindole was used and 10 μM paroxetine was used for HEK-SERT. After incubation at room temperature, reactions were stopped by the addition of 100 μL of ice-cold KHB. Finally, cells were lysed with 300 μL of 1% SDS and released radioactivity was measured by a liquid scintillation counter (Tri-carb-2300TR, Perkin Elmer).
Results:
[0175] Compound C1, Compound C2 and Compound C3 were characterized in HEK293 cells expressing cloned human transporters. A reuptake inhibition assay was used to determine the efficacy of Compound C1/Compound C2/Compound C3 to block the uptake of substrates by different monoamine transporters DAT, NET and SERT ([3H]DA, [3H]MPP+ and [3H]5-HT, respectively). It was found that Compound C.sub.1 very strongly inhibits DAT-mediated dopamine reuptake (IC50=2.8 μM±0.4) whilst inhibition of NET and SERT is negligible. It was found that Compound C3, i.e., the racemate, is much less effective in inhibiting DAT-mediated dopamine reuptake (IC50=4.6 μM±0.6), and that Compound C2, i.e., the R-enantiomer, showed a much lower effect in inhibiting DAT-mediated dopamine reuptake (IC50=22.3 μM±1.6).
[0176] Although modifications and changes maybe suggested by those skilled in the art, it is the intention of the applicant to embody within the patent warranted hereon all changes and modifications as reasonably and probably come within the scope of this contribution to the art. The features of the present disclosure which are believed to be novel are set forth in detail in the appended claims. The features disclosed in the description, the figures as well as the claims could be essential alone or in every combination for the realization of the disclosure in its different embodiments.
[0177] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
[0178] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.