Because they have fewer side effects, newly discovered chemicals may have significant medicinal promise. The lessened side effects of newly discovered chemicals hold great medicinal potential. 2-amino-4-substituted thiazoles have previously been shown to exhibit anthelmintic, anti-leukotriene, anticonvulsant, antimalarial, and fungicidal properties.⁹

A higher risk of PD is linked to both "passive" and occupational exposure due to closeness of residence with pesticide-treated fields. Pesticide related with PD such as organochlorines and dithiocarbamates, paraquat, rotenone, 2,4-D, induce experimental Parkinson’s in laboratory examination, reinforcing an idea that these pesticides are connected with causative effects of PD¹⁰.

As an illustration of how genes and the environment interact, genetically predisposed deficiency in toxicant management can enhance PD risk when individuals are exposed to pesticides. On the other hand, engaging in actions like maintaining proper hygiene or having a nutritious food may guard against negative consequences of pesticide exposure.^11,12

MATERIAL AND METHOD:

Materials:

Analytical grade substances were used in the inquiry. The supplier of chemicals was Sigma Aldrich Chemicals Private Limited, located in Bangalore, India.

Method:

The study is based on the synthesis of novel derivatives of carboxylate and hydrazides from Ethyl 2-[2-chloroacetamido]-thiazole-4-carboxylate.

Production of ethyl 2-aminothiazole-4-carboxylate new derivatives:

The combination of 20 mmol ethyl 2-[(Z)-(4-oxo-1,3-thiazolidin-2-ylidene) amino]-1,3-thiazole-4-carboxylate (1) and furan-2-carboxaldehyde (2) was added in absolute ethanol and piperidine was added in catalytic quantity to the reaction mixture. The mixture was refluxed for 4 hrs while being stirred. A precoated TLC plate was used to track the reaction's progress. When reaction was done, mixture was poured in ice-cold water and acidified to a pH of 3–4 using glacial acetic acid, resulting in a crude solid. After filtering, the solid was washed extensively with water, dried and recrystallized from methanol to yield ethyl 2-((5-(furan-2-ylmethylene)-4-oxothiazolidin-2-ylidene) amino)-thiazole-4-carboxylate (SH-4).

An equimolar mixture of 2-acetamido thiazole-4-carbohydrazide (2mmol, 3) and the substituted benzaldehyde (2mmol, 4) was refluxed in the presence of few drops of glacial acetic acid in absolute ethanol for 5 hrs. After the completion of reaction as indicated by the TLC, the mixture was poured into ice water to afford a solid, which was then collected through filtration and washed with water to remove traces of acid. The obtained solid was recrystallized from aqueous alcohol to afford the final product (SH-9) in good yield and purity.

Experimental Design:

Molecular docking study was carried out on the library of compounds to determine the affinity of NCE’s towards alpha synuclein (a protein responsible for Parkinson’s Disease) in reducing the symptoms of Parkinson’s disease. In house library of substituted thiazole derivatives were subjected to molecular docking against MAO–B enzyme. The crystal structure of the MAO-B was downloaded from the Protein Data Bank (PDB ID: α-synuclein). Docking calculations were performed to predict the binding affinities of various compounds and protein of interest α-synuclein by using AutoDock 4.2 tool. The generated maps were calculated by using the auxiliary program AutoGrid. Molecular docking study and MMGBSA binding free energy revealed that the maximum binding energy was calculated and the generated scores lead to the identification of two hit compounds i.e. SH-4 and SH-9, which were used for further studies. These hit compounds with highest binding scores were then tested in vivo for anti-Parkinson’s activity in animal models. The compounds were also tested in a range of disease-related behavioural scenarios.^13,14

Animals:

Up-and-Down method was utilized to assess the acute toxicity of derivatives of hydrazide and carboxylate in Swiss albino mice. In this investigation, animals weighing between 20 to 24g at 12–14 weeks of age were employed. The study design was accredited by Institutional Animal Ethics Committee. The study design was approved by the Institutional Animal Ethics Committee. Standard housing conditions for the animals included a 12-hour light/dark cycle, 65±10% humidity, and 28±30°C room temperature. Group I administered with (10mL/kg, p.o.) normal saline; Group II animals were administered with Rotenone and Paraquat which is used as Parkinson’s inducing agent. Group III had Llevodopa (100mg/kg, p.o.) and Groups IV to V were administered at 100mg/kg dose, p.o. with test drugs (SH-4 and SH-9). The animals were given Paraquat and Rotenone once daily for 55 days, based on their individual groups, following a 48-hour induction period.

Behaviour Assessment:

a) Catalepsy bar test:

The stiffness of the muscles was measured using the catalepsy bar test. The steel bar used for testing was horizontal, 15cm long, 0.9cm in diameter, and maintained at 5.5cm height above ground. The mice were to be held by the tail, with their hind legs on a level surface and their front paws resting on a steel bar. Time taken by animals to become acclimated to externally enforced position was used to calculate the descending latency. The experiment ended after 180 seconds if the animal did not move its paws in an active manner.¹⁵

b) Open field test:

An open-field test was utilized to determine animal's spontaneous locomotor function. A closed room with sides measuring 50cm and a 40cm height was utilized for open field test. The animals were put in the centre of arena and allowed to watch for three minutes. Before usage and again after every test, to eliminate any odour indications, the device was cleansed with ethanol 95% (v/v). The video tracking software ANY-maze^TM was used to record the data of total distance travelled (m) and average velocity.¹⁶

c) Rotarod activity:

Using the rotarod apparatus, the animals' grip performance and motor coordination were assessed. For the purpose of preparing the mice for rotarod performance, they had already had training. Mice were put on a spinning rod with a 7cm diameter (speed 25 rpm). An average time of descent was determined and cut-off time was 180s.¹⁷

d) Pole test:

On a wooden pole, the mice were placed (1cm diameter, 50cm long). It was timed how long it took the mice to descend to the floor. For further analysis, each mice had three trials of testing, and the average testing time was determined.¹⁶

Neurochemical Assessment:

Dissection and homogenization:

Following the therapy time, animals were decapitated while under a light anesthetic in order to scarify them. The cerebellum was thrown away, the brains were cut out, and the forebrain was removed. To eliminate blood, brains were placed on ice and then rinsed by using ice cold isotonic saline. A homogenate of 10% (w/v) tissue was made using 0.1M phosphate buffer of pH 7.4. After centrifuging homogenate for 15 minutes at 10,000g, supernatant aliquots were taken and utilized for biochemical analysis.

Biochemical Assessment:

a) Malondialdehyde (MDA) level:

An amount of malondialdehyde was measured by reaction with thiobarbituric acid (TBA) and utilized as an lipid peroxidation indirect indicator. The test tubes were filled with 1mL of supernatant aliquots and 3mL of TBA reagent (trichloroacetic acid [TCA 15%], hydrochloric acid (HCl) 0.25 M and TBA 0.38% (w/w). After shaking the mixture and letting it sit for 15 minutes, it was cooled on ice bath. After cooling, solution was centrifuged for ten minutes at 3500rpm. Upper layer of the centrifuged compound was separated and evaluated at 532nm by using spectrophotometer. Every decision was made three times. Nanomoles per milligram of protein was used to express the results.¹⁸

b) GSH level (Decreased glutathione):

Using 1ml of solution, tissue homogenate 1 ml was precipitated in TCA 10%. The precipitate was eliminated by centrifugation. An aliquot of supernatant was mixed with DTNB reagent 0.5 ml and phosphate solution 4ml. A colour produced was read at 412nm. Using a standard curve made using standard decreased glutathione, GSH amount in supernatant was determined and represented in nM/mg of protein.¹⁹

c) Dopamine level determination:

Dopamine quantity in entire brain was calculated utilizing UV-visible spectroscopy. The typical linear dopamine curve was eliminated by serially diluting standard dopamine (from 50 to 500ng/mL). The concentration v/s. absorbance linear curve was generated, and dopamine determination range was chosen at 240-280 nm. To determine whether dopamine was present, supernatant from every test group was diluted 10 times and absorbance at 278nm was employed. Equation y=mx+c was developed utilizing linear curve and sample absorbance to quantify the total amount of dopamine in brain in ng/g of tissue.¹⁹

d) Monoamine oxidase activity:

As suggested by Puka-Sundvall et al., From the striatum, mitochondrial fractions were isolated for the MAO-B test. Homogenate 10% (w/v) was prepared in ice cold buffer A (pH 7.4), that comprises bovine serum albumin 0.1% (w/v), 10 mM EDTA, 0.44 M sucrose and 10mM Tris-HCl using a Potter-Elvehjam-style glass homogenizer. Subsequently, homogenate was centrifuged for 15 minutes at 4°C at 2100 g. Additional centrifugation was performed on the supernatant for 15 minutes at 4°C and 14,000g. The rough mitochondrial pellet should be separated, cleaned with buffer A, and centrifuged again for 15 minutes at 7000g and 4°C. Buffer B (pH 7.4), which contains Tris-HCl 10mM and sucrose 0.44 M was utilized to re-suspension final mitochondrial pellet. To quantify MAO-B activity, the procedure was slightly altered. The specific MAO-B substrate used in the assay was 4 mM benzylamine, and sodium phosphate buffer 2.75 ml (pH 7.4) of 0.1 M were included in mixture. At a wavelength of 249.5 nm, mitochondrial fraction was suspended to start reaction as a MAO-B source. Absorbance was measured for 5 minutes against a control. The enzymes activity was determined in nmol/min/mg protein.^20,21

Statistical Analysis:

The standard error mean (SEM) for each value is displayed as mean. The results were evaluated using Tukey's multiple comparison test and a one-way analysis of variance (ANOVA). Values with p < 0.05 were regarded as significant in statistics.

RESULTS:

New compounds synthesis:

In this study, Carboxylate and Hydrazide derivatives were prepared in good yield and purity.

Behavioural assessment:

The SH-4 and SH-9 effect on paraquat induced PD in locomotor activity: All of the animals exhibited comparatively similar activity during the pre-treatment. Following treatment, the Paraquat-induced control group's line-crossing performance was lower than that of normal control. Compared to PD group, line-crossing performance gradually improved in treatment group at a dose of 100 mg/kg (***p<0.0001). When compared to mice given solely with Paraquat, levodopa (100 mg/kg), an antidepressant, significantly enhanced line-crossing activity (****p<0.00001). Results were depicted in (figure 1).

Figure 1: SH-4 and SH-9 treatment effect on motor functions determined by using open field test in paraquat induced PD mice model. Number of line crossed were expressed as mean ± SEM; n = 6/ group. A significant difference was seen in SH-4 group (***p<0.0001) while compared with paraquat group.

The SH-4 and SH-9 effect on paraquat induced Parkinson’s disease in rotarod performance: During the pre-treatment phase, the rotarod activity of each animal was comparatively equal. Following treatment, the Paraquat-induced control group's rotarod performance was lower than that of normal control. Rotarod activity gradually improved in the treatment group with SH-4, 100mg/kg (***P<0.0001). This improvement was dose-related. Standard anti-Parkinson's medication levodopa (100mg/kg) considerably enhanced performance of rotarod (****P<0.00001) when compared to mice treated with Paraquat only. Result is shown in (figure 2).

Figure 2: SH-4 and SH-9 effect on treatment on activity of rotarod in paraquat induced PD model. Values are expressed as mean ± SEM; n = 6/group; Levodopa (****p<0.00001) and SH-4 (***p<0.0001) showed difference from paraquat group.

SH-4 and SH-9 effect on climbing time in rotenone induced PD model:

Climbing time in control group was 10.5±1.72 sec. Rotenone-induced mice PD model climbing time was increased (25.18±1.16 sec). SH-4 and SH-9 (100mg/kg) treatment significantly decreased the climbing time i.e., 18.26±1.52 sec (***p<0.0001) and 22.51±1.28 sec. (p=ns) with SH-4 and SH-9, respectively, compared with the rotenone group. Results were depicted in (figure 3).

Figure 3: SH-4 and SH-9 effect on treatment on motor functions determined by utilizing Pole Test in Rotenone induced PD mice model. Climbing time was expressed as mean ± SEM; n = 6/ group. A significant difference was seen in SH-4 group (***p<0.0001) when compared with rotenone group.

SH-4 and SH-9 effect on the catalepsy score in rotenone-induced PD mice model:

After 2 weeks of rotenone treatment, the enhancement in catalepsy was significantly higher in rotenone group. The standard drug showed significant fall in rigidity (****p<0.00001). Similarly, the treatment groups also represented reduction in catalepsy score. However, SH-4 showed more significant reduction (***p<0.0001) in muscular rigidity comparable to standard drug. The outcomes were depicted in (figure 4).

Figure 4: SH-4 and SH-9 effect on treatment on catalepsy score in rotenone induced PD mice model. Mean fall time was showed as mean±SEM; n = 6/group. A significant difference was seen in SH-4 group (***p<0.0001) when compared with rotenone group.

SH-4 and SH-9 effect on treatment on open field (locomotor function) test in rotenone induced PD mice model:

When compared to the vehicle-treated group, daily administration of rotenone for 28 days significantly decreased locomotor activity in an open field test. Levodopa treatment significantly and dose-dependently (****p<0.00001) reduced the motor activity drop caused by rotenone and returned it to normal. The test compounds significantly restored the motor activity. The data was represented in (figure 5).

DISCUSSION:

PD is a long-term neurodegenerative condition marked by SNpc dopamine neurons death. Oxidative stress, protein buildup like as ɑ-synuclein, mitochondrial malfunction, apoptosis, and neuronal excitotoxicity are among pathophysiology associated with PD. The most important pathogenic mechanism for PD of all is oxidative stress. Since it contains more dopamine, SNpc is more susceptible to reactive oxygen species. Today, PD treatment involves levodopa and certain MAO-B inhibitors. Two generated compounds, SH-4 and SH-9, were examined for their inhibitory potential using an in silico molecular docking approach.^22,23

According to the current study, paraquat has been shown to cause neuronal degeneration and motor impairments. These pesticide-induced PD have been proposed as a possible cause of PD because they cause the nigrostriatal pathway's dopaminergic neurons to die. Pesticide exposure during development has been shown in multiple animal models to cause cumulative, persistent, and progressive neurotoxicity of nigrostriatal system. The current investigation demonstrated neuroprotective function of recently synthesized drugs against PD phenotype in mice produced by paraquat. The characteristics of PD is rigidity and diminished motor movements, which are caused by an overabundance of free radicals that raise oxidative stress and dopaminergic neurons' degradation in the brain.²⁴

We assessed memory impairment and stress management using the rotarod test. The capacity of the mice to grip onto rotarod while it is spinning at a controlled speed is the primary criterion to be examined in this test. The paraquat-induced group of mice exhibited a consistent decline in their behavioral activities, which is thought to be the cause of dopaminergic neuron impairment and loss. In contrast, the SH-4 (100 mg/kg p.o.) dose produced more effective outcomes which were comparable to those of standard group (levodopa). In the current investigation, levodopa therapy significantly reduced GSH levels and the activity of reactive oxygen species scavenging enzymes such LPO in brain tissue.

Furthermore, compared to control group, paraquat-induced group produced considerably more MDA, indicating enhanced lipid peroxidation. GSH levels were found to be considerably higher after receiving NCEs (SH-4 and SH-9) treatments than after paraquat-induced mice. Compared to the SH-9 (100 mg/kg p.o) group, SH-4 (100 mg/kg p.o) group more successfully decreased the MDA levels, hence reducing the extent of lipid peroxidation.

The paraquat-induced group displayed oxidation occurring in multiple regions due to a deficiency of dopamine, the SH-9 (100mg/kg p.o) group demonstrated treatment occurring in a few regions but was not entirely effective, and the SH-4 (100mg/kg p.o) group demonstrated even better outcomes for treatment that were significant to the group receiving levodopa. The behavioral characteristics and biochemical assessment revealed that the SH-9 (100mg/kg p.o) and SH-4 (100 mg/kg p.o) groups outperformed the Levodopa-treated group in terms of effectiveness.

Neuroprotective impact of NCEs in PD rotenone model was also examined in the current research. To cause a gradual dopaminergic degeneration in the SNPc area, we subcutaneously gave rotenone for 4 weeks at a dose of 1.5mg/kg body weight.

Our research findings on effects of rotenone administration on motor coordination included reduced grip strength activity, a drop in rotarod activity, and hypokinetic movement in an open field test. The injection of SH-4 and SH-9 resulted in a considerable increase in locomotor activity, but it also decreased the number of foot slips and increased latency time. The injection of rotenone resulted in a considerable increase in both nitrosative and oxidative damage as compared to normal animals. Animals with lipid peroxidation higher levels (higher MDA) and lower GSH levels were found to be affected. On the other hand, rotenone-induced oxidative damage was significantly and dose-dependently reduced by treatment with SH-4 and SH-9.

CONCLUSION:

Taking into account the aforementioned information, it was determined that synthesized compounds demonstrated antioxidant properties and showed promise in treating Parkinson's disease in mice. However, more thorough research on the pharmacology and toxicology of the chosen compounds in anti-Parkinson's is needed.

CONFLICT OF INTEREST:

Regarding this study, the authors have no conflicts of interest.

ACKNOWLEDGMENTS:

The authors would like to express their gratitude to Amity University for their kind assistance with their research.

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Received on 16.06.2024 Revised on 12.10.2024

Accepted on 15.01.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2639-2646.

DOI: 10.52711/0974-360X.2025.00379

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