Modification of 4-(4-chlorothiophen-2-yl)thiazol-2-amine derivatives for the treatment of analgesia and inflammation: synthesis and in vitro, in vivo, and in silico studies

Inflammation is a protective response to a variety of infectious agents. To develop a new anti-inflammatory drug, we explored a pharmacologically important thiazole scaffold in this study. In a multi-step synthetic approach, we synthesized seven new thiazole derivatives (5a–5g). Initially, we examined the in vitro anti-inflammatory potentials of our compounds using COX-1, COX-2, and 5-LOX enzyme assays. After in vitro confirmation, the potential compounds were subjected to in vivo analgesic and anti-inflammatory studies. The hot plate method was used for analgesia, and carrageenan-induced inflammation was also assayed. Overall, all our compounds proved to be potent inhibitors of COX-2 compared to celecoxib (IC50 0.05 μM), exhibiting IC50 values in the range of 0.76–9.01 μM .Compounds 5b, 5d, and 5e were dominant and selective COX-2 inhibitors with the lowest IC50 values and selectivity index (SI) values of 42, 112, and 124, respectively. Similarly, in the COX-1 assay, our compounds were relatively less potent but still encouraging. Standard aspirin exhibited an IC50 value of 15.32 μM. In the 5-LOX results, once again, compounds 5d and 5e were dominant with IC50 values of 23.08 and 38.46 μM, respectively. Standard zileuton exhibited an IC50 value of 11.00 μM. Based on the COX/LOX and SI potencies, the compounds 5d and 5e were subjected to in vivo analgesic and anti-inflammatory studies. Compounds 5d and 5e at concentrations of 5, 10, and 20 mg/kg body weight were significant in animal models. Furthermore, we explored the potential role of compounds 5d and 5e in various phlogistic agents. Similarly, both compounds 5d and 5e were also significantly potent in the anti-nociceptive assay. The molecular docking interactions of these two compounds with the target proteins of COX and LOX further strengthened their potential for use in COX/LOX pathway inhibitions.


Introduction
Inflammation is a physiological response that occurs naturally in response to a variety of infectious agents, trauma, autoimmune diseases, injury, tissue ischemia, and an imbalance.It is a process that is required for the body to heal from infection and damage.Inflammation causes biochemical changes that result in swelling, redness, pain, itching, and an increase in body temperature.However, if inflammation is not appropriately treated, leukocytes, lymphocytes, or damaged collagen can infiltrate the tissue and cause long-lasting tissue damage (Nathan, 2002;Mahmood et al., 2022).A significant current public health concern is chronic inflammatory diseases (Killeen et al., 2014;Shin et al., 2014).Specific illnesses such as psoriasis, rheumatoid arthritis, periodontal disease, asthma, and atherosclerosis are caused by the dysregulation of inflammatory processes.Additionally, it plays a significant role in the development of several degenerative ailments, including diabetes, Alzheimer's disease, immunological disorders, and periodontal disease (Libby, 2002;Hansson and Peter, 2006;Sadiq et al., 2018).Additionally, the primary contributor to the harm caused by autoimmune illnesses is the inflammatory response (Jan et al., 2020).As a result, controlling inflammatory processes is a crucial strategy for treating a variety of illnesses.The quest for novel anti-inflammatory substances is still a crucial topic of research, despite the fact that there have been several previous attempts in this direction.This is because conventional medicines employing steroidal or nonsteroidal drugs are frequently associated with ineffectiveness and unfavorable side effects.In the traditional medicine of many cultures, plants have been utilized for generations to treat inflammation-related pain (Alam et al., 2020;Mahnashi et al., 2021).Chronic inflammation causes an overproduction of inflammatory mediators by activating Toll-like receptors found in macrophages.These mediators activate transcription factors that are either directly or indirectly controlled by mitogen-activated protein kinases (MAPKs), including nuclear factor kappa B (NF-kB) and nuclear factor erythroid 2-related factor 2 (Nrf2) (He et al., 2020).Additionally, a number of enzymes are activated, including COX-2 (cyclooxygenase), I kappa B kinase (IKK), inducible nitric oxide synthase (iNOS), and 5-LOX (lipoxygenase) (Ko et al., 2013;Tambuwala, 2016).
An endogenous enzyme called cyclooxygenase (COX) is primarily responsible for turning arachidonic acid into prostaglandins.COX-1 and COX-2 are two prevalent isoforms of this enzyme.COX-2 is an inducible enzyme that is expressed in response to an inflammatory stimulus and promotes inflammation, whereas COX-1 is a constitutive enzyme (Munir et al., 2020;Sadiq et al., 2021).Inflammation can be treated effectively by blocking the enzyme COX-2 (Osama et al., 2024).The untreated inflammation causes an increase in free radicals around the swelling area.Furthermore, the increase in temperature of the inflammatory site and even the whole body is due to the inflammatory response.Therefore, the analgesic and anti-inflammatory drugs work side by side due to their common enzymatic targets (Javed et al., 2022).Due to the uncoupling effects on oxidative phosphorylation, anti-inflammatory, analgesic, and antipyretic effects have been related to the suppression of prostaglandin formation.The pro-inflammatory cytokine tumor necrosis factor (TNF), which is released by a variety of cells and has a wide range of cellular effects, is significant (Montgomery and Bowers, 2012;Zelová and Jan 2013).TNF-α has been linked to a variety of disease states in people, including cancer, psychological disorders, and immunological and inflammatory diseases.IL-1 is a different cytokine that primarily promotes inflammation (Fenton, 1992;Alshehri et al., 2024).
Inflammation has typically been treated with steroids, although the use of these medications has increasingly decreased because of their negative effects (Poetker and Reh, 2010).One of the main classes of medications used to treat inflammation is nonsteroidal NSAIDs, which operate on the afflicted tissues by blocking COX, an enzyme involved in the manufacture of prostaglandins (Javed et al., 2021).Celecoxib (1), rofecoxib (2), and etoricoxib (3) are/were some of the very efficient COX-2 inhibitors, and they represent the COX-2 category of medications (Alqahtani et al., 2022).Rofecoxib and etoricoxib had to be taken off the market due to their damage to the gastrointestinal tract and increased risk of cardiotoxicity and hepatotoxicity (Dvorakova and Landa, 2017).Most of the currently used anti-inflammatory drugs, particularly those with established clinical efficacy, such as aspirin and ibuprofen, have an acidic character.It is, therefore, necessary to develop new antiinflammatory drugs with great therapeutic efficacy and minimal adverse effects.
The researchers working on organo-medicinal compounds provide effective methods for the development of new drugs, natural products, or building blocks of bioactive compounds (Ahmad et al., 2019;Qayyum et al., 2024).Various synthetic reactions provide baseline structural units for pharmacologically important molecules.Previously, we reported the synthetic approaches for the synthesis of various medicinally important compounds (Bibi et al., 2020;Qayyum et al., 2022).The medicinal compounds, particularly the nitrogenous moieties, have been explored for various medicinal purposes (Nugent et al., 2012;Sadiq et al., 2015;Sadiq and Nugent, 2020;Sadiq et al., 2022).The thiazole nucleus has been regarded as one of the prospective bioactive scaffolds in a variety of biological systems based on the literature that is currently accessible (Shah et al., 2023).Thiazole derivatives have a wide range of medicinal uses, including anti-Alzheimer, anticancer, antibacterial, and anti-diuretic properties.Certain medicines have thiazole rings (Tacconelli et al., 2002;Kovala-Demertzi, 2006).For many years, various natural and synthesized compounds containing the thiazole core moiety have been investigated for their anticancer, antibacterial, anti-HIV, anticoagulant, anti-inflammatory, and antioxidant effects.The bioactivities of thiazole scaffolds are further supported by the literature (Kamat et al., 2020).Anti-inflammatory medications lessen pain by completely or partially halting prostaglandin synthesis (Kawabata, 2011).Well-known anti-inflammatory medications (Figure 1), like meloxicam (5), are hybrid substances containing a thiazole core (Kamat et al., 2020).This study has been designed to synthesize new derivatives of thiazole for the treatment of analgesia and inflammation.

COX-1 activity
The anti-cyclooxygenase-1 activities of the compounds (5a-5g) were assessed using a modified protocol of the Glassman and White experiment.In brief, the COX-1 enzyme was added to a co-factor solution containing glutathione, hematin, and N, N, N-tetramethylp-phenylenediamine dihydrochloride (TMPD) prepared in a Tris-HCl buffer (pH 8.0).Then, test samples were added to the COX-1 enzymes and co-factor solution mixture.After 5 min, arachidonic acid was added and the solution was incubated.After incubation, the absorbance of the final reaction was measured at 570 nm using a UV spectrophotometer.Aspirin was used as a positive control for cyclooxygenase-1 enzyme inhibition (Ahmad et al., 2021).

COX-2 activity
The anti-cyclooxygenase-2 activities of the compounds (5a-5g) were assessed using a slightly modified protocol of the Glassman and White experiment.In short, COX-2 enzymes (300 units per ml) were added to a co-factor solution containing glutathione, hematin, and N, N, N-TMPD prepared in a Tris-HCl buffer (pH 8.0).Then, test samples were added to the COX-2 enzymes and co-factor solution mixture.After 5 min, arachidonic acid was added and incubated.After incubation, the absorbance of the final reaction was measured at 570 nm using a UV spectrophotometer.Celecoxib was used as a positive control for cyclooxygenase-2 enzyme inhibition (Jan et al., 2020).The selective COX-2 inhibitors are normally determined Marketed and reported anti-inflammatory drugs and agents.
Frontiers in Pharmacology frontiersin.orgusing the selectivity index (SI) of the compounds in both COX-1 and COX-2 assays.A greater SI value shows that the compound is a more selective COX-2 inhibitor.The SI of the compounds can be calculated from the IC 50 values of their respective COX-1 and COX-2 inhibitors as per the procedure.

5-LOX activity
The 5-lipoxygenase inhibitory activities of the compounds (5a-5g) were evaluated using different concentrations of the compounds against the standard drug zileuton.In brief, a concentration of 10,000 U per ml of the 5-LOX enzyme was used in the experiment.Moreover, linoleic acid was added as a substrate.Afterward, we dissolved different concentrations of test samples in a phosphate buffer (with pH of 6.3), followed by the addition of the 5-LOX enzyme solution.The mixture was then incubated for 5 min at 25 °C.After incubation, we added and mixed the linoleic acid solution with it and kept it for 5 min.Finally, the absorbance was measured at 234 nm using a UV spectrophotometer, and the experiment was repeated three times for optimal results (Jan et al., 2020).

Experimental animals and ethical approval
The experimental animals used in this study were obtained from the breeding house and were used as per the Animal By-Laws of the University of Malakand.The animals were maintained at a controlled temperature, humidity, and a 12-h dark/light cycle.The animals were provided food and water as per the approval of the ethical committee.The experimental animals used in this study were reviewed and approved by the Research Ethics Committee of the Department of Pharmacy, University of Malakand, Pakistan (Ref No. /055A).

Carrageenan-induced in vivo inflammation
For the in vivo anti-inflammatory test, the potential compounds (5d and 5e) were put to the test with carrageenan in albino mice that weighed 25-30 g.In total, there were 5 groups of 40 mice.Each group had eight animals.For group I, the negative control was 10 mL of 1% DMSO per kg of body weight.Group II was the positive control and received 100 mg of aspirin per kg of body weight.Compounds 5d and 5e were administered to groups III, IV, and V at amounts of 5, 10, and 20 mg/kg, respectively.Every 30 min, a newly prepared saline solution (0.05 mL) with 1% carrageenan was applied to the sub-planter surface of each mouse.A plethysmometer (LE 7500 Plan Lab SL) was used to measure the paw edema volume every 1-5 h after carrageenan was given.We measured the paw edema of the tested samples and the usual medicine at different times and compared them with the negative control group (Jan et al., 2020).The following method was used to obtain the percentage inhibition of inflammation: where "EC" is the average edema of the control group and "ET" is the edema of the group that was measured.

Mechanism of inflammation of the potent compounds
The potent synthesized compounds (5d and 5e) might help reduce inflammation by using paw edema tests that were triggered by prostaglandin E 2 , bradykinin, leukotriene, and histamine.It was done by injecting 10% DMSO, a Bradykinin inhibitor (HOE 140) at a dose of 1 mg/kg, a lipoxygenase inhibitor (montelukast) at a dose of 25 mg/kg, an antihistamine at a dose of 100 mg/kg, the tested compound at a dose of 100 mg/kg, or a cyclooxygenase inhibitor (celecoxib) at a dose of 50 mg/kg.After 1 h, doses of prostaglandin E 2 (0.01 mg/mL), leukotriene (10 mg/mL), bradykinin (20 mg/mL), or histamine (0.1 mg/mL) under the planter caused paw swelling.The volume of the paw of each mouse was measured immediately before and after irritants (inflammatory agents) were injected under the planter at 1-5 h (Jan et al., 2020).

Hot plate test
All of the animals were used to being in the laboratory for at least 2 h before the experiments began.An analgesiometer was used to test how well the painkillers worked on a hot plate (Shah et al., 2012).It was kept at 54.0 ± 0.1 °C on the hot plates.The animals were given a pre-test, and those with a delay time of less than 30 s were chosen.After the animals were chosen, they were divided into different groups, such as the normal saline group, the standard group, and other groups that would be tried on more powerful samples.Normal water, a standard drug, and different doses of the test sample were all given via abdominal administration.It was possible to determine the duration for which the animals remained on the hot plates at various time intervals.

Docking study
To investigate the anti-inflammatory inhibition potential of our newly synthesized 4-(4-chlorothiophen-2-yl) thiazole-based compounds, all the compounds were docked deep inside the receptors of the target proteins using Molecular Operating Environment (MOE) software.The crystal structures of the target proteins COX-2 and 5-LOX were retrieved from the Protein Data Bank (PDB) using the four-letter alphanumeric access codes 1CX2 and 6N2W, respectively (Sadiq et al., 2021).All compounds and target proteins were protonated, minimized, and prepared according to the previously reported standard procedure.The docking protocol was verified by the redocking method with RMSD >2.0.After docking calculations, all the orientations were evaluated based on the binding energy of the protein-ligand complex (ΔE = S-score) and the non-covalent interaction deep inside the pocket of target proteins.

In vitro COX/LOX results
The in vitro results of cyclooxygenases (COX-1 and COX-2) and lipoxygenase (5-LOX) of the compounds (5a-5g) are shown in Table 1.Compared to standard zileuton, our compounds (5a-5g) were less potent as inhibitors of 5-lipoxygenase.
The observed IC 50 value of zileuton in 5-LOX inhibition was 11.00 μM.Among the compounds, 5d had the highest activity,   The selectivity index is one of the major parameters for cyclooxygenase inhibitors.A greater value of SI shows that the compound is a more selective inhibitor of COX-2.Among our compounds, 5d and 5e had the highest SI values of 112 and 124, respectively.Based on the potencies of these compounds (5d and 5e) and their selectivity indexes, we further used these two compounds for in vivo experiments.

Anti-inflammatory mechanism of the potent synthesized compounds 5d and 5e
After observing potential anti-inflammatory effects in vitro and in vivo, we further investigated the synthesized compounds 5d and 5e to understand how they work to fight inflammation.We used different phlogistic agents, such as histamine, bradykinin, prostaglandin, and leukotriene, to test the process.It was found that compound 5d had a slight antihistaminic effect (27.12%) at the highest dose (20 mg/kg) at 3 h.This may be because it stopped mast cells from releasing mediators.In the same way, compound 5e showed 35% suppression at 3 h.In addition, the standard drug chlorpheniramine maleate greatly reduced swelling observed from the first to the fifth hour, resulting in a reduction of 71.62%-64.02%caused by histamine (Figure 3A).In the second part, swelling in the mouse paw reached its peak 60 min after the injection of bradykinin.The swelling of the mouse paw was not alleviated by compound 5d.At the highest doses (20 mg/kg), compound 5d stopped paw swelling by 9.20% in the first hour.It then decreased but not as much as the normal drug (HOE 140).Similarly, compound 5e did not yield excellent results either.Its effect peaked at 21.30% in the first hour and then decreased from the second to the fifth hour, as shown in Figure 3B. Figure 3C shows a notable increase in the amount of prostaglandin E 2 in the paw tissue after the injection of the prostaglandin E 2 mediator.When mice were given compound 5d (20 mg/kg) or celecoxib (50 mg/kg), the level of increased PGE 2 significantly decreased.At the fourth hour, compound 5d had the strongest effect (76.56 ± 0.58), about the same as a normal drug.It was still significant (***p < 0.001) at the fifth hour.In the same way, compound 5e was stronger than compound 5d and showed a suppression of 66.50% ± to 88.0% ± 0.66% from the first to the fourth hour.At the 4th hour after inflammogen, the usual drug celecoxib showed an 88% reduction.Furthermore, leukotriene caused inflammation, and the mouse paws swelled up after it was applied.The swelling reached its highest point 30 min after it was applied.It was found that compound 5d was very effective at treating mouse paw edema.Compound 5d showed good results, causing a percentage inhibition of 51.80 ± 0.58, 55.90 ± 0.66, 59.46 ± 0.52, 64.56 ± 0.78, and 59.90 ± 0.68.The compound 5d result was significant (**p < 0.01) at the first hour and reached its highest level at the fourth hour after the leukotriene mediator was given.It remained significant (***p < 0.001) until the fifth hour.Similarly, compound 5e showed excellent results, showing 69.84% inhibition at the fourth hour.At the fourth hour (Figure 3D), the usual drug montelukast showed a percentage reduction of 72.79 ± 1.10.Frontiers in Pharmacology frontiersin.org07 Mahnashi et al. 10.3389/fphar.2024.1366695Evaluation of anti-nociceptive activity on the hot plate model The results of the hot plate method of analgesia are presented in Table 3.By the administration of the positive control group after 30 min, the delay time was 17.23, and the painkilling effect was 47.59% ± 0.71% at 100 mg/kg body weight.Synthesized compound 5e changed the delay time by 14.33 ± 0.44 (p < 0.01) and stopped thermal shocks 36.99% ± 0.94% of the time at a dose of 20 mg/kg I/p.Furthermore, compound 5d greatly reversed the effect, stopping 29.62% ± 0.84% (p < 0.05) of the activity at a dose of 20 mg/kg (Table 3).Similarly, after 60 min, the delay time was significantly longer in animals that had been given the usual drug (5 mg/kg tramadol I/P) than in those that had been given the vehicle control group (p < 0.001).At a dose of 20 mg/kg I/p, compound 5e significantly changed the pain-relieving effect, stopping 44.16% ± 0.70% of it (p < 0.01).Synthesized compound 5d that was tested had a delay time of 14 s and blocked the good effects of heat stimuli with a significance level of 35.50% ± 0.50% (p < 0.05) at a dose of 20 mg/kg (Table 3).In addition, the latency time was significantly longer in animals that were given the usual drug (5 mg/kg tramadol  The information shows pain-relieving effects using a hot plate test on mice.The results are shown as the mean ± SEM (n = 6) and were analyzed using a two-way ANOVA, followed by Bonferroni's post hoc test.pM < 0.05; ppp < 0.01, and pppp < 0.001; ns; not important.I/P) than in those given the vehicle control group (latency time 13.27 s; p < 0.001).The pain effect was greatly offset by compound 5e, which had a delay time of 11.25 (p < 0.01) and caused percentage analgesic inhibitions of 22.93 ± 0.60 at a dose of 20 mg/kg I/p.Compared to the negative control at the same amount, compound 5d had a delay time of 8.8 and had a lesser effect than compound 5e.
Compound 5d displayed important binding affinities similar to those of native ligands, including vital hydrophilic hydrogen bond interactions with His90 and Ser353 through the hydrogen of the NH group.It also displayed pi-sulfur interaction (hydrophobic) with Tyr355 and Phe518 by the sulfur of the thiazole and thiophene regions.Other hydrophobic interactions include the π-π stacked interaction with Asp515 and the π-alkyl interaction with Val523.
Compound 5e also displayed binding affinities with important residues similar to the native ligand.It includes pi-sulfur hydrophobic interaction with His90 through the sulfur of the thiophene region.The aromatic region in the benzyl region showed pi-sulfur interaction with Met522 and Met113.Pi-sigma interaction was also displayed between the Ser353 amino acid residue and the aromatic region of thiophene.The halogen attached to the thiophene group also displayed interaction with Gln192.The aromatic region of one terminal side of the molecule displayed binding affinity (hydrophobic interaction) with Trp387 (Figure 4).
Compound 5d displayed binding affinities with amino acid residues similar to those of native ligands, including hydrophobic interactions with His367, Trp599, and Phe359 (pi-pi T-shaped) through the thiophene aromatic region and aromatic region present at the one terminal end.The halogen group attached to the thiophene ring displayed halogen interactions.Pi-sulfur hydrophobic binding affinity was displayed with the His372 residue through the sulfur of the thiophene group.Other hydrophobic binding affinities include Leu607 and Ala410.
Compound 5e also displayed binding affinities similar to those of native ligands, including hydrophobic interactions with His367 (pi-pi T-shaped), His 432, and Trp599 (pi-pi stacked) with an aromatic region present on both terminals of the synthesized molecule and thiophene aromatic region.It also displayed pi-alkyl hydrophobic interactions with Leu268 and Ala603 (Figure 5).
Analgesia and its major cause, inflammation, are medical complications that go side by side in many ailments (Zhang and An, 2007;Mahnashi et al., 2021).Both of these ailments are protective responses to injuries and external stimuli (Antonelli and Kushner, 2017).Hotness, or analgesia, is among the major symptoms of inflammation.The current marketed analgesic and Frontiers in Pharmacology frontiersin.org09 anti-inflammatory drugs are associated with severe unwanted effects (Carter et al., 2014).To minimize toxicity and improve the activity profile, medicinal chemists synthesize various potent molecules for the treatment of analgesia and inflammation (Zayed, 2022).Previously, we synthesized simple pyrrolidinedione and other Michael products as potential analgesic and antiinflammatory agents (Ahmad et al., 2021;Sadiq et al., 2021).In this research, we found that bis-succinimide efficiently inhibits the COX/LOX pathway for the management of analgesia and inflammation.

Conclusion
In this study, we synthesized 7 new 4-(4-chlorothiophen-2-yl) thiazol-2-amine derivatives (5a-5g) as potential anti-inflammatory and analgesic agents.The compounds were synthesized efficiently through multistep chemical reactions.The pharmacologically important building blocks of thiazoles, specifically, compounds 5d and 5e, have been proven to be potential inhibitors of COX/ LOX pathways.Compared to the respective standard drugs (zileuton for 5-LOX, aspirin for COX-1, and celecoxib for COX-2), compounds 5d and 5e were potent anti-COX/LOX agents.Moreover, our compounds also proved to be safe in experimental animals.The compounds proved to be very active analgesic and anti-inflammatory agents in in vivo models.The molecular docking of the selected compounds in the enzymes COX-1, COX-2, and 5-LOX further supported our results and confirmed that our synthesized compounds effectively bind to the amino acid residues of the target site.In this study, we explored the thiazole scaffold as a potential analgesic and anti-inflammatory building block.Frontiers in Pharmacology frontiersin.org10
FIGURE 3 (A) Percentage of blockage caused by histamine and compounds 5d and 5e (20 mg/kg) in a mouse model of histamine-induced paw edema.(B) Amount of blockage caused by bradykinin and compounds 5d and 5e (20 mg/kg) in a mouse model of bradykinin-induced paw edema.(C) Amount of inhibition that the studied compounds 5d and 5e (20 mg/kg) created in a mouse model of prostaglandin E 2 -induced paw edema.(D) Amount of reduction that tested compounds 5d and 5e (20 mg/kg) caused in a mouse model of leukotriene-induced paw edema.The mean (±SEM) for a group of eight mice is shown by each percentage point.ANOVA and Dunnett's test were used to analyze the data.*p < 0.05, **p < 0.01, and ***p < 0.001 are all significant, and n.s.means that there was no difference between the experimental and normal drugs.

TABLE 1
In vitro cyclooxygenase inhibition results.

TABLE 2
Percentage inhibitions of the synthesized compounds in carrageenan-induced paw edema.The percentage of carrageenan-induced paw edema model in mice was blocked by tested molecules at doses of 5, 10, and 20 mg/kg.The mean (±SEM) for a group of eight mice is shown by each percentage point.Two-way repeated measures ANOVA and Bonferroni's post hoc test were used to analyze the data.Asterisks indicate numbers that are important from the control (car).*p < 0.05, **p < 0.01, and ***p < 0.001; n.s.stands for "not significant."There are eight mice in each group.

TABLE 3
Analgesic activity of the synthesized compounds assessed using the hot plate method.