Synthesis, characterization, biochemical, and molecular modeling studies of carvacrol‐based new thiosemicarbazide and 1,3,4‐thiadiazole derivatives

A series of carvacrol‐based thiosemicarbazide (3a–e) and 1,3,4‐thiadiazole‐2‐amine (4a–e) were designed and synthesized for the first time. The structures were characterized by nuclear magnetic resonance and high resolution mass spectroscopy techniques. All compounds were examined for some metabolic enzyme activities. Results indicated that all the synthetic molecules exhibited powerful inhibitory actions against human carbonic anhydrase I and II (hCAI and II), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes compared to the standard molecules. Ki values of five novel thiosemicarbazides and five new 1,3,4‐thiadiazole‐2‐amine derivatives (3a–e and 4a–e) for hCA I, hCA II, AChE, and BChE enzymes were obtained in the ranges 0.73–21.60, 0.42–15.08 µM, 3.48–81.48, 92.61–211.40 nM, respectively. After the experimental undertaking, an extensive molecular docking analysis was conducted to scrutinize the intricate details of interactions between the ligand and the enzyme in question. The principal focus of this investigation was to appraise the potency and efficacy of the most active compound. In this context, the calculated docking scores were noted to be remarkably low, with values of −8.65, −7.97, −8.92, and −8.32 kcal/mol being recorded for hCA I, hCA II, AChE, and BChE, respectively. These observations suggest a high affinity and specificity of the studied compounds toward the enzymes, as mentioned earlier, which may pave the way for novel therapeutic interventions aimed at modulating the activity of these enzymes.

Natural products have historically been considered an invaluable inspiration for chemistry, biology, and medicine.These molecules are produced by plants, microorganisms, and animals and have a variety of chemical structures that naturally fall within the respective chemical domains. [1][4] As well as being used for flavor, fragrance, and preservative supplement in food or cosmetics, [5] it is known that carvacrol has many biological effects such as antioxidant, [6,7] antinociceptive, [8] antibacterial, [9][10][11] anti-influenza, [12] antiviral, [7,13,14] antifungal, [15,16] anticancer, [17] enzyme inhibitor, [18][19][20][21] antidepressant, and so on. [22]iosemicarbazides can be evaluated as a target's starting, intermediate, or final compound.[25] They can form different dinucleophilic centers because of C, N, and S bonds in their structures.Therefore, they synthesize various heterocyclic compounds containing nitrogen and sulfur, such as 1,3,4-thiadiazole, pyrazole, oxadiazole. [26,27]The 1,3,4-thiadiazole nucleus is a heterocyclic nucleus frequently encountered, especially in medicinal chemistry. [28]ere are many 1,3,4-thiadiazole derivative drugs in the market such as acetazolamide, methazolamide, megazole, sulfametizole and cefazolin. [29]2] Carbon dioxide (CO 2 ) and water (H 2 O) can be interconverted to bicarbonate (HCO 3− ) by metalloenzymes called carbonic anhydrases (CAs).Six families of CAs exist, each defined by a unique gene and the presence of metal ions in the catalytic sites.For instance, the Zn 2+ metal ion is a distinctive component of the α, β, and δ type gene families; the family of CAs is saturated with Zn 2+ , Fe 2+ , and Co 2+ metal ions, whereas the ζ-CAs family is rich in Zn 2+ or Cd 2+ in the catalytic domains. [33,34] Numerous physiological processes, including pH control, respiration, the secretion of electrolytes, bone resorption, Na + ion retention, signal transduction, lipogenesis, ureagenesis, and gluconeogenesis, are all thought to be affected by the roles played by CAs. [35,36]e primary function of the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is to convert acetylcholine into choline acetic acid.Hydrolysis links a lack of acetylcholine products to critical psychological processes in the brain's hippocampus and cortex.Additionally, it is a chronic, lifelong brain disorder in which the cholinergic system in the brain is perpetually out of balance. [37,38]is might result from confusion, difficulty thinking, cognitive Because of this, it is still necessary to reduce enzyme potentials using a stronger medication. [38,39]lecular docking, a computational method that predicts the binding mode and affinity between a small molecule, typically a drug candidate, and a receptor, such as a protein target or a nucleic acid, has emerged as an indispensable tool in the field of drug discovery due to its high significance and versatility. [33,34]It provides a rational and efficient approach for identifying and optimizing potential drug candidates by predicting their interactions with the target biomolecule.Molecular docking is recommended as an alternative strategy to the traditional in vitro screening methods, which are costly, time-consuming, and often limited to a few compounds. [35]The remarkable achievements of molecular docking in drug discovery include identifying novel lead compounds, optimizing existing drugs with improved efficacy, selectivity, and pharmacokinetic properties, and exploring new therapeutic targets. [36,37]Therefore, molecular docking has become essential in the drug discovery pipeline, accelerating drug development and improving human health.
42][43] Moreover, acetazolamide and methazolamide, prototypical CA inhibitors, contain thiadiazole nuclei. [44]7] Our previous studies showed that carvacrol derivative compounds have inhibitory properties against several metabolic enzymes (human carbonic anhydrase [hCA] I, hCA II, AChE, and BChE). [48]In similar studies conducted by our study group, 1,3,4-triazole and thiosemicarbazide compounds were proved to be potential enzyme inhibitors against AchE and BCHE, as well as hCA I and hCA II. [49,50]In this study, new 1,3,4triazole and thiosemicarbazide derivatives with carvacrol structure were designed and studied against enzymes predicted to inhibit.
In this study, five new thiosemicarbazide derivatives (4a-e) were prepared, based on the natural product carvacrol, in three steps.Then, by cyclizing these derivatives in an acidic environment with a practical method, five new 1,3,4-thiodizaol-2-amine derivatives (4a-e) compounds were synthesized.The biological activities of all synthesized compounds on some metabolic enzymes were investigated.

| Chemistry
In the first step, ethylphenoxy acetate derivative (1) was synthesized by refluxing the carvacrol compound with ethyl chloroacetate in acetone in a basic medium.In the second step, this compound was reacted with hydrazine hydrate to convert the ester group to acetohydrazide, and the acetohydrazide (2) derivative was obtained.
Later then, 2 was treated with different aryl isocyanates to obtain thiosemicarbazide derivatives (3).Finally, triazole-3-thiol derivatives (4) were obtained by intramolecular cyclization reactions of 3 in an acidic medium.The applied synthesis plan is given in Scheme 1, and the structures and yields of all synthesized compounds are shown in Table 1.

| Biochemical studies
The selective inhibition of CA enzymes is a frequently employed strategy in designing new therapeutic drugs that can be used to treat various diseases since they are improperly expressed or activated in many pathological circumstances.Clinical applications for several CA inhibitors include the treatment of glaucoma, cancer, and obesity. [51,52]Compound 4e (hCA I, K i = 0.73 ± 0.07 µM; IC 50   almost 34%, respectively. [54]ditionally, the only Food and Drug Administration-approved cholinesterase inhibitors now in the market are galantamine, tacrine, donepezil, and rivastigmine, but they all have substantial adverse effects such as hepatotoxicity and anxiety.[57] Tacrine served as the positive control, and the anticholinesterase effects of five new thiosemicarbazide and five new 1,3,4-thiadiazole-2-amine derivatives (3a-e and 4a-e) were assessed using Ellman's technique.

| Molecular docking
Molecular docking is an essential manner that has significant implications for the drug innovation fields.It is suggested as a replacement method for in vitro assessment and has had significant success in drug/inhibitor design. [58]Therefore, molecular docking was investigated to interact with the thiadiazole derivatives with metabolic enzymes.carbon-hydrogen bond were observed with Trp439 and His447, respectively.Gly122, Gly121, Ser125, Tyr72, Val73, and Tyr124 were found at the AChE active site. [59]4e was bound to the AChE active site, consistent with the prior report.Ser287, Gln119, Phe118, Phe398, Tyr332, Pro285, Gly115, Glu197, and Gly439 residues bonded using van der Waals bond.Only 1 π-π shaped and carbon-hydrogen bonds were observed with Trp82 and Ser198, respectively.Furthermore, 11 π-π-alkyl interactions were seen in the bound 3e-BChE complex.Phe398, Tyr332, Pro285, Gly115, Glu197, and Ser198 were found at the BChE active site. [59] bound to the BChE active site, which was consistent with the prior report.Phe91, and Pro202 residues bonded using π-alkyl bond.Only one conventional H-bond was observed with His200 residue.Furthermore, Ala135, Gln92, His67, Pro201, Leu203, Ser197, Trp209, Val207, and Val120 residues bonded using the van der Waals bond.

| CONCLUSION
The ethylphenoxy acetate derivative (1) was synthesized in 92% yield by refluxing the carvacrol compound in acetone with ethyl chloroacetate in a basic medium.This compound was reacted with hydrazine hydrate to convert the ester group to acetohydrazide and acetohydrazide derivative (2) was obtained in 88% yield.Then, thiosemicarbazide derivatives were obtained by treatment of 2 with different aryl isocyanates (3a-e).These compounds were prepared practically in very high yields (77%-93%).Finally, triazole-3-thiol derivatives (4a-e) were obtained by intramolecular cyclization of 3a-e in sulfuric acid.Yields were also quite high in this step (78%-96%).days.After the reaction, the mixture was cooled, and the solvent was removed on a rotary evaporator.The crude product was dissolved with diethyl ether and extracted with water (5 × 30 mL).
The ether phase was washed with 0.1 M sodium hydroxide solution (1 × 30 mL) followed by water (1 × 30mL).The organic phase was dried with sodium sulfate, and the solvent was removed in a rotary evaporator.The obtained light brown viscous liquid product 1 (8.65 g, 92%) was used in further reactions without any purification.2] Light brown viscous liquid, 92% yield; Then it was cooled to room temperature, and the solvent was removed in a rotary evaporator.The crude product was recrystallized from diethyl ether.2] White solid, 88% yield; General procedure for the synthesis of thiosemicarbazide derivatives (3a-e): 2 (0.5 g, 2.25 mmol) in ethanol (10 mL) was added to the related isocyanate (2.24 mmol) derivative and refluxed for 4 h.After the reaction, the mixture was cooled to room temperature and poured onto ice.The precipitate was filtered and washed with ether, then dried in an oven and used in further reactions without further purification.The resulting structures were characterized by spectroscopic methods. [62][2-(5-Isopropyl-2-methylphenoxy)acetyl]-N--nitrophenyl)hydrazinecarbothioamide (3a): Light yellow solid, 77% yield; 1 H NMR frequently evaluated as inhibitors of AChE and BuChE.Examples in which triazole and thiosemicarbazide derivatives inhibit choline esterase enzymes are also available in the literature.Although there are examples in the literature where triazole and thiosemicarbazide derivatives inhibit choline esterase enzymes, studies are still limited.

Table 2 )
. Another compound, 3d (hCA I, K i = 11.14 ± 1.04 µ M; IC 50 = 9.18 µM with r 2 : 0.946), having a benzyl group, was the second less potent analog of the series.The test complex' K The structures and yields of all synthesized compounds.
[53]lues showed that 4e was the most effective compound against hCA I and II (hCA I, K i = 0.73 ± 0.07 µM; hCA II, K i = 0.42 ± 0.03 µM).Derivatives 3e and 4d, with K i values of 0.97 ± 0.06 and 1.02 ± 0.09 µM, were the second and third most effective molecules against hCA I among the synthetic derivatives.Indeed, CA II, IV, XII, and XIV are inhibited by CA inhibitors used as diuretic medications.While CA II, VII, and XIV inhibitors are utilized as antiepileptic medications, CA inhibitors that affect CA II, IV, and XII are used to treat glaucoma.Additionally, the transmembrane CA isoenzymes CA IX and CA XII, which control pH, have emerged as prospective cancer indicators.[53]RarelyaretheseCAsassociatedwithcancer present in healthy tissues.Theyare, S C H E M E 1 Synthesis plan.Reaction conditions: [a] Chloroethyl acetate, acetone, K 2 CO 3 , 4 days, reflux; [b] N 2 H 2 .H 2 O, EtOH, 4 h, reflux; [c] R-SCN, EtOH, 4 h reflux; [d] concentrated H 2 SO 4 , 90°C, 5 h.T A B L E 1 T A B L E 2The enzyme inhibition results of novel compounds (3a-e, 4a-e) against hCA I and II.Abbreviations: AZA, acetazolamide; CA, carbonic anhydrase.a AZA has been under investigation since 1956 and is the best-known clinically used CA inhibitor, and its conjugates are nominated as promising agents for the treatment of metastatic kidney cancer.This study was used as a standard.however, overexpressed or upregulated in tumor cells, including those from malignancies of the breast, colon, pancreatic, brain, head, and neck because CA I and II isozymes expressed in red blood cells resemble CA IX and XII isozymes by about 34% and CA II isozyme by

Table 3
The enzyme inhibition results of novel compounds (3a-e and 4a-e) against AChE, BChE.Details of interacting thiadiazole derivatives and metabolic enzymes.
The docking scores of the thiadiazole derivatives docked into AChE, BChE, hCA I, and hCA II are revealed in Table 4. Docking energy algorithms guess the enzyme molecules binding with the synthetic compound's atoms and calculate the most stable possible state.From the binding energy, it can be understood that the binding of synthetic compounds to AChE, BChE, hCA I, and hCA II enzymes has T A B L E 3 T A B L E 4 negative docking energy.The docking score of TAC (standard compound) with AChE and BChE were −7.28 and −6.86 kcal/mol, respectively.Furthermore, the docking score of AZA (standard compound) with hCA I and hCA II were −7.23 and −6.93 kcal/mol, respectively.Docking energy outputs display that among the thiadiazole derivatives, 4e (for hCA I, hCA II, AChE) and 3e (for BChE) show more negative docking scores than standard inhibitors and signify great binding abilities for interaction with the AChE, BChE, CA I, and CA II enzymes, respectively.