4.3.4. Evaluation of the Motor Activity

## Rota-Rod Test

The Rota-Rod test device (Ugo Basile, 47600, Varese, Italy) was used to evaluate the possible effects of test compounds on the motor coordination of animals. Before experiments, mice were trained on the rotating rod of the apparatus set at 16 rpm for 3 consecutive days. Animals that could stay on the rotating mill for more than 180 s were used for the tests. The falling time of the mice was considered as a parameter for motor coordination. The cut-off time for this test was accepted as 10 min [40,41,57].

#### 4.3.5. Statistical Analysis

Statistical evaluation of the experimental data was carried out using GraphPad Prism ver. 8.4.3 software (GraphPad Software, La Jolla, CA, USA). The data used in the statistical analyses were acquired from seven animals in each group. The differences between experimental groups were determined by one-way analysis of variance (ANOVA) followed by a post-hoc Tukey's test. The results were presented as mean ± standard error of the mean (SEM) and considered statistically significant when *p* < 0.05.

GraphPad Prism ver. 8.4.3 software were used for creating the figures.

#### *4.4. Molecular Docking Studies*

Molecular docking studies were performed using an in silico procedure to define the binding modes of obtained compounds in active regions of opioid receptors. X-ray crystal structures of µ-opioid receptor (PDB ID: 5C1M) [33], δ-opioid receptor (PDB ID: 4N6H) [34], and k-opioid receptor (PDB ID: 6B73) [35] were retrieved from the Protein Data Bank server (www.pdb.org, accessed date 28 April 2021). Active conformations of these receptors were used.

The structures of proteins were built using the Schrödinger Maestro [58] interface and were then submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [59]. The ligands were prepared using LigPrep 3.8 [60] to correctly assign the protonation states at pH 7.4 ± 1.0, as well as the atom types. Bond orders were assigned, and hydrogen atoms were added to the structures. The grid generation was formed using the Glide 7.1 [61] program and docking runs were performed with standard precision docking mode (SP).

#### **5. Conclusions**

To the best of our knowledge, this is the first study showing that molecules designed to carry thiazole and piperazine moieties together on their structures have convenient pharmacokinetic profiles and show notable antinociceptive efficacies mediated by the opioid receptors at the spinal, supraspinal, and peripheral sites.

**Supplementary Materials:** The following are available online. Figure S1. IR spectra of compound **3a**. Figure S2. <sup>1</sup>H-NMR spectra of compound **3a**. Figure S3. <sup>13</sup>C-NMR spectra of compound **3a**. Figure S4. LCMSMS spectra of compound **3a**. Figure S5. IR spectra of compound **3b**. Figure S6. <sup>1</sup>H-NMR spectra of compound **3b**. Figure S7. <sup>13</sup>C-NMR spectra of compound **3b**. Figure S8. LCMSMS spectra of compound **3b**. Figure S9. IR spectra of compound **3c**. Figure S10. <sup>1</sup>H-NMR spectra of compound **3c**. Figure S11. <sup>13</sup>C-NMR spectra of compound **3c**. Figure S12. LCMSMS spectra of compound **3c**. Figure S13. IR spectra of compound **3d**. Figure S14. 1H-NMR spectra of compound **3d**. Figure S15. 13C-NMR spectra of compound **3d**. Figure S16. LCMSMS spectra of compound **3d**. Figure S17. IR spectra of compound **3e**. Figure S18. <sup>1</sup>H-NMR spectra of compound **3e**. Figure S19. <sup>13</sup>C-NMR spectra of compound **3e.** Figure S20. LCMSMS spectra of compound **3e**. Figure S21. IR spectra of compound **3f.** Figure S22. <sup>1</sup>H-NMR spectra of compound **3f**. Figure S23. <sup>13</sup>C-NMR spectra of compound **3f.** Figure S24. LCMSMS spectra of compound **3f**. Figure S25. IR spectra of compound **3g.** Figure S26. <sup>1</sup>H-NMR spectra of compound **3g**. Figure S27. <sup>13</sup>C-NMR spectra of compound **3g**. Figure S28. LCMSMS spectra of compound **3g**. Figure S29. IR spectra of compound **3h.** Figure S30. <sup>1</sup>H-NMR spectra of compound **3h**. Figure S31. <sup>13</sup>C-NMR spectra of compound **3h**. Figure S32. LCMSMS spectra of compound **3h.** Figure S33. The two-dimensional interacting mode of compound **3a** in the active region of µ-opioid receptor (PDB Code: 5C1M). Figure S34. The three-dimensional interacting mode of compound **3a** in the active region of µ -opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with purple and white, respectively (PDB Code: 5C1M). Figure S35. The two-dimensional interacting mode of compound **3b** in the active region of µ-opioid receptor. (PDB Code: 5C1M). Figure S36. The three-dimensional interacting mode of compound **3b** in the active region of µ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with orange and white, respectively (PDB Code: 5C1M). Figure S37. The two-dimensional interacting mode of compound **3c** in the active region of µ-opioid receptor. (PDB Code: 5C1M). Figure S38. The three-dimensional interacting mode of compound **3c** in the active region of µ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with blue and white, respectively (PDB Code: 5C1M). Figure S39. The two-dimensional interacting mode of compound **3d** in the active region of µ-opioid receptor. (PDB Code: 5C1M). Figure S40. The three-dimensional interacting mode of compound **3d** in the active region of µ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with red and white, respectively (PDB Code: 5C1M). Figure S41. The two-dimensional interacting mode of compound **3e** in the active region of µ-opioid receptor. (PDB Code: 5C1M). Figure S42. The three-dimensional interacting mode of compound **3e** in the active region of µ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with red and white, respectively (PDB Code: 5C1M). Figure S43. The two-dimensional interacting mode of compound **3h** in the active region of µ-opioid receptor. (PDB Code: 5C1M). Figure S44. The three-dimensional interacting mode of compound **3h** in the active region of µ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with red and white, respectively (PDB Code: 5C1M). Figure S45. The two-dimensional interacting mode of compound **3a** in the active region of δ-opioid receptor. (PDB Code: 4N6H). Figure S46. The three-dimensional interacting mode of compound **3a** in the active region of δ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with blue and white, respectively (PDB Code: 4N6H). Figure S47. The two-dimensional interacting mode of compound **3b** in the active region of δ-opioid receptor. (PDB Code: 4N6H). Figure S48. The three-dimensional interacting mode of compound **3b** in the active region of δ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with blue and white, respectively (PDB Code: 4N6H). Figure S49. The two-dimensional interacting mode of compound **3c** in the active region of δ-opioid receptor. (PDB Code: 4N6H). Figure S50. The three-dimensional interacting mode of compound **3c** in the active region of δ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with green and white, respectively (PDB Code: 4N6H). Figure S51. The two-dimensional interacting mode of compound **3d** in the active region of δ-opioid receptor. (PDB Code: 4N6H). Figure S52. The three-dimensional interacting mode of compound **3d** in the active region of δ-opioid receptor. The

ligand and significant residues of the active site of the receptor are presented by a tube model colored with red and white, respectively (PDB Code: 4N6H). Figure S53. The two-dimensional interacting mode of compound **3e** in the active region of δ-opioid receptor. (PDB Code: 4N6H). Figure S54. The three-dimensional interacting mode of compound **3e** in the active region of δ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with red and white, respectively (PDB Code: 4N6H). Figure S55. The two-dimensional interacting mode of compound **3h** in the active region of δ-opioid receptor. (PDB Code: 4N6H). Figure S56. The three-dimensional interacting mode of compound **3h** in the active region of δ-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with red and white, respectively (PDB Code: 4N6H). Figure S57. The two-dimensional interacting mode of compound **3a** in the active region of k-opioid receptor. (PDB Code: 6B73). Figure S58. The three-dimensional interacting mode of compound **3a** in the active region of k-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with green and white, respectively (PDB Code: 6B73). Figure S59. The two-dimensional interacting mode of compound **3b** in the active region of k-opioid receptor. (PDB Code: 6B73). Figure S60. The three-dimensional interacting mode of compound **3b** in the active region of k-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with pink and white, respectively (PDB Code: 6B73). Figure S61. The two-dimensional interacting mode of compound **3c** in the active region of k-opioid receptor. (PDB Code: 6B73). Figure S62. The three-dimensional interacting mode of compound **3c** in the active region of k-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with orange and white, respectively (PDB Code: 6B73). Figure S63. The two-dimensional interacting mode of compound **3d** in the active region of k-opioid receptor. (PDB Code: 6B73). Figure S64. The three-dimensional interacting mode of compound **3d** in the active region of k-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with yellow and white, respectively (PDB Code: 6B73). Figure S65. The two-dimensional interacting mode of compound **3e** in the active region of k-opioid receptor. (PDB Code: 6B73). Figure S66. The three-dimensional interacting mode of compound **3e** in the active region of k-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with blue and white, respectively (PDB Code: 6B73). Figure S67. The two-dimensional interacting mode of compound **3h** in the active region of k-opioid receptor. (PDB Code: 6B73). Figure S68. The three-dimensional interacting mode of compound **3h** in the active region of k-opioid receptor. The ligand and significant residues of the active site of the receptor are presented by a tube model colored with pink and white, respectively (PDB Code: 6B73).

**Author Contributions:** Conceptualization, N.T.Y., D.O., and Ü.D.Ö.; methodology, D.O., N.T.Y., and A.E.E.; software, A.E.E., D.O., and N.T.Y.; validation, N.T.Y. and Ü.K.; formal analysis, Ö.D.C. and Ü.D.Ö.; investigation, D.O., N.T.Y., and Ü.K.; resources, D.O., N.T.Y., Ö.D.C., and Ü.D.Ö.; data curation, D.O., N.T.Y., and Ü.K.; writing—original draft preparation, N.T.Y., Ö.D.C., Ü.D.Ö., D.O., A.E.E.; writing—review and editing, Ö.D.C.; visualization, Ö.D.C. and Ü.D.Ö.; supervision, Ö.D.C. and Ü.D.Ö.; project administration, D.O. and N.T.Y.; funding acquisition, Ü.D.Ö. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was approved by the Local Ethical Committee on Animal Experimentation of Anadolu University, Eski¸sehir, Turkey (protocol code 2018-22 and 13 April 2018).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All relevant data are included within the article or Supplementary Materials. The raw data are available upon request from the corresponding author.

**Acknowledgments:** This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are available from the authors.
