New Paracyclophanylthiazoles with Anti-Leukemia Activity: Design, Synthesis, Molecular Docking, and Mechanistic Studies

A new series of methyl 2-(2-(4′-[2.2]paracyclophanyl)-hydrazinylidene)-3-substituted-4-oxothiazolidin-5-ylidene)acetates 3a–f were synthesized from the reaction of paracyclophanyl-acylthiosemicarbazides 2a–f with dimethyl acetylenedicarboxylate. Based upon nuclear magnetic resonance (NMR), infrared (IR), and mass spectra (HRMS), the structure of the obtained products was elucidated. X-ray structure analysis was also used as unambiguous tool to elucidate the structure of the products. The target compounds 3a–f were screened against 60 cancer cell lines. They displayed anticancer activity against a leukemia subpanel, namely, RPMI-8226 and SR cell lines. The activity of compound 3a was found as the most cytotoxic potency against 60 cancer cell lines. Consequently, it was selected for further five doses analysis according to National Cancer Institute (NCI) protocol. The cytotoxic effect showed selectivity ratios ranging between 0.63 and 1.28 and between 0.58 and 5.89 at the GI50 and total growth inhibition (TGI) levels, respectively. Accordingly, compound 3a underwent further mechanistic study against the most sensitive leukemia RPMI-8226 and SR cell lines. It showed antiproliferation with IC50 = 1.61 ± 0.04 and 1.11 ± 0.03 µM against RPMI-8226 and SR cell lines, respectively. It also revealed a remarkable tubulin inhibitory activity, compared to colchicine with IC50 = 4.97 µM/mL. Caspase-3, BAX, and Bcl-2 assays for 3a using annexin V-FITC staining revealed significant pro-apoptotic activity. Furthermore, multidrug-resistant leukemia SR cells were used to show better resistance indices (1.285 ng/mL, 1.15-fold) than the reference. Docking studies with β-tubulin indicate that most of the tested compounds illustrated good binding at the colchicine binding site of the enzyme, especially for compound 3a, which made several interactions better than that of the reference colchicine.


Introduction
Cyclophane chemistry is rapidly growing in the field of stereoselective synthesis with its incorporation into heterocyclic and/or polymer chemistry [1][2][3][4][5][6][7][8]. A great deal of attention is focused on developing new synthetic tools for synthesizing functionalized [2.2]paracyclophanes. Substituted [2.2]paracyclophanes can also serve as chiral templates and/or as auxiliaries [9]. The synthesis and application of heterocycles based on [2.2]paracyclophane [10,11] can be organized into five structural classes ( Figure 1): heterocycle derived by paracyclophanyl group (type I), heterocycle derived by bridge (type II), heterocycle fused to ethano bridge (type III), fused heterocycle to the benzene moiety (type IV), and heterocycle between the two benzene rings of paracyclophane (type V).
-Cytotoxic activity of the synthesized compounds toward the NCI-60 panel of cancer cell lines was determined, and the cellular mechanism of the most potent inhibitors was further investigated in leukemia cell lines. -Compound 3a was found as the most cytotoxic one, and it was selected for further five-dose analysis according to NCI protocol. -Compound 3a, in comparison to other derivatives, exhibited high specificity against leukemia RPMI-8226 and SR cell lines, and it also showed a remarkable tubulin inhibitory activity in relation to colchicine with IC50 = 4.97 µ M/mL. -Docking studies with β-tubulin revealed that most of the tested compounds showed good binding at the colchicine binding site of the enzyme, especially for compound 3a, which made several interactions better than that of the reference colchicine.
The thiazole scaffold possesses potent cytotoxic activity in cancer cell lines. For example, thiazolidine-4-carboxylic acid amides (ATCAA) I showed activity against prostate cancer cells with an average IC50 in the range from 0.7 to 1.0 μM, while the average IC50 against melanoma cells ranged from 1.8 to 2.6 μM ( Figure 2) [25].
Moreover, gold(I) complexes of phosphino [2.2]paracyclophane ligands III exhibited their cytotoxic activity in the HeLa S3 cell line (LD50 = 22.15 μm) in comparison to cisplatin (LD50 = 7.65 μm). Their cytotoxicity and their mechanisms of action are different and involve apoptosis, necrosis, and DNA damage ( Figure 2) [26]. Studies reported that methoxylbenzoylaryl-thiazole (SMART) II compounds showed nanomolar activity in inhibiting melanoma and prostate cancer cell growth [27][28][29]. Due to the interest in developing new anti-cancer agents, we designed a strategy of gathering both thiazole and paracyclophane skeletons for designing new anti-tumor agents (Figure 3). A [2.2]paracyclophanyl acetic acid enantiomer was tested as anti-inflammatory agent [12]. Recent studies reported that 1,3-thiazoles derivatives showed in vitro α-glucosidase inhibitory activity [13,14].
The thiazole scaffold possesses potent cytotoxic activity in cancer cell lines. For example, thiazolidine-4-carboxylic acid amides (ATCAA) I showed activity against prostate cancer cells with an average IC 50 in the range from 0.7 to 1.0 µM, while the average IC 50 against melanoma cells ranged from 1.8 to 2.6 µM ( Figure 2) [25].
Synthesis of compound 1 can be achieved from the reported route starting with the commercial hydrocarbon 5 (Scheme 2). The procedure consisted firstly of the conversion of 5 into the keto acid chloride 6 with oxalyl chloride/aluminum trichloride. Heating of 6 in chlorobenzene caused decarbonylation to give 7, and, when the resulting acid chloride 7 was quenched with ethanol, the ester 8 [31] was obtained (Scheme 2). On refluxing the α-ketoester 8 with hydrazine hydrate in different solvents, the reaction failed to give the target hydrazide 1 in good yields (Scheme 2). However, heating 8 in an excess of hydrazine hydrate afforded the corresponding compound carbohydrazide 1 in 80% yield (Scheme 2). Moreover, gold(I) complexes of phosphino [2.2]paracyclophane ligands III exhibited their cytotoxic activity in the HeLa S3 cell line (LD 50 = 22.15 µm) in comparison to cisplatin (LD 50 = 7.65 µm). Their cytotoxicity and their mechanisms of action are different and involve apoptosis, necrosis, and DNA damage ( Figure 2) [26]. Studies reported that methoxylbenzoylaryl-thiazole (SMART) II compounds showed nanomolar activity in inhibiting melanoma and prostate cancer cell growth [27][28][29]. Due to the interest in developing new anti-cancer agents, we designed a strategy of gathering both thiazole and paracyclophane skeletons for designing new anti-tumor agents ( Figure 3).
The X-ray structure analysis of compounds 2a,b,d strongly confirmed the proposed structures as shown in Figures 4-6, respectively. One can note that the dihedral angle of CS-NH-NH-CO was nearly 90 • , and that angle was also seen in an example reported in reference [32].
The abnormal behavior of 2c toward 9 might be attributed to the expected resonance structure of 2c that would decrease the basicity of the N 3 -thioamide compared with the N 2 -hydrazide and, therefore, the N 2 -hydrazide would be more reactive toward nucleophilic addition (Scheme 5). The methodology of the NCI anticancer screening was described in detail elsewhere (http://www.dtp.nci.nih.gov). Briefly, the primary anticancer assay was performed using  The methodology of the NCI anticancer screening was described in detail elsewhere (http://www.dtp.nci.nih.gov). Briefly, the primary anticancer assay was performed using approximately 60 human tumor cell lines derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda. Tested compounds were added to the culture at a single concentration (10 −5 M) and the cultures were incubated for 48 h. End-point determinations were made with a protein binding dye, SRB. Results for each tested compound were reported as the percentage growth of the treated cells when compared to the untreated control cells. The percentage growth was evaluated spectrophotometrically versus controls not treated with test agents. All experiments were repeated three times (Table 1). Compounds 3a-f revealed that compound 3a achieved complete cell death on the nine tested cancer cell lines. It is noteworthy that compounds 3a, 3b, 3d, and 3e were the most potent tested derivatives on the leukemia cell line. They showed growth inhibition percentages greater than 100%, which means that they were cytotoxic and displayed complete cancer cell death that killed all cells, including cancer cells. They may stop cancer cells from dividing and growing and may cause tumors to shrink in size. The complete cell death was against leukemia RRMI-8226 with inhibitions of 120.89%, 147.00%, 109.36%, and 114.28%, respectively, and against SR with inhibitions of 115.60%, 114.70%, 98.21%, and 113.40%, respectively. Compound 3e showed remarkable activity on the other tested cell lines. Although 3f showed moderate to weak activity on most of tested cancer cell lines, compound 3b exhibited significant inhibition against non-small-cell lung cancer NCI-H522, colon cancer HT29 and SW-620, melanoma LOX IMVI, ovarian cancer OVCAR-3, renal cancer CAKI-1, prostate cancer PC-3, and breast cancer BT-549, T-47D, and MDA-MB-468 with inhibitions of 84.06%, 87.62%, 81.83%, 93.79%, 79.71%, 84.43%, 82.23%, 89.68%, 106.25%, and 78.91%, respectively. Furthermore, compounds 3c and 3d displayed mild to moderate activity on most of the cancer panel cell lines

Structure Activity Relationship (SAR)
It is notable that the new prepared paracyclophane/thiazole conjugates showed significant anti-cancer activity with different growth inhibition percentages. This disparity among the different derivatives may be attributed to the type of substitution on the thiazole ring, whether on nitrogen (compounds 3a,b,d-f) or at position 2 (compound 3c). It is expected that increasing thiazole flexibility through inserting a phenyl or benzyl group (compounds 3a and 3b, respectively) into the structure would improve binding to the target protein and, hence, show higher antiproliferative activity on all tested cell lines than those containing an aliphatic group, i.e., either compound 3d (allyl) or 3e (ethyl) of the same series, which provides less flexibility. Interestingly, the other paracyclophane/thiazole derivative 3c bearing a pyridinyl amine moiety at position 2 of the thiazole ring altered the activity, showing lower cytotoxicity.

In Vitro Five-Dose Full NCI 60 Cell Panel Assay
Compound 3a was selected by NCI for five-dose investigation against 60 human tumor cell lines that were incubated at five different concentrations (0.01, 0.1, 1, 10, and 100 µM) ( Figure 9). The outcomes were used to form log concentration vs. percentage growth inhibition curves and three response parameters (GI 50 , total growth inhibition (TGI), and LC 50 ) were calculated for each cell line ( Table 2). The GI 50 value (growth inhibitory activity) corresponds to the concentration of the compound causing 50% decrease in net cell growth, the TGI value (cytostatic activity) is the concentration of the compound resulting in total growth inhibition (TGI), and the LC 50 value (cytotoxic activity) is the concentration of the compound causing a net 50% loss of initial cells at the end of the incubation period of 48 h.  2.55 >100 The criterion for selectivity of a compound depends upon the ratio obtained by dividing the full-panel MID (the average sensitivity of all cell lines toward the test agent) (µM) by the individual subpanel MIDs (µM). Ratios between 3 and 6 refer to moderate selectivity, and ratios greater than 6 indicate high selectivity toward the corresponding cell line, while compounds not meeting either of these criteria are rated non-selective.  Compound 3a under investigation exhibited remarkable anticancer activity against most of the tested cell lines representing nine different subpanels with GI 50 ranging from 1.52-7.56 µM (Table 2). Results indicate that 3a showed high activity against renal cancer RXF-393, melanoma LOX IMIV, colon cancer HCC-2998, non-small-cell lung cancer EKVX, and leukemia RPMI-8226 with GI 50 values of 1.52, 1.69, 1.78, 1.91, and 2.15 µM, respectively. An obvious sensitivity profile was seen toward the leukemia subpanel (GI 50 values ranging from 2.15 to 3.15 µM), colon cancer subpanel (GI 50 values ranging from 1.78 to 2.13 µM), breast cancer subpanel (GI 50 values ranging from 1.54 to 1.87 µM), and ovarian cancer subpanel (GI 50 values ranging from 1.66 to 3.55 µM). In this context, compound 3a was found to have broad-spectrum antitumor activity against the nine tumor subpanels tested with selectivity ratios ranging from 0.63-1.28 and 0.58-5.89 at the GI 50 and TGI levels, respectively; however, it exhibited noteworthy antiproliferative activity against all cancer cell lines with low selectivity ratios.

Evaluation of In Vitro Antiproliferative Activities against Leukemia RPMI-8226 and SR
Since the antiproliferative investigation results against 60 cell lines at the NCI revealed greater activity toward leukemia cancer, especially leukemia RPMI-8226 and SR, we were encouraged to perform further in vitro antiproliferative studies against those two cell lines. Compounds 3a-e were evaluated for their antiproliferative activity by performing an MTT assay against a panel of two human tumor cell lines, leukemia RPMI-8226 and SR, compared with colchicine as a reference. As shown in Table 3, the antiproliferative activities of the tested compounds were generally more pronounced against the two panels of leukemia cancer cells as compared with the reference. The calculated results were subjected to statistical analysis using GraphPad Prism 7 with the one-way ANOVA and non-parametric program. The difference in the results was considered significant when the p-values were less than 0.05. All the tested compounds were significant, as well as the reference (*** p < 0.05), in comparison to control. Compound 3a exhibited the highest antiproliferation compared to reference and the other tested compounds, whereas it showed IC 50 values 1.61 and 1.11 µM better than colchicine (i.e., the reference compound) of 4.05 and 1.81 µM against leukemia RPMI-8226 and SR, respectively. On the other hand, compound 3e showed a significant antiproliferative activity with an IC 50 value 3.17 µM better than the reference of 4.05 µM against leukemia RPMI-8226 only. This may be attributed to both compounds 3a and 3b having electron-withdrawing substitution of phenyl and benzyl, respectively, which positively affected their permeability to cancer cells. Compound 3b showed comparable IC 50 values of 4.62 and 2.02 µM to colchicine. Table 3. MTT assay for the antiproliferative IC 50 ± SD (µM) activity of compounds 3a-e and colchicine.
Additionally, compound 3c bearing a pyridinyl amine moiety at position 2 of the thiazole ring showed weak anti-proliferation activity with IC 50 values of 9.69 and 4.84 µM, which explains its low cytotoxicity. It is interesting to mention that the proliferation inhibitory results were positively correlated with the anticancer results obtained from NCI.

Evaluation of In Vitro Tubulin Polymerization Inhibitory Activity
To investigate whether the antiproliferative activities of these target compounds 3a-e were related to their interaction with tubulin, these compounds were tested for their ability to inhibit tubulin polymerization at their IC 50 concentrations using an ELISA assay for β-tubulin.
The in vitro kinetics of microtubule assembly was measured using an ELISA kit for TUBb (Cloud-Clone. Corp.) on the leukemia SR cell line. The compounds tested were 3a-f and colchicine. Briefly, growing cells from the SR cell line were trypsinized, counted, and seeded at the appropriate densities into 96-well microtiter plates. Cells were then incubated in a humidified atmosphere at 37 • C for 24 h.
The assay revealed that all the tested compounds 3a-e showed tubulin polymerization inhibitory activity compared to colchicine as a reference (Table 4). Again, compound 3a showed the highest ability to inhibit tubulin polymerization with an IC 50 value of 4.97 µM compared to the reference with an IC 50 value 3.76 µM and the other tested compounds. On the other hand, compounds 3e and 3c showed remarkable tubulin polymerization inhibition with IC 50 values of 6.61 and 8.38 µM, while 3d displayed weak inhibition with an IC 50 value of 14.79 µM. Results are in agreement with the previous mentioned anti-proliferative activity Table 4. Inhibition of tubulin polymerization displaying IC 50 ± standard error of the mean (SEM) (µM) for compounds 3a-e and colchicine as a reference.

Cell Cycle Analysis
Cell cycle analysis was performed using cytometers from Becton Dickinson Immunocytometry Systems, Beckman/Coulter Inc., DACO/Cytomation, and PARTEC GmbH. Regulation of cell growth is mainly controlled through cell-cycle control mechanisms. Proliferation inhibition can be triggered by cell-cycle arrest in cancer cells. During the cell cycle, the G2/M checkpoint is a potential target for cancer therapy.
This prevents DNA-damaged cells from entering mitosis and allows for the repair of DNA that was damaged in late S or G2 phases prior to mitosis ( Table 5). Induction of cell-cycle arrest is a common mechanism proposed for the cytotoxic effects of anticancer drugs containing paracyclophane/thiazole derivatives. The analysis indicated that leukemia SR cells treated with compound 3a showed significant growth arrest at the G2/M phase compared to control cells, where the S-phase progression of SR cells was substantially delayed ( Figure 10). The annexin V/PI flow cytometry of SR cells was repeated three times after treatment with the IC 50 value (1.11 µM) of 3a, which showed an increase in percentage of the necrotic cells in late apoptosis to 19% (upper right quadrant of the cytogram) ( Figure 11). Hence, compound 3a showed a considerable ability to dissipate cell membrane integrity, whereas the lower right quadrant illustrating the early apoptotic cells which kept their membrane integrity indicated the ability of 3a to initiate apoptosis.

Cell Cycle Analysis
Cell cycle analysis was performed using cytometers from Becton Dickinson Immunocytometry Systems, Beckman/Coulter Inc., DACO/Cytomation, and PARTEC GmbH. Regulation of cell growth is mainly controlled through cell-cycle control mechanisms. Proliferation inhibition can be triggered by cell-cycle arrest in cancer cells. During the cell cycle, the G2/M checkpoint is a potential target for cancer therapy.  This prevents DNA-damaged cells from entering mitosis and allows for the repair of DNA that was damaged in late S or G2 phases prior to mitosis ( Table 5). Induction of cell-cycle arrest is a common mechanism proposed for the cytotoxic effects of anticancer drugs containing paracyclophane/thiazole derivatives. The analysis indicated that leukemia SR cells treated with compound 3a showed significant growth arrest at the G2/M phase compared to control cells, where the S-phase progression of SR cells was substantially delayed (Figure 10). The annexin V/PI flow cytometry of SR cells was repeated three times after treatment with the IC50 value (1.11 µ M) of 3a, which showed an increase in percentage of the necrotic cells in late apoptosis to 19% (upper right quadrant of the cytogram) ( Figure 11). Hence, compound 3a showed a considerable ability to dissipate cell membrane integrity, whereas the lower right quadrant illustrating the early apoptotic cells which kept their membrane integrity indicated the ability of 3a to initiate apoptosis.

Compound 3b Induced Mitochondrial Depolarization and ROS Production
Mitochondria play an essential role in the propagation of apoptosis [33]. It is reported that, at an early stage, apoptotic stimuli are capable of modifying the mitochondrial transmembrane potential (∆ψmt). ∆ψmt was recorded by the fluorescence of the dye JC-1.20. SR cells treated with 3a displayed a non-significant shift in fluorescence compared with control cells, explaining the weak depolarization of the mitochondrial membrane potential. In contrast, colchicine showed significant results ( Figure 12). The disruption of ∆ψmt is associated with the appearance of annexin V positivity in the treated cells when they are in an early apoptotic stage. It is documented that the dissipation of ∆ψmt is characteristic of apoptosis, and this was indicated with both microtubule-stabilizing and -destabilizing agents in different cell types [34]. Induction of cell-cycle arrest is a common mechanism proposed for the cytotoxic effects of anticancer drugs containing paracyclophane/thiazole derivatives. Mitochondrial membrane depolarization is associated with mitochondrial production of ROS [35]. Therefore, we investigated whether ROS production increased after treatment with the test compounds.
The presented results in Figure 13 show that 3a induced the production of significant amounts of ROS (109.84%) in comparison with control cells and the colchicine reference. This result is compatible with the dissipation of ∆ψmt described above.
Δψmt is characteristic of apoptosis, and this was indicated with both microtubule-stabilizing anddestabilizing agents in different cell types [34]. Induction of cell-cycle arrest is a common mechanism proposed for the cytotoxic effects of anticancer drugs containing paracyclophane/thiazole derivatives. Mitochondrial membrane depolarization is associated with mitochondrial production of ROS [35]. Therefore, we investigated whether ROS production increased after treatment with the test compounds.   The presented results in Figure 13 show that 3a induced the production of significant amounts of ROS (109.84%) in comparison with control cells and the colchicine reference. This result is compatible with the dissipation of Δψmt described above.
2.2.7. Effect of Compound 3a on Multidrug-Resistant (MDR) Leukemia SR Cells A previous study reported that multidrug resistance was observed for numerous chemotherapeutic agents and could be identified through overexpression of P-glycoprotein (Pgp) Figure 13. Mitochondrial production of ROS in SR cells with 3a and colchicine reference compared to control. Mean ± standard deviation plotted for three replicates per condition. Results are significantly different from control at *** p < 0.05. The substrate was incubated for 30 min at 37 • C protected from light.

Effect of Compound 3a on Multidrug-Resistant (MDR) Leukemia SR Cells
A previous study reported that multidrug resistance was observed for numerous chemotherapeutic agents and could be identified through overexpression of P-glycoprotein (Pgp) [36,37].
Compound 3a and tubulin-targeting agent colchicine were tested on the MDR-SR Leukemia cell line. Compound 3a showed Pgp-mediated MDR expression at 1.285 ng/mL (1.15-fold change), which means that it had much better resistance indices comparable to control at 1.121 ng/mL (1-fold) than colchicine at 1.726 ng/mL (1.54-fold change). Results of compound 3a (Table 6) showed the ability of overcoming the assumed glycoprotein overexpression of the cell line; thus, 3a could be considered as an important substrate candidate against MDR cells. Since caspase-3 is important for spreading the apoptotic signal after exposure to antimitotic compounds [38], the effect of compound 3a on the caspase-3 activated enzyme was evaluated using ELISA and replicated three times. The reference compound and 3a were incubated for 30 min at room temperature in the dark on the leukemia SR cell line. Results revealed that 3a is a potential caspase-3 activator with a slight increase in the level of active caspase-3 at a concentration of 471.2 ng/mL (8.84-fold) compared to colchicine at a concentration of 428.9 ng/mL (8.05-fold) as shown in Table 7. The proteins of the Bcl2 family [39] play a major role in controlling apoptosis through the regulation of mitochondrial processes and the release of mitochondrial proapoptotic molecules that are important for the cell death pathway [40]. Compound 3a caused nearly 7.98-fold upregulation (Table 8), while it showed markedly higher levels of the antiapoptotic Bcl-2 proteins up to 0.59-fold compared to the control untreated cells (Figure 14). The proteins of the Bcl2 family [39] play a major role in controlling apoptosis through the regulation of mitochondrial processes and the release of mitochondrial proapoptotic molecules that are important for the cell death pathway [40]. Compound 3a caused nearly 7.98-fold upregulation (Table 8), while it showed markedly higher levels of the antiapoptotic Bcl-2 proteins up to 0.59-fold compared to the control untreated cells (Figure 14).

Docking Studies
The molecular modeling of the possible binding modes for the newly synthesized thiazole/paracyclophane hybrids 3a-e and colchicine as a reference was done to predict the binding interactions between them and β-tubulin at the colchicine binding site, which was obtained from the protein data bank (PDB: 3HKC). Docking studies were carried out using Molecular Operating Environment (MOE® ) version 2014.09 (Chemical Computing Group Inc., Montreal, QC, Canada) using colchicine as a reference for validation of the method. The theoretical predictions from the molecular docking study agreed for some highly active derivatives such as 3a, 3b, 3d, and 3e with the experimentally observed tubulin polymerization inhibition. All derivatives were successfully docked into the colchicine binding site of β-tubulin. The binding free energies from the major docked poses are listed in Table 9, and the most favorable poses of the tested compounds are shown in

Docking Studies
The molecular modeling of the possible binding modes for the newly synthesized thiazole/paracyclophane hybrids 3a-e and colchicine as a reference was done to predict the binding interactions between them and β-tubulin at the colchicine binding site, which was obtained from the protein data bank (PDB: 3HKC). Docking studies were carried out using Molecular Operating Environment (MOE ® ) version 2014.09 (Chemical Computing Group Inc., Montreal, QC, Canada) using colchicine as a reference for validation of the method. The theoretical predictions from the molecular docking study agreed for some highly active derivatives such as 3a, 3b, 3d, and 3e with the experimentally observed tubulin polymerization inhibition. All derivatives were successfully docked into the colchicine binding site of β-tubulin. The binding free energies from the major docked poses are listed in Table 9, and the most favorable poses of the tested compounds are shown in Figures 15-20. Most of the tested compounds had high binding affinity to the enzyme as the binding free energy (∆G) values of them ranged from −0.5 to −3.4 kcal/mol, which was comparable to the reference colchicine (∆G = −0.6 to −2.3 kcal/mol). The docking result of the reference compound colchicine was completely consistent with the mode of action of thiazole/paracyclophane derivatives ( Figure 15). Table 9. Energy scores for the complexes formed by the tested compounds 3a-e and the reference colchicine in the active site of β-tubulin enzyme (PDB: 3HKC). Compounds 3a, 3c, 3d, and 3e exhibited the same interaction as the reference with Mg 601; however, both 3d and 3b showed hydrogen bonding interactions with Glu 71 (Figures 16-20). On the other hand, compound 3d possessed greater interactions than the reference with the same amino acids with additional one H-pi bond with Arg 2 ( Figure 20); however, compounds 3a and 3c displayed an extra H-pi interaction with Ala 247 (Figures 16 and 18). Compound 3c (Figure 18) did not feature hydrogen binding interactions with the amino acid residue Glu 71, but kept an H-Pi interaction with Tyr 224.

Materials and Methods
Melting points were taken in open capillaries on a Gallenkamp melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. The IR spectra were recorded using the attenuated total reflection (ATR) technique with a FT device (FT-IR Bruker IFS 88, Bruker, Leiderdorp, The Netherlands), Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe, Germany. The NMR spectra were measured in DMSO-d6 and acetone-d6 on a Bruker AV-400 spectrometer at 400 MHz for 1 H and 100 MHz for 13 C; the chemical shifts are expressed in δ (ppm) versus internal tetramethylsilane (TMS) = 0 for 1 H and 13 C, and external liquid ammonia = 0. The description of signals includes s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, ddd = doublet of dd, dt = doublet of triplet, td = triplet of doublet, bs = broad singlet, and m = multiplet. Mass spectra were recorded on a FAB (fast atom bombardment) Thermo Finnigan Mat 95 (70 eV). For the high-resolution mass, the following abbreviations were used: calc.= theoretical calculated mass; found = mass found in analysis at the Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. Thin Layer Chromatography (TLC) was performed on analytical Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf254 indicator; TLCs were viewed at λmax = 254 nm. Crude products were purified by flash chromatography with silica gel 60 (0.040 × 0.063 mm, Geduran® ) (Merck, Darmstadt, Germany). Compounds 2 and 5-8 were prepared according to the methodology mentioned in References [30] and [31], respectively.

Preparation of Paracyclophanyl-N-substituted Hydrazinecarbothioamides 2a-f
A mixture of carbohydrazide paracyclophane (1) (1.00 g, 3.7 mmol) in 60 mL of ethanol and different derivatives of isothiocyanate (3.7 mmol) was refluxed for 4-8 h. The reaction mixture was poured into a beaker and was allowed to stand until a precipitate was formed. Then, the precipitate was filtered and washed with heptane several times (3 × 100 mL).  The two-dimensional (2D) diagram showed crucial binding involving Ala 247, Tyr 224, and Mg 601 through the [2.2]paracyclophane ring, thiazole ring, and ester C=O functionality. Moreover, stabilization of the reference colchicine within the active site occurred through one strong hydrogen bond interaction with amino-acid residue Glu 71 and metal intercalation with Mg 601. Docking results with the colchicine binding site revealed that most of the tested compounds showed good binding with the enzyme and made several interactions comparable to that of the reference colchicine ( Figure 15).
Compounds 3a, 3c, 3d, and 3e exhibited the same interaction as the reference with Mg 601; however, both 3d and 3b showed hydrogen bonding interactions with Glu 71 (Figures 16-20). On the other hand, compound 3d possessed greater interactions than the reference with the same amino acids with additional one H-pi bond with Arg 2 ( Figure 20); however, compounds 3a and 3c displayed an extra H-pi interaction with Ala 247 (Figures 16 and 18). Compound 3c (Figure 18) did not feature hydrogen binding interactions with the amino acid residue Glu 71, but kept an H-Pi interaction with Tyr 224. For the high-resolution mass, the following abbreviations were used: calc.= theoretical calculated mass; found = mass found in analysis at the Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. Thin Layer Chromatography (TLC) was performed on analytical Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf 254 indicator; TLCs were viewed at λ max = 254 nm. Crude products were purified by flash chromatography with silica gel 60 (0.040 × 0.063 mm, Geduran ® ) (Merck, Darmstadt, Germany). Compounds 2 and 5-8 were prepared according to the methodology mentioned in References [30] and [31], respectively.

Preparation of Paracyclophanyl-N-substituted Hydrazinecarbothioamides 2a-f
A mixture of carbohydrazide paracyclophane (1) (1.00 g, 3.7 mmol) in 60 mL of ethanol and different derivatives of isothiocyanate (3.7 mmol) was refluxed for 4-8 h. The reaction mixture was poured into a beaker and was allowed to stand until a precipitate was formed. Then, the precipitate was filtered and washed with heptane several times (3 × 100 mL).

Reactions of Hydrazinecarbothioamide Derivatives 2a-f with 9: Preparation of Compounds 3a-f
A mixture of hydrazinecarbothioamide derivatives (2a-f, 1 mmol) and 9 (0.142 g, 1 mmol) in 40 mL of absolute methanol was refluxed for 3-4 h (the reaction was monitored by thin-layer chromatography). After removal of the solvent on vacuum, the crude residue was purified by column chromatography (cyclohexane/ethyl acetate 10:6) as eluent to afford 3a-f.

3a-f
A mixture of hydrazinecarbothioamide derivatives (2a-f, 1 mmol) and 9 (0.142 g, 1 mmol) in 40 mL of absolute methanol was refluxed for 3-4 h (the reaction was monitored by thin-layer chromatography). After removal of the solvent on vacuum, the crude residue was purified by column chromatography (cyclohexane/ethyl acetate 10:6) as eluent to afford 3a-f.

3a-f
A mixture of hydrazinecarbothioamide derivatives (2a-f, 1 mmol) and 9 (0.142 g, 1 mmol) in 40 mL of absolute methanol was refluxed for 3-4 h (the reaction was monitored by thin-layer chromatography). After removal of the solvent on vacuum, the crude residue was purified by column chromatography (cyclohexane/ethyl acetate 10:6) as eluent to afford 3a-f.   zinecarbothioamide Derivatives 2a-f with 9: Preparation of Compounds zinecarbothioamide derivatives (2a-f, 1 mmol) and 9 (0.142 g, 1 mmol) in 40 ol was refluxed for 3-4 h (the reaction was monitored by thin-layer removal of the solvent on vacuum, the crude residue was purified by column exane/ethyl acetate 10:6) as eluent to afford 3a-f. de Derivatives 2a-f with 9: Preparation of Compounds e derivatives (2a-f, 1 mmol) and 9 (0.142 g, 1 mmol) in 40 for 3-4 h (the reaction was monitored by thin-layer ent on vacuum, the crude residue was purified by column e 10:6) as eluent to afford 3a-f. azinecarbothioamide Derivatives 2a-f with 9: Preparation of Compounds azinecarbothioamide derivatives (2a-f, 1 mmol) and 9 (0.142 g, 1 mmol) in 40 nol was refluxed for 3-4 h (the reaction was monitored by thin-layer removal of the solvent on vacuum, the crude residue was purified by column hexane/ethyl acetate 10:6) as eluent to afford 3a-f.