N-Naphthoyl Thiourea Derivatives: An Efficient Ultrasonic-Assisted Synthesis, Reaction, and In Vitro Anticancer Evaluations.

This work demonstrates the optimization of an efficient, mild, and environmentally friendly synthetic approach to access a diverse library of N-naphthoyl thioureas. These derivatives could be exploited as precursor scaffolds for designing valuable heterocycles with anticipated biological activities. Additionally, the utilization of a copper complex derived from the newly synthesized N-naphthoyl thiourea ligand in the photodegradation of methyl orange (MO) dye was explored. The antiproliferative effect of the synthesized derivatives was examined against MCF-7, HCT116, and A549 cancer cell lines. Most of the assembled derivatives revealed a significant cytotoxic effect, in some cases, greater than doxorubicin. Of these, the copper complex demonstrated significant antitumor activities (IC50 < 1.3 μM) and lesser cytotoxic impact (IC50 > 76 μM), indicating its possibility as a pioneering candidate for future carcinogenic pharmaceutics. Relations between the structure and activity also have been addressed.

Introduction

For many years, thioureas and their comprising compounds were attracting attention as they are considered to be versatile chemicals that possess a plethora of applications. They have outstanding medicinal applications along with non-medicinal activities in the analytical industry, agriculture, and metallurgy.[1−4] Thiourea-containing compounds have exhibited plenty of biological virtues such as anti-inflammatory, anti-bacterial, anti-fungal, anti-allergens, anti-hypertensive, anti-epileptic drugs, anti-thyroid drugs, and anti-cancer drugs.[5−15] Also, they are used as powerful enzyme inhibitors and DNA binders.[16−18] Interestingly, many of thiourea derivatives are in clinical use already: isoxyl is used for TB treatment, while trovirdine is used for the treatment of HIV. Moreover, thiourea derivatives are used as anion sensors and supramolecular assemblies.[19−21] Recently, the utilization of chiral thiourea-based organocatalysis in the synthesis of enantiomerically enriched compounds is of utmost importance.[22,23] It is worth to mention that cinchona-thiourea-based catalysts play a pivotal role in an assortment of asymmetric organocatalytic conversions.[24,25] Such ingrained merits of thiourea derivatives, in addition to the prevalent research on their utilization in the assembly of assorted heterocycles, make them outstanding building scaffolds for organic chemists. Thence, a considerable amount of endeavors has been spent to develop newer synthetic routes for the formation of diverse thiourea building blocks. So far, one of the most conventional protocols for the preparation of thioureas is the treatment of isothiocyanate derivatives with secondary or primary amines.[26−30] As conventional method processes, many drawbacks comprise the usage of toxic reactants, such as hydrogen sulfide and thiophosgene, and drastic reaction conditions, and they are predominantly restricted to the design of symmetrical thioureas. Herein, various user-friendly synthesis strategies have been assembled to access a diverse library of N-naphthoyl thioureas and their derivatives for cytotoxicity testing against several cancer cell lines in accordance with other studies.[31−35]

The cytotoxic activities of the newly assembled compounds were assessed toward three tumorigenic cells and a non-cancerous cell. In addition, the performance of a promising copper complex was evaluated in wastewater remediation.

Results and Discussion

It is well known that the utilization of ammonium thiocyanate (NH4SCN) or potassium thiocyanate (KSCN) in the synthesis of N-aroyl thioureas is one of the most versatile synthetic routes.[15,36,37] Herein, 2-naphthoyl chloride was treated with an equimolar amount of KSCN and cyclohexanamine in acetone to have an access to the aroyl thiourea derivative 2. Upon spectroscopic analysis, the chemical structure was identified as N-(cyclohexylcarbamothioyl)-2-naphthamide (2a, Table , entry 1).

Table 1

Screening of the Reaction Parameters for the Assembly of 2a

entry	solvent	method	temp (°C)	time (min)	yield (%)
1	acetone	stirring	23	300	63
2	acetonitrile	stirring	23	300	87
3	EtOH	stirring	23	300	12
4	DMF	stirring	23	300	25
5	DMA	stirring	23	300	15
6	1,4-dioxane	stirring	23	300	18
7	solvent-free	stirring	23	300	10
8	acetonitrile	USa	23	45	95
9	acetonitrile	US	50	90	92

Ultrasonic irradiation with 80% intensity.

Although 2-naphthoyl chloride was selected to be the aroyl chloride of our choice, as it revealed a good reaction profile and an excellent yield, further tuning was essential to enhance the yield and increase the reaction rate. Various reaction parameters, such as temperature, solvents, and energy sources, were adjusted in order to enhance the productivity of derivative 2a, and the results are tabulated in Table . With the exception of acetonitrile, the other employed polar solvents, including DMA, DMF, and EtOH, as well as non-polar solvents including 1,4-dioxane, were found to be inappropriate for such a reaction (Table , entries 2–6). Unfortunately, upon performing the reaction under solvent-free conditions, the reaction yield was dramatically diminished (Table , entry 7). Surprisingly, when the aforementioned reaction was employed under sonication (US), both reaction yield and rate were enhanced (Table , entry 8). The use of US in the reaction is important since it helps in reducing the reaction duration and enhancing the reaction yield. Notably, elevating the temperature from 23 (rt) to 50 °C was found to have an insignificant influence on both reaction yield and rate (Table , entry 9). This conclusion might be explained by the fact that while more bubbles are formed at high temperatures (50 °C), their collisions are weaker, resulting in a limitation in conversion efficiency, and no significant enhancement in percentage yield was recorded.[38] In addition, these additional bubbles function as barriers, and as a result, slowing the rate at which ultrasonic signals penetrate the reaction phase. Consequently, both ultrasonic efficient energy and cavitation intensity are significantly diminished, and this contributes to the reduction of the yield.[39] Accordingly, the sonication (intensity of 80%) of model reactants (equimolar) in acetonitrile solvent at 23 °C was found to be optimal. Noteworthy, for this kind of transformations, microwave-assisted synthetic methods can improve the reaction times drastically,[40] but as we are interested in the utilization of US, we used the ultrasonic irradiation as our sustainable energy source.[41] Recently, visible-light irradiation of the photocatalyst N–ZnO nanorods was also exploited to have an access to thiourea derivatives but only moderate yields were accessible.[42]

Utilizing the optimized reaction conditions, the substrate scope study was achieved. Thus, various substrates were explored leading to the formation of mono- and bis-2-naphthamides (Scheme ). Assorted possible permutations and combinations of substituents (primary and secondary amines) were next investigated. When the substituents were varied with either primary or secondary amines, all underwent an effective reaction and afforded the desired products in excellent yields (91–96%). Interestingly, there was no discernible relationship between the product yields and the electronic nature of the substituents, with the exception of a somewhat prolonged reaction period needed for a precursor comprising an electron-withdrawing motif (2h) (Scheme ). Additionally, the reaction was also successful with the diamines and good yields (91–95%) of the corresponding bis-derivatives (2i–k) were accessible. Several spectral methods were used to validate the structure of the prepared derivatives (2a–k), which were verified to be in accordance with the predicted data (see the Supporting Information). The 1H NMR spectra exhibited the NH protons of the thiourea motif: CONH and CSNH as singlets around 11 and 8 ppm, while aromatic ring protons were revealed between 7.5 and 8.9 ppm based on their electronic structure. In the 13C NMR spectra of derivatives 2a–k, thione carbon (C=S) was observed above 160 ppm. Moreover, the results of the mass spectra, elemental analyses, and FT-IR were in agreement with the proposed structures (see the Supporting Information).

Scheme 1

Substrate Scope for the Synthesis of Derivatives 2a–k

Later, N-(cyclohexylcarbamothioyl)-2-naphthamide (2a) was used as a building block for designing valuable heterocyclic compounds of anticipated biological activities. Accordingly, the desired thiazolidine derivative (3) was accessible, but in a poor yield (38%), through the reaction of 2a with chloroacetic acid and triethylamine as an acid scavenger. The chemical structure of compound 3 was established based on spectral analyses (Scheme ).

Scheme 2

Sonosynthesis of Derivatives 3 and 4

Obtaining discouraging yield of thiazolidine derivative 3 encourages us to explore an alternative route to synthesize derivative 3via a sequential reaction involving the sonication of 2-naphthoyl isothiocyanate and cyclohexanamine for 15 min followed by the addition of chloroacetic acid. Thereafter, as an attempt to optimize the yield, the sequential reaction was carried out without the acid scavenger. Surprisingly, the reaction yield was improved (88%). Therefore, the acid scavenger had no positive effect, but rather an impeding effect, on the reaction’s yield. In this case, the generated hydrochloric acid or the iminium ions might be neutralized by the employed acid scavenger. However, such a basic catalyst could also create a carboxylate anion from either chloroacetic acid or the pre-formed thiourea-chloroacetic acid. As a consequence, both the rate of amine–carboxylic acid interaction and reaction yield were remarkably reduced.[45] Interestingly, compound 3 could be also obtained in excellent yield by utilizing ethyl bromoacetate instead of chloroacetic acid (Scheme ). In addition, the stereoselective generation of the (Z)-stereoisomer could be explained depending on the allylic strain, as displayed in Figure .

Figure 1

Stereoselectivity of the reaction directed by the A1,3 strain.

Additionally, the active methylene derivative 3 underwent a Knoevenagel condensation reaction with p-chloro benzaldehyde easily to afford the isolable arylidene 4 under ultrasonic irradiation conditions (Scheme ). The 1H NMR spectrum of the obtained arylidene derivative 4 exhibited a downfield singlet at 7.6 ppm assigned for the vinylic proton, which confirms the isolation of the stereospecific Z-isomer.

Moreover, an efficient and clean method for the preparation of thiazole-2-imine (6) by a cyclization reaction of an equimolar amount of 2a and α-bromoacetophenone in the presence of triethylamine (Et3N) was described (Scheme ). The reaction proceeded smoothly under sonication and afforded the desired product in an excellent yield (98%). Interestingly, the expected isomeric imidazole derivative (5) was not formed and the thiazole derivative (6) was isolated instead. The proposed structure of compound 6 was confirmed based on the spectral data. For example, the 13C NMR spectrum did not show any signals of carbon related to the presence of C=S. In addition, the 1H NMR spectrum exhibited a distinguished signal at δ 7.1 ppm assigned to the CH of the thiazole nucleus (see the Supporting Information).

Scheme 3

Sonosynthesis of Derivative 6

As one of the most interesting protocols for designing thiazoles is the reaction of thiourea derivatives with acetylene derivatives, therefore dialkyl acetylenedicarboxylates were added dropwise to a stirred solution of thiourea derivative 2a in ethanol, and the resulting homogenized mixture was then vigorously stirred at room temperature to obtain the corresponding thiazole derivative 7 after 45 min. Alternatively, thiazole derivative 7 was accessible, in only 7 min, via sonication of the aforementioned reactants at room temperature (Scheme ). The characterization of proposed structures (7a,b) was carried out by FT-IR, NMR, and HRMS analyses. The IR spectra displayed a C=N absorption band at about 1610 cm–1 with additional carbonyl stretching bands in the range of 1711–1660 cm–1, while the absorption bands corresponding to NH groups disappeared. In addition, the 1H NMR spectrum of 7a exhibited two distinguished signals at 4.0 and 7.0 ppm that were assigned for the methoxy and the exocyclic vinylic protons, respectively. Further, the 1H NMR spectrum revealed no signals for the NH protons, confirming the proposed structure (7a). The presence of a signal at 122.6 ppm in the 13C NMR spectrum also confirmed the existence of such an exocyclic vinylic motif. Furthermore, the peaks at 172.6, 168.1, 166.9, 158.6, and 53.8 ppm were attributed to three carbonyl, an imino, and methoxy carbons, respectively, which obviously confirmed the suggested structure. The HRMS of derivative 7a displayed the molecular ion peak as [M + Na] at m/z 445.1199 corresponding to C23H22N2NaO4S.

Scheme 4

Sonosynthetic Route to Derivatives 7a,b

An attempt for the preparation of the triazole derivative (8) via the reaction between thiourea derivative 2a and hydrazine hydrate resulted in the formation of a complicated reaction profile (TLC). Thus, the synthesis of 1,2,4-triazole (8) by hydrazine condensation required a preliminary condition screening. Therefore, a study involving the effect of the solvent, number of hydrazine equivalents, reaction temperature, and time on the selected substrates was conducted (Scheme ). The suitable conversion was observed with the following conditions: utilizing two equivalents of hydrazine hydrate in DCM and refluxing for 90 min. Under these conditions, 1,2,4-triazole (8) was obtained in a good yield (82%) as the sole product. By performing this reaction under ultrasound irradiation at room temperature, an attempt was made to improve the reaction yield and rate; unfortunately, the denaphthoylation of derivative 2a occurred and the corresponding thiourea derivative 9 was obtained as the only product (Scheme ).

Scheme 5

Synthesis of Derivatives 8 and 9

On the other hand, the reaction of derivative 2a with sodium azide under basic conditions and using CuSO4·5H2O (50.0 mol %) as a catalyst afforded the corresponding tetrazole derivative 10. Different solvents were screened for this reaction including EtOH, dioxane, DMF, and DCM. Of them, EtOH afforded the desired product in an acceptable yield (95%). In addition, organic bases such as pyridine and Et3N could generate the required product 10 in high yield. This might be due to the fact that copper salt (CuSO4.5H2O) could form a complex with Et3N or pyridine (1.0 equiv), thus increasing the copper salt solubility, which results in an improvement in the desired product’s yield. With the exclusion of a base, the controlled test demonstrated that the reaction did not yield the required product; instead, the reactants were retrieved unchanged. Interestingly, derivative 10 could be also synthesized in a sequential protocol including the slow addition of naphthoyl chloride to a solution of acetonitrile and KSCN then sonication for 15 min. Afterward, cyclohexanamine was introduced to the preceding mixture and the obtained solution was sonicated for 30 min at ambient temperature. Subsequently, CuSO4·5H2O (50.0 mol %) and Et3N (1.0 equiv) were added to the resulting mixture containing a thiourea derivative before being sonicated for another 30 min at ambient temperature. In this reaction, the copper(I) species (could be formed from Cu(II) salt)[46] could be coordinated to a sulfur atom followed by desulphurization (CuS was generated as a side product and the excess sulfur may be transformed to polysulfides),[47] then nucleophilic substitution with NaN3 occurred, and finally electrocyclization took place to afford the target product 10 (Scheme ). In this sequential reaction, EtOH was not recommended: as such protic solvents could be reacted with naphthoyl chloride, diminishing the yield of naphthoyl isothiocyanate.

Scheme 6

Sonosynthesis of Tetrazole Derivative 10

The roles of naphthoyl thioureas in coordination chemistry have been thoroughly investigated and well addressed. The naphthoyl thiourea derivatives possessed two superior chelating motifs (C=S and C=O), improving their capability to encapsulate various metal ions within their coordinating moieties. Consequently, naphthoyl thiourea derivative 2a was investigated as a valuable ligand for copper ions, and the resulting complex was also studied for the degradation of methyl orange dye (MO) in water. Thus, the mononuclear copper(II) complex 11 (Scheme ) was obtained by the reaction between ligand 2a (2.0 mmol) and acidified solution of CuCl2 (1.0 mmol) in EtOH under ultrasonic irradiation. Immediately, yellow crystals were isolated, which were collected via filtration and washed with ethanol to afford the required product in 97% yield. The structure of complex 11 was confirmed by FT-IR, HRMS, and elemental analyses. A comparative absorption pattern study of the complex (11) with the values of the free ligand (2a) suggested a significant effect in stretching frequencies of (C=O) and (C=S) groups due to the coordination of the ligand to the Cu ions. The FT-IR spectrum of ligand 2a exhibited absorption bands at 3217 and 3170 cm–1 that are attributed to the N–H groups. One of these bands disappeared upon metal–complex formation from the ligand. This behavior suggested that the ligand was coordinated to the metal atom and lost one of the amine protons. The delocalization of the C=O stretching vibration caused a slightly negative shift (ca. 13 cm–1) of the corresponding band of the ligand, which appeared in the 1653 cm–1 region, confirming the involvement of the carbonyl group in coordination, which confirmed the formation of the desired compound.[48] A shift in frequency was also to be expected for the C=S stretching vibration, but this stretching vibration could not be assigned unambiguously because it is located in the fingerprint zone of the IR spectrum. The bands observed for the complex between 457–442 and 393–355 cm–1 were metal-sensitive and are assigned to ν(Cu–O) and ν(Cu–S), respectively.[49]

Scheme 7

Synthesis of Cu-Complex 11

Photodegradation of Methyl Orange (MO) Using the Copper Complex (11)

The prepared mononuclear Cu(II) complex was then investigated as a catalyst in the photodegradation of methyl orange (MO) dye (Figure ). Owing to the complete solubility of the copper complex in water, the degradation process was performed in an aqueous medium. In the beginning, the impact of the catalyst (11), UV radiation, and H2O2 on dye (MO) degradation was assessed. The system comprising (UV + H2O2 + complex) was investigated, and the degradation efficiency of the catalyst reached an excellent value (99%, Table & Figure ). In order to establish an acceptable explanation for the influence of the ligand and copper ions on the previously mentioned photocatalytic process, four other systems were considered. The first one comprising the ligand, H2O2, and UV afforded an efficient dye degradation of up to 58% under the same conditions (Table & Figure ). Photon absorption or the existence of active oxygen products in the reaction solution, including *OH from UV-induced H2O2 photolysis, could be the cause of the oxidation of the ligand, which in turn might directly interact with MO, enhancing the photodegradation efficiency of the employed dye. The second system, (Cu(II), H2O2, and UV), demonstrated a degradation efficiency of 94%, confirming that the pure copper(II) ions could facilitate the dye’s photodegradation as well. Moreover, the other two systems, comprising H2O2 + UV or complex + UV, failed to degrade MO efficiently (Table and Figure ). These results indicate that the designed complex (11) could participate efficiently in the photocatalysis process and significantly degrade the evaluated pollutant (MO).

Figure 2

Structure of the studied dye (MO).

Table 2

Photodegradation of MO under Different Conditions

entry	conditionsa	degradation (100%)b
1	H2O2 + UV	26
2	11 + UV	11
3	11 + H2O2 + UV	99
4	CuCl2 + H2O2 + UV	94
5	2a + H2O2 + UV	58

[H2O2] = 3.1 × 10–2, [11] = 3.3 × 10–5, and [MO] = 5.0 × 10–5 mol L–1.

120 min.

Figure 3

Photodegradation of MO under different conditions.

In the area of anticancer drugs, thioureas and N-aroyl thioureas have received much attention. The stark cytotoxic activities of thiourea derivatives against various types of cancers make them valuable structural motifs.[50−52] Likewise, to have an access to novel therapeutic metal-based anticancer drugs with profound cytotoxicity, it is frequent to combine pharmacophores with metals. Accordingly, various metal complexes exhibited potent antiproliferative activities and are in clinical use as anticancer drugs.[53,54] In the same context, copper complexes are of paramount importance as anticancer agents revealing promising in vitro and in vivo antiproliferative activities.[55,56] Therefore, combining thiourea pharmacophores with Cu(II) became a very favorable approach for developing compounds with antitumor activities.[57,58] With the N-naphthoyl thiourea library in hand, attention was now turned toward testing it for cytotoxicity against a range of cancer cell lines. In addition, the antiproliferative activity of Cu-complex 11 will be examined.

In Vitro Antiproliferative Activities toward cancer Cells (MCE-7, HCT116, and A549), and a Representative Normal Cell (MCF-10A)

Globally, cancer is considered to be a major cause of death. According to the WHO, among different types of cancers, lung cancer is responsible for the most deaths, whereas colorectal cancer comes in the second place. On the other hand, for women, breast cancer is the leading cause of deaths related to cancer worldwide. Based on the abovementioned facts, the anti-proliferative activities of all newly synthesized compounds were evaluated by the MTT method toward three human tumor cell lines, namely, human breast carcinoma cell line (MCF-7), human colon carcinoma cell line (HCT-116), and human lung adenocarcinoma cell line (A549), in addition to (MCF–10A) as a normal cell line. The results are expressed as IC50 values in Table and Figure .

Table 3

In Vitro Antiproliferative Activities of the Assembled Derivatives toward MCF-7, HCT116, and A549 Cancer Cells and a Normal Cell, MCF-10Aa

	IC50/μM
compound	MCE-7	HCT116	A549	MCF-10A
2a	5.22 ± 0.13	4.62 ± 0.11	5.72 ± 0.18	NT
2b	6.26 ± 1.01	4.14 ± 0.13	5.01 ± 0.17	NT
2c	4.99 ± 0.12	4.55 ± 0.15	5.53 ± 0.13	NT
2d	4.72 ± 0.21	5.07 ± 0.49	4.76 ± 0.16	NT
2e	7.32 ± 1.63	9.12 ± 2.01	5.95 ± 0.54	NT
2f	3.62 ± 0.11	6.22 ± 1.63	4.22 ± 0.62	NT
2g	5.11 ± 0.45	5.77 ± 0.92	6.86 ± 0.82	NT
2h	7.70 ± 0.15	6.09 ± 0.11	5.24 ± 0.13	NT
2i	1.20 ± 0.17	1.31 ± 0.23	2.73 ± 0.29	86.11 ± 1.67
2j	1.12 ± 0.25	1.23 ± 0.23	2.41 ± 0.44	90.22 ± 2.45
2k	1.19 ± 0.17	1.35 ± 0.23	2.82 ± 0.12	91.38 ± 3.17
3	7.96 ± 2.08	9.12 ± 2.52	18.84 ± 2.17	NT
4	6.93 ± 2.91	9.28 ± 3.28	17.64 ± 2.16	NT
6	17.30 ± 2.62	19.22 ± 2.44	16.38 ± 3.82	NT
7a	14.34 ± 3.43	12.22 ± 1.82	13.40 ± 1.99	NT
7b	14.92 ± 2.92	11.38 ± 2.03	13.77 ± 1.30	NT
8	1.75 ± 0.62	2.53 ± 0.38	1.22 ± 0.50	86.57 ± 2.28
10	2.38 ± 0.37	3.06 ± 1.44	1.12 ± 0.61	81.02 ± 2.39
11	1.22 ± 0.09	0.95 ± 0.05	0.68 ± 0.11	76.46 ± 2.72
doxorubicin	1.20 ± 0.07	1.02 ± 0.14	0.56 ± 0.16	77.01 ± 1.46

NT = not tested.

Figure 4

In vitro antiproliferative activities of the assembled derivatives toward MCF-7, HCT116, and A549 cancer cells.

Most of the naphthalene derivatives (2–11) showed potent activities (IC50 values <20 μM) using doxorubicin as a positive control. Derivatives 2a–h displayed good anti-proliferative activities against the screened cell lines. As depicted in Table , derivatives 2i–k exhibited profound antiproliferative activities against the three tested cancer cells (IC50 < 2.9 μM). Further, compounds 3–7 exhibited relatively high and similar selectivity against tested tumor cells with the IC50 values of 6.93–19.22 μM. Compounds 8 and 10 showed significant inhibitions against all the tested cell lines with IC50 values from 1.12 to 3.06 μM. Notably, compound 11, Cu-complex, displayed the most potent anti-proliferative effects against three cell lines among the tested samples. In addition, compounds with outstanding activities (2i–k, 8, 10, and 11) were chosen to assess their cytotoxicity against a normal cell (MCF-10A, Table ). As displayed in Table , such derivatives exhibited promising results (IC50: 76.46–91.38 μM), indicating preferential cytotoxicity toward the tumor cells. Compound 11 demonstrated the least cytotoxic impact on the investigated normal cell line, with an IC50 of 76.46 μM. Consequently, this derivative was evidenced to be safer than doxorubicin, indicating that it has the possibility to be a pioneer molecule in the development of prospective antitumor drugs. A preliminary relationship between the chemical structure and anti-proliferative activity (SAR) of the synthesized compounds could be analyzed. The skeletons of compounds 2a–h are similar to monosubstituted thiourea. These candidates, 2a–h, displayed comparable results in comparison with the reference material. This might be owing to the existence of a polar thiocarbonyl moiety in which its attraction with the hydrophilic active sites can be enhanced.[38] Further, within this series, derivatives possessing electron-releasing motifs, viz., piperidinyl, cyclohexyl, pyrrolidinyl, and ethyl (2a–f), displayed pioneer cytotoxic influences. Noteworthy, such electron-releasing motifs could tremendously enhance the lipotropy that leads to an improvement in the cytotoxic activities. Meanwhile, derivatives 2g and 2h bearing an electron-withdrawing group (Ph and p-nitrophenyl, respectively) exhibited a small decrease in the cytotoxic activities with IC50 values in the range 5.11–7.70 μM. To our delight, bis-substituted thioureas (2i–k) showed superior cytotoxic activity against the evaluated cancer cells. In comparison with the inhibitory activities of the thiourea derivatives (2a–h) with that of bis-thioureas (2i–k), the results indicated that the additional naphthoyl motif may have an essential effect on the anticancer efficacy. In this series, compound 2j with a propyl linker exhibited the highest activities against the evaluated cells (IC50: 1.12–2.41 μM), achieving superior behavior against MCE-7 than doxorubicin. It may be deduced from the series (2i–2k) that the cytotoxic effects of substances improve as the linker length between the two thiourea motifs increases. According to the obtained results, the anticancer activities diminished in the sequence 2j > 2k–2i. Next, SAR analyses were performed in the cyclized derivatives of compound 2a. Thus, compounds 3–11 were assembled and their anticancer behaviors were evaluated aiming to determine if other heterocyclic moieties besides the thiourea motif had an influence on activity. It is noticed that the cyclization of thiourea to thiazolidine derivative (3) slightly reduces the anticancer activity. Similar results were observed for the other thiazolidine derivatives (6 and 7). Interestingly, the presence of the chlorophenyl arylidene motif (4) resulted in a remarkable improvement in the anticancer behavior (Table ). The obtained results are in agreement with the reported one, in which the presence of the lipophilic chloro-substituent enhanced the anticancer activities via the improvement of the interaction with the hydrophilic active sites.[59] Interestingly, the cyclization of thiourea derivative 2a to triazole (8) or tetrazole (10) improved the activity throughout all evaluated cancer cells (IC50: 1.12–3.06 μM). Complexation of derivative 2a with CuCl2 resulted in complex 11 that was found to be the most potent product of all series against the three investigated cells (IC50: 0.68–1.22 μM) and exhibited stronger in vitro inhibition activity than doxorubicin. The aforementioned observation could be attributed to the copper complex mechanism of action that depends on many biological features including induction of programmed cell death (apoptosis), pro-apoptotic cell death due to endoplasmic reticulum stress, DNA damaging, and ROS production. In addition, copper complexes are considered to be potent cancer stem cell inhibitors, proteasome inhibitors, and human DNA-topoisomerase I and II inhibitors.[55,60,61] Importantly, copper has several merits including adopting an ideal geometry in interacting with DNA molecules, being better tolerated, and can be preferably managed in vivo than other metals that are used in anticancer drugs.[62,63] As a result, it can be inferred that cyclization of thioureas to thiazolidine rings decreased anticancer activity. However, cyclization to triazole or tetrazole or complexation with copper salt remarkably increased the anticancer activity toward all the cancer cells.

Conclusions

In this study, a versatile approach for rapidly assembling novel mono- and bis-thioureas from easily available starting materials has been achieved. Such promising thioureas were employed as a building block for the preparation of an assortment of biologically active heterocycles such as thiazolidines, triazole, and tetrazole. Further, the thiourea derivative 2a smoothly formed a single-site Cu-complex that could be used effectively as a catalyst in the photodegradation of methyl orange (MO) dye. Employing an ecofriendly energy source such as an ultrasound technique successfully improved the reaction rate and yield in comparison to the conventional methodologies. Moreover, the cytotoxic effect of the newly established compounds against three cancer cells, MCF-7, A549, and HCT116, was also studied. Further, compounds with outstanding activities represented lower cytotoxicity against one chosen normal cell (MCF-10A) in comparison with doxorubicin. Of these, compounds 2i–k and 7–11 have both superior anticancer behavior (IC50 < 2.9 μM) and lower cytotoxic effect (IC50 > 76 μM) on non-cancerous cells, indicating their promising potential as candidate compounds for future antitumor drug development.

Experimental Section

The open capillary approach was used to determine the melting points using the Electrothermal IA9100 melting point equipment (U.K.), and the results were presented uncorrected. The IR spectra (KBr) on a Perkin-Elmer Spectrum One spectrometer were reported. The NMR spectra were performed on the Bruker AV instrument operating at 400 MHz. The high-resolution mass spectra (HRMS) were determined employing a Bruker Daltonics microTOF spectrometer. The purity of compounds was evaluated by analytical thin-layer chromatography (TLC) that was proceeded on an EM silica gel F254 sheet (0.2 mm). Ultrasound irradiation was performed in a SY5200DH–T ultrasound cleaner.

General Procedure for the Preparation of Naphthoyl Thioureas 2a–k

Potassium thiocyanate (1.0 mmol) was added to a solution of naphthoyl chloride (1.0 mmol) in acetonitrile (15.0 mL) at room temperature and sonicated for 15 min. Substituted amines (1a–k) were added to the above reaction mixture, and sonication was continued at ambient temperature for about 20 min until completion (monitored by TLC). The solvent was removed in vacuo and the remaining mass was washed with 10% NaHCO3 solution (3 × 5.0 mL) and brine (3 × 5.0 mL), dried, and crystallized from ethanol to afford the products as solids in 91–96% yield.

N-(Cyclohexylcarbamothioyl)-2-naphthamide (2a)

Yield: 96%; mp 167–168 °C. IR (KBr): 3217, 3170 (NH), 1666 cm–1 (C=O). 1H NMR (400 MHz, CDCl3) δ: 11.68 (s, 1H, NH), 9.20 (s, 1H, NH), 8.77 (s, 1H, Ar–H), 7.76–7.69 (m, 3H, Ar–H), 7.48–7.40 (m, 3H, Ar–H), 4.99 (m, 1H, CH), 1.91–1.87 (m, 2H, CH2), 1.71–1.69 (m, 4H, CH2), 1.40–1.25 ppm (m, 4H, CH2). 13C NMR (100 MHz, CDCl3) δ: 173.0 (C=S), 166.0 (C=O), 154.0, 146.7, 136.2, 134.1, 131.3, 130.7, 129.7, 129.4, 128.5, 127.3 (Ar–C), 58.4, 33.9, 27.5, 26.0, 25.0 ppm. HRMS m/z: 335.0819 (M + Na). Anal Calcd for C18H20N2OS: C, 69.20; H, 6.45; N, 8.97. Found: C, 69.29; H, 6.41; N, 8.89.

N-(Ethylcarbamothioyl)-2-naphthamide (2b)

Yield: 92%; mp 96 °C. IR (KBr): 3220, 3161 (NH), 1668 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ: 11.18 (s, 1H, NH), 8.34 (d, J = 8.3 Hz, 1H, Ar–H), 7.85 (d, J = 7.9 Hz, 2H, Ar–H), 7.74 (s, 1H, NH), 7.42–7.30 (m, 4H, Ar–H), 4.18 (q, J = 7.2 Hz, 2H, CH2), 1.20 ppm (t, J = 7.2 Hz, 3H, CH3). HRMS m/z: 257.0756 (M – H). Anal. Calcd for C14H14N2OS: C, 65.09; H, 5.46; N, 10.84. Found: C, 64.97; H, 5.53; N, 10.78.

N-(Benzylcarbamothioyl)-2-naphthamide (2c)

Yield: 95%; mp 122 °C. IR (KBr): 3310, 3193 (NH), 1667 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6): δ = 11.46 (s, 1H, NH), 9.56 (s, 1H, NH), 8.32 (s, 3H, Ar-H), 8.18 (s, 3H, Ar-H), 8.07 (s, 3H, Ar-H), 7.63 (s, 3H, Ar-H), 4.65 ppm (s, 2H, CH2). HRMS, m/z: 343.0889 (M + Na). Anal. Calcd for C19H16N2OS: C, 71.22; H, 5.03; N, 8.74. Found: C, 71.29; H, 5.00; N, 8.71.

N-(Diethylcarbamothioyl)-2-naphthamide (2d)[43]

Yield: 95 (86)%; mp 108 (106) °C. IR (KBr): 3328 (NH), 1660 cm–1 (C=O).

N-(Piperidine-1-carbonothioyl)-2-naphthamide (2e)

Yield: 93%; mp 109 °C. IR (KBr): 3207 (NH), 1672 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ: 10.44 (s, 1H, NH), 8.89 (s, 1H, Ar–H), 7.98–7.92 (m, 3H, Ar–H), 7.81 (d, J = 7.9 Hz, 1H, Ar–H), 7.72–7.68 (m, 1H, Ar–H), 7.55 (d, J = 8.3 Hz, 1H, Ar–H), 3.46–3.40 (m, 4H, CH2), 1.37–1.33 ppm (m, 6H, CH2). HRMS, m/z: 321.1034 (M + Na). Anal. Calcd for C17H18N2OS: C, 68.43; H, 6.08; N, 9.39. Found: C, 68.40; H, 6.11; N, 9.32.

N-(Pyrrolidine-1-carbonothioyl)-2-naphthamide (2f)

Yield: 95%; mp 113 °C. IR (KBr): 3210 (NH), 1667 cm–1 (C=O). 1H NMR (400 MHz, CDCl3) δ: 10.19 (s, 1H, NH), 9.96 (s, 1H, Ar–H), 8.40 (d, J = 7.7 Hz, 2H, Ar–H), 8.06–8.00 (d, J = 7.9 Hz, 2H, Ar–H), 7.81–7.77 (m, 2H, Ar–H), 2.81–2.78 (m, 4H, CH2), 1.89–1.70 ppm (m, 4H, CH2). HRMS m/z: 284.0981 (M). Anal. Calcd for C16H16N2OS: C, 67.58; H, 5.67; N, 9.85. Found: C, 67.51; H, 5.71; N, 9.78.

N-(Phenylcarbamothioyl)-2-naphthamide (2g)[44]

Yield: 94 (70)%; mp 155 (152) °C. IR (KBr): 3367, 3195 (NH), 1676 cm–1 (C=O).

N-((4-Nitrophenyl)carbamothioyl)-2-naphthamide (2h)

Yield: 95%; mp 211–212 °C. IR (KBr): 3309, 3283 (NH), 1670 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ: 11.46 (s, 1H, NH), 10.79 (s, 1H, NH), 8.76 (s, 1H, Ar–H), 8.35 (d, J = 7.7 Hz, 2H, Ar–H), 8.05–7.97 (m, 4H, Ar–H), 7.86 (d, J = 7.8 Hz, 2H, Ar–H), 7.59–7.56 (m, 1H, Ar–H), 7.40–7.36 ppm (m, 1H, Ar–H). HRMS m/z: 374.0576 (M + Na). Anal. Calcd for C18H13N3O3S: C, 61.53; H, 3.73; N, 11.96. Found: C, 61.51; H, 3.79; N, 11.87.

N,N′-((Ethane-1,2-diylbis(azanediyl))bis(carbonothioyl))bis(2-naphthamide) (2i)

Yield: 91%; mp 199–201 °C. IR (KBr): 3228, 3201 (NH), 1669 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ: 11.18 (s, 2H, NH), 8.33 (s, 2H, Ar–H), 7.85 (d, J = 8.6 Hz, 4H, Ar–H), 7.74 (s, 2H, NH), 7.42–7.30 (m, 8H, Ar–H), 3.89 (s, 4H, CH2). 13C NMR (100 MHz, CDCl3) δ: 171.2 (C=S), 166.1 (C=O), 146.8, 136.1, 134.1, 131.3, 130.6, 129.8, 129.5, 128.6, 127.3, 126.5 (Ar–C), 47.0 ppm (CH2). HRMS m/z: 509.1083 (M + Na). Anal. Calcd for C26H22N4O2S2: C, 64.18; H, 4.56; N, 11.51. Found: C, 64.11; H, 4.59; N, 11.47.

N,N′-((Propane-1,3-diylbis(azanediyl))bis(carbonothioyl))bis(2-naphthamide) (2j)

Yield: 95%; mp 182–183 °C. IR (KBr): 3319, 3272 (NH), 1679 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ: 11.04 (s, 2H, NH), 8.37–7.44 (m, 16H, Ar–H + NH), 3.70–3.64 (m, 4H, CH2), 1.70–1.60 ppm (m, 2H, CH2). HRMS m/z: 500.1343 (M). Anal. Calcd for C27H24N4O2S2: C, 64.78; H, 4.83; N, 11.19. Found: C, 64.81; H, 4.80; N, 11.12.

N,N′-(((1,4-Phenylenebis(methylene))bis(azanediyl))bis(carbonothioyl))bis(2-naphthamide) (2k)

Yield: 92%; mp 190–1193 °C. IR (KBr): 3411, 3296 (NH), 1678 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ: 11.04 (s, 2H, NH), 8.71 (s, 2H, NH), 8.63 (s, 2H, Ar–H), 8.25–8.19 (m, 6H, Ar–H), 8.11–8.08 (m, 3H, Ar–H), 7.72–7.68 (m, 3H, Ar–H), 7.25 (s, 4H, Ar–H), 4.79 ppm (s, 4H, CH2). HRMS m/z: 585.1390 (M + Na). Anal. Calcd for C32H26N4O2S2: C, 68.30; H, 4.66; N, 9.96. Found: C, 68.25; H, 4.71; N, 9.88.

Synthesis of (Z)-N-(3-Cyclohexyl-4-oxothiazolidin-2-ylidene)-2-naphthamide (3)

A mixture of 2-naphthoyl isothiocyanate (1.0 mmol) and cyclohexanamine (1.0 mmol) in acetonitrile (15.0 mL) was sonicated for 15 min at room temperature to provide the thiourea derivative (2a) as white crystals. To the separated solid, chloroacetic acid (1.0 mmol) was added, and the mixture was further sonicated for 45 min at 50 °C. After completion of the reaction as monitored by TLC, crushed ice was added to the reaction mixture, and the resulting solid was filtered off, washed with water (3 × 5.0 mL), and purified by recrystallization from dioxane to afford the pure product 3.

Yield: 88%; mp 219 °C. IR (KBr): 1710, 1677 cm–1 (C=O). 1H NMR (400 MHz, DMSO-d6) δ: 8.70 (s, 1H, Ar–H), 8.13 (d, J = 7.9 Hz, 2H, Ar–H), 7.95 (d, J = 7.7 Hz, 2H, Ar–H), 7.62 (t, J = 7.4 Hz, 2H, Ar–H), 4.32 (s, 2H, CH2–thiazole), 3.98–3.91 (m, 1H, CH), 2.19–2.01 (m, 2H, CH2), 1.65–1.59 (m, 3H, cyclohexane), 1.31–1.01 ppm (m, 5H, cyclohexane). 13C NMR (100 MHz, DMSO-d6) δ: 163.3 (C=O), 162.7 (C=O), 148.8, 148.7, 148.6, 143.7, 138.4, 138.2, 137.5, 130.5, 127.5, 127.4, 124.2 (Ar–C), 61.0, 35.1, 33.4, 24.8, 24.2 ppm. HRMS m/z: 375.1144 (M + Na). Anal. Calcd for C20H20N2O2S: C, 68.16; H, 5.72; N, 7.95. Found: C, 68.20; H, 5.66; N, 7.89.

Synthesis of N-((Z)-5-((Z)-4-Chlorobenzylidene)-3-cyclohexyl-4-oxothiazolidin-2-ylidene)-2-naphthamide (4)

The solution of thiazolidine derivative (3, 1.0 mmol) and 4-chlorobenzaldehyde (1.0 mmol) in EtOH (20.0 mL) containing a catalytic amount of TEA was sonicated at 50 °C for 30 min. The progress of the reaction was monitored by TLC utilizing DCM:MeOH (9:1) as an eluent. After completion of the reaction, the reaction mixture was cooled to ambient temperature, and the separated solid was suction filtered, washed with ethanol (3 × 5.0 mL), and recrystallized from EtOH to obtain the pure product.

Yield: 92%; mp 259–261 °C. IR (KBr): 1682, 1664 cm–1 (C=O). 1H NMR (400 MHz, CDCl3) δ: 8.84 (s, 1H, Ar–H), 7.92 (d, J = 7.7 Hz, 2H, Ar–H), 7.71 (d, J = 7.8 Hz, 2H, Ar–H), 7.61 (s, 1H, =CH), 7.46 (t, J = 7.8 Hz, 2H, Ar–H), 7.25 (t, J = 7.8 Hz, 2H, Ar–H), 7.01 (d, J = 8.1 Hz, 2H, Ar–H), 4.02–3.91 (m, 1H, CH), 2.09–1.98 (m, 2H, CH2), 1.77–1.60 (m, 3H, cyclohexane), 1.31–0.92 ppm (m, 5H, cyclohexane). Anal. Calcd for C27H23ClN2O2S: C, 68.27; H, 4.88; N, 5.90. Found: C, 68.32; H, 4.80; N, 5.85.

Synthesis of (Z)-N-(3-Cyclohexyl-4-phenylthiazol-2(3H)-ylidene)-2-naphthamide (6)

A mixture of N-(cyclohexylcarbamothioyl)-2-naphthamide (2a, 1.0 mmol), phenacyl bromide (1.0 mmol), and a catalytic amount of TEA in water (20.0 mL) was sonicated at 50 °C for 50 min (TLC). The obtained product was separated and crystallized from dioxane to give the derivative 6.

Yield: 98%; mp 231–232 °C. IR (KBr): 1657 cm–1 (C=O). 1H NMR (400 MHz, CDCl3) δ: 8.90 (s, 1H, Ar–H), 8.58–8.51 (m, 2H, Ar–H), 8.33–8.27 (m, 2H, Ar–H), 8.18–8.15 (m, 2H, Ar–H), 7.95–7.89 (m, 2H, Ar–H), 7.57–7.46 (m, 3H, Ar–H), 7.10 (s, 1H, Ar–H), 4.88–4.82 (m, 1H, CH), 2.34–2.22 (m, 2H, CH2), 1.89–1.81 (m, 2H, cyclohexane), 1.69–1.61 (m, 3H, cyclohexane), 1.40–1.19 ppm (m, 3H, cyclohexane). 13C NMR (400 MHz, CDCl3) δ: 170.6 (C=O), 158.2 (C=N), 150.8, 150.6, 149.4, 149.3, 146.5, 146.4, 133.5, 129.6, 128.1, 127.2, 127.1, 124.7, 123.7, 123.5, 122.4, 122.3, 108.6 (Ar–C), 58.0, 31.1, 26.0, 25.1 ppm (cyclohexane). HRMS m/z: 411.1577 (M – H). Anal. Calcd for C26H24N2OS: C, 75.70; H, 5.86; N, 6.79. Found: C, 75.75; H, 5.80; N, 6.71.

Synthesis of Alkyl (Z)-2-((Z)-2-((2-Naphthoyl)imino)-3-cyclohexyl-4-oxothiazolidin-5-ylidene)acetate (7a,b)

A mixture of N-(cyclohexylcarbamothioyl)-2-naphthamide (2a, 1.0 mmol) and dialkyl acetylenedicarboxylate (1.0 mmol) in 20.0 mL of ethanol was sonicated for 7 min at ambient temperature. The obtained solid was filtered off, dried, and then crystallized from EtOH.

Methyl (Z)-2-((Z)-2-((2-Naphthoyl)imino)-3-cyclohexyl-4-oxothiazolidin-5-ylidene)acetate (7a)

Yield: 88%; mp 116–118 °C. IR (KBr): 1702, 1669, 1660 (C=O), 1610 cm–1 (C=N). 1H NMR (400 MHz, CDCl3) δ: 8.60 (s, 1H, Ar–H), 7.98–7.93 (m, 1H, Ar–H), 7.75–7.61 (m, 3H, Ar–H), 7.50–7.42 (m, 2H, Ar–H), 7.02 (s, 1H, =CH), 4.01 (s, 3H, OCH3), 3.57–3.48 (m, 1H, CH), 2.26–2.15 (m, 5H, cyclohexane), 1.90–1.86 ppm (m, 5H, cyclohexane). 13C NMR (400 MHz, CDCl3) δ: 172.6, 168.1, 166.9, (C=O), 158.6 (C=N), 149.9, 148.2, 137.5, 136.4, 130.3, 127.4, 126.9, 126.5, 125.0, 124.8, 122.7, 122.6 (Ar–C), 62.0 (cyclohexane), 53.8 (CH3), 31.5, 27.0, 25.9 ppm (cyclohexane). HRMS m/z: 445.1199 (M + Na). Anal. Calcd for C23H22N2O4S: C, 65.39; H, 5.25; N, 6.63. Found: C, 65.44; H, 5.19; N, 6.60.

Ethyl (Z)-2-((Z)-2-((2-Naphthoyl)imino)-3-cyclohexyl-4-oxothiazolidin-5-ylidene)acetate (7b)

Yield: 93%; mp 105–106 °C. IR (KBr): 1711, 1686, 1662 (C=O), 1619 cm–1 (C=N). 1H NMR (400 MHz, DMSO-d6) δ: 8.32 (s, 1H, Ar–H), 7.89–7.75 (m, 3H, Ar–H), 7.49–7.28 (m, 4H, Ar–H & =CH), 4.32 (q, J = 7.2 Hz, 2H, CH3CH2), 3.68–3.55 (m, 1H, CH), 2.28–1.56 (m, 10H, cyclohexane + CH3), 1.18–1.13 ppm (m, 3H, cyclohexane). HRMS m/z: 436.1454 (M). Anal. Calcd for C24H24N2O4S: C, 66.04; H, 5.54; N, 6.42. Found: C, 66.11; H, 5.50; N, 6.35.

Synthesis of N-Cyclohexyl-5-(naphthalen-2-yl)-4H-1,2,4-triazol-3-amine (8)

To the solution of N-(cyclohexylcarbamothioyl)-2-naphthamide (2a, 1.0 mmol) in DCM (20.0 mL), hydrazine hydrate (2.0 mmol) was added. The reaction was refluxed for 90 min; the conversion was monitored by TLC. The obtained crude compound was purified by crystallization from EtOH.

Yield: 82%; mp 239–240 °C. IR (KBr): 3411, 3370 cm–1 (NH). 1H NMR (400 MHz, CDCl3) δ: 12.10 (s, 1H, NH), 8.91 (s, 1H, Ar–H), 8.28–8.20 (m, 1H, Ar–H), 7.88–7.75 (m, 3H, Ar–H), 7.58–7.47 (m, 2H, Ar–H), 6.51 (s, 1H, NH), 3.59–3.41 (m, 1H, CH), 1.99–1.87 (m, 3H, cyclohexane), 1.69–1.57 (m, 4H, cyclohexane), 1.29–1.18 ppm (m, 3H, cyclohexane). HRMS m/z: 292.1686 (M). Anal. Calcd for C18H20N4: C, 73.94; H, 6.89; N, 19.16. Found: C, 73.90; H, 6.95; N, 19.05.

Synthesis of N-(1-Cyclohexyl-1H-tetrazol-5-yl)-2-naphthamide (10)

The solution having N-(cyclohexylcarbamothioyl)-2-naphthamide (2a, 1.0 mmol) was treated with CuSO4·5H2O (50 mol %, 249 mg) and TEA (1.0 mmol) in ethanol (15.0 mL), and the resulting mixture was sonicated for 30 min at room temperature. During this period, a black color precipitate (CuS) was observed. The reaction solution was transferred to centrifuge vials and centrifuged for 5 min with a centrifuge apparatus. The isolated black solid was separated from the centrifuged vials. Gradually, NaN3 (1.0 mmol) was introduced to the clear solution. The reaction mixture was then sonicated at ambient temperature for 45 min. The progress of the reaction was investigated by TLC (10% ethyl acetate in hexane). After completion of the reaction, the formed solid product was separated and purified by crystallization from dioxane.

Yield: 95%; mp 211–212 °C. IR (KBr): 3353 (NH), 1664 cm–1 (C=O). 1H NMR (400 MHz, CDCl3) δ: 9.47 (s, 1H, NH), 8.57 (s, 1H, Ar–H), 8.11–8.09 (m, 2H, Ar–H), 8.02–7.95 (m, 2H, Ar–H), 7.74–7.73 (m, 1H, Ar–H), 7.68–7.67 (m, 1H, Ar–H), 3.71–3.63 (m, 1H, CH), 1.91–1.86 (m, 2H, cyclohexane), 1.72–1.67 (m, 4H, cyclohexane), 1.44–1.19 ppm (m, 4H, cyclohexane). HRMS m/z: 320.1510 (M – H). Anal. Calcd for C18H19N5O: C, 67.27; H, 5.96; N, 21.79. Found: C, 67.25; H, 5.90; N, 21.69.

Synthesis of Complex 11

To a 100 mL round-bottom flask containing the thiourea ligand (2a, 2.0 mmol) dissolved in 15.0 mL of acidified (2.0 mL of 0.5% HCl) ethanol was added powdered CuCl2 (1.0 mmol). The suspension was sonicated at 50 °C for 90 min. The yellow crystals were separated by filtration, washed with EtOH (2 × 5.0 mL), and dried under vacuum.

Yield: 97%; mp 385–388 °C. IR (KBr): 3102 (NH), 1653 (C=O), 457, 449, 442 (CuO), 393, 381, 367, 355 cm–1 (CuS). HRMS m/z: 685.1734 (M). Anal. Calcd for C36H38CuN4O2S2: C, 63.00; H, 5.58; Cu, 9.26; N, 8.16. Found: C, 63.09; H, 5.50; Cu, 9.23; N, 8.07.

Cytotoxicity Evaluation

The cell lines MCF-7, A549, HCT116 (tumorigenic cells), and MCF-10A (non-tumorigenic cell) were received from ATCC via the Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. Potential anticancer activity and cytotoxicity of synthesized compounds were evaluated utilizing the sulforhodamine B (SRB) technique. Tumor cell lines were plated for 24 h in 96-multi-well plates (104 cells/well) before being treated with substances to facilitate cell adhesion to the plate wall. After being prepared for each individual dosage, multiple quantities of each substance (0, 2.5, 5, 10, and 15 mg/mL) were applied to the cell monolayer triplicate wells. Every sample had a final concentration of dimethyl sulfoxide (DMSO) of less than 1% v/v. Doxorubicin was employed as a standard antitumor drug for comparison. The compounds were cultured with monolayer cells for 48 h at 37 °C in a 5% CO2 atmosphere. Cells were fixed, washed, and stained with SRB dye after 48 h. The excess stain was removed using acetic acid, and the attached stain was retrieved using Tris-EDTA buffer. The concentration that induced a 50% loss of monolayer was defined as cytotoxicity. To test the newly produced compounds, the assay performed on cells was used.[64]

Photodegradation Tests

In a typical procedure, 400 μL of aqueous 5.0 × 10–4 mol L–1 complex stock solution, 600 μL of aqueous 5.0 × 10–4 mol L–1 stock solution of methyl orange (MO), 20 μL of aqueous H2O2 (9.23 mol L–1) stock solution, and 4.98 mL of H2O, to accomplish a total reaction volume of 6.0 mL, were successively mixed in a glass capped vial. The preliminary concentrations were as follows: [MO] = 5.0 × 10–5 mol L–1, [complex] = 3.3 × 10–5 mol L–1, and [H2O2] = 3.1 × 10–2 mol L–1, producing a 1:1.5:939 complex:dye:H2O2 molar ratio. The UV–vis spectra were taken at predetermined times during the reaction, which was performed under magnetic stirring at 25 °C. A vapor Hg lamp (250 W) to imitate UV light was placed 25.0 cm from the samples to preserve uniformity in the photon flux distribution. Control experiments were employed with no light irradiation to emphasize that the evaluated complex does not have any catalytic impact in the dark. The MO decolorization was calculated employing the following equation:[65]