Synthesis, Anticancer, Antimicrobial and Antioxidant Potential of Novel 4-(Substituted phenyl-1,3,4-oxadiazol/thiadiazol-2-yl)-4-(4-substituted phenyl) Azetidin-2-One Derivatives

By exploiting the ample biological potential of 1,3,4-oxadiazole/thiadiazole ring, 4-substitutedphenyl-1,3,4-oxadiazol/Thiadiazol-2-yl)-4-(4-substitutedphenyl) azetidin-2-one derivatives were prepared. Various substituted azetidin-2-one derivatives have been identified as immunostimulating and antimicrobial, as well as their antioxidant activity. 2-amino 1,3,4 oxadiazole/thiadiazole conjugates were synthesized by mixing semi/thio carbazides and sodium acetate with water and stirring well, followed by adding aldehydes in methanol at room temperature. Acetate (glacial) was used as the catalyst to produce Schiff’s bases (intermediates) by treating substituted aldehydes with 2-amino 1,3,4 oxadiazole/thiadiazole(s). Using the mixture of triethylamine (dropwise) and chloroacetylchloride with vigorous stirring, 4-substitutedphenyl-1,3,4-oxadiazol/Thiadiazol-2-yl)-4-(4-substitutedphenyl) azetidin-2-one derivatives were prepared. The newly synthesized conjugates were evaluated for their anticancer potential using MCF-7 cell lines. Amoxicillin and fluconazole were used as reference drugs to determine their antimicrobial activity. Synthesized derivatives were evaluated for their antioxidant properties using 2-diphenyl-1-picrylhydrazyl (DPPH). In vitro cytotoxicity screening (MTTS assay) revealed that derivatives AZ-5, 9, 10, 14 and 19 demonstrated high efficacy with the percentage of inhibition at different concentration ranges (0.1 μM, 0.5 μM, 1 μM, 2 μM) of 89% to 94% μM as compared to doxorubicin as standard drug. The antimicrobial study indicated that compounds AZ-10, 19, and AZ-20 were found to have significant antimicrobial potential with MIC ranges of 3.34 µM to 3.71 µM in comparison to reference drugs having 4.29 µM to 5.10 µM. Based on antioxidant screening, most of the synthetic derivatives showed greater stability and effectiveness than the standard drug. According to the antioxidant screening, compounds AZ-5 and AZ-15 (IC50 = 45.02 μg/mL and 42.88 μg/mL, respectively) showed the greatest potency, as compared to ascorbic acid (IC50 = 78.63 μg/mL). Structure-activity relationship (SAR) studies of synthesized novel derivatives revealed that para-substituted halogen and nitro derivatives have remarkable potential against MCF-7 cancer cell lines and different microbial strains. Current evidence indicates that the synthesized derivatives may be promising candidates for use in the prevention and treatment of these infections. These synthesized compounds require further mechanism-based research to understand how they interact with the cells.


Introduction
Cancer is an illness that can be fatal, and it is one of the leading causes of mortality worldwide. Breast cancer is one of the most common malignancies among women. Most breast cancer deaths are caused by metastasis, which is the primary cause of treatment failure. Breast cancer affects approximately 40 million women worldwide, mostly in regions with high human development indices [1,2].
In breast cancer, tumor cells have the same characteristics and can be treated as if they were basal-like cells. Gene transcription and translation alterations can lead to functional defects that destabilize cell homeostasis and occur in oncogenes or tumor suppressor genes [3,4]. Mutation usually affects genes involved in the growth, proliferation, or death of cells ( Figure 1). Detecting and treating the localized disease at an early stage generally results in 99% overall survival for patients with a low to intermediate risk of recurrence [5,6]. A lumpectomy or mastectomy can be performed to remove the tumor or the entire breast. Certain kinds of breast cancer may be treated with hormones through medications such as Tamoxifen and aromatase inhibitors. Breast cancer research relies heavily on the development and testing of novel drugs. Novel compounds, however, are often needed to fill this gap because the rate of discovery of new drugs is relatively slow. Thus, the search for new derivatives that can combat breast cancer is essential [7,8].

Introduction
Cancer is an illness that can be fatal, and it is one of the leading causes of mortality worldwide. Breast cancer is one of the most common malignancies among women. Most breast cancer deaths are caused by metastasis, which is the primary cause of treatment failure. Breast cancer affects approximately 40 million women worldwide, mostly in regions with high human development indices [1,2].
In breast cancer, tumor cells have the same characteristics and can be treated as if they were basal-like cells. Gene transcription and translation alterations can lead to functional defects that destabilize cell homeostasis and occur in oncogenes or tumor suppressor genes [3,4]. Mutation usually affects genes involved in the growth, proliferation, or death of cells ( Figure 1). Detecting and treating the localized disease at an early stage generally results in 99% overall survival for patients with a low to intermediate risk of recurrence [5,6]. A lumpectomy or mastectomy can be performed to remove the tumor or the entire breast. Certain kinds of breast cancer may be treated with hormones through medications such as Tamoxifen and aromatase inhibitors. Breast cancer research relies heavily on the development and testing of novel drugs. Novel compounds, however, are often needed to fill this gap because the rate of discovery of new drugs is relatively slow. Thus, the search for new derivatives that can combat breast cancer is essential [7,8].  Investigations demonstrate that inflammatory factors can damage breast cells by releasing ROS and RNS with oxidative stress. This causes a pattern of cells to continually shift and experience distress. Cells can become cancerous as a result of the damage from free radicals [9][10][11][12]. To prevent cancer, antioxidants neutralize free radicals that can damage cells and make them cancerous. To ward off infection and inflammation in cells, new antimicrobial and antioxidant agents must be developed [13][14][15][16][17][18].
Investigations demonstrate that inflammatory factors can damage breast cells by releasing ROS and RNS with oxidative stress. This causes a pattern of cells to continually shift and experience distress. Cells can become cancerous as a result of the damage from free radicals [9][10][11][12]. To prevent cancer, antioxidants neutralize free radicals that can damage cells and make them cancerous. To ward off infection and inflammation in cells, new antimicrobial and antioxidant agents must be developed [13][14][15][16][17][18].
It is essential that a drug has specific and targeted pharmacological action in order to qualify as highly effective, since these actions can treat a variety of diseases, including cancer. Therefore, nitrogen, sulfur, and oxygen-containing heterocyclic scaffolds have attracted significant attention in the chemotherapeutic domain due to their importance in medicinal chemistry. Therefore, 1,3,4-oxadiazole/thiadiazole derivatives have wide-spectrum pharmacological actions. There have been some reports suggesting that a 1,3,4oxadiazole/thiadiazole moiety may be effective as an anticancer [19][20][21], antimicrobial [22,23], antioxidant [24,25], tyrosinase inhibitor [26], cathepsin K inhibitor [27], anti-tubercular [28,29], anti-diabetics [30], anti-inflammatory [31][32][33], anticonvulsant [34,35], antihypertensive activities [36], etc. These heterocyclic scaffolds are the main structural backbone of a wide range of drugs. As such, these scaffolds occupy the focal point of many commercial drugs ( Figure 2). This research paper highlights the anti-breast cancer and antimicrobial potential of azetidin-2-one derivatives of 1,3,4-oxadiazole/thiadiazole ring by inhibiting specific biological targets. Therefore, using these two isomers (1,3,4-oxadiazole/thiadiazole derivatives) in the pharmaceutical industry could be advantageous because of their chemical and thermal strength. This research paper highlights the anti-breast cancer and antimicrobial potential of azetidin-2-one derivatives of 1,3,4-oxadiazole/thiadiazole ring by inhibiting specific biological targets. Therefore, using these two isomers (1,3,4-oxadiazole/thiadiazole derivatives) in the pharmaceutical industry could be advantageous because of their chemical and thermal strength.

Antimicrobial Screening
Comp.     Antibacterial results of the synthesized molecules were comparable to those of the reference drug (amoxicillin), whereas antifungal screening of the synthetic conjugates were evaluated against both strains T. harzianum and A. niger compared to the standard

AZ-1 AZ-2 AZ-3 AZ-4 AZ-5 AZ-6 AZ-7 AZ-8 AZ-9
AZ-10    Antibacterial results of the synthesized molecules were comparable to those of the reference drug (amoxicillin), whereas antifungal screening of the synthetic conjugates were evaluated against both strains T. harzianum and A. niger compared to the standard

AZ-1 AZ-2 AZ-3 AZ-4 AZ-5 AZ-6 AZ-7 AZ-8 AZ-9
AZ-10  Antibacterial results of the synthesized molecules were comparable to those of the reference drug (amoxicillin), whereas antifungal screening of the synthetic conjugates were evaluated against both strains T. harzianum and A. niger compared to the standard drug (fluconazole). The antifungal screening outcomes revealed that derivatives AZ-4, 5, and AZ-6, 11, 18, and AZ-20 were moderately active against both fungal strains, i.e., T. harzianum and A. niger with MIC values 3.43 µM-7.51 µM, respectively. Compound AZ-11 and AZ-20 were remarkably effective against A. niger with MIC values 4.19 and 3.42 µM, whereas derivative AZ-19 was found as the most potent antifungal against T. harzianum in comparison to standard drugs. Overall compounds AZ-10, 19, and AZ-20 show promising antimicrobial potential in comparison to the reference drug.
The results from the minimum bactericidal concentration (MBC) test indicate that the synthesized compounds have both bacterial-inhibiting and bacterial-killing effects against the selected bacterial strains. MBC of synthesized azetidin-2-one derivatives of the 1,3,4-oxadiazole/thiadiazole ring was often three times greater than the MIC values, demonstrating that the synthesized compounds were bacteriostatic. (Table 3). All derivatives have shown mild to moderate antibacterial potential against bacterial strains. The MBC screening outcomes revealed that derivatives AZ-5, 9,11, and AZ-20 were moderately active against all Gram +ve as well Gram -ve bacterial strains with MIC values 4.19 µM-13.74 µM, respectively. Out of all active derivatives, AZ-20 showed remarkable antibacterial potential against Gram +ve as well as Gram -ve bacterial strains. Therefore, these prepared conjugates could be used as lead structures to develop new antimicrobial agents with improved activity. Additionally, they can help reduce the risk of developing drug-resistant bacteria, which can be a major problem in cancer patients.

In-Vitro Cytotoxic Assay (MTT Assay)
MCF-7 cell lines (breast cancer) were used to assess the efficacy of synthesized derivatives (Table 4). Synthesized conjugates (AZ-1-AZ-20) produced a significant amount of apoptosis and cytotoxicity. The preliminary screening of a total of 20 synthesized compounds from the chemical scheme shown as 01 led to only a few derivatives shown cytotoxic potential against the breast cancer cell line (MCF-7). The data obtained from pre-Pharmaceuticals 2023, 16, 517 9 of 19 liminary screening showed highly cytotoxic effects against the MCF-7 cell line with values ranging from 89.84% to 94.76%. Cell viability was measured by MTT assay at different concentrations (0.1 µM, 0.5 µM, 1 µM, 2 µM) and time points. Using synthesized conjugates (AZ-1-AZ-20) as anticancer potential with MCF-7 found that there was a significant increase in cell viability with higher concentrations of synthesized derivatives. In vitro cytotoxicity screening (MTTS assay) revealed that derivatives AZ-5, 9, 10, 14, and AZ-19 demonstrated high efficacy with the percentage of inhibition at different concentration ranges (0.1 µM, 0.5 µM, 1 µM, 2 µM) of 93.28%, 90.56%, 93.14%, 89.84%, and 94.76%, respectively, as compared to doxorubicin (99.98%) as a standard drug, for treating breast cancer. Morphological changes in the cells treated with these agents were assessed by staining with 0.05% crystal violet solution followed by microscopic examination. The anticancer activity measured using MTT assay showed that almost all derivatives exhibited significant anticancer activity as compared to control cells.

In-Vitro Antioxidant Evaluation
The antioxidant potentials of the synthesized conjugates were evaluated by free radical oxidation assay mechanisms (DPPH assay (methanolic) at 517 nm absorption band), respectively. The antioxidant assay is based on the chain-breaking mechanism of DPPH the hydroxyl radical, a natural regulator of the antioxidant defense system. Free radicals such as DPPH can be changed into a diamagnetic state by accepting H + ions or electron radicals from antioxidants. With the formation of a bond between DPPH radicals and antioxidant agents, the color strength of the solution decreases. Increased antioxidant strength increases the DPPH radical's ability to absorb electrons, decreasing the intensity of the purple solution to colorless, as measured by spectrophotometry at 517 nm. Calculations were made for all synthesized molecules to determine their IC 50 (µg/mL). The antioxidant screening revealed that most of the synthesized derivatives were more potent than the standard drug, indicating greater stability and effectiveness. As a result of the antioxidant screening, compounds AZ 05 and AZ-15 (IC 50 = 45.02 µg/mL and 42.88 µg/mL respectively) were found to be the greatest potential. Figure 6 illustrates the results of the antioxidant evaluation.

Structure-Activity Relationship
Analyzing the structures of synthesized derivatives for anticancer, antimicrobial, and antioxidant activities reveals the following SAR study (Figure 7

Structure-Activity Relationship
Analyzing the structures of synthesized derivatives for anticancer, antimicrobial, and antioxidant activities reveals the following SAR study (Figure 7):

3.
The presence of EWGs (Cl/dichloro) at the para position in the synthesized compounds showed significant antibacterial potential against gram (-ve) bacterial strains (AZ-5 and AZ-10).

4.
Similarly, the presence of the NH 2 group at the para position in the synthesized compounds (AZ-15) showed remarkable antioxidant action. 5.
EWGs-Cl and Br at the para position in the synthesized compound AZ-19 and AZ-20 increased the antifungal potential against Aspergillus niger and T. harzianum.

6.
The presence of OCH 3 in the synthesized AZ-8 and AZ-9 resulted in antibacterial activity against K. pneumoniae.

7.
The overall presence of a Cl and NO 2 combination at the para position of synthesized conjugates showed remarkable anticancer activity against the MCF-7 cancer cell line and potent antimicrobial action (AZ-19).
Therefore, these potent molecules can serve as lead compounds to create novel antimicrobial and anticytotoxic drugs containing less toxicity and high efficacy.

Material and Methods
The derivatives were synthesized without purification using commercially available analytical grade chemicals, i.e., (E. Merck (Germany) and S. D. Fine Chem. lmt (India). On a melting point apparatus, open glass capillary measurements were made to determine the melting point (M. Pt). TLC glass plates containing silica gel G were used to monitor each synthetic step using the mobile phases, ethyl acetate: petroleum ether (3:1) *, chloroform: methanol: (7:3) **. TMS was used as an internal standard in the NMR measurement of 1 H spectra on a Bruker Advance III 400 spectrometer. Mass spectra were obtained using Agilent mass spectrometers. A CHN analyzer was used to analyze the elements. Synthesis: Step 1: Synthesis of substituted 2 amino 1,3,4, (Oxa/Thia) diazoles (3) [37] A solution containing Semicarbazide hydrochloride (0.5 mol)/thiosemicarbazides (0.5 mol) (02) was added to water (10 mL) and stirred well before being added to methanol at room temperature. Under reduced pressure, the string was continued until the solvent had not completely evaporated. The residue was then treated with 1,4-dioxane and K2CO3 (1.5 mol) and iodine (0.5 mol) for 4-6 h at 80-85 °C. A TLC analysis was used to check and monitor the entire reaction step by step. The solution was treated with 5% Na2S2O3 (30 mL)

Material and Methods
The derivatives were synthesized without purification using commercially available analytical grade chemicals, i.e., (E. Merck (Germany) and S. D. Fine Chem. lmt (India). On a melting point apparatus, open glass capillary measurements were made to determine the melting point (M. Pt). TLC glass plates containing silica gel G were used to monitor each synthetic step using the mobile phases, ethyl acetate: petroleum ether (3:1) *, chloroform: methanol: (7:3) **. TMS was used as an internal standard in the NMR measurement of 1 H spectra on a Bruker Advance III 400 spectrometer. Mass spectra were obtained using Agilent mass spectrometers. A CHN analyzer was used to analyze the elements. Synthesis: Step 1: Synthesis of substituted 2 amino 1,3,4, (Oxa/Thia) diazoles (3) [37] A solution containing Semicarbazide hydrochloride (0.5 mol)/thiosemicarbazides (0.5 mol) (02) was added to water (10 mL) and stirred well before being added to methanol at room temperature. Under reduced pressure, the string was continued until the solvent had not completely evaporated. The residue was then treated with 1,4-dioxane and K 2 CO 3 (1.5 mol) and iodine (0.5 mol) for 4-6 h at 80-85 • C. A TLC analysis was used to check and monitor the entire reaction step by step. The solution was treated with 5% Na 2 S 2 O 3 (30 mL) after cooling, and the solution was extracted with CH 2 Cl 2 /MeOH (5:1). Anhydrous sodium sulfate was used to dry and concentrate the combined organic layer. The given residue was purified through a recrystallization process to obtain the corresponding 2 amino 1,3,4, (oxa/thia) diazoles (03) in 80-90% yield.
Step 2: General procedure for the synthesis of Schiff bases (5) In ethanolic solution, 2-amino-1,3,4, (oxa/thia) diazoles (03) (0.5 mol) were refluxed with different aromatic aldehydes (4) (0.5 mol). To complete the reaction, a small amount of GAA (2-3 mL) (dehydrating agent) was mixed into the reaction solution and the whole solution was refluxed for 7-8 h, or until the reaction was complete. The completion of the reaction was identified by TLC. After the reaction was complete, the excess ethanol as a solvent was distilled off and the residue mixture was stirred for 20 min on ice. After filtrating, washing with ice-cold water, drying, and recrystallizing in ethanol, the precipitates were collected.

Biological Procedure (s): MTT Assay
The MTT assay is a cytotoxic test which measures the metabolic activity of the cells. It is a colorimetric assay and based on the reduction of the yellow tetrazolium salt, i.e., MTT into purple formazan product through active mitochondria. The number of active cells present is directly proportional to the total quantity of MTT cleaved and quantification of the same is done by measuring the absorbance using the colorimeter [38].
individually, and added equal quantities of methanolic DPPH solution (0.0039%). A UVvisible double-beam spectrophotometer measuring 517 nm absorbance was used to measure the above solution's absorbance after 30 min in the dark. Data presented here represent the mean IC 50 based on the average of at least three observations.

Conclusions
The goal of this research was to develop new anticancer, antimicrobial, and antioxidant drugs by using azetidin-2-one derivatives of the 1,3,4-oxadiazole/thiadiazole ring. Because of their versatile biological action, the goal of this research has been to integrate the azetidin-2-one moiety into the oxadiazole ring to achieve 1-(5-(4-substituted phenyl)-1,3,4oxadiazol/thiadiazol-2-yl)-4-(4-substituted phenyl) for the exploration of altered biological interactions. In vitro cytotoxicity screening (MTTS assay) revealed that derivatives AZ-5, 9, 10, 14, and AZ-19 demonstrated high efficacy with the percentage of inhibition at different concentration ranges (0.1 µM, 0.5 µM, 1 µM, 2 µM) of 89% to 94% µM as compared to doxorubicin as the standard drug. The antimicrobial evaluation showed that compounds AZ-10, 19, and AZ-20 were found to have significant antimicrobial potential with MIC ranges of 3.34 µM to 3.71 µM in comparison to reference drugs having 4.29 µM to 5.10 µM. Based on antioxidant screening, most of the synthetic derivatives showed greater stability and effectiveness than the standard drug. According to the antioxidant screening, compounds AZ 05 and AZ-15 (IC 50 = 45.02 µg/mL and 42.88 µg/mL, respectively) showed the greatest potency, as compared to ascorbic acid (IC 50 = 78.63 g/mL). SAR studies of a synthesized novel series revealed that para-substituted halogen and nitro derivatives have remarkable potential against MCF-7 cancer cell lines and different microbial strains.