Design, Synthesis of New 1,2,4-Triazole/1,3,4-Thiadiazole with Spiroindoline, Imidazo[4,5-b]quinoxaline and Thieno[2,3-d]pyrimidine from Isatin Derivatives as Anticancer Agents

The current work aims to design and synthesis a new series of isatin derivatives and greatly enhances their cytotoxic activity. The derivatives 3-((bromophenyl) imino)-1-(morpholino (pyridine) methyl) indolin-2-one, 2-((oxoindoline) amino) benzoic acid, 3-(thiazolo-imino) indolinone, ethyl-2-((oxoindolin-3-ylidene)amino)-benzothiophene-3-carboxylate, 1-(oxoindoline)-benzo[4,5] thieno [2,3-d]pyrimidin-4(1H)-one, ethyl-2-(2-oxoindoline) hydrazine-1-carboxylate, N-(mercapto-oxo-pyrimidine)-2-(oxoindoline) hydrazine-1-carboxamide, N-(oxo-thiazolo[3,2-a] pyrimidine)-2-(oxoindolin-ylidene) hydrazine-carboxamide, 3-((amino-phenyl) amino)-3-hydroxy- indolinone, 3-((amino-phenyl) imino)-indolinone, 2-(2-((oxoindoline) amino) phenyl) isoindolinone, 2-(oxoindoline) hydrazine-carbothioamide, 5′-thioxospiro[indoline-3,3′-[1,2,4]triazolidin]-one, 5′-amino-spiro[indoline-3,2′-[1,3,4]thiadiazol]-2-one and 3-((2-thioxo-imidazo[4,5-b]quinoxaline) imino) indolinone were synthesized from the starting material 1-(morpholino (pyridine) methyl) indoline-2,3-dione and evaluated for their in vitro cytotoxic activity against carcinogenic cells. The new chemical structures were evidenced using spectroscopy (IR, NMR and MS) and elemental analysis. The results show that compounds imidazo[4,5-b]quinoxaline-indolinone, thiazolopyrimidine-oxoindoline, pyrimidine-oxoindoline-hydrazine-carboxamide, spiro[indoline-3,2′-[1,3,4] thiadiazol]-one and spiro[indoline-3,3′-[1,2,4]triazolidin]-one have excellent anti-proliferative activities against different human cancer cell lines such as gastric carcinoma cells (MGC-803), breast adenocarcinoma cells (MCF-7), nasopharyngeal carcinoma cells (CNE2) and oral carcinoma cells (KB).


Introduction
Recently, cancer has been spreading among people at a high rate. Thus, cancer is one of the main causes of death. In 2017, the death of nearly 9.6 million people due to different types of cancer made it the second leading cause of death, second only to cardiovascular diseases [1,2].
Hence, in the last decade, many researchers have worked to prepare various heterocyclic compounds that are used in the treatment of most cancer cells in the human body. For instance, isatin derivatives are an interesting category of heterocyclic molecules that have different biological and pharmacological activities [3].
In addition, spiro compounds have been showing distinguished pharmacological activity, especially in the chemistry of natural products. Many studies have been demonstrating the synthesis of spiro derivatives of isatin at the (C-3) position as α-methylene γ-butyrolactone spirocyclic, which is derived from isatin and is the proper core for optimization to identify new anticancer agents [4]. The spiro pyrazole-oxindole analogues are considered as valuable anticancer agents against human cancer cells [5].
Furthermore, the oxindolylidene acetate derivatives showed potent anticancer activities against a diversity of human cancer cell lines [6]. The indoline-2,3-dione moiety is a significant constituent of many pharmaceutically essential compounds such as sunitinib and hesperidin.
Based on the above, isatin continues to be one of the more researched areas in synthetic and medicinal chemistry, aiding us in continuing our research work on the synthesis and design of biologically and pharmacological active heterocyclic compounds .
The 13 C-NMR spectrum of (2) displayed absorption signals at δ 51.2 and 58.8 ppm conforming to four carbon atoms of the (4CH 2 ) morpholine and 95.4 ppm corresponding to one carbon atom of the methine proton (CH) group.
Likewise, the IR spectrum of (4) indicated absorption bands at ν 3750 cm −1 for the one hydroxyl group and at ν 1740 and 1688 cm −1 for two carbonyl groups, and the 1 H-NMR spectrum of (4) evidenced one singlet signal at δ 12.60 ppm for the one proton of (OH) group (D 2 O exchangeable). The 13 C-NMR spectrum of (4) exposed absorption signals at δ 52.2 and 59.1 ppm matching four carbon atoms of the (4CH 2 ) morpholine, at δ 95.3 ppm corresponding for one carbon atom of the methine proton (CH) group and at δ 164.7 and 167.1 ppm corresponding to two carbonyl groups.
Similarly, 1 H-NMR spectrums of (5) discovered multiplet signals at δ 2.72-3.18 ppm conforming to eight protons of morpholine, one singlet at δ 6.67 ppm corresponding to one proton of methine proton and two doublet signals at δ 7.78 and 7.85 ppm corresponding for two protons of (J = 6.92 Hz) thiazole ring. The 13 C-NMR spectrum of (5) demonstrated absorption signals at δ 52.5 and 59.7 ppm corresponding to four carbon atoms of the (4CH 2 ) morpholine, at 94.4 ppm corresponding to one carbon atom of the methine proton (CH) group and at δ 168.2 ppm conforming to one carbonyl group.
Furthermore, the same compound (15) can be prepared in another way, which is to use cycloaddition reactions of the latter compound (14) in dry chloroform or glacial acetic acid with a few drops of triethylamine as catalysis for a long time under reflux and (TLC) control. The IR spectrum compound 15 showed absorption bands at ν 3350-3230 cm −1 compatible with three (3NH) groups and ν 1688 cm −1 conforming to one carbonyl group and ν 1450 cm −1 for one (C=S) group. 1 H-NMR spectrum of 15 revealed three broad singlets at δ 10.10, 10.40 and 11.55 ppm corresponding to three protons of (3NH) groups (D 2 O exchangeable). The 13 C-NMR spectrum of 15 showed absorption signals at δ 97.1 ppm conforming to one spiro-carbon atom of (1C, 5 -thioxospiro[indoline-3,3 -[1,2,4] triazolidin]-2-one) and δ 168.2 ppm for one carbon atom of the one carbonyl group.
In addition, 3,4]thiadiazol]-2-one (16) was prepared separately using compound (14) and iodine with potassium carbonate in boiling dioxane or DMF for long periods of time.
Another method for synthesizing a compound (16) is refluxing of compound (14) in ethanol containing a few drops of triethylamine as catalysis under (TLC) control. The IR spectrum of (16) displayed absorption bands at ν 3420 and 3335 cm −1 matching NH 2 and NH groups and ν 1682 cm −1 for one carbonyl group. 1 H-NMR spectrum of (16) proved one singlet at δ 6.85 ppm corresponding to two protons of (NH 2 ) and 10.25 ppm corresponding to one proton of (NH) groups exchangeable with D 2 O. The 13 C-NMR spectrum of (16) indicated absorption signals at δ 78.5 ppm corresponding to one spiro-carbon atom of (1C, 3 H-spiro[indoline-3,2 -[1,3,4]thiadiazol]-2-one) and δ 167.5 ppm for one carbon atom of the one carbonyl group.
was synthesized by nucleophilic aromatic substitution reactions of compound (14) with 2,3-dichloro-quinoxaline by refluxing in absolute ethanol in the presence of triethylamine as catalysis. The IR spectrum of (17) exposed absorption bands at ν 3340 cm −1 corresponding to NH group, 1686 cm −1 compatible with one carbonyl group and ν 1480 cm −1 conforming to one (C=S) group. 1 H-NMR spectrum of (17) revealed one singlet at δ 10.60 ppm conforming to one proton of (NH) group (D 2 O exchangeable).
The IC 50 values are summarized in (Table 1) and compared to 5-Fluorouracil as a positive control. IC 50 values are located over a wide range of concentrations, from 9 µM to over 50 µM, showing a significant disparity in the cytotoxicity of the isatin compounds of various cancer cell lines.
(III) Isatin derivatives are used for their anti-proliferative and antitumor properties and are considered are drugs. They deserve special attention, due to the following reasons: • Isatin derivatives are used as free ligands or coordinated bonds with many metal and organic ions. Hence, they provide promising anti-proliferative properties against different cancer cells.

•
Isatin-containing ligands have been fused with drug delivery systems for anti-tumor application.

•
Isatin and its derivatives have the ability to bind to nucleic acid (DNA) and generate different types of ions that act as antioxidants and also inhibit selected proteins.
(IV) Isatin (1H-indol-2, 3-Dione) is extracted as a red-orange powder from plants of the Isatis genus (Isatis tinctoria) and is a natural compound of alkaloids. Furthermore, indolic compounds are natural compounds found in these plants that have wide-ranging biological activity as anti-inflammatory and anti-tumor.

•
In addition, isatin has been detected in the human body as a metabolite of tryptophan or epinephrine, and it is widely distributed in the central nervous system (CNS) [55].

Experimental Section
Through cooperation with the teamwork of researchers, a research plan was made for the synthesis of new heterocyclic compounds. These new compounds were planned to study their biological activity as anticancer, and this plan was successfully implemented.

General Information
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Tokyo, Japan). Elemental analyses were performed on Vario EL (Elementar, Langenselbold, Germany). Microanalytical data were processed in the microanalytical center of the Faculty of Science at Cairo University and National Research Centre. The IR spectra (KBr disc) were recorded using a Perkin-Elmer 1650 spectrometer (Waltham, MA, USA). NMR spectra were determined using JEOL 270 MHz and JEOL JMS-AX 500 MHz (JEOL, Tokyo, Japan) spectrometers with Me4Si as an internal standard. Mass spectra were recorded on an EI Ms-QP 1000 EX instrument (Shimadzu, Tokyo, Japan) at 70 eV. Biological evaluations were done by the anticancer unit of Mansoura University, Faculty of Pharmacy (Department of Pharmacognosy), 35516, Egypt. All starting materials and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

Human and Animal Rights
No humans or animals were used in the study. The research was conducted according to ethical standards in vitro.

Chemicals and Drugs
Types of human carcinoma cancer cell line (MGC-803, MCF-7, CNE2 and KB) are derived from the National Cancer Institute, Cairo University, Cairo, Egypt, and 5-Fluorouracil and DMSO were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

Materials and Methods (In Vitro Cytotoxicity)
The in vitro cytotoxicity of the synthesized compounds against different cancer cell lines was measured with the MTT assay, in agreement with the method found in [22][23][24][25][26][27][28][29][30][31][32][33]54]. The MTT assay is based on the reduction of the soluble 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazoliumbromide (MTT) into a blue-purple formazan product, mainly by mitochondrial reductase activity inside living cells. The cells used in the cytotoxicity assay were cultured in the suitable cell culture medium (RPMI 1640) supplemented with 10% fetal calf serum. Cells suspended in the medium (2Y' 104/mL) were plated in 96-well culture plates and incubated at 37 • C in a 5% CO 2 incubator. After 12 h, the test sample (2 mL) was added to the cells (2Y' 104) in 96-well plates and cultured at 37 • C for 3 days. The cultured cells were mixed with 20 mL of MTT solution and incubated for 4 h at 37 • C. The supernatant was carefully removed from each well, and 100 mL of DMSO was added to each well to dissolve the formazan crystals, which were formed by the cellular reduction of MTT. After mixing with a mechanical plate mixer, the absorbance of each well was measured by a microplate reader using a test wavelength of 570 nm. The results were expressed as the IC 50 , which is the concentration of the drugs inducing a 50% inhibition of cell growth of treated cells, when compared to the growth of control cells. Each experiment was performed at least three times. There was good reproducibility between replicate wells with standard errors below 10%. Each experiment was repeated on three different days and conducted in triplicate. The relative cell anti-proliferative was measured according to the following equation: % cytotoxicity = (1 − As/Ab) × 100, where As = absorbance of each sample and Ab = absorbance of the blank. The probity analysis using the SPSS software program (version 20, SPSS Inc., Chicago, IL, USA) was used to determine each IC 50 .