Sequential Four-Component Synthesis And Antitumor Activity Screening of Spiro[chromene-indolo[2,1-b]quinazoline] And Spiro[indolo[2,1-b]Quinazoline-pyrano[3,2-c]chromene]

Chemotherapy is one of the most common types of treatment among cancer patients and by using potent chemicals and agents, tumor promotion was inhibited. Despite the usage of many chemical agents in cancer therapy, cancer is still incurable. It seems that the synthesis of new compounds with high eciency on cancer cells and low side effects on normal cells will remain a critical challenge among researchers in this area. In the present work, a fast and straightforward process for the transformations involving tryptanthrins, malononitrile, some types of CH-acids such as 1,3-cyclohexanedione, dimedone, and 4-hydroxycumarin resulting in preparing spiro[chromene-indolo[2,1-b]quinazoline] and spiro[indolo[2,1-b]quinazoline-pyrano[3,2-c]chromene] derivatives through sequential Knoevenagel/Michael/intramolecular cyclization sequences was reported. at room temperature. This protocol benets some notable advantages including short reaction time, mild reaction condition, and simple purication, which make it interesting. Furthermore, it was carried out at room temperature, so it is according to green chemistry procedures. Also, antitumor screening of our new synthetic compounds (4a-i) was evaluated on pancreatic cancer cells (Panc1), breast cancer cells (MDA-MB-231), prostate cancer cells (PC3), and normal human adult dermal broblast cells (HDF) by using MTT assay using etoposide as a positive control. We found that 50% growth inhibitory concentration (IC 50 ) values of our synthetic compounds were not lower than etoposide against three cancer cell lines.


Introduction
Cancer is a major global problem with a high mortality rate. Chemotherapy is considered an effective platform in cancer therapy. However, the resistance to drugs and side effects of compounds on normal cells are the main unsolved problems. Therefore, the synthesis of new compounds with potent antitumor properties is required. In this respect, chromenes are an attractive group of compounds that are synthesized or occur in nature. Heterocyclic compounds bearing chromene moiety display a wide spectrum of biological properties including antileishmanial, antibacterial, antiproliferation, antioxidant, and antifungal. These compounds, due to their outstanding selective inhibitory activities of acetylcholinesterase and monoamine oxidase B enzymes exhibit excellent activity in the treatment of neurodegenerative diseases such as anti-Parkinson's anti-Alzheimer's and diseases [1][2][3][4][5][6][7][8][9][10][11][12][13].
On the other hand, alkaloids, characterized by an indolo [2,1-b]quinazoline core, present a broad range of potential biological activities too. In this regard, tryptanthrins, as natural alkaloids, are fascinating structures that have been considered as outstanding structures in drug discovery. These compounds are observed to be an aryl hydrocarbon receptor agonist, IDO-1 inhibitor, anti-cancer agent, antiparasitic, antineoplastic, and antileishmanial agent and have a wast range of immune-modulatory e cacies [17][18][19][20][21].
Owing to the importance and potential utility of the two above-mentioned structures, signi cant progress has been made in the synthesis of them which have been reported in the literature [22][23]. It is valuable to say that the connection of two structural motifs into one molecular structure might have importance and brings new enhancement properties, increasing their medicinal performance.
Numerous publications reported the synthesis of spiropyran derivatives through the reaction of isatin derivatives, malononitrile, and CH-acids such as dimedone and 4-hydroxycoumarin in presence of different catalysts [24][25][26].
To extend these works and intend to have two above-mentioned constructions in one molecule, we applied tryptanthrins instead of isatins. A few reports using tryptanthrin as a starting material are found Tryptanthrins 1a-c were formed via a reaction between isatoic anhydride and isatin derivatives in the presence of K 2 CO 3 in dimethylformamide (Scheme 3) [30].
Initially, the reaction of tryptanthrin 1a, malononitrile 2, and 1,3-cyclohexadione 3a were used as a model reaction in several solvents and temperatures to obtain an optimized condition. First, this reaction was examined in CH 2 Cl 2 at room temperature (25°C) ( Table 1, entry 1). Adding a base as a catalyst was necessary for the formation of the tryptanthrin-malononitrile adduct in this solvent and the Knoevenagel product did not form without the basic catalyst. Et 3 N as a catalyst was chosen and the reaction was carried out in a very short time, with a very good yield (Table 1, entry 2). The other solvents including water and ethanol were investigated and compared. In these solvents, the target compound did not form or the yield was very low. After that, the optimal reaction condition was applied to extend the domain of the synthetic procedure using tryptanthrin derivatives 1, malononitrile 2, and CH-acids 3a-c leading to the construction of derivatives 4a-i in very good yields (Table 2). These results showed that the time of tryptanthrins reaction with 1,3 cyclohexadione as a ketone was lower than dimedone and 4-hydroxycumarin and the yields of products were higher than the other used CH-acids. It's supposed that the steric effect has an effective role in this reaction. In these reactions, the substituent on tryptanthrin motif did not have remarkable e cacy on the reaction time. A possible mechanism is shown in Scheme 4. First, tryptanthrin molecule 1a and malononitrile 2, in the basic medium, take part in the Knoevenagel condensation reaction to produce the adduct 5. Next, dimedone 3a attacks adduct 5 via its nucleophilic center to construct the Michael adduct 6, followed by intermolecular O-cyclization to yield the nal product 4a.

Cytotoxic activity
First, to nd antitumor properties of the synthesized compounds, cell lines were cultured, and next, the MTT colorimetric assay was performed. Fifty percent of growth inhibitory concentration (IC 50 ) values were determined after 48 h treatment (Table 3) [31]. DMSO (1%) as a negative control and Etoposide (a reference drug) as a positive control were used. Among ten compounds, no IC 50 values lower than etoposide against three cancer cell lines were found. However, 4g and 4h showed some antitumor activities against PC3, Panc1, and MDA-MB231 cells respectively. Therefore, more studies will be required to nd the potent agents in cancer therapy. Then, a mixture of tryptanthrin 1 (1 mmol), malononitrile 2 (1 mmol) in the presence of Et 3 N (10 mol%) was stirred in CH 2 Cl 2 (5 mL) at room temperature. At once, an orange precipitate of tryptanthrinmalononitrile adduct was formed (followed by TLC). Afterward, 1,3-cyclohexanedione 3a was added to the mixture. A solid product was formed after 7 minutes and completion of the reaction was con rmed by TLC. Then, the precipitated product was ltered and washed with diluted acidic water to obtain the pure product.

Biological activity
Cell culture and cell viability assay Pancreatic cancer cells (Panc1), breast cancer cells (MDA-MB-231), prostate cancer cells (PC3), and, human adult dermal broblast cells (HDF) were received from Pasture Institute, Tehran, Iran. Cell culture condition was performed according to our previous report [30]. By using MTT assay, IC 50 values of the synthesized compounds were investigated after 48 h treatment and the results are presented in Table 3.