Synthesis and Biological Evaluation of 2,4-Diaminoquinazolines as Potential Antitumor Agents

In the present study, a set of 2-anilino-4-alkylaminoquinazoline derivatives were synthesized and tested for their antitumor activities in vitro against a panel of four human cancer cell lines and for their DNA-binding affinity. Among the synthesized compounds, 4c and 5b with 4-substitution at the phenyl ring were found to have the highest inhibitory effects against breast adenocarcinoma (MCF-7), colon cancer (HCT-116), hepatocellular carcinoma (HePG-2), and human skin cancer (HFB4). Further investigation revealed that compounds 4a and 5d exhibited better affinity to bind with DNA than other tested compounds.


Introduction
Although the development of novel targeted antitumor drugs has made important progress in recent years, cancer remains the major leading cause of death in the world due to drug resistance or undesirable toxic e ects [1]. erefore, continued commitment to the laborious task of discovering new anticancer agents remains critically important [2].
In the course of nding new bioactive molecules that may serve as potent antitumor agents, quinazoline derivatives are of particular interest [3]. ese quinazolines have been identi ed as a new class of cancer chemotherapeutic agents against solid tumors and exert their antitumor activity through the inhibition of the receptor tyrosine kinases (RTKs) [4], speci cally epidermal growth factor receptor (EGFR), such as PD153035 [5], and ge tinib [6], or dihydrofolate reductase, such as trimetrexate (TMQ) [7] (Figure 1). Moreover, the anticancer activity of quinazolinebased drugs is related to their DNA-binding a nity to show inhibition of topoisomerases, such as Luotonin A [8], or inhibition of telomerase such as 05 9 (Figure 1), leading to cell death by inhibition of replicative enzymes and DNA repair systems.
In the current study, we have synthesized a series of 2,4disubstituted quinazolines 4a-d and 5a-d ( Figure 1), each of which contains a simple alkyl amino side chain at position 4 instead of the diaminopropyl moiety of 05 [9]. In addition, an aryl amino moiety was introduced at position 2 of the quinazoline ring instead of the substituted aryl fragment.
is can signi cantly increase the binding ability of these compounds with the relevant desired receptors, such as EGFR tyrosine kinase or DNA, through the formation of hydrogen bonding. An evaluation of their antiproliferative activity was carried out using four human tumor cell lines, in addition to their DNA-binding a nity.

In Vitro Anticancer Screening.
e newly synthesized compounds 4a-d and 5a-d were tested for their potencies for inhibition of breast adenocarcinoma (MCF-7), colon cancer (HCT-116), hepatocellular carcinoma (HePG-2), and human skin cancer (HFB4) by applying the MTT colorimetric assay. 5-Fluorouracil (5-FU) was chosen as a reference cytotoxic agent range of 5.3-8.8 μg/ml. Compounds were tested over a range of concentrations from 1.56 to 100 μg/ml, which we used to determine the inhibitory effects of the compound on cell growth using the MTT assay, this colorimetric is based on the conversion of the yellow MTT to a purple formazan derivative by mitochondrial succinate in viable cells. e cells were cultured in RPMI-1640 medium with 10% fetal bovine serum. Also, the calculated IC 50 values, that is, the concentration (μg/mL) of a compound that was able to cause 50% growth inhibition with respect to the control culture, were reported differently according to different cancer cells [13].
As shown in Table 1, the results of in vitro antitumor activity of the tested compounds indicated that compound 5b (with 4-NO 2 substitution) exhibited the highest cytotoxic activity against MCF-7, HCT-116, HePG-2, and HFB4 with an IC 50 ranging from 9.1 to 10.9 μM. Moreover, compound 4c (with 4-NO 2 substitution) showed similar activity against the four cancer cell lines, with an IC 50 ranging from 10.8 to    12.0 μM. As shown in Table 2, the replacement of the 4-NO 2 substituent in 4c and 5b with 3-NO 2 , F, or Br resulted in compounds 4d, 5c, or 5d, all of which exhibited a decrease in the activity against the four cell lines. ese results indicate that the 4-NO 2 substitution of the phenyl ring connected to the N 2 position is more favorable for anticancer activity than the 3-substitution.

DNA-Binding
Assay. Some anticancer agents block DNA synthesis, leading to cell cycle arrest [14]. We thus sought to determine if these compounds would have an impact on the cell cycle progression through measuring the affinity of these compounds to bind to DNA. A DNAbinding colorimetric assay was applied for the determination of the interaction of the small synthesized molecules with DNA. e results showed that all the tested compounds have a moderate affinity for DNA, which was demonstrated by a decrease in the absorbance of the DNA/methyl green. Compounds 4a and 5d exhibited the best DNA-binding affinity among other compounds.

Conclusion
A new series of 2,4-diaminoquinazoline compounds 4a-d and 5a-d were synthesized and tested against four cell lines belonging to different tumor subpanels. Some of the synthesized compounds 4c and 5b showed promising antiproliferative properties against MCF-7, HCT-116, HePG-2, and HFB4 with an IC 50 range of 9.1-12.0 μg/ml, in comparison to 5-FU with an IC 50 range of 5.3-8.8 μg/ml as reference agent. From the obtained results, we can conclude that the incorporation of the 4-NO 2 group into the phenylamino moiety improved the antitumor activity, which can be considered a valuable lead for future studies to obtain more potent antitumor agents. e wave numbers of maximum absorption peaks in IR spectroscopy are presented in cm −1 . Microanalytical data (C, H, and N) were found within ± 0.4% of the calculated values of the proposed structures. All dried solvents were stored over anhydrous Na 2 SO 4 (EtOH, CH 2 Cl 2 , and DMF). Moisture or air-sensitive reactions were conducted under the protection of the CaCl 2 tube in oven-dried glassware. Reagents were purchased from commercial suppliers and used without purification. Mass spectra were recorded on a ermo Scientific DSQ II spectrometer. Elemental analysis was performed at the Microanalytical Center, Cairo University, Egypt. (2). A suspension of anthranilic acid (0.073 mol) in dist. water (350 ml) was stirred at room temperature, and acetic acid (0.094 mol) was added dropwise until (pH � 4-5), the mixture changed to a greenishwhite suspension. An aqueous solution of KCNO (0.015 mol) was added dropwise to the wall of the breaker for 30 min, and the mixture turned to a heavy white suspension. e reaction was treated with an aqueous solution of NaOH (0.35 mol) till pH was 11-12 and left for stirring for 2 days. Conc. H 2 SO 4 was added to the cooling orange solution to give heavy off-white solids. e solid was filtered, washed with water, and dried to get the pure quinazoline-2,4-dione 1 as a white solid (m.p � > 290°C, Yield � 90%). [14] Compound 1 (0.03 mol) and POCl3 (0.85 mol) were refluxed for 13h. A mixture was cooled to room temperature and poured over a mixture of ice and water while stirring. e separated solids were filtered, washed with water, and dried to give a yellow solid product 2 with (m.p � 121-123°C, Yield � 90%) [11].

Synthesis of N-Butyl-2-Chloroquinazolin-4-Amine (3b).
To a solution of compound 3b (1.5 mmol) in CH 2 Cl 2 (15 ml), n-buthylamine (6 mmol) was added and the mixture was refluxed for 6 h. After cooling the solvent was evaporated under reduced pressure to give oil and leave it for 24 h, then added to a mixture of ice and water to give an off-white solid (m.p � 109-111°C, Yield � 90%) [12].

Synthesis of Compounds 5c-d.
In the solution of compound 3b (0.43 mmol) in a mixture of absolute EtOH and DMF (18 ml, 5 : 1), the appropriate aryl amine (0.43 mmol) was added, and the mixture was refluxed for 24 h. e separated solids were filtered, washed with EtOH, and the solvent was evaporated under reduced pressure. e crude was purified by column chromatography (SiO 2 : CH 2 Cl 2 /MeOH � 10 : 1) to give the desired products 5a-d.

In Vitro Anticancer Screening.
e cytotoxicity activity of the synthesized compounds 4a-d, and 5a-d are tested on four human tumor cell lines namely; MCF-7 (breast adenocarcinoma), HCT-116 (colon cancer), HePG-2 (hepatocellular carcinoma), and HFB4 (human skin cancer). e cell lines were purchased from ATCC via a holding company for biological products and vaccines (VACSERA), Cairo, Egypt. e used reagents are RPMI-1640 medium, MTT, DMSO, and 5-fluorouracil (Sigma Co., St. Louis, USA), and Fetal Bovine serum (GIBCO, UK). 5-fluorouracil (5-FU) was used as a standard anticancer drug for comparison. e previously mentioned cell lines were used to determine the inhibitory effects of the synthesized compounds on the growth of the tested cell lines using the MTT assay [15,16]. e colorimetric assay is based on the conversion of the yellow tetrazolium bromide (MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells. e cells were cultured in RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in a 5% CO 2 incubator. e cells were seeded in a 96-well plate at a density of (1.0 × 10 4 cells/well) at 37°C for 48 h under 5% CO 2 . After incubation, the cells were treated with different concentrations of compounds and incubated for 24 h. After 24 h of drug treatment, 20 μl of MTT solution at 5 mg/ml was added and incubated for 4 h. Dimethyl sulfoxide (DMSO) in a volume of 100 μl is added into each well to dissolve the purple formazan formed. e colorimetric assay is measured and recorded at an absorbance of 570 nm using a plate reader (EXL 800, USA). e relative cell viability expressed as a percentage was calculated as (A570 of treated samples/A570 of the untreated sample) X 100 [17]. e relative cell viability % = (A570 of treated samples/ A570 of untreated samples) * 100.

DNA-Binding
Assay. DNA methyl green (20 mg) was suspended in 100 ml of 0.05 M Tris-HCl buffer (pH � 7.5) containing 7.5 mM MgSO 4 ; the mixture was stirred at 37°C with a magnetic stirrer for 24 h. Test samples (10,100, and 1000 mg) were dissolved in ethanol in Eppendorf tubes, the solvent was removed under vacuum, and 200 μl of the DNA/ methyl green solution was added to each tube. Samples were incubated at in the dark at ambient temperature. After 24 h, the final absorbance of the samples was determined at 642.5-645 nm. Readings were corrected for initial absorbance and normalized as the percentage of the untreated standard [18].

Data Availability
All data used to support the findings of the study (NMR, IR, MASS and biological evaluation) are available from the corresponding author upon request (by email).

Conflicts of Interest
e authors declare that they have no conflicts of interest.