Synthesis and cytotoxic evaluation of some new 3-(2-(2-phenylthiazol-4-yl) ethyl)-quinazolin-4(3H) one derivatives with potential anticancer effects.

Quinazolinones are a group of heterocyclic compounds that have important biological activities such as cytotoxicity, anti-bacterial, and anti-fungal effects. Thiazole-containing compounds have also many biological effects including antitumor, antibacterial, anti-inflammatory, and analgesic activities. Due to significant cytotoxic effects of both quinazoline and thiazole derivatives, in this work a group of quinazolinone-thiazol hybrids were prepared and their cytotoxic effects on three cell lines were evaluated using MTT assay. Compounds A3, A2, B4, and A1 showed highest cytotoxic activities against PC3 cell line. Compounds A3, A5, and A2 were most active against MCF-7 and A3, A5, and A6 showed good cytotoxic effect on HT-29 cell line. According to the results, A3 efficiently inhibited all cell growth tested in a dose dependent manner. The IC50 of A3 was 10 M, 10 μM, and 12 μM on PC3, MCF-7, and HT-29 cells, respectively.

INTRODUCTION

Cancer is a life threatening health problem in developing and undeveloped countries. Although great progress in the treatment of this disease is made with respect to the problems associated with drug resistance, more research is essential for discovery of new anticancer agents. Combining two or more pharmacophores into one molecule is an approach to discover new targets. Therefore, there is more than one pharmacophore in a single molecule, each with a different mechanism of action, which can be effective for cancer treatment. Hybrid pharmacophores may be attached to different locations in the active site leading to elimination of drug resistance. Also, this method can reduce anticancer side effects (12).

Quinazoline and their derivatives are structural units for 150 natural alkaloids isolated from a number of families of the plant kingdom, from microorganisms and animals (3).

There are many reports on biological activities of synthetic and natural quinazolines including sedative (4), anticonvulsant (456), anti-inflammatory (47), antitumor (48), antibacterial (491011), antifungal (56), antitubercular (57912), antimalarial (1013), antiviral (57), anti-HIV (49101114), and antihyperlipidimic activities (1516).

Some drugs have been synthesized with quinazoline structure such as chloroqualone (antitussive), diproqualone (analgesic) (17), gefitinib, lapatinib (anticancer) (1), piriqualone (anticonvulsant) (18), doxazocin (antihypertensive) (19), prazosin (antihypertensive) (20), trimetrexate (antibacterial), thymitaq (anticancer) (21) and raltitrexed (anticancer) (22).

Thiazole-containing compounds also have valuable biological activities such as antitumor, anti-inflammatory, analgesic, antibacterial, and antifungal effects (23242526). Thiazole, an important heterocyclic ring, is widely used in anticancer drug development. Several anticancer agents containing thiazole moiety have been discovered, like tiazofurin and bleomycin. Ritonavir (anti-HIV), meloxicam (anti-inflammatory), nizatidine (antipeptic ulcer), and penicillin (antibiotic) are some examples of other thiazole compounds with biological activities (2327). Due to the valuable cytotoxic effects of both thiazole and quinazoline compounds, in this work a group of quinazolinone-thiazole hybrids were synthesized and their antiproliferative activities were determined using tumor cells in culture.

MATERIALS AND METHODS

Instrumentation

All starting materials, reagents, and solvents were purchased from commercial suppliers like Merck (Germany) and Aldrich (USA) companies. The purity of the synthesized compounds was proved by thin layer chromatography (TLC) using various solvents. Merck silica gel 60 F254 plates were applied for analytical TLC. 1H-NMR spectra were recorded using a Bruker 400 MHz spectrometer (Germany), and chemical shifts are expressed as δ (ppm) with tetramethylsilane as internal standard. The IR spectra were obtained on a Shimadzu 470 spectrophotometer (Japan) (potassium bromide disks). Melting points were determined using electrothermal melting point analyzer apparatus and are uncorrected. The mass spectra were run on a Finigan TSQ-70 spectrometer (Finigan, USA) at 70 EV. All cell lines were purchased from Pasteur Institute of Iran.

Preparation of compounds

To produce 3-(2-(2-phenylthiazol-4-yl)ethyl)-quinazolin-4(3H)- one derivatives, the primary amine G was synthesized through a five step procedure. In the first step, 4-phthalimido-2-butanone (B), was prepared through the addition of methyl vinyl ketone to phthalimide (A). In the second step, 1-bromo-4-N-phthalimido-2-butanone (C) was synthesized by bromination of the methyl group of compound B. Nucleophilic substitution of thiobenzamide (E) to the brominated intermediate (C) resulted in compound F which was reacted with hydrazine hydrate and deprotected to produce the 2-phenyl-4-(2-aminoethyl) thiazole G (1). A group of benzoxazinones with different substituents at position 2 were synthesized. Reaction of the primary amine G with these benzoxazinones yielded the final compounds as presented in Fig. 1. The structure of final compounds are presented in Figs. 2 and 3.

Fig. 1

General reaction scheme for preparation of the final compounds.

Fig. 2

General structure of final compounds (group A).

Fig. 3

General structure of final compounds (group B).

Cell culture conditions

PC3 (human prostate cancer cell line), MCF7 (human breast adenocarcinoma cell line), and HT-29 (human colon adenocarcinoma cell line) cells were maintained at 37 °C in a humidified atmosphere (90%) containing 5% CO2. PC3, MCF7, and HT-29 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM-F12) with 10% v/v fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The medium was changed every two to three days and sub-cultured when the cell population density reached to 70-80% confluence. Cells were seeded at an appropriate density according to each experimental design (28).

Cytotoxicity assay

HT-29, MCF-7, and PC-3 cells were seeded in triplicate in 96-well tissue culture plates (15 × 103 cells/well) and incubated overnight. Doxorubicin was used as positive control and the wells containing DMSO (1%) and cell suspension was regarded as the negative control. The blank wells were consisted of 200 μL of the culture medium. Cells were treated with different concentrations of the derivatives (0-275 μM).

The microplates were further incubated for 48 h. To evaluate cell survival, each well was then incubated with 20 μL of MTT solution (5 mg/mL in PBS) for 3 h and the media in each well was replaced with 200 μL of DMSO and pipetted up and down to dissolve the formazan crystals.

The absorbance of each well was measured at 540 nm using an ELISA reader. Each experiment was repeated three times. The percentage of cell viability was calculated using the following formula:

IC50 values were calculated by plotting the log10 of percent cell viability against compound concentrations (2829).

RESULTS

Details of preparation procedures and chemistry of synthesized compounds

N-Acyl anthranilic acid (I)

Acyl chloride (0.37 mol) was added dropwise to a mixture of compound H (0.25 mol) in dimethyl formamide (125 mL) at such rate that the temperature of the mixture did not rise above 40 °C.

The mixture was stirred at room temperature for at least an additional 3 h. Completion of the reaction was determined by TLC and the mixture was poured into water (1 L) and stirred for 1 h.

The precipitated product was collected by filtration, washed with cold water, and dried under reduced pressure yielding I as a white powder (50-70%).

2-Substituted-3,1-benzoxazin-4-one (J)

Compound I (0.125 mol) was dissolved in acetic anhydride (90 mL) and slowly heated to 170-180 °C in a round-bottom flask equipped with a magnetic stirrer bar and a claisen-distillation head. Completion of the reaction was confirmed by TLC, and the produced acetic acid was distilled under reduced pressure. The residue was then cooled and product was washed by n-hexan to give compound J as yellow crystals (65-80%).

3-(2-(2-phenylthiazol-4-yl) ethyl) -quinazolin-4 (3H)-one derivatives

To prepare 3-(2-(2-phenylthiazol-4-yl)ethyl)-quinazolin-4(3H)-one derivatives, 0.5 mmol of related benzoxazone was refluxed with 1 mmol of amine G in chloroform (5 mL) for 6-7 h. After completion of the reaction, chloroform was evaporated under reduced pressure and the residue was treated with ethylene glycol (2 mL) and NaOH pellets (0.003 g) in a flask equipped with a claisen-distillation head.

The mixture was reheated to 130-140 °C for 5 h. After completion of the reaction, the clear solution was allowed to cool to room temperature and kept overnight to precipitate which was then crystallized from 2-propanol to obtain final products.

2-methyl-3-(2-(2-phenylthiazol-4-yl)ethyl) quinazolin-4(3H)-one (A1)

Yield: 55%. m.p 119 °C, (Found: M 347, C20H17N3OS requires 347), IR (KBr, cm-1) νmax = 1671, 1595, 1474. 1H-NMR (400 MHz, CDCl3) δ: 1.60 (3H, s, H-CH3:R1), 3.29 (2H, t, J = 6.8 Hz, H-C10), 4.51 (2H, t, J = 6.8 Hz, H-C9), 6.93 (1H, s, H-C12), 7.4-7.5 (4H, m, H-C17, H-C18, H-C19, H-C6), 7.60 (1H, d, J = 7.2 Hz, H-C8), 7.70-7.76 (1H, m, H-C7), 7.88-7.94 (2H, m, H-C16, H-C20), 8.28 (1H, dd, J = 6.4 Hz, J = 1.6 Hz, H-C5).

2-ethyl-3-(2-(2-phenylthiazol-4-yl)ethyl) quinazolin-4(3H)-one (A2)

Yield: 48%. m.p 122 °C, (Found: M 361, C21H19N3OS requires 361), IR (KBr, cm-1) νmax = 1671, 1593, 1473. 1H-NMR (400 MHz, CDCl3) δ: 1.34 (3H, t, J = 7.5, CH3:R1), 2.81 (2H, q, J = 7.5 Hz, CH2:R1), 3.28 (2H, t, J = 7.5 Hz, H-C10), 4.51 (2H, t, J = 7.5 Hz, H-C9), 6.94 (1H, s, H-C12), 7.40-7.50 (4H, m, H-C17, H-C18, H-C19, H-C6), 7.62-7.67 (1H, m, H-C8), 7.70-7.76 (1H, m, H-C7), 7.87-7.96 (2H, m, H-C16, H-C20), 8.26-8.31 (1H, m, H-C5).

3-(2-(2-phenylthiazol-4-yl)ethyl)-2-propyl quinazolin-4(3H)-one (A3)

Yield: 35%. m.p 184 °C, (Found: M 375, C22H21N3OS requires 375), IR (KBr, cm-1) νmax = 3093, 2873, 1678, 1523. 1H-NMR (400 MHz, CDCl3) δ: 0.95 (3H, t, J = 7.2 Hz, CH3:R1), 1.75 (2H, Hex, J = 7.2Hz, CH2:R1), 2.66 (2H, t, J = 7.6 Hz, CH2:R1), 3.21 (2H, t, J = 7.2 Hz, H-C10), 4.44 (2H, t, J = 7.2 Hz, H-C9), 6.86 (1H, s, H-C12), 7.30-7.40 (4H, m, H-C17, H-C18, H-C19, H-C6), 7.56 (1H, d, J = 8.0 Hz, H-C8), 7.65 (1H, t, J = 5.6 Hz, H-C7), 7.80-7.90 (2H, m, H-C16, H-C20), 8.20 (1H, d, J = 5.6 Hz, H-C5).

2-phenyl-3-(2-(2-phenylthiazol-4-yl)ethyl) quinazolin-4(3H)-one (A4)

Yield: 50%. m.p 140 °C, (Found: M 409, C25H19N3OS requires 409), IR (KBr, cm-1) νmax = 1672, 1605, 1472. 1H-NMR (400 MHz, CDCl3) δ: 3.17 (2H, t, J = 7.2 Hz, H-C10), 4.46 (2H, t, J = 7.2 Hz, H-C9), 6.72 (1H, s, H-C12), 7.24-7.42 (8H, m, H-C-17, H-C18, H-C19, H-C6, H-C8, 3H-Ph:R1), 7.52 (1H, t, J = 7.2 Hz, H-C7), 7.66-7.80 (4H, m, H-C16, H-C20, 2H-Ph:R1), 8.36 (1H, d, J = 8.0 Hz, H-C5).

2-(4-chlorophenyl)-3-(2-(2-phenylthiazol-4-yl) ethyl)quinazolin-4(3H)-one (A5)

Yield: 36%. m.p 170 °C, (Found: M 443, C25H18ClN3OS requires 443), IR (KBr, cm-1) νmax = 3120, 2920, 1678, 1566. 1H-NMR (400 MHz, CDCl3) δ: 3.10 (2H, t, J = 6.4 Hz, H-C10), 4.41 (2H, t, J = 6.4 Hz, H-C9), 6.67 (1H, s, H-C12), 7.22-7.28 (4H, m, 4H-ClPh:R1), 7.30-7.38 (4H, m, H-C17, H-C18, H-C19, H-C6), 7.46 (1H, t, J = 6.8 Hz, H-C7), 7.58-7.64 (2H, m, H-C16, H-C20), 7.69-7.73 (1H, m, H-C8), 8.30 (1H, t, J = 6.8 Hz, H-C5).

2-(4-nitrophenyl)-3-(2-(2-phenylthiazol-4-yl) ethyl)quinazolin-4(3H)-one (A6)

Yield: 30%. m.p 145 °C, (Found: M 454, C25H18N4O3S requires 454), IR (KBr, cm-1) νmax = 3329, 3066, 1674, 1600. 1H-NMR (400 MHz, CDCl3) δ: 3.05 (2H, t, J = 6.8 Hz, H-C10), 3.75 (2H, t, J = 6.4 Hz, H-C9), 6.99 (1H, s, H-C12), 7.05 (1H, t, J = 6.4 Hz, H-C18), 734-7.40 (2H, m, H-C17, H-C19), 7.50 (1H, t, J = 5.6 Hz, H-C7), 7.66 (1H, d, J = 6.8 Hz, H-C8), 7.83-7.90 (2H, m, H-C16, H-C20), 8.14 (2H, d, J = 8.4 Hz, 2H-NO2Ph:R1), 8.28 (2H, d, J = 8.8 Hz, 2H-NO2Ph:R1), 8.32-8.38 (1H, m, H-C6), 8.76 (1H, d, J = 8.4 Hz, H-C5).

6-bromo-2-methyl-3-(2-(2-phenylthiazol-4-yl) ethyl)quinazolin-4(3H)-one (B1)

Yield: 75%. m.p 156 °C, (Found: M 425, C20H16BrN3OS requires 425), IR (KBr, cm-1) νmax = 3390, 2922, 1670, 1593. 1H-NMR (400 MHz, CDCl3) δ: 2.44 (3H, s, CH3:R1), 3.21 (2H, t, J = 6.8 Hz, H-C10), 4.42 (2H, t, J = 6.8 Hz, H-C9), 6.85 (1H, s, H-C12), 7.32-7.42 (5H, m, H-C16. H-C17, H-C18, H-C19, H-C20), 7.72 (1H, d, J = 6.4 Hz, H-C8), 7.8-7.86 (1H, m, H-C7), 8.33 (1H, d, J = 2.4 Hz, H-C5).

6-bromo-2-ethyl-3-(2-(2-phenylthiazol-4-yl) ethyl)quinazolin-4(3H)-one (B2)

Yield: 55%. m.p 125 °C, (Found: M 439, C21H18BrN3OS requires 439), IR (KBr, cm-1) νmax = 3298, 3062, 1670, 1508. 1H-NMR (400 MHz, CDCl3) δ: 1.25 (3H, t, J = 7.2 Hz, CH3:R1), 2.71 (2H, q, J = 7.2 Hz, CH2:R1), 3.19 (2H, t, J = 7.2 Hz, H -C10), 4.43(2H, t, J = 7.2 Hz, H-C9), 6.67(1H, s, H-C12), 7.30-7.40 (3H, m, H-C17, H-C18, H-C19),7.44 (2H, d, J = 8.8 Hz, H-C16, H-C20), 7.72 (1H, d, J = 6.4 Hz, H-C8), 7.81-7.83 (1H, m, H-C7), 8.33 (1H, d, J = 2.4 Hz, H-C5).

6-bromo-2-phenyl-3-(2-(2-phenylthiazol-4-yl) ethyl)quinazolin-4(3H)-one (B3)

Yield: 40%. m.p 137 °C, (Found: M487, C25H18BrN3OS requires 487), IR (KBr, cm-1) νmax = 3321, 3059, 1678, 1083. 1H-NMR (400 MHz, CDCl3) δ: 3.08(2H, t, J = 6.8 Hz, H-C10), 4.40 (2H, t, J = 7.2 Hz, H-C9), 6.65 (1H, s, H-C12), 7.30-7.40 (6H, m, H-C17, H-C18, H-C19, Ph-3H:R1), 7.50 (2H, d, J = 8.8 Hz, H-C16, H-C20), 7.59 (2H, d, J = 6.4 Hz, Ph-2H:R1), 7.76 (1H, d, J = 6.4 Hz, H-C8), 7.83-7.87 (1H, m, H-C7), 8.41 (1H, d, J = 2.0 Hz, H-C5).

6-bromo-2-(4-chlorophenyl)-3-(2-(2-phenyl thiazol-4-yl)ethyl)quinazolin-4(3H)-one (B4)

Yield: 36%. m.p 195 °C, (Found: M 521, C25H17BrClN3OS requires 521), IR (KBr, cm-1) νmax = 3066, 2924, 1643, 1516, 1091. 1H-NMR (400 MHz, CDCl3) δ: 3.22 (2H, t, J = 6.4 Hz, H-C10), 3.75 (2H, t, J = 6.4 Hz, H-C9), 6.86 (1H, s, H-C12), 7.30-7.40 (5H, m, H-C16, H-C17, H-C18, H-C19, H-C20), 7.70-7.80 (4H, m, Cl-Ph-4H:R1), 7.84-7.90 (2H, m, H-C7, H-C8), 8.64 (1H, d, J = 8.8 Hz, H-C5).

6-bromo-2-(4-nitrophenyl)-3-(2-(2-phenyl thiazol-4-yl)ethyl)quinazolin-4(3H)-one (B5)

Yield: 62%. m.p 185 °C, (Found: M 532, C25H17BrN4O3 S requires 532), IR (KBr, cm-1) νmax = 3309, 3066, 1685, 1585. 1H-NMR (400 MHz, CDCl3) δ: 3.04 (2H, t, J = 6.0 Hz, H-C10), 3.75 (2H, t, J = 6.4 Hz, H-C9), 6.98 (1H, s, H-C12), 7.30-7.50 (4H, m, H-C16, H-C17, H-C19, H-C20), 7.60 (1H, d, J = 6.8 Hz, H-C18), 7.75-7.90 (2H, m, NO2 -Ph-2H:R1), 8.11 (2H, d, J = 8.8 Hz, NO2 -Ph-2H:R1), 8.28 (1H, d, J = 8.8 Hz, H-C8), 8.34-8.40 (1H, m, H-C7), 8.69 (1H, d, J = 8.8 Hz, H-C5).

Antiproliferative effects of the dervitivaves

Results of MTT assay for evaluation of cytotoxic effects of the fractions are listed in Table 1.

Table 1

The IC50 (μM) of tested compounds against PC-3, MCF-7, and HT-29 cancer cell lines.

DISCUSION

In this study, quinazolinones as biologically active compounds were conjugated with another well-known moiety (thiazole ring) in a multi-step reaction procedure to produce interesting novel compounds. Next, all synthesized compounds were tested for their cytotoxic effects on three human carcinoma cell lines including MCF-7, HT-29, and PC-3.

The primary synthesis of quinazolinones may be performed by cyclization of benzene substrates which have appropriate substituents. This could be achieved by substitution of proper groups on COOH or NH2 of antranilic acid or its derivatives to provide one or more of the ring atoms required to complete the pyrimidine ring. Modification of appropriate derivatives of other heterocyclic systems such as benzodiazepines is another procedure for preparation of quinazolinones.

In another synthetic method, 3,1-benzoxazin-4-one was used to prepare quinazolinones. This procedure has been used extensively to make a large number of 4(3H)-quinazolinones with good yields. In these reactions almost any primary amine may be added to 3,1-benzoxazin-4-one to achieve overall replacement of ring-O by ring-N with the formation of 4(3H)-quinazolinone. Preparation of benzoxazinone has been reported in several literatures using different methods. It can be produced via one or two step(s) procedures using anthranilic acid or its derivatives as starting materials in high yields.

Benzoxazinones are highly reactive and should be used immediately after preparation. In this study benzoxazinones were prepared by a two steps procedure.

The synthesized 4(3H)quinazolinones in this study contained 4-ethyl-2-phenylthiazole group on position 3 of quinazolinone structure. For preparation of these quinazolinones, a primary amine containing thiazole (compound G, Fig. 1) was synthesized.

The most practical method to prepare thiazoles is Hantzsch reaction which involves the condensation of α-haloketones and thiourea or thioamides in refluxing alcohol. Phthalimide as an NH2-synthon was used here for the preparation of the amine. Application of phthalimide in Gabriel synthesis for preparation of primary amines is well documented. After alkylation, the resulting alkyl phthalimide is reacted with hydrazine. The desired primary amine could be generated by reacting with hydrazine hydrate. Consequently phthalazine as a stable cyclic product is formed and precipitated.

The reaction of 2-phenyl-4-(2-aminoethyl)thiazole (compound G) with different benzoxazinones resulted in the production of new 4(3H)quinazolinones (Fig. 1). The results of the in vitro experiments revealed that the compounds A3, A2, B4, and A1 showed the highest cytotoxic activities against PC3 cell line. Compounds A3, A5, and A2 potentially inhibited proliferation of MCF-7 cell line. Moreover, in the HT-29, A3, A5, and A6 are able to inhibit strongly growth of the cells. In the next study, more details and methods will be used to explore the molecular mechanisms that mediated the cytotoxic effects of the compounds.

CONCLUSION

According to the results, A3 efficiently inhibited the growth of all cell tested in a dose dependent manner. The IC50 of A3 was 10 M, 10 μM, and 12 μM in PC3, MCF-7, and HT-29 cells, respectively. The IC50 values for doxorubicin as positive control was 3.7, 7.2, and 5.6 in PC3, MCF-7, and HT-29 cell lines, respectively. None of the compounds were found as effective as doxorubicin. According to cytotoxic results, presence of Br in position 6 resulted in reduction of cytotoxicity as well as nitro group in another group of similar quinazolinones (1), so it can be concluded that substitution on position 6 is not beneficial for cytotoxicity in this group of quinazolinones.