Anticancer Activity and High Content Screening of New 6-Substituted-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline Derivatives

 

Hiba Ali Hasan1*, Afrah Salman2, Emilia Abdulmalek3,4

1Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, Mustansiriyah University, Baghdad, Iraq.

Department of Pharmacology and Toxicology, College of Pharmacy, Mustansiriyah University, Baghdad, Iraq

3Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,

Selangor, Malaysia.

4Integrated Chemical BioPhysics Research, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

*Corresponding Author E-mail: hibaalichemist@uomustansiriyah.edu.iq

 

ABSTRACT:

Pursuing our interest in bioactive heterocyclic compound, two benzoimidazoquinazoline derivatives were synthesised using both microwave-assisted and classical heating methods. Structures of the compounds were confirmed by standard spectroscopic methods and elemental analysis. The target scaffolds were incidentally found to emit blue light when exposed to ultraviolet light. Consequently, a photoluminescence characterization was carried out as a part of characterization protocol. The anticancer activities of the benzimidazoquinazoline compounds were investigated using both methylthiazol tetrazolium (MTT) and the high content screen (HCS) assays against liver hepatocellular cells. The results showed a significant reduction in the inhibitory concentration of the cancer cells by 1 and 2.6 fold when exposed to compounds (3a) and (3b), respectively. The high content screen (HCS) was conducted for compound (3b) and the results showed high toxicity towards the cancer cells.

 

KEYWORDS: Microwave, dihydrobenzo[4,5]imidazo[1,2-c]quinazoline, anticancer, MTT, HCS.

 

 


INTRODUCTION:

The heterocyclic compounds are important molecules in medicinal and organic chemistry and their syntheses have garnered a lot of attention1-11. The quinazoline derivatives are a type of the N-containing heterocyclic organic compounds, which have become very popular because of their distinct and numerous biopharmaceutical activities. Many studies have investigated the different therapeutic activities of the quinazoline derivatives. On the other hand, the benzimidazole derivatives were found to be important nitrogen-containing heterocyclic compounds, which could be used as intermediates in the organic chemical synthesis.

 

The quinazoline and the benzimidazole derivatives showed many therapeutic activities like anti-tumour, anti-viral, anti-tuberculosis, anti-inflammatory, anti-spasmodic, and anti-cytotoxic. These compounds have been used as PI3-kinase 6 inhibitors and potent anti-staphylococcal agents since they display a dual inhibitory mechanism against the DNA gyrase enzyme12-18. Several published studies described the pharmaceutical properties of the quinazoline-fused benzimidazole skeleton19-21. The benzimidazole fused quinazoline molecules results benzimidazoquinazoline nucleus, which is described in previous studies19-21.

 

Since quinazoline and benzimidazole have shown many interesting properties in separate occasions, together they are expected to exhibit superior activity. Therefore, it is the research's main interest in synthesising a series of benzimidazoquinazoline derivatives. In the past few years, there has been an increasing demand for designing and synthesising novel anticancer molecules for medicinal purposes. Reviewing previously published literatures regarding benzimidazoquinazolines had disclosed its anticancer activity22-25.

 

In this study, two new dihydrobenzimidazoquinazolines were synthesised and characterised using the microwave-assisted and the classical heating processes. It was predicted that these compounds would inhibit tumor activity accordingly the anticancer activity of those two compounds have to be evaluated using MTT assay, as well as investigation of the cell death pathway against Hep G2 cell line in comparison with the normal cell for the derivative with high toxicity against tumor cells.

 

MATERIAL AND METHODS:

Chemicals and reagents:

The chemicals used in the study were of an analytical grade and commercially available. Glacial acetic acid was obtained from J.T. Baker, USA, while silica gel aluminium 60 F254 plates, 1-butanol, methanol, were obtained from Merck, Germany. Furthermore, 2-(2-aminophenyl)-1H-benzimidazole, 3-ethoxy-4-hydroxybenzaldehyde, and 3-methoxy-4-hydroxybenzaldehyde were obtained from Sigma-Aldrich. Toluene, n-hexane, ethyl acetate, and DMSO were purchased from Fisher Scientific, UK. The solvents were dried using 3Ĺ molecular sieves obtained from Acros Organics, USA. The American Type Culture Collection provided Hep G2 cell line (ATCC, Manassas, VA, USA). Dulbeccos modified Eagle medium was purchased from Life Technologies, Inc, Rockville, MD, USA and used for growing the cell line with the adding of 10% heat inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). This media is also supplemented with 1% of penicillin, 2 mM of glutamine, and streptomycin. The high content screening (HCS) assay was performed using Thermo Scientific Cytotoxicity 3 Multiparameter Kit (Thermo Fisher Scientific).

 

Instrumentation:

Synthesis and characterisation processes were conducted using all of the following instruments: the microwave-enhanced synthesis process was conducted with the aid of a single mode Bench top CEM microwave reactor, while the complete reaction profile was monitored via the Synergy software. Moreover, the melting points for all the samples were measured using the Barnstead Electrothermal apparatus. Furthermore, the 1H NMR (500 MHz) and the 13C NMR (125 MHz) spectra for all compounds were analysed using the JEOL JNM ECA 500. On the otherhand, the CHN elemental analysis was carried out with the employment of LECO TruSpec Micro CHNS instrument.

 

Spectroscopical studies include the following instruments: GCMS QP5050A (Shimadzu, Japan) was used for determining the MS spectra of all the synthesised compounds, while the Fourier Transform Infrared (FTIR) analysis was done with the IR Tracer-100 (Shimadzu, Japan). UV-Vis absorbance of the compounds was measured using UV-1650 PC (UV-VISIBLE spectrophotometer, Shimadzu/Japan). 

 

Optical rotation was measured by Autopol VI, Automatic Polarimeter which manufactured by Rudolph Research Analytical/Hackettstown, NJ, USA, whereas the fluorescence study was measured at room temperature in 5 cm3 quartz cuvette by using Perkin Elmer LS 55 Fluorescence Spectrometer/UK.

 

The Anticancer activities were done using the following instruments. The ELISA reader (Bio-Rad, Germany) was used to measure the absorbance at 570 nm for the MTT assay. The morphological alterations of Hep G2 cell line nuclear of was determined using Zeiss Axio Observer microscope (Thermo Scientific) that attached to the screening system.

 

Synthesis and Characterization:

In this study, two dihydro-benzimidazoquinazoline derivatives were synthesised according to the following two protocols as shown in Fig. 1. The microwave-assisted synthesis was conducted according to Hasan et al., 201919 and the crystals were grown by slow evaporation of butanol or toluene to give crystalline solid with yield of 95-97%, whereas the conventional reflux method was performed according to Hasan et al., 201820; then, the target derivative obtained after vacuum drying and vigorously washing of the crude product with suitable solvents to produce the precipitate which was recrystallized from butanol or toluene to furnish shiny crystals of 92% yield. 

 

Fig. 1: General scheme for synthesis of dihydro-benzimidazoqunazolines (3a and 3b).

 

4-(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-2-ethoxyphenol (3a):

Following the microwave-assisted method (3.3.1) using 3-ethoxy-4-hydroxybenzaldehyde (0.2 g, 1.2 mmole), new compound (3a) was obtained and characterized directly without any further purification as a white solid (0.35 g, 97%), while following the conventional heating method (3.3.2) the same compound was obtained in 0.32 g, 92% ; m.p.: 219-221°C; Rf: 0.63 in hexane: ethyl acetate (1:1) solvent system.  -433.945 (c=0.01, DMSO). IR spectrum (UATR), ʋmax, cm-1: 3039 (N-H), 2971 (O-H, =C-H sp2, -C-H sp3), 1608 (C=N), 1504 (C=C and N-H), 1261 (C-O and C-N), 742 (C-H). UV-Vis spectrum (DMSO) λmax, nm (ɛ, L mole-1 cm-1): 360 (ɛ, 0.288 ×104), 305 (ɛ, 0.401 ×104). 1H NMR spectrum (500 MHz, DMSO-d6), δ, ppm (J, Hz):  1.25 (3H, t, J=6.87 Hz, CH3), 3.92 (2H, td, J=13.17, 6.88 Hz, CH2), 6.71 - 6.79 (2H, m, H-5",6"), 6.87 (1H, t, J=7.5 Hz, H-8), 6.92 (1H, d, J=8.2 Hz, H-10), 6.99 (1H, s, H-6), 7.02 (1H, d, J=8.0 Hz, H-5'), 7.13 (1H, d, J=1.1, H-2"), 7.19 (1H, t, J=7.3 Hz, H-4'), 7.29 (1H, t, J=7.3 Hz, H-3'), 7.34 (1H, ddd, J=1.3, 7.5, 8.2 Hz, H-9), 7.70 (1H, d, J=8.0 Hz, H-2'), 7.77 (1H, br. s., N-H), 8.03 (1H, dd, J=1.3, 7.5 Hz, H-7), 9.27 (1H, br. s., O-H). 13C NMR spectrum (125 MHz, DMSO), δ, ppm: 14.6 (CH3), 63.9 (CH2), 68.8 (C-6), 111.6 (C-5'), 112.4 (C-3, 8), 115.0 (C-4'), 115.6 (C-3'), 118.3 (C-2', 10), 119.4 (C-7), 123.3 (C-6"), 123.7 (C-5"), 125.2 (C-1"), 129.7 (C-9), 131.6 (C-6'), 133.3 (C-4"), 144.3 (C-7'), 146.2 (C-4), 146.8 (C-2, 3"), 148.1 (C-4"). Mass Spectrum, m/z (Irel, %): 357 [M]+ (20), 220 [C14H10N3]+ (20), 194 [C13H10N2]+ (100), 92 [C6H6N]+ (7). Found, %: C 73.87; H 5.44; N 11.70. C22H19N3O2 Calculated, %: C 73.93; H 5.36; N 11.7.

 

4-(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-2-methoxyphenol (3b):

Following the microwave-assisted method (3.3.1) using 4-hydroxy-3-methoxybenzaldehyde (0.18 g, 1.2 mmole), new compound (3b) was obtained and characterized directly without any further purification as a white solid (0.37 g, 95%), while following the conventional heating method (3.3.2) the same compound was obtained in 0.35 g, 91%; m.p.: 258-259°C. Rf: 0.25 in hexane: ethyl acetate (2:1) solvent system.  -335.4 (c=0.01, DMSO). IR spectrum (UATR), ʋmax, cm-1: 3046 (N-H), 2917 (O-H and -C=H Sp2), 2831 (-C-H Sp3), 1600 (C=N), 1489 (C=C), 1450 (N-H), 1249 (C-O and C-N), 733 (C-H). UV-Vis spectrum (DMSO) λmax, nm (ɛ, L mole-1 cm-1): 361 (ɛ, 0.210 ×104), 305 (ɛ, 0.312 ×104), 292 (ɛ, 0.274 ×104). 1H NMR spectrum (500 MHz, DMSO-d6), δ, ppm (J, Hz): 3.68 (3H, s, OCH3), 6.66 (1H, dd, J=1.43, 7.65 Hz, H-10), 6.71 - 6.74 (1H, m, H-5'), 6.84 (1H, ddd, J=1.43, 7.65, 8.49 Hz, H-8), 6.87 (1H, d, J=1.44 Hz, H-6), 6.89 (1H, d, J=8.13 Hz, H-6"), 6.96 (1H, d, J=8.13 Hz, H-5"), 7.07 (1H,ddd, J=0.96, 7.65, 8.60 Hz, H-3'), 7.11 (1H, d, J=1.91 Hz, H-2"), 7.16 (1H, ddd, J=0.96, 7.64, 8.60 Hz, H-4'), 7.26 (1H, ddd, J=1.44, 7.65, 8.49 Hz, H-9), 7.46 (1H, s, N-H), 7.64 (1H, d, J=7.65 Hz, H-2'), 7.96 (1H, dd, J=1.44, 7.65 Hz, H-7), 9.24 (1H, s, OH). 13C NMR spectrum (125 MHz, DMSO), δ, ppm: 55.6 (OCH3); 68.4 (C-6); 110.7 (C-5'); 110.9 (C-3); 112.0 (C-8); 114.7 (C-2'); 115.3 (C-10); 118.1 (C-6"); 118.5 (C-5"); 119.0 (C-2"); 121.8 (C-4'); 122.0 (C-3'); 124.6 (C-7); 130.8 (C-9); 131.5 (C-6'); 133.0 (C-1"); 143.6 (C-7'); 143.8 (C-4); 147.2 (C-2); 147.3 (C-3"); 147.7 (C-4"). Mass Spectrum, m/z (Irel, %): 343 [M]+ (23), 220 [C14H10N3]+ (17), 194 [C13H10N2]+ (100), 92 [C6H6N]+ (5). Found, %: C 73.69; H 5.03; N 12.51. C21H17N3O2 Calculated, %: C 73.45; H 4.99; N 12.24. Supplementary materials 2a-2f.

 

Photoluminescence Study:

The UV-Vis absorbance for the target derivatives was measured according to Hasan et al., 201919, while the fluorescence study was measured following the method of Hasan et al., 201820.

 

Optical Activity:

The optical rotations of the target derivatives were measured following the method of Hasan et al., 201820.

 

Anticancer Activity Assay:

Cell Culture and MTT assay:

MTT assay was employed to measure the cytotoxicity of the potential anticancer chemical compounds according to both of manufacturer’s instructions and Duellman et. al., 201526. 200mL per well of cells with concentration (1×104 - 1×106 cell per mL) were added in to each well using 96-well plates. These plates were covered and gently stirred then incubated in the presence of 5% CO2 at 37˚Ϲ overnight, following the incubation the media were replaced by the same volume of two fold dilution of the synthesised chemical compounds from 12.5 µg/mL- 200µg/mL in triplicate, plates were incubated for 48 hr in the same previous condition. After the incubation time 10µL of MTT solution was added to each well and incubated at 37˚Ϲ under 5% CO2 for 4 hr. Then, the solution of MTT was replaced by 100µL of dissolution solution and the plates were incubated for 5 minutes. Finally, the absorbance at a wavelength of 570 nm was determined by an ELISA reader, eventually the optical density data collected from the ELISA reader and used to calculate the viability percentage using equation (1). The results were statistically analyzed to estimate the effectiveness of the synthesised compounds to inhibit 50% of the target cancer cells in term of measuring the IC50.

 

                     Optical density of sample

Viability % = ------------------------------------ × 100      (1)

                         Optical density of control

 

High-Content Screening:

The assay of a high content screening (HCS) was conducted at Department of Pharmacology, The Centre for Natural Product Research and Drug Discovery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. A computerized imaging microscope was connected to the HCS system (using Zeiss 40×, 0.75 NA, Plan-Neofluar objective lens) to measure different parameters by imaging the intensity and the distribution of fluorescence within cell line HCS system. These parameters which included cell count and permeability, nuclear intensity, mitochondrial membrane potential, and cytochrome C were measured using Cellomics Multi parameter cytotoxicity 3 Kit. This kit contained primary antibody DyLight 649 conjugated goat anti-mouse IgG. The dyes used within this kit included three types of dyes Hoechst, MMP (mitochondrial membrane potential) and permeability. It was also provided with thin plate seal assembly and contained three different buffers permeabilization buffer (10× Dulbecco’s PBS with 1% Triton® X-100), blocking buffer (10X), and wash buffer (10×Dulbecco’s PBS). The first step before screening was treating the cells with the synthesised compounds for 24 hours. Then, added both of cell permeability and MMP dyes, and incubated for half an hour at 37°C. After the incubation time, the fixation and permeabilization process of the cells were performed according to Duellman et. al., 201526. Finally, the alterations of the nuclear morphology of the cancer cell line Hep G2 that grown overnight in ninety six-well plates was investigated via Zeiss Axio Observer microscope using the Hoechst 33342 staining assay.

 

RESULTS AND DISCUSSION:

Synthesis and Characterisation:

Microwave-enhanced and traditional reflux processes were used for synthesising the target compounds in which 2-(2-Aminophenyl)-1H-benzimidazole was condensed with two aldehydes for generating dihydro-benzimidazoquinazoline derivatives, thereafter the microwave-assisted technique was used. The derivative compounds could be synthesised within a short time period (15-20 min) with an excellent yield (95-97%). Therefore, this technique enabled the reduction in the reaction time by 75-89% with 3-5% increase in the product percent yield, as shown in Table 1.


 

Compounds

Classical reflux

Microwave assisted

Increase in Yield (%)

MW/CR*

Decrease in Reaction time (%) MW/CR**

Yield (%)

Time (min.)

Yield (%)

Time (min.)

3a

92

180

97

20

5

89

3b

92

60

95

15

3

75

Table 1: Reaction time and yield, % of dihydro-benzimidazoquinazoline (3a and 3b) under conventional reflux and microwave irradiation.

*: Microwave over Classical Reflux increase in yield (%)=;

**: Microwave over Classical Reflux decrease in reaction time=


After synthesis, the compounds were filtered and rinsed using an appropriate solvent. However, the use of the microwave condensation process yielded extremely pure compounds which could be directly used for characterisation, after filtration and washing, without any additional purification steps. The mechanism of the reaction followed standard addition reaction of amines to aldehydes, yet the expected Schiff bases were not recovered, instead the reaction proceed further to give the benzoimidazolequinazoline derivatives. Detailed mechanistic scheme was published in the earlier report of similar compounds19-21.

The two dihydro-benzimidazoquinazolines were characterised using different techniques such as the 13C NMR, 1H NMR, GC-MS, FTIR, and elemental analysis. Table 2 summarises all the physical and elemental analytical results for the synthesised compounds. As shown in the table, the melting point values indicate the purity of all the compounds as they could melt within the range of 1-2 degree Celsius, indicating that the compounds were impurity-free. Furthermore, the estimated CHN values were also comply with all experimental results.


 

Table 2: Physical and elemental analysis of compounds (3a and 3b).

Compounds

Molecular

Formula

Molecular weight

Melting point °C

Elemental analysis % found (calculated)

%C

%H

%N

3a

C22H19N3O2

357.15

219-221

73.87 (73.93)

5.44 (5.36)

11.70 (11.76)

3b

C21H17N3O2

343

258-259

73.69 (73.45)

5.03 (4.99)

12.51 (12.24)

 


Benzimidazoquinazoline derivatives were also characterised using the 1H NMR analysis. The spectra showed significant resonances within the range of 7.46 to 7.77 ppm for N-H protons and from 6.87 to 6.99 ppm for the H-6 proton (Table 3). From these 1H NMR data, cyclisation was confirmed and novel diazine ring was formed rather than the expected imine in the same reaction conditions. After comparison, it was observed that the synthesised dihydrobenzimidazoquinazoline derivatives did not show any singlet peak for the N=C-H azomethine distinctive functional groups in the 8.5-9 ppm range. These results confirmed that no Schiff’s base was formed. The N-H and H-6 chemical shifts for all the derivative compounds in this family are summarised in Table 3. The 1H NMR spectrum for compound (3a) is demonstrated in Fig. 2, while the spectrum for compound (3b) is intensively described in the Supplementary material file 2a.

 

Fig. 2: 1H NMR of 4-(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-2-ethoxyphenol (3a).

 

Table 3: Important protons and carbons in 1H NMR and 13 C NMR spectra of dihydro-benzimidazoquinazolines (3a and 3b).

Compound

 

R=

 

 

N-H

 

 

H-6

 

 

C6

3a

 

7.77 (br. s)

 

6.99 (s)

 

68.8

3b

 

 

7.46 (s)

 

6.87 (d)

 

68.4

The 13C NMR spectral analysis for the benzimidazoquinazoline derivatives was carried out as well. The aliphatic C-6 peaks that appeared between 68.4 and 68.8 ppm (as mentioned in Table 3) were the significant peaks that confirmed the cyclisation process for all derivatives. Fig. 3 displays the 13C NMR spectrum for compound (3a), while the 13C NMR spectrum for compound (3b) is available in Supplementary materials file 2b.

 

Fig. 3: 13C NMR of of 4-(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-2-ethoxyphenol (3a).

 

The synthesised dihydro-benzimidazoquinazoline derivatives were then analysed with the aid of the FTIR technique for identifying functional groups presented in the studied derivatives. The results are summarised in Table 4, whereas The FTIR spectrum of the two compounds are available in Supplementary materials files 1a and 2c.


 

Table 4: Infrared frequencies of the major functional groups.

Compounds

N-H stretching

O-H and =C-H sp2 stretching

-C-H sp3 stretching

C=N stretching

C=C aromatic stretching

N-H bending

C-O and C-N stretching

C-H aromatic

3a

3039

2971

1608

1504

1261

742

3b

3046

2917

2831

1600

1489

1450

1249

733

 


The found molecular weights of the studied derivatives were the same as the calculated molecular weights as proved by mass spectroscopy (GC-MS). The GC-MS for dihydrobenzimidazoquinazoline 3a and 3b are available in Supplementary materials files 1b and 2d.

 

Fluorescence Study:

Handling these derivatives during conducting the experimental work, unexpectedly disclose that they fluoresce and emit bright blue light under ultraviolet either from the sun (as a natural source) or from UV-lamp (as an artificial source). Therefore, the study of photoluminescence nature of these compounds is significant.

 

Electronic Spectral Analysis:

UV-Vis absorption spectra for the two compounds were measured at RT for 1×10-4 M concentration in DMSO. The spectra displayed one - two bands ranging between 292-305 nm that recognized for π→π* transitions from HOMO to LUMO. Moreover, broad band at 360 nm was rose and attributed to n→π* transitions of the non-bonding electrons of the chromophores. The UV-Vis spectrum of the two compounds are available in Supplementary materials files 1c and 2e.

 

Emission Spectral Data:

Recently, chemists’ attention have attracted by the fluorescent compounds for their wide applications27. When visible, or ultraviolet light, was absorbed by molecule’s electrons it causes transition of these electrons from ground to excited state, eventually when they returns back to the lower state they produce light. This process called photoluminescence, which has two types: fluorescence and phosphorescence in which the difference between them is the time interval28.

 

Fluorescence study was performed for very diluted prepared solutions which were colourless under ambient light and bright blue under UV-light and the resulted emission spectra had broad band within range of 423-424nm as cleared in Table 5. Furthermore, the emission spectrum for the two compounds are available in Supplementary materials files 1d and 2f.

 

Quantum yield for the two derivatives were calculated by equation (2), which reviled that compound (3b) emit more light (29%) than compound (3a) (26%) because it has higher quantum yield.

 

 

Where, subscripts indices “sam” and “ref” refer to sample and reference, respectively;  when excited at 350 nm; I= integrated area of emission peak at the excitation wavelength; A=UV-vis abortion correction factor which is = ; n= refractive index for both water and DMSO.

 

Table 5: Absorption and emission maxima and quantum yields (Φ) for compounds (3a and 3b).

Compound

λex (nm)

λem (nm)

Stock Shifts (nm)

Quantum Yield (Φ)

Quantum Yield (%)

3a

360

424

64

0.256

26

3b

360

423

63

0.289

29

 

Anticancer Study:

Viability Assay:

The cytotoxic effect of the synthesised compounds (3a) and (3b), as potential drugs, was determined by measuring the viability of normal and cancer cell lines (WRL 68, Hep G2, respectively). The results showed that half maximal inhibitory concentration (IC50) of the synthesised compound (3b) against WRL 68 was 196.40 µg/mL, while the IC50 against the Hep G2 was 75.32 μg/mL. The IC50 of compound (3b) against cancer cells was significantly decreased by 2.6fold (P value < 0.0001) in comparison to its effect of against normal cell line (as shown in Fig. 4).

 

Fig. 4: The inhibitory logarithmic concentration of compound (3b) against Hep G2 cells; * Hep G2: Hepatic cancer cells; *WRL68: Normal cell line as a control.

On the other hand, compound (3a) which has the same backbone of compound (3b), yet consists of ethoxy instead of methoxy functional group, showed only one fold reduction of IC50 towards Hep G2 (96.98μg/mL) in comparison to the normal cell line (141.70μg/mL) as depicted in Fig. 5.

 

Fig. 5: The inhibitory logarithmic concentration of compound (3a) against Hep G2 cells; *Hep G2: Hepatic cancer cells; *WRL68: Normal cell line as a control.

 

According to the results of MTT assay demonstrated in Figs. 4 and 5, compound (3b) showed a higher cytotoxic activity against Hep G2 cell line than that of compound (3a). This might be attributed to different functional groups substitution on the meta-position of the benzene ring accordingly the replacement of methoxy by ethoxy group significantly enhance the selective cytotoxicity towards cancer cell line rather than the normal cell line. Further subsequent tests were carried out in order to understand these results including the high content screening parameters. These assays were performed in the presence and absence of the potential anticancer compound (3b) in comparison with a positive control doxorubicin (11.6µg/mL).

 

The Analysis of High Content Screening:

The high content screening parameters were performed to investigate whether the synthesised compound with a high cytotoxic effect can induce apoptosis or necrosis activity towards the Hep G2 cell line. Those parameters include: cancer viable cell count, measurement of the cell nuclear intensity, permeability of the cancer cell membrane, variations in mitochondrial membrane potential (MMP), and release of cytochrome C.

 

The cancer viable cell count was performed to observe the inhibition rate of the cancer cells that being treated with compound (3b) with different concentrations ranging from 12.5µg/mL to 200µg/mL. The results showed that the viable count of Hep G2 cell line was inversely proportional to the concentration of compound (3b) and it decreased slightly starting from concentration of 25µg/mL to reach its significant reduction at 200 µg/mL (P value < 0.01). This was approximately resembling the effect of positive control doxorubicin. The effect of compound (3b) on the hepatic cancer viable cell count is available in Supplementary materials file 2g.

.

The viable cell count reflects the toxic effect of the potential anticancer drug and this result is highly compatible with the result of the MTT assay, that this effect of the potential compound used in this project against cancer cells could be attributed to the presence of benzoimidazoquinazoline nucleus, which complies with a previous study which demonstrated that the benzoimidazoquinazoline derivatives showed anticancer effects29.

 

The second parameter involved the measurement of cell nuclear intensity exhibited significant increase (P value < 0.02) of nuclear intensity by 1.3 fold in treated cells (with 200 µg/mL of potential anticancer compound (3b)) comparing to the untreated cells. Furthermore, it composed 68% of the Hep G2 nuclear intensity that been treated with 11.6µg/mL doxorubicin (positive control) as illustrated in Supplementary materials file 2h. The elevation of the nuclear condensation is considered as a feature for the apoptosis pathway according to previous studies30,31.

 

The permeability of the Hep G2 cell membrane showed no effect when treated with compound (3b) comparing to the cells that been treated with doxorubicin. This is an evidence that this compound showed no necrosis activity as illustrated in Supplementary materials file 2i.

 

Another parameter of high content is monitoring of the variation occurs in mitochondrial membrane potential (MMP), which reflects the mitochondrial function. This assay gives information about cells’ health condition as the mitochondria are considered as the major organelles that provide cells with ATP for metabolism. The penetration of the dye into the mitochondria was measured by florescence intensity, which is inversely proportional to the mitochondrial membrane potential which means the low florescence intensity reflects that the mitochondria were highly affected. The results (as shown in Supplementary materials file 2j) display that the treatment of cancer cells with 100 and 200µg/mL of compound (3b) significantly reduced the potential of the mitochondrial membrane of the hepatic cancer cells with the P value < 0.0026 and 0.0002, respectively.

 

The last parameter that used as part of multiparametric cytotoxic activity and related to the potential drug treatment of the Hep G2 cells is the release of cytochrome C, that Cytochrome C is one of a group of cytokines that located in the space between the inner and outer mitochondrial membrain. The results of the study revealed that the high concentration of compound (3b) is significantly inducing the release of cytochrome C from Hep G2 by 1.4 fold compared to untreated group. It was approximately in the same trend effect of doxorubicin which stimulates the release by 1.5 fold compared to the negative control as shown in Supplementary materials file 2k. This result also can be seen in Fig. 6 which cleared the highly staining cytochrome C around the nucleus of the treated cells with high concentration of compound (3b). This remarkably proved the release of the cytochrome C into the cytosol. On the other hand, cytochrome C of the untreated cells was weakly stained and this emphasis it remained in its normal place between the inner and outer mitochondrial membrane.

 

Fig. 6: High content screening images of human Hep G2 cells treated with the potential anticancer compound (3b) and doxorubicin in comparison to untreated cell culture using multiparameter which included: nuclear intensity, cell membrane permeability, mitochondrial membrane permeability (MMP), and   cytochrome C.

 

The above two results of the MMP and cytochrome C have the main role for the induction of apoptosis pathway due to reduction in the MMP which causes high release of cytochrome C out of the mitochondria. This cytochrome is known as a caspase activator that activate a series of biochemical reactions that induce caspase cascade and leading to activate the apoptosome32.

These results might be due to the presence of benzoimidazoquinazoline backbone as previous literatures demonstrated, that these derivatives had anticancer properties against MCF-7 cell line33.

 

CONCLUSIONS:

In conclusion, a microwave-assisted process was used for successfully synthesising two 6-substituted-5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazoline compounds, with an excellent yield and high purity. All the synthesised benzimidazoquinazolines could be further characterised by using many elemental analytical and spectroscopic methods, without requiring any additional purification. The anticancer activities of these two compounds were investigated against Hep G2 cell line. Compound (3b) showed cytotoxic effect against the hepatocellular cancer cells higher than that of compound (3a) as determined by MTT assay. Interestingly, the treatment of the Hep G2 cells with compound (3b) induces the apoptosis pathway by its ability to reduce the MMP of the Hep G2 cells. These results are highly promising and encouraging to test its effect in-vivo using laboratory animals to evaluate its effect accurately.

 

ACKNOWLEDGMENT:

First two authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad-Iraq for its support in the present work. All authors thank the Malaysia government and Universiti Putra Malaysia for research grant Initiative Putra Siswazah (9474300).

 

SUPPLEMENTARY MATERIALS:

Full analysis data of the synthesised compounds can be found in supplementary files.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 29.05.2020           Modified on 01.07.2020

Accepted on 30.07.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(5):2397-2405.

DOI: 10.52711/0974-360X.2021.00423