INTRODUCTION:

The heterocyclic compounds are important molecules in medicinal and organic chemistry and their syntheses have garnered a lot of attention^1-11. 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 enzyme^12-18. Several published studies described the pharmaceutical properties of the quinazoline-fused benzimidazole skeleton^19-21. The benzimidazole fused quinazoline molecules results benzimidazoquinazoline nucleus, which is described in previous studies^19-21.

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 activity^22-25.

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 3Å 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; R_f: 0.63 in hexane: ethyl acetate (1:1) solvent system. -433.945 (c=0.01, DMSO). IR spectrum (UATR), ʋ_max, 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) λ_max, 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. R_f: 0.25 in hexane: ethyl acetate (2:1) solvent system. -335.4 (c=0.01, DMSO). IR spectrum (UATR), ʋ_max, 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) λ_max, 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.

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	λ_ex (nm)	λ_em (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 studies^30,31.

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

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

DOI: 10.52711/0974-360X.2021.00423

Compounds	Molecular Formula	Molecular weight	Melting point °C	Elemental analysis % found (calculated)
Compounds	Molecular Formula	Molecular weight	Melting point °C	%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)

Compounds	Classical reflux		Microwave assisted		Increase in Yield (%) MW/CR^*	Decrease in Reaction time (%) MW/CR^**
Compounds	Yield (%)	Time (min.)	Yield (%)	Time (min.)	Increase in Yield (%) MW/CR^*	Decrease in Reaction time (%) MW/CR^**
3a	92	180	97	20	5	89
3b	92	60	95	15	3	75

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