Introduction

Many mediators have interfered in the inflammatory process, such as cytokines, cell adhesion molecules, and autacoids, including prostaglandins (PGs). The pain and tissue blood flow principally increase in the presence of PGs, especially PGE2 [1]. PGs are biosynthesized by cyclooxygenase (COX) enzyme, so COX enzyme inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) decrease inflammation. The COX isoforms that have been discovered are COX-1, COX-2, and COX-3 [2]. COX-1 and COX-2 are more important than COX-3, and NSAIDs effects are attributed to the inhibition of these isoforms [3]. The COX-1 isoform has a constitutive role and expresses PGs responsible for maintaining physiological functions such as gastric mucosa protection and vascular homeostasis. In contrast, COX-2 is an inducible enzyme and over-expressed in response to the release of several pro-inflammatory mediators. The COX-2 isoform is over-expressing in the inflammatory sites and introduces the PGs produced in pathologic conditions [4]. However, NSAIDs as anti-inflammatory drugs not only inhibit COX-2 but also these drugs inhibit COX-1 isoform and cause many side effects, such as gastrointestinal problems associated with the inhibition of COX-1 as a protector of the gastric mucosa. To prevent the side effects of NSAIDs, the difference between COX-1 and COX-2 isoforms should be identified to design the compounds with high selectivity for inhibition COX-2 enzyme [5, 6]. In general, the active site of these isoforms are very similar, but the significant difference between these isoforms is a second pocket in the binding site of the COX-2 isoform due to the replacement of the bulk isoluesin523 amino acid in COX-1 by valine at the binding site of COX-2. So, the large compounds place properly into the COX-2 active site while they do not in the COX-1 active site [7]. Therefore, compounds can be designed that are more desirable to COX-2 than COX-1 and do not show the side effects of this isoform inhibition. The COX-2 role is investigated in diverse diseases, such as Alzheimer’s disease [8,9,10], several types of cancer [11,12,13,14,15], and Parkinson’s disease [16]. Also, the upregulation of COX-2 in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been studied [17]. Some well-known COX-2 inhibitors, like Rofecoxib, have been withdrawn from the market due to cardiovascular effects [18]. Therefore, the introduction of new COX-2 inhibitors with high potency and low side effects is desirable. Diarylheterocycles are abundantly present in the structure of selective COX-2 inhibitors. The hetero or carbocyclic ring systems or acylic structures constitute the central core of this group and consist of two aryl substitutions. One of the two phenyl rings carries a pharmacophore group that replaces the secondary hydrophobic pocket at the COX-2 active site and is responsible for selectivity. According to recent studies, the COX-2 inhibitors with tricyclic rings as central skeletons are introduced [19,20,21]. Benzimidazole rings have been appeared as the prominent scaffold in medicinal chemistry and also used as the central core in COX-2 inhibitors [22,23,24,25,26,27,28]. Our previous work introduced a new potent and selective COX-2 inhibitors class with tricyclic benzimidazole central core (A, Fig. 1) [21]. In this regard, we designed a new series of imidazo[1,2-a]pyrimidine compounds with a tricyclic central core. To design these compounds, we hybrid molecules comprising benzimidazole and pyrimidine ring as new COX-2 inhibitors. Pyrimidine rings are interesting structures widely used in the design of COX-2 inhibitors and have been shown anti-cancer effects (B, Fig. 1) [29,30,31,32,33,34]. Based upon this, we designed, synthesized, and evaluated a series of novel 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine derivatives with high potency and selectivity. The newly synthesized compounds were employed to assess their ability to inhibit COX-1 and COX-2 enzymes. Also, the anti-cancer effects of these series were assayed on the MCF-7 breast cancer cell line. In addition, an in vivo assay of these compounds has also been performed and showed promising results.

Fig. 1
figure 1

COX-2 inhibitors containing tricyclic benzimidazole central core (A), pyrimidine ring (B), and designed compounds

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Results and discussion

Chemistry

The synthetic procedure of the target molecules is described in Scheme 1. Accordingly, the synthesis of this series was carried out by reaction of two main precursors: substituted chalcones and 2-aminobenzimidazole. To synthesize related chalcones, at first, 4’-(methylsulfonyl) acetophenone (1) was synthesized from thioanisole as starting substance in two steps reaction as explained in our previous work [35]. Accordingly, first 4’-(methylthio)acetophenone was obtained from the reaction of thioanisole with acetyl chloride in the presence of AlCl3 and acetyl chloride in chloroform at room temperature. Then, oxidation step was performed in THF/water as the solvent, and 4’-(methylsulfonyl)acetophenone was produced using Oxone reagent. The 4’-(methylsulfonyl)acetophenone was treated with different aldehyde derivatives at room temperature in an alcoholic NaOH solution to give desired chalcones (3) [36]. Finally, 2-aminobenzimidazole (4) reacted with substituted chalcones (3) in DMF as solvent under microwave irradiation and the presence of a catalytic amount of piperidine to produce 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine derivatives [37] (5a-o).

Scheme 1
scheme 1

The synthesis procedure of target benzo[4,5]imidazo[1,2-a]pyrimidine derivatives

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Molecular docking study

A docking study was performed to investigate the probable interaction between 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine derivatives and the COX-2 active site. This study shows the interaction of compounds and active site, and partly explains the reasons for the existence of different derivatives in potency and selectivity. Generally, the SO2Me group of all derivatives was inserted in the secondary pocket of the COX-2 enzyme that is responsible for the selectivity. Also, the superimposition of compound 5a with celecoxib confirmed the correct placement of the pharmacophore group in this hydrophobic pocket. The oxygen atoms of the SO2Me group in compound 5a have formed hydrogen bonds with NH of His-90 (distance: 2.30 Å) and NH of Arg-513 (distance: 5.53 Å). Also, hydrogen bonds between N-atoms of 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine structure and Arg-120 (distances: 3.87, 2.25, and 2.14 Å) were observed Fig. 2.

Fig. 2
figure 2

A model of an efficient compound (5a) in the COX-2 isoform active site: A Celecoxib and 5a superimposition (B) placement of SO2Me as pharmacophore group in the secondary pocket, (C) Sc-558 and 5a superimposition, D The most important interactions distance. PyMol created pictures

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Biological evaluations

In vitro cyclooxygenase activity

A new series of 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine with SO2Me pharmacophore group at the para-position of the C-2 phenyl ring and C-4 phenyl ring having various substituents were synthesized, and the COX-2 inhibitory activity of these compounds was determined. COX-2 inhibition assay was performed accordingly to our previously reported method using a fluorescent enzyme kit (Table 1) [38]. The COX-2 inhibition results indicated that compound 5a had the highest potency, even more than celecoxib as a known drug, and also showed the highest selectivity among all compounds. These results demonstrated that the presence of any substitution, especially on the para- and meta- position of the C-1 phenyl ring, reduces the efficiency and selectivity of COX-2 inhibitory activity. This effect may be attributed to the presence of steric hindrance around these positions. According to these results, compounds with ortho- methoxy or fluoro substituents showed considerable in vitro activities, apparently due to the formation of hydrogen bonds with the COX-2 active site. The higher potency and selectivity of compound 5e compared with compound 5j may be explained by the presence of a hydrogen acceptor area at this position.

Table 1 The COX-1 and COX-2 enzyme inhibition assay
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In vivo anti-inflammatory assay

The anti-inflammatory of synthesized compounds possessing high potency and selectivity on the COX-2 enzyme was also screened in mice. The in vivo acetic acid-induced writhing test is widely used as a non-selective model for determining pain [39, 40]. The results of the acetic acid-induced writhing test for the investigation of the nociceptive responses are reported in Table 2. As shown in Table 2, all compounds had high efficiency in decreasing inflammation. Moreover, some compounds have been shown to have better effects compared to celecoxib as, a well-known anti-inflammatory drug. Compound 5a had a high potency and selectivity on COX-2 inhibition as well as an anti-inflammatory effect. However, compound 5d showed the highest efficacy in the in vivo test, which may be attributed to the pharmacokinetic properties. These results indicated that the compounds with hydrophilic substitution are more effective than compounds having hydrophobic substitution.

Table 2 The anti-inflammation activities of target compounds
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In vitro anti-cancer study

The synthesized compounds have been screened against the MCF-7 breast cancer cell line. As shown in Table 2, all compounds demonstrated considerable anti-proliferative activities against the MCF-7 cell line compared with celecoxib as a reference, probably due to their COX-2 and PGs inhibitory effects Table 3.

Table 3 The cytotoxic effects of synthesized compounds
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Conclusion

This study introduced a new series of benzo[4,5]imidazo[1,2-a]pyrimidine derivatives as selective COX-2 inhibitors. The biological evaluation results, including COX-2 inhibition activities, in vivo anti-inflammatory and anti-cancer effects on the MCF-7 breast cancer cell line, indicated that the majority of the synthesized compounds had selectivity for COX-2 inhibitory activity and demonstrated considerable anti-proliferative activities against the MCF-7 cell line. These results indicated that 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine possessing SO2Me pharmacophore group is a suitable structure having a tricyclic central core for designing new selective COX-2 inhibitors.

Materials and methods

Experimental

All chemicals and solvents were commercially available and prepared from Aldrich Chemical Co., Acros Co., and Merck AG Chemical Co., and any further purification was not used. The MicroSYNTH (Milestone company) was used for the synthesis of final derivatives. Through Thomas–Hoover capillary instrument, melting points were measured. Using a Perkin Elmer Model 1420 spectrometer on the ʋmax (cm−1) scale, all IR spectra were recorded in a KBr disk. All the 1H NMR and 13C NMR spectra were recorded in CDCl3 on a Bruker FT-500 MHz instrument (Bruker Biosciences, USA) with internal reference trimethylsilane (TMS). High-resolution mass spectra were recorded on a 6410 Agilent LC-MS triple quadrupole mass spectrometer (LC-MS) with an electrospray ionization (ESI) interface. The absorbance was determined using a spectrophotometer ELISA reader (Infinite® M200, TECAN Austria GmbH, Grodig, Austria).

General procedure for the synthesis of chalcones (3)

The synthesis of target compounds begins with the production of substituted chalcone. Synthesis of related chalcones was accomplished by the treatment of 4’-(methylsulfonyl)acetophenone 1 (1 mmol) with aryl aldehyde 2 (1 mmol) and 10 wt % sodium hydroxide solution (3 mL) in ethanol; and for 30 min, continue stirring at room temperature. With TLC, the completion progress of the reaction was monitored, and after finishing starting substances, the precipitates were filtered and rinsed with cold ethanol; finally, the pure products were obtained.

General synthesis procedure of 2,4-diphenylbenzo[4,5]imidazo[1,2-a]pyrimidine derivatives (5a-o)

2-Aminobenzimidazole (1.5 mmol) was added to DMF (3 ml) as a solvent, and then a catalytic amount of piperidine (10% mmol) was added to this mixture. Then, substituted chalcone was added (1 mmol), and the reaction was accomplished under microwave irradiation at power 450 W for 10 min. For the purity process, methanol (2 ml) was added to the mixture, and after 10 min, the precipitate was filtered and washed with cold methanol. The product was purified by column chromatography on silica gel with dichloromethane:methanol (respectively 8:1).

2-(4-(Methylsulfonyl)phenyl)-4-phenylbenzo[4,5]imidazo[1,2-a]pyrimidine (5a)

Yellow solid, 80%; M. P. 221 °C; IR (KBr) ʋmax1170, 1329 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.06 (s, 3H, SO2CH3), 6.66 (d, J = 7.6 Hz, 1H, benzimidazole H7), 7.01 (t, 1H, benzimidazole H6), 7.24 (s, 1H, pyrimidine ring), 7.43 (br, 1H, benzimidazole H5), 7.62–7.69 (m, 5H, phenyl), 7.93 (d, J = 7.6 Hz, 1H, benzimidazole H4), 8.03 (d, J = 7.6 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.42 (d, J = 7.6 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 43.47, 104.25, 113.73, 119.41, 120.83, 125.45, 126.25, 126.97, 127.27, 127.66, 128.54, 129.48, 130.37, 131.12, 140.59, 141.37, 149.14, 157.70. HRMS (ESI+) m/z calc for C23H17N3O2S 399.11, found 400 (M + H); Anal. Calcd. for C23H17N3O2S: C, 69.16; H, 4.29; N, 10.52. Found: C, 69.02; H, 4.50; N, 10.71.

2-(4-(Methylsulfonyl)phenyl)-4-(o-tolyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5b)

Yellow solid, 83%; M. P. 278 °C; IR (KBr) ʋmax 1170, 1328 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 2 2.44 (s, 3H, CH3), 3.04 (s, 3H, SO2CH3), 6.67 (d, 1H, J = 8.4 Hz, benzimidazole H7), 6.99 (t, 1H, benzimidazole H6), 7.21 (s, 1H, pyrimidine ring), 7.38–7.50 (m, 5H, 2-methylphenyl & benzimidazole H5), 7.89 (d, J = 8.4 Hz, 1H, benzimidazole H4), 8.00 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.40 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 20.52, 43.43, 104.15, 113.80, 119.26, 120.74, 124.24, 125.38, 126.22, 126.90, 127.61, 127.68, 128.36, 131.04, 138.59, 140.58, 141.30, 144.35, 149.37, 150.43, 157.63; HRMS (ESI+) m/z calc for C24H19N3O2S 414.12, found 414 (M + H); Anal. Calcd. for C24H19N3O2S: C, 69.71; H, 4.63; N, 10.16. Found: C, 69.95; H, 4.82; N, 10.29.

2-(4-(Methylsulfonyl)phenyl)-4-(m-tolyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5c)

Yellow solid, 79%; M. P. 270 °C; IR (KBr) ʋmax 1162, 1323 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 3H, CH3), 3.04 (s, 3H, SO2CH3), 6.67 (d, J = 8.4 Hz, 1H, benzimidazole H7), 6.99 (t, 1H, benzimidazole H6), 7.21 (s, 1H, pyrimidine ring), 7.38–7.52 (m, 5H, 3-methylphenyl & benzimidazole H5), 7.88 (d, J = 8 Hz, 1H, benzimidazole H4), 7.99 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.39 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) 20.52, 43.43, 104.16, 113.81, 119.25, 120.73, 124.24, 125.37, 126.22, 126.90, 127.60, 127.68, 128.35, 130.98, 138.59, 140.57, 141.30, 144.34, 149.37, 150.43, 157.63; HRMS (ESI+) m/z calc for C24H19N3O2S 413.12, found 414 (M + H); Anal. Calcd. for C24H19N3O2S: C, 69.71; H, 4.63; N, 10.16. Found: C, 69.97; H, 4.78; N, 10.11.

2-(4-(Methylsulfonyl)phenyl)-4-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5d)

Yellow solid, 79%, M. P. 265 °C; IR (KBr) ʋmax 1167, 1328 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 2.50 (s, 3H,CH3), 3.04 (s, 3H, SO2CH3), 6.73 (d, J = 8.4 Hz, 1H, benzimidazole H7), 6.99 (t, 1H, benzimidazole H6), 7.18 (s, 1H, pyrimidine ring), 7.37-7.42 (m, 3H, 4-methylphenyl H3 & H5 & benzimidazole H5), 7.49 (d, J = 8 Hz, 2H, 4-methylphenyl H2 & H6), 7.88 (d, J = 8 Hz, 1H, benzimidazole H4), 7.99 (d, J = 8 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.38 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 21.73, 44.48, 105.36, 114.92, 120.27, 121.71, 126.43, 127.29, 127.94, 128.19, 128.64, 129.22, 130.17, 141.63, 141.85, 142.33, 145.38, 150.47, 151.53, 158.67; HRMS (ESI+) m/z calc for C24H19N3O2S 413.12, found 414 (M + H); Anal. Calcd. for C24H19N3O2S: C, 69.71; H, 4.63; N, 10.16. Found: C, 69.55; H, 4.40; N, 10.31.

4-(2-Methoxyphenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5e)

Yellow solid, 80%; M. P. 273 °C; IR (KBr) ʋmax 1152, 1310 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.059 (s, 3H, SO2CH3), 3.58 (s, 3H, OCH3), 6.65 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.40 (t, 1H, benzimidazole H6), 7.10 (d, J = 8.4 Hz, 1H, 2-methoxyphenyl H3), 7.18–7.22 (m, 1H, 2-methoxyphenyl H5), 7.31 (s, 1H, pyrimidine ring), 7.42-7.46 (m, 2H, 2-methoxyphenyl H4 & H6), 7.66 (t, 1H, benzimidazole H6), 7.96 (d, J = 8.4 Hz, 1H, benzimidazole H4), 8.03 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.45 (d, 2H, 4-methylsulfonylphenyl H3 & H5 J = 8.4 Hz); 13C NMR (100 MHz, CDCl3) δ 44.50, 55.65, 96.12, 106.18, 111.33, 114.07, 119.83, 121.01, 121.54, 122.22, 126.63, 127.57, 127.99, 128.82, 130.27, 133.23, 141.60, 142.47, 148.23, 157.30; HRMS (ESI+) m/z calc for C24H19N3O3S 429.12, found 430 (M + H); Anal. Calcd. for C24H19N3O3S: C, 67.12; H, 4.46; N, 9.78. Found: C, 67.33; H, 4.22; N, 9.51.

4-(3-Methoxyphenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5f)

Yellow solid, 83%; M. P. 251 °C; IR (KBr) ʋmax 1157, 1324 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.05 (s, 3H, SO2CH3), 3.83 (s, 3H, O CH3), 6.72 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.02 (t, 1H, benzimidazole H6), 7.10 (s, 1H, 3-methoxyphenyl H2), 7.20 (t, 2H, 3-methoxyphenyl H4 & H6), 7.24 (s, 1H, pyrimidine ring), 7.41 (t, 1H, benzimidazole H5), 7.53 (t, 1H, 3-methoxyphenyl H5), 7.90 (d, J = 8 Hz, 1H, benzimidazole H4), 8.00 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.40 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3): 43.43, 54.63, 104.17, 112.58, 113.85, 116.00, 119.24, 119.30, 120.90, 125.49, 126.11, 126.93, 127.64, 129.79, 132.14, 140.52, 141.38, 144.19, 148.94, 150.30, 157.70, 159.26. HRMS (ESI+) m/z calc for C24H19N3O3S 429.12, found 430 (M + H); Anal. Calcd. for C24H19N3O3S: C, 67.12; H, 4.46; N, 9.78. Found: C, 67.01; H, 4.29; N, 9.59.

4-(4-Methoxyphenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5g)

Yellow solid, 84%; M. P. 275 °C; IR (KBr) ʋmax 1152, 1310 (SO2) cm−1; 1H NMR NMR (400 MHz, CDCl3) δ 3.05 (s, 3H, SO2CH3), 3.92 (s, 3H, OCH3), 6.82 (d, J = 8.8 Hz, 1H, benzimidazole H7), 7.03 (t, 1H, benzimidazole H6), 7.12 (d, J = 8.4 Hz, 2H, 4-methoxyphenyl H3 & H5), 7.22 (s, 1H, pyrimidine ring), 7.42 (t, 1H, benzimidazole H5), 7.55 (d, J = 8.4 Hz, 2H, 4-methoxyphenyl H2 & H6), 7.92 (d, J = 8 Hz, 1H, benzimidazole H4), 8.01 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.41 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 44.49, 55.66, 105.69, 114.89, 114.94, 120.15, 121.84, 124.13, 126.60, 127.23, 127.98, 128.71, 129.89, 141.56, 142.43, 150.40, 151.40, 158.91, 181.96; HRMS (ESI+) m/z calc for C24H19N3O3S 429.12, found 430 (M + H); Anal. Calcd. for C24H19N3O3S: C, 67.12; H, 4.46; N, 9.78. Found: C, 66.99; H, 4.60; N, 9.89.

4-(3,4-Dimethoxyphenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5h)

Yellow solid, 87%; M. P. 249 °C; IR (KBr) ʋmax 1157, 1314 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.06 (s, 3H, SO2CH3), 3.84 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.83 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.04–7.10 (m, 3H, benzimidazole H6, 3,4-dimethoxyphenyl H2 & H5), 7.20 (d, J = 7.6 Hz, 1H, 4-methoxyphenyl H6), 7.26 (s, 1H, pyrimidine ring), 7.44 (t, 1H, benzimidazole H5), 7.94 (d, J = 8 Hz, 1H, benzimidazole H4), 8.02 (d, J = 8 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.42 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 43.44, 55.18, 55.26, 104.63, 109.86. 110.58. 113.96, 119.10, 120.27, 120.89, 123.18, 125.64, 126.12, 126.96, 127.70, 140.50, 141.45, 148.62, 149.23, 150.29, 150.42, 157.95; HRMS (ESI+) m/z calc for C25H21N3O4S 459.12, found 460 (M + H); Anal. Calcd. for C25H21N3O4S: C, 65.35; H, 4.61; N, 9.14. Found: C,65.77; H, 4.81; N, 9.01.

4-(3,4,5-Trimethoxyphenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a] pyrimidine (5i)

Yellow solid, 85%; M. P. 225 °C; IR (KBr) ʋmax 1157, 1322 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.04 (s, 3H, SO2CH3), 3.82 (s, 6H, OCH3), 3.96 (s, 3H, OCH3), 6.73 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.08 (t, 1H, benzimidazole H6), 7.28 (s, 1H, pyrimidine ring), 7.34 (s, 1H, 3,4,5-trimethoxyphenyl H2), 7.36 (s, 1H, 3,4,5-trimethoxyphenyl H6), 7.44 (t, 1H, benzimidazole H5), 7.88 (d, J = 7.6 Hz, 1H, benzimidazole H4), 7.97 (d, J = 7.68 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.34 (d, J = 7.88 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 44.40, 56.52, 61.43, 65.69, 105.35, 114.92, 115.82, 120.09, 121.79, 126.70, 127.08, 127.96, 128.67, 132.36, 140.23, 141.50, 142.41, 150.09, 151.47, 154.16, 158.87, 169.07; HRMS (ESI+) m/z calc for C26H23N3O5S 489.12, found 490 (M + H); Anal. Calcd. for C26H23N3O5S: C, 63.79; H, 4.74; N, 8.58. Found: C, 63.52; H, 4.49; N, 8.42.

4-(2-Flourophenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5j)

Yellow solid, 81%; M. P. 254 °C; IR (KBr) ʋmax 1154, 1317 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.04 (s, 3H, SO2CH3), 6.66 (d, 1H, J = 8.4 Hz, benzimidazole H7), 7.05 (t, 1H, benzimidazole H6), 7.30 (s, 1H, pyrimidine ring), 7.33 (t, 1H, 2-flourophenyl H5), 7.43 (m, 2H, 2-flourophenyl H3 & H4), 7.60 (t, 1H, benzimidazole H5), 7.69 (m, 1H, 2-flourophenyl H6), 7.91 (d, J = 8 Hz, 1H, benzimidazole H4), 8.00 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.40 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 44.48, 106.24, 113.71, 116.73, 120.43, 122.43, 125.53, 126.64, 127.36, 128.00, 128.71, 130.60, 133.76, 141.40, 142.48, 144.40, 145.20, 151.10, 158.53, 161.03; HRMS (ESI+) m/z calc for C23H16FN3O2S 417.12, found 418 (M + H); Anal. Calcd. for C23H16FN3O2S: C, 66.17; H, 3.86; N, 10.07. Found: C, 66.29; H, 4.01; N, 10.02.

4-(3-Flourophenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5k)

Yellow solid, 81%; M. P. 290 °C; IR (KBr) ʋmax 1157, 1316 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.13 (s, 3H, SO2CH3), 6.76 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.11 (t, 1H, benzimidazole H6), 7.27 (s, 1H, pyrimidine ring), 7.32 (s, 1H, 3-flourophenyl H2), 7.41–7.74 (m, 4H, 3-flourophenyl H4 & H5 & H6, benzimidazole H5), 8.01 (d, J = 8 Hz, 1H, benzimidazole H4), 8.11 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.49 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 44.48, 105.40, 114.50, 115.71, 115.94, 118.51, 118.72, 120.53, 122.23, 124.21, 124.25, 126.74, 126.99, 128.06, 128.71, 131.56, 131.64, 141.37, 142.59, 148.51, 151.58, 158.80, 161.73; HRMS (ESI+) m/z calc for C23H16FN3O2S 417.12, found 418 (M + H); Anal. Calcd. for C23H16FN3O2S: C, 66.17; H, 3.86; N, 10.07. Found: C, 66.01; H, 3.68; N, 10.20.

4-(4-Flourophenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5l)

Yellow solid, 81%; M. P. 290 °C; IR (KBr) ʋmax 1165, 1325 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.06 (s, 3H, SO2CH3), 6.69 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.06 (t, 1H, benzimidazole H6), 7.23 (s, 1H, pyrimidine ring), 7.34 (t, 2H, 4-flourophenyl H3 & H5), 7.45 (t, 1H, benzimidazole H5), 7.61–7.64 (m, 2H, 4-flourophenyl H2 & H6), 7.95 (d, J = 8.4 Hz, 1H, benzimidazole H4), 8.41 (d, J = 8 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.43 (d, J = 8 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 43.44, 104.51, 113.47, 115.84, 116.05, 119.51, 121.03, 125.63, 127.00, 127.66, 129.51, 129.60, 140.62, 141.72, 148.29, 151.40, 159.25, 161.99; HRMS (ESI+) m/z calc for C23H16FN3O2S 417.12, found 418 (M + H); Anal. Calcd. for C23H16FN3O2S: C, 66.17; H, 3.86; N, 10.07. Found: C, 66.40; H, 3.99; N, 10.21.

4-(2-Chlorophenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5m)

Yellow solid, 81%; M. P. 290 °C; IR (KBr) ʋmax 1165, 1326 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.04 (s, 3H, SO2CH3), 6.42 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.02 (t, 1H, benzimidazole H6), 7.28 (s, 1H, pyrimidine ring), 7.42 (t, 1H, benzimidazole H5), 7.56–7.65 (m, 4H, 2-chlorophenyl), 7.90 (d, J = 8 Hz, 1H, benzimidazole H4), 8.00 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.41 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 44.48, 105.45, 113.65, 120.37, 122.53, 126.62, 127.19, 127.98, 128.09, 128.72, 130.48, 131.24, 132.75, 133.61, 141.46, 142.46, 145.27, 147.05, 150.97, 154.97, 154.16, 158.62; HRMS (ESI+) m/z calc for C23H16ClN3O2S 432.12, found 433 (M + H); Anal. Calcd. for C23H16ClN3O2S: C, 63.67; H, 3.72; N, 9.68. Found: C, 63.76; H, 3.99; N, 9.83.

4-(3-Chlorophenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5n)

Yellow solid, 81%; M. P. 290 °C; IR (KBr) ʋmax 1164, 1323 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.14 (s, 3H, SO2CH3), 6.78 (d, J = 8.8 Hz, 1H, benzimidazole H7), 7.14 (t, 1H, benzimidazole H6), 7.26 (s, 1H, pyrimidine ring), 7.33 (s, 1H, 3-chlorophenyl H2), 7.53–7.38 (m, 4H, 2-chlorophenyl H4 & H5 & H6 & benzimidazole H5), 8.03 (d, J = 8.4 Hz, 1H, benzimidazole H4), 8.12 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.50 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 44.48, 105.74, 114.52, 119.62, 120.54, 122.54, 126.52, 128.09, 128.51, 128.75, 130.96, 131.66, 133.68, 136.06, 139.89, 142.66, 148.66, 156.88, 162.72; HRMS (ESI+) m/z calc for C23H16ClN3O2S 432.12, found 433 (M + H); Anal. Calcd. for C23H1Cl6N3O2S: C, 63.67; H, 3.72; N, 9.68. Found: C, 63.80; H, 3.99; N, 9.84.

4-(4-Chlorophenyl)-2-(4-(methylsulfonyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (5p)

Yellow solid, 81%; M. P. 290 °C; IR (KBr) ʋmax 1152, 1312 (SO2) cm−1; 1H NMR (400 MHz, CDCl3) δ 3.13 (s, 3H, SO2CH3), 6.80 (d, J = 8.4 Hz, 1H, benzimidazole H7), 7.13 (t, 1H, benzimidazole H6), 7.28 (s, 1H, pyrimidine ring), 7.51 (t, 1H, benzimidazole H5), 7.65 (d, J = 8.8 Hz, 2H, 4-chlorophenyl H2 & H6), 7.70 (d, J = 8.8 Hz, 2H, 4-chlorophenyl H3 & H5), 7.99 (d, J = 8.4 Hz, 1H, benzimidazole H4), 8.10 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H2 & H6), 8.48 (d, J = 8.4 Hz, 2H, 4-methylsulfonylphenyl H3 & H5); 13C NMR (100 MHz, CDCl3) δ 44.48, 105.42, 114.55, 120.59, 122.10, 126.66, 127.09, 128.03, 128.67, 129.80, 129.97, 130.46, 137.81, 141.43, 142.53, 145.37, 148.91, 151.35, 158.68; HRMS (ESI+) m/z calc for C23H16ClN3O2S 432.12, found 433 (M + H); Anal. Calcd. for C23H16ClN3O2S: C, 63.67; H, 3.72; N, 9.68. Found: C, 63.58; H, 3.45; N, 9.54.

Molecular modeling

AutoDock software version 4.0 was used to find the suitable placement of ligands in the active site and investigation important interactions between flexible ligands and the rigid COX-2 enzyme. A high-resolution PDB format file of the 3D crystallized enzyme was retrieved from the protein data bank server with PDB code 6COX. To prepare the enzyme for ducking, the SC-588 molecule as internal ligand and water molecules were first removed from this protein. The next steps were adding Kollman charges and, in the following, merging non-polar hydrogens. Finally, AutoDock 4 was used to gain the PDBQT format of the enzyme. To achieve the conformation with minimized energy, HyperChem 8.0 program through the MM + method was used, and then with AutoDock tools, the PDBQT format of ligands was obtained. A grid box with dimensions 40*40*40 around the active site of protein was built. Docking run was put to 100, and the Lamarckian genetic search algorithm was used. For more efficiency, the ligands-enzyme interactions with distances greater than 7.0 Å were removed. At last, for each compound, the structure with lower energy was chosen as the best conformation [41, 42].

Biological assays

In vitro cyclooxygenase inhibition evaluations

The in vitro COX inhibition assay was carried out with a Fluorescent enzyme kit (Cayman Chemical, MI, USA) (37). This Fluorescent inhibitor screening kit utilizes the production of fluorescence using the heme-catalyzed peroxidase activity of cyclooxygenase (COX-1: ovine, COX-2: human recombinant). This assay is based on the generation of resorufin as a fluorescent compound in the reaction PGG2 with ADHP (10-acetyl-3,7-dihydroxyphenoxazine). High potency compounds are determined due to the less intensity of fluorescence arising from resorufin [43].

In vivo anti-inflammatory assay

This experiment used male NMRI mice (18–22 g), obtained from the Animal House of School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. They were housed in eight-mouse cages with a standard diet (temperature: 22 ± 2 °C, light-dark cycle: 12 h, humidity: 45–55%). All novel compounds in the vehicle (DMSO) were dispersed and injected at the doses of 5, 10, 20, and 40 mg/kg body weight of mice intraperitoneally (i.p.). This experiment, based on a protocol approved by the “Animal Experimentation Ethics Committee” of the University of Shahid Beheshti Medical Sciences, was performed, and each animal only once was used. For investigation of the nociceptive responses in mice, the acetic acid-induced writhing test was used. Thirty minutes after administration of different doses of the novel compounds, an acetic acid solution of 1% to each animal was injected to cause abdominal constriction, a typical writhing response, and used celecoxib and vehicle as a positive and negative control, respectively. The number of writhing responses during a 30 min period by observation was counted. The mean ± SEM of eight mice for each group was represented. The estimated ED50 values were calculated with a 95% confidence interval by using Graph Pad Prism software by nonlinear regression analysis of the log dose-response curve.

In vitro anti-cancer assay

Anti-cancer effects of these compounds against MCF-7, breast cancer cell line, were evaluated based on the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)) assay.

MCF-7 cancerous cell line was prepared from the Iranian Biological Resource Center (IBRC), Tehran, Iran. The cancerous cells were seeded in 96-well culture plates with the RPMI1640 medium under a high-humidity atmosphere with 5% CO2 at 37 °C. One day after, the culture medium present in the wells was replaced with a medium containing celecoxib as a reference compound, synthesized compounds, and RPMI culture medium as a control. Under the above situation, the cells for 72 h were allowed to incubate. Then 10 µL MTT was added, and incubation was continued for 4 h at 37 °C. After 4 h, the supernatant was removed, and cells for 20 min at 37 °C were exposed to 100 μL DMSO. The spectrophotometer plate reader determined the absorbance of cells at 570 nm [44].