*3.5. Antiproliferative Assay*

The antiproliferative activity evaluation of the synthesized triphenyl imidazole derivatives was made with the sulforhodamine B (SRB) assay. The tumoral cell lines employed were adherent epithelial cells from different anatomic origins. All results were expressed as growth inhibition 50 (GI50), as the concentration needed to inhibit the 50% of cell population, and calculated and expressed as micromolar (μM). As positive controls, different antitumor drugs were employed such as cisplatin, etoposide, and camptothecin, and imidazole was used as the structural reference of the synthesized compounds.

The obtained results from the evaluation of the 13 synthesized compounds with the SRB assay against the tumor cell lines (Table 2) showed no selectivity by any specific line. Based on the results in Table 2 and the GI50 range (Figure 3), the most active compounds of the series were **10** and **11**. As initial highlights, imidazole had no inhibitory activity in the compounds employed as controls; on the other hand, one of the most resistant cell lines against the synthesized compounds and drugs was A549, which corresponds to lung adenocarcinoma, and this is in agreemen<sup>t</sup> with the literature, as it has been documented that lung type cancers are usually chemotherapy resistant, even to one of the most used antitumor drugs, taxol [43].

From the 13 synthesized compounds, lower activity was shown from derivative **1**, this being the triphenyl imidazole bearing no substitutions, as against five of the six evaluated cell lines, it showed no significant activity, and a low one against SW1573 (89 μM). Likewise, between the molecules with one methoxy substitution **4** (*p*-OMe), **5** (*m*-OMe) and **6** (*o*-OMe), which are position isomers, only **5** showed a low activity against SW1573 with an GI50 of 76 μM, while **4** and **6** presented no significant activity against all of the evaluated cell lines.

Following these general low active compounds, derivatives **9** (*o*-Cl) and **12** (*o*-NO2) were partially active, as they presented different degrees of activity, but against only a couple of cell lines. Compound **9** showed good to moderate activity only against two cell lines, which were HeLa with a GI50 of 7.7 μM and SW1573 with 17 μM; compound **12** presented activity against the same cell lines with GI50 of 6.1 and 66 μM respectively.

The following molecules with better results were the compounds **3** (*p*-OH), **7** (*m*-OMe, *p*-OH), **8** (*m*-OMe, *p*-OMe), and **13** (anthracene), as these molecules presented activity against all evaluated cell lines, the only exception being **13** against lines T-47D and WiDr; however, **13** also showed one of the best particular results, this being a GI50 of 4.2 μM against SW1573. In the case of **3**, activity was shown against all analyzed cell lines, with HeLa being the most sensitive with a GI50 of 13 μM, followed by SW1573 and HBL-100 with 15 and 16 μM, respectively, while in the rest of the lines, the results were between 19 and 22 μM.


**Table 2.** Antiproliferative activity of compounds **1**–**13** against six human solid tumor cell lines a.

a GI50 values are given in μM. Standard deviation was calculated from two to four independent experiments. Cisplatin (CDDP) and etoposide (VP-16) were used as reference antiproliferative drugs. Values in bold represent the best anti-proliferative data against tumor cell lines with GI50 values < 10 μM. b Only one experiment was performed.

**Figure 3.** GI50 range plot of the tested compounds.

With molecules **7** and **8**, very similar GI50 values could be seen between them. Comparing them against monosubstituted compounds **4**, **5**, and **6** (which have a methoxy group in different positions), **7** and **8** showed that di-substitution enhanced the antiproliferative activity against these cell lines, these being substitutions of the hydroxy and methoxy type. In the literature, compound **8** was reported

to show antiproliferative activity against the breast cancer cell line MDA-MB-231 with a GI50 of 21 μM [44]. This value is consistent with the results obtained in our study.

The best set of synthesized molecules were **2** (*o*-OH), **10** (*p*-N(CH3)2) and **11** (*p*-NO2) as they showed the overall lowest GI50 values. In particular, **2** presented significant activity against lines SW1573 and HeLa with values of 3.6 and 4.3 μM; **10** showed significant activity against the majority of cell lines, where the outstanding results were against A549 with GI50 of 3.8 μM, and against SW1573 with 4.4 μM. The most active compound against all of the analyzed cell lines was **11**, as its GI50 was between 2.9 to 6.3 μM, with SW1573 being the most sensitive line.

These two most active compounds had a substitution in their A ring in the *para* position, where the second most active was a tertiary amine nitrogen, and the most active had a nitro group. One of the most sensitive cell lines to the synthesized compounds and the evaluated drugs was SW1573, which is from alveolar carcinoma. This is despite the line belonging to a lung cancer lineage which, as mentioned before with A549, are pharmacotherapy resistant carcinomas. However, these two lung related cell lines showed that they were sensitive to compound **11**, with GI50 values of 6.3 for A549 and 2.9 μM for SW1573, which were the second lowest and the lowest values, respectively, for these lines.

In 2017, Dake's research team [45] reported the synthesis and evaluation of triphenyl imidazole derivatives with substitutions in their A ring against the A549 line, where their compound 6f showed an IC50 of 15 μM. This molecule has *m*-I, *m*-OMe, and *p*-OH substitutions, where the iodine is structurally similar to **7**. The presence of this heteroatom improved activity by a 2 μM di fference compared to not having it (17 μM for molecule **7**).

The *p*-NO2 substitution in compound **11** bears an important role in antiproliferative activity, which could be due to the nitroaromatic structure. Nitroaromatic compounds have gained interest as chemotherapeutic agents against cancer because molecules with nitro groups in their metabolism can go through bio-reduction, which generates reactive species that cause damage to cell components by oxidative stress; additional reductions are favored in hypoxic conditions, which generates highly cytotoxic species [46]. Even though molecule **12** is an isomer of **11**, in comparison, it showed much lower activity. This could be due to **12** having the nitro group in the *ortho* position, where it could interact with the hydrogen in the imidazole ring, diminishing the generation of the reactive species needed for the antiproliferative activity.
