3.2.2. Metal-Chelating Activity (MChA) on Ferrous Ions

Fe(II) is a transition metal well known for its increased propensity for the Fenton reaction, but it also makes it one of the most important pro-oxidants involved in lipid peroxidation, i.e., the oxidation of lipids leading to cell membrane damage [13,26]. This, in turn, accelerates the aging process. Chelation is one of the antioxidant methods for reducing the catalytic action of the transition metals Fe(II) and Cu(II). Chelating agents create sigma bonds with the metal and are regarded as strong secondary antioxidants [13,29] because they can lower the redox potential and stabilize the oxidized form of the iron ion.

The metal-chelating properties of the newly synthesized furan derivatives (**H1–4**) on ferrous iron are shown in Table 1 and Figure 4B. The results for the metal-chelating activity are presented as IC50. Chelating activity on ferrous iron is an important step in preventing lipid peroxidation. Therefore, we investigated the ability of compounds **H1–4** to form chelate complexes with Fe(II) ions. The concentration gradient of **H1–4** affects the chelating activity. As the concentration of compounds **H1–4** increases, their ability to chelate Fe(II) increases. At low concentrations, **H1–4** show low chelating activity. From the analysis, we found that compounds **H3** (1.02 mg/mL) and **H4** (1.46 mg/mL) showed significant metal-chelating activity four times higher than compounds **H1** and **H2**.

Here, we must clarify that there is a connection between the two methods (HPSA and MChA). In living tissues, competing reactions are most likely taking place—both hydrogen peroxide deactivation and Fe(II) chelation. The research shows that there is a good correlation dependence of 0.9672 between the two methods. This relationship is beneficial because the chelation of Fe(II) will prevent the Fenton reaction from occurring. This approach provides more information about the antioxidant activity of the compounds, proving them to be reliable exogenous antioxidants.

#### 3.2.3. Inhibition of Albumin Denaturation (IAD)

Living tissues' response to harm is inflammation. Cell migration, tissue breakdown, mediator release, enzyme activation, fluid extravasation, and tissue healing are only a few

of the numerous complicated processes involved [30]. Inflammation in rheumatoid arthritis is well known to be brought on by the denaturation of proteins. The ability of certain anti-inflammatory medications to prevent thermally induced protein denaturation has been demonstrated to depend on dose [31]. The obtained furan hybrids were examined for their ability to inhibit albumin denaturation. This method determines the extent to which albumin is protected from denaturation when heated. We used human albumin for this purpose. Figure 5A shows the percentages of inhibition of synthetic furan derivatives. The obtained results of the analysis are presented as IC50. Because ibuprofen and ketoprofen have well-established properties, we decided to utilize them as a standard to compare the activities of the newly synthesized furan derivatives. The IC50 values of ibuprofen and ketoprofen estimated as IAD are 81.50 μg/mL and 126.58 μg/mL, respectively (Table 1, Figure 5A). All of the obtained results show that the IC50 values of furan hybrid molecules are in the range of 114.31 to 150.99 μg/mL (Table 1, Figure 5A).

**Figure 5.** In vitro biological activity was assessed as inhibition of albumin denaturation (IAD) (**A**) and antitryptic activity (ATA) (**B**). As benchmarks, anti-inflammatory medications such as ibuprofen and ketoprofen were utilized. The results of both methods are presented as IC50.

In general, the obtained compounds (**H1–4**) exhibited high IAD activity as profens. The in silico anti-inflammatory activity (cAnti-A) results show that the standards (ibuprofen and ketoprofen) have higher activity than the synthesized derivatives (**H1–4**), indicating that there is a directly proportional dependence between in vitro and in silico for ibuprofen and a reverse dependence for ketoprofen (Table 1).

In addition, IAD analysis reveals that lipophilicity is a significant physicochemical parameter. The lipophilicity (RM) of the synthetic furan derivatives studied ranges from 0.87 to 1.04, which influences albumin protection to some extent (Table 1).

For the stability of albumin, the hydrophobic pocket of subdomain IIA and IIIA plays an important key role, popularly known as Sudlow's sites I and II, respectively. Due to the hydrophobic nature of the interior of Sudlow's sites I pocket, the drug primarily formed hydrophobic interactions with Phe211, Trp214, Leu219, Phe223, Leu234, Leu238, Leu260, Ile264, and Ile290 [32].

As the interior of Sudlow's sites I pocket is hydrophobic in nature, the drug predominantly formed hydrophobic interactions with Phe211, Trp214, Leu219, Phe223, Leu234, Leu238, Leu260, Ile264, and Ile290 [32]. The stabilization of the albumin molecule in this study is due to hydrophobic interactions between Sudlow's sites I and the furan derivatives.

#### 3.2.4. Antitryptic Activity (ATA)

Proteinases have been linked to the development of arthritic symptoms. Neutrophils are known to be a good source of proteinase because their lysosomal granules contain many serine proteinases. It has previously been reported that leukocyte proteinase is important in the development of tissue damage during inflammatory reactions and that proteinase inhibitors provide significant protection [16,31]. In vitro anti-arthritic activity was assessed as antitryptic activity [16]. The IC50 results for the ATA range from 60.21 to 85.33 μg/mL. The results reveal that the furan derivatives **H1–4** show better antitryptic activity compared to ibuprofen and ketoprofen (Table 1, Figure 5B).
