## 3 Results and Discussion
### Fluorescence Spectroscopic Investigation of the Binding Mechanism  
Serum albumins often enhance the apparent solubility of hydrophobic drugs in plasma and modulate their delivery to the cells in vivo and in vitro. The pharmacokinetic characteristics, stability, and toxicity of drugs can be significantly altered as a result of their binding to serum albumins. Bovine serum albumin (BSA) and human serum albumin (HSA) are the major components in plasma protein for cows and humans, respectively. BSA is homologous to HSA, displaying about 88% sequence homology, and consists of three linearly arranged domains (I-III) that are composed of two subdomains (A and B). The use of HSA may be favored over BSA for the studies carried out on clinically used drugs. However, in the present study, for such a compound in the early stages of development, BSAwas employed as a model representing the serum albumins family. Several earlier studies have utilized spectroscopic techniques, particularly fluorescence spectral determination, to explore the different aspects of protein binding to various ligands [34; 35; 36]. Fluorescence quenching shows decline protein intrinsic fluorescence occurring through diverse molecular interactions [37; 38]. The BSA fluorescence emission spectra with and without the addition of compound **3** were monitored in the wavelength range 290-500 nm upon excitation at 280 nm. The addition of compound **3** caused the BSA fluorescence intensity to be quenched in a concentration dependent way (Figure 3), with no BSA peak shift or shape alteration. The experimental quenching effect can be either classified as a dynamic or a static type of quenching [22; 39].  
Based on their temperature dependence, the two types of quenching can be further identified, i.e., higher temperatures yield higher quenching constants in dynamic quenching, while the opposite is always true in the case of static quenching. Hence, the quenching results at the three investigated temperatures were analyzed using the Stern-Volmer (Equation (2)) [40] and Lineweaver-Burk equations (Equation (3)) [41].  
\[\frac{F_{0}}{F}=1+K_{\text{SV}}C_{Q}=1+K_{q}\tau_{0}[Q] \tag{2}\]  
\[(F_{0}-F)^{-1}=\ F_{0}^{-1}+K_{\text{LB}}{}^{-1}F_{0}^{-1}[Q]^{-1} \tag{3}\]  
In the two Equations, \(F_{0}\) and \(F\) represent the BSA fluorescence intensity alone and when bound to compound **3**, respectively, and \([Q]\) stands for the concentration of compound **3**. The Stern-Volmer and Lineweaver-Burk constants are symbolized by \(K_{\text{SV}}\) and \(K_{\text{LB}}\), respectively, with \(K_{\text{q}}\) representing the quenching rate constant, and \(\tau_{0}\) is the mean protein lifetime without compound **3** (quencher) that equals (2.7 \(\times\) 10\({}^{-9}\) s\({}^{-1}\)) [42]. Figure 4a,b show that the compound **3**-BSA binding system yields linear Stern-Volmer and Lineweaver-Burk relations with their constants summarized in Table 1. They decline with the gradual temperature rise that then fits well with a static quenching proposal [43]. Furthermore, Equation (4) was employed to estimate the \(K_{\text{q}}\) values listed in Table 1, which are shown to be higher than the formerly reported values for numerous quenchers with the biopolymer of 2 \(\times\) 10\({}^{10}\) LM\({}^{-1}\cdot\)s\({}^{-1}\)[42]. Hence, the observed results propose the formation of a complex between compound **3** and BSA [44; 45].  
\[K_{\text{q}}=K_{\text{SV}}/\tau_{0} \tag{4}\]  
Figure 3: Emission spectra of (**1**) BSA (1.5 \(\upmu\)M) and the BSA-compound **3** system succeeding the addition of compound **3** at the concentrations of (**2**) 0.65 \(\upmu\)M; (**3**) 1.0 \(\upmu\)M; (**4**) 1.5 \(\upmu\)M; (**5**) 3.0 \(\upmu\)M; (**6**) 4.5 M; (**7**) 6.0 \(\upmu\)M; (**8**) 9.0 \(\upmu\)M; (**9**) 12.0 \(\upmu\)M; (**10**) 18 \(\upmu\)M; (**11**) 25 \(\upmu\)M.