# _In Silico_ Development of SARS-CoV-2 Non-Covalent Mpro Inhibitors: A Review
## 2 Mpro Inhibitors for SARS-CoV-2
### Discovery Strategies for Non-Covalent Inhibitors of Mpro
In parallel, ML300 was also subjected to optimisation, ultimately leading to the development of compound 41 (CCF981). Notably, this derivative extended into the hydrophilic S2c subsite, significantly enhancing its inhibitory potency, as reflected by an IC\({}_{50}\) of 0.068 \(\upmu\)m and an EC\({}_{50}\) of 0.497 \(\upmu\)m in Vero E6 cells [16]. Scaffold refinement emerged as a central strategy for enhancing potency, as demonstrated by the advancement of ML188 to its optimised analogue 23R (IC\({}_{50}\) = 0.20 \(\upmu\)m)  
Figure 5: Structural optimisation and representative compounds in the development of ensitrelvir [17]. Figure 6: Chemical optimisation of ML188 [46]. and the progression of ML300 to CCP981 (IC\({}_{50}\) = 0.068 \(\upmu\)m). These improvements reflect the value of targeted modifications to key substituents, particularly those that enhance interactions within the S1\({}^{\prime}\), S2, and S4 subsites. Nonetheless, these efforts remain mostly cell-line-bound, lacking validation in animal models and pharmacokinetic studies, which hinders the assessment of in vivo efficacy. For example, while 23R induced a new binding pocket, the absence of ADME or toxicity profiling remains a critical gap. In pursuing effective SARS-CoV-2 Mpro inhibitors, Table 1 displays some representative medicinal chemistry strategies and active compounds included in this review. In the screening of structure-related compounds based on a series of N-substituted isatin derivatives, compound 26 was identified as a potent Mpro inhibitor, demonstrating the highest potency (IC\({}_{50}\) = 0.045 \(\upmu\)m) [43]. However, high cytotoxicity hindered further antiviral evaluation. Additionally, Benzoxaborole-based inhibitors were explored for their dual activity against SARS-CoV-2 and dengue virus proteases [47]. The resulting compound 18 exhibited an IC\({}_{50}\) of 6.1 \(\upmu\)m against SARS-CoV-2 Mpro with minimal cytotoxicity. \begin{table} \begin{tabular}{c c c c} \hline \hline
**Inhibitor** & **Structure** & **Strategies for the Discovery of Non-Covalent Mpro Inhibitors** & **Mpro IC\({}_{50}\) (\(\upmu\)m)** & **Ref.** \\ \hline \hline \end{tabular} \begin{tabular}{c c c} \hline \hline
**N** & **N** & **N** \\ \hline \hline \end{tabular} \begin{tabular}{c c} \hline \hline
**N** & **N** \\ \hline \hline \end{tabular} 
\begin{tabular}{c c} \hline \hline \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Inhibitor** & **Structure** & **Strategies for the Discovery of Non-Covalent & **Mpro IC\({}_{\mathbf{50}}\) (\(\mathbf{\mu}\)m)** & **Ref.** \\  & & **Mpro Inhibitors** & \\ \hline
23R & Coupling structure-based drug design with the one-pot Ugi four-component reaction. & 0.20 & [46] \\ \hline
3C & \(\mathbf{Cl}\) & \(\mathbf{N}\) & Structure-based optimisations. & 0.068 & [16] \\ \hline
3N-substituted isatin derivative compound 26 & \(\mathbf{O}\) & In-house synthetic compound library screening. & 0.045 & [43] \\ \hline Benzoxaborole-based inhibitor compound 18 & \(\mathbf{O}\) & In-house synthetic compound library screening. & 6.1 & [47] \\ \hline
4H & \(\mathbf{O}\) & \(\mathbf{O}\) & Fluorescence resonance energy transfer (FRET) biochemical assay. & 1.55 & [48] \\ \hline
5H & \(\mathbf{O}\) & \(\mathbf{O}\) & Fluorescence resonance energy transfer (FRET) biochemical assay. & 1.81 & [48] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Inhibitor** & **Structure** & \multicolumn{3}{c}{**Strategies for the Discovery of**} \\
**Non-Covalent** & **Mpro Inc\({}_{\text{50}}\) (\(\mu\)m)** & **Ref. ** \\ \hline HL-3-68 & \(O\)=HN & \(Cl\) & Large-scale virtual screening and structural optimisation. & 0.29 & [49] \\ \hline MCule-CSR-494190-S  
Moreover, non-peptiddomimetic 9,10-dihydrophenanthrene derivatives, specifically C1 and C2, demonstrated potent Mpro inhibition with IC\({}_{50}\) values of 1.55 \(\upmu\)m and 1.81 \(\upmu\)m, respectively [48]. Compound C1 also exhibited excellent metabolic stability in the gastrointestinal tract, plasma, and liver microsomes.