Design, Synthesis, Anti-TMV Activity, and Structure–Activity Relationships of <i>Seco</i>-pregnane C₂₁ Steroids and Their Derivatives

<i>seco</i>-pregnane C₂₁ steroids exhibit high antiviral activity against the tobacco mosaic virus (TMV). However, the structural modification of <i>seco</i>-pregnane C₂₁ steroids and the structure-activity relationship (SAR) of the modified compounds remain unevaluated. Hence, the present study investigated how variations in the original skeletons of natural <i>seco</i>-pregnane C₂₁ steroids affect their antiviral activity. A series of glaucogenin C and A derivatives were designed and synthesized for the first time, and their anti-TMV activity was evaluated. Bioassay results showed that most of the newly designed derivatives exhibited good to excellent antiviral activity; among these derivatives, <b>5g</b>, <b>5j</b>, and <b>5l</b> with higher antiviral activity than that of ningnanmycin emerged as new antiviral candidates. Reverse transcription-polymerase chain reaction and Western blotting assay revealed reduced levels of TMV coat protein (TMV-CP) gene transcription and TMV-CP protein expression, which confirmed the antiviral activity of these derivatives. These compounds also downregulated the expression of <i>NtHsp70-1</i> and <i>NtHsp70-061</i>. Computational simulations indicated that <b>5l</b> displayed strong van der Waals energy and electrostatic with the TMV coat protein, affording a lower binding energy (Δ_Gbind = -56.2 kcal/mol) compared with Ribavirin (Δ_Gbind = -47.6 kcal/mol). The SAR of these compounds was also evaluated, which demonstrated for the first time that substitutions at C-3 and double bonds of C-5/C-6 and C-13/C-18 are crucial for maintaining high anti-TMV activity.