In Silico Design, Synthesis, and Molecular Mechanism of Chlorothiophene-based Chalcone and Pyrazoline as Anticancer Agents

Fia Fathiana Wulan, Prof. Dra Tutik Dwi Wahyuningsih, M.Si., Ph.D; Dr. Endang Astuti, M.Si.; Dr.rer.nat Niko Prasetyo, S.Si., M.Sc

2025 | Disertasi | S3 Ilmu Kimia

Penelitian ini bertujuan mengembangkan turunan kalkon dan pirazolina berbasis klorotiofena sebagai agen antikanker untuk mengatasi resistensi agen antikanker, dengan pendekatan komputasi dan eksperimen. Senyawa pirazolina banyak dilaporkan memiliki potensi aktivitas antikanker, dan modifikasi hibridisasi dengan struktur klorotiofena diharapkan dapat meningkatkan aktivitas maupun selektivitasnya. Rangkaian penelitian mencakup: (1) desain in silico melalui penambatan molekul dan simulasi dinamika molekuler, (2) sintesis senyawa antara kalkon, (3) sintesis turunan pirazolina, (4) uji sitotoksisitas, dan (5) analisis ekspresi protein menggunakan imunositokimia.

Desain in silico dilakukan melalui penambatan molekul pada 30 senyawa pirazolina yang ditargetkan terhadap EGFR (PDB ID: 4HJO), COX-2 (PDB ID: 3LN1), MDM2 (PDB ID: 4HG7), dan Bcl-2 (PDB ID: 6QGK) menggunakan AutoDock Vina. Senyawa dengan performa terbaik kemudian dianalisis lebih lanjut melalui simulasi dinamika molekuler (MD) menggunakan GROMACS, diikuti dengan perhitungan energi bebas. Senyawa pirazolina terpilih disintesis melalui senyawa antara kalkon. Sintesis kalkon dilakukan menggunakan kondensasi Claisen-Schmidt dengan katalis basa NaOH pada suhu ruang, kemudian dilanjutkan dengan siklisasi berbasis NaOH menggunakan fenilhidrazin untuk memperoleh turunan pirazolina. Senyawa yang telah disintesis diuji sitotoksisitasnya terhadap berbagai lini sel kanker, dan sel normal Vero menggunakan uji MTT. Senyawa paling potensial diuji lebih lanjut menggunakan imunositokimia tidak langsung untuk mengevaluasi mekanisme kerjanya terhadap ekspresi protein H-Ras, p53, dan Bcl-2.

Hasil studi penambatan molekul menunjukkan bahwa senyawa pirazolina berbasis klorotiofena memiliki potensi untuk menghambat EGFR, MDM2, dan Bcl-2, tetapi memiliki daya inhibisi yang lemah terhadap COX-2. Dalam hal stabilitas, P25 mampu membentuk kompleks stabil dengan protein target selama simulasi MD. Dari penelitian ini, sebanyak 15 senyawa kalkon berhasil disintesis dengan rendemen 60–96%, sementara 12 senyawa pirazolina diperoleh dengan rendemen 56–89%. Seri senyawa pirazolina menunjukkan toksisitas tinggi tetapi selektivitas sedang terhadap sel kanker paru A549. Namun, P4 dan P25 menjadi senyawa paling potensial terhadap sel kanker payudara MCF-7, dengan nilai IC50 masing-masing sebesar 1,18 dan 0,14 ?g/mL dengan SI > 100. Analisis imunositokimia menunjukkan bahwa P25 menghambat proliferasi sel MCF-7 cenderung dengan menurunkan ekspresi H-Ras dibandingkan menginduksi apoptosis dengan meningkatkan ekspresi p53 atau menurunkan ekspresi Bcl-2. Temuan ini menunjukkan bahwa mekanisme utama efek antikanker P25 adalah melalui penghambatan sinyal proliferasi yang berkelanjutan.

Given the urgent need to overcome resistance commonly observed in existing anticancer treatments, this study explores novel chlorothiophene-based chalcones and pyrazolines as multi-target anticancer agents through computational and experimental approaches. Pyrazolines have shown significant potential as anticancer agents, and further hybridization with chlorothiophene is expected to improve their activity and selectivity. The workflow includes: (1) in silico design through molecular docking and molecular dynamics simulations, (2) synthesis of chalcone intermediates, (3) synthesis of pyrazoline derivatives, (4) cytotoxicity screening, and (5) analysis of protein expression via immunocytochemistry.

The in silico design was conducted using molecular docking studies on 30 proposed pyrazoline compounds targeting EGFR (PDB ID: 4HJO), COX-2 (PDB ID: 3LN1), MDM2 (PDB ID: 4HG7), and Bcl-2 (PDB ID: 6QGK) with AutoDock Vina. The best-performing compound underwent MD simulations using GROMACS, followed by free energy calculations. Selected pyrazoline compounds were synthesized via chalcone intermediates. Chalcone synthesis was carried out using Claisen-Schmidt condensation with NaOH as a base catalyst at room temperature, followed by another NaOH-catalyzed cyclization with phenylhydrazine to obtain N-phenyl pyrazoline derivatives. The synthesized compounds were evaluated for cytotoxicity against various predominant cancer cells and normal Vero cells, using the MTT assay. The most potent compound underwent indirect immunocytochemistry to assess its mechanism of action on the expression of H-Ras, p53, and Bcl-2 proteins.

Docking results indicated that chlorothiophene-based pyrazoline compounds have the potential to inhibit EGFR, MDM2, and Bcl-2 but exhibit weak inhibition of COX-2. In terms of stability, P25 demonstrated the ability to form stable complexes with target proteins during the MD simulation. From this research, 15 chalcone compounds were successfully synthesized with yields ranging from 60–96%, while 12 pyrazoline compounds were obtained with yields ranging from 56–89%. The pyrazoline series displayed high cytotoxicity but moderate selectivity against lung cancer cells A549, but P4 and P25 emerged as the most potent compounds against MCF-7, with IC50 values of 1.18 and 0.14 ?g/mL against MCF-7, respectively, and excellent selectivity (SI > 100). The immunocytochemistry analysis revealed that P25 inhibits MCF-7 cell proliferation relatively by reducing H-Ras expression significantly than inducing apoptosis by increasing p53 expression or decreasing Bcl-2 expression. These findings suggest that P25 exerts its anticancer effects primarily by blocking sustained proliferation signals rather than promoting apoptosis.

Kata Kunci : chalcone, pyrazoline, chlorothiophene, anticancer.