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. 2023 Dec 14;8(51):48994-49008.
doi: 10.1021/acsomega.3c06612. eCollection 2023 Dec 26.

ZIKV Inhibitors Based on Pyrazolo[3,4- d]pyridazine-7-one Core: Rational Design, In Vitro Evaluation, and Theoretical Studies

Quang De Tran  1   2 , Cuong Quoc Nguyen  1   2 , Quang Le Dang  3   4 , Thi Hong Minh Nguyen  5 , Bui Thi Buu Hue  1 , Minh Uyen Thi Le  6 , Nguyen Trong Tuan  1 , Nguyen Quoc Chau Thanh  1 , Tran Thanh Men  7 , Pham Minh Quan  4   8 , Nguyen Duy Tuan  9 , Thai Thi Cam  9 , Nguyen Thi Thu Thuy  10 , Vu Thi Bich Hau  10 , Tran Duy Binh  7 , Hong Phuong Nguyen  11
Affiliations

ZIKV Inhibitors Based on Pyrazolo[3,4- d]pyridazine-7-one Core: Rational Design, In Vitro Evaluation, and Theoretical Studies

Quang De Tran et al. ACS Omega. .

Abstract

The Zika virus (ZIKV) is believed to cause birth defects, and no anti-ZIKV drugs have been approved by medical organizations to date. Starting from antimicrobial lead compounds with a pyrazolo[3,4-d]pyridazine-7-one scaffold, we synthesized 16 derivatives and screened their ability to interfere with ZIKV infection utilizing a cell-based phenotypic assay. Of these, five compounds showed significant inhibition of ZIKV with a selective index value greater than 4.6. In particular, compound 9b showed the best anti-ZIKV activity with a selectivity index of 22.4 (half-maximal effective concentration = 25.6 μM and 50% cytotoxic concentration = 572.4 μM). Through the brine shrimp lethality bioassay, 9b, 10b, 12, 17a, and 19a showed median lethal dose values in a range of 87.2-100.3 μg/mL. Compound 9b was also targeted to the NS2B-NS3 protease of ZIKV using molecular docking protocols, in which it acted as a noncompetitive inhibitor and strongly bound to five key amino acids (His51, Asp75, Ser135, Ala132, Tyr161). Utilizing the pharmacophore model of 9b, the top 20 hits were identified as prospective inhibitors of NS2B-NS3 protease, and six of them were confirmed for their stability with the protease via redocking and molecular dynamics simulations.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Pyrazolo-pyridazine structures with different ring fusion patterns (A) and representative compounds containing a pyrazolo-pyridazine unit (B).
Figure 2
Figure 2
Rational design of the pyrazolo[3,4-d]pyridazin-7-one scaffold of lead compounds (A–D).
Scheme 1
Scheme 1. General Procedure for the Synthesis of Compounds Containing the Pyrazolo[3,4-d]pyridazin-7-one Moiety
(i) Conc. HCl/H2O (1:1), NaNO2/H2O, 0 °C, 1 h; (ii) EtOH, NaOAc, 0 °C, 1 h; (iii) NaOEt, EtOH, rt, 1 h; (iv) hydrazine hydrate, EtOH, reflux, 1 h; (v) DMF, NaH, 0 °C, 30 min; (vi) SnCl2·H2O, EtOH, reflux, 1 h; (vii) MC, TEA, 0 °C, 1 h; (viii) CuI, K2CO3, DMSO, microwave, 150 °C; (ix) LiOH 1 M, rt, overnight; (x) DMF, HOBT, EDC·HCl, rt, −50 °C, overnight.
Figure 3
Figure 3
Dose–response curves of compounds 9b, 10b, 12, 17a, and 19a against ZIKV and the reference inhibitor (MPA-mycophenolate acid). The percentage of ZIKV inhibition is shown in the black line. Percentage cell viability is shown in the red line. A compound shows good activity if the SI value is greater than 10 (SI of 10–6: activity; SI of 6–4: low activity; and SI < 4: no activity). A full antiviral data set for all compounds can be found in the Supporting Information, Table S1.
Figure 4
Figure 4
Binding interaction between 9b (pink) and 17b (cyan) at the active site of NS2B-NS3.
Figure 5
Figure 5
Molecular dynamics result for the 9b-NS2B-NS3 complex. (A) Protein at the center of the water box in simulation protocol using Desmond Academic license 2018–4. (B) The RMSD values for complex during the molecular dynamic simulation. (C) Protein interactions with the ligand 9b (hydrogen bonds, green; hydrophobic, violet; ionic, pink; and water bridges-blue). (D) Ligand torsion profile torsion and flexibility. (E) Ligand torsion angles.
Figure 6
Figure 6
HOMO and LUMO orbitals of compound 9b in the gas phase and water.
Figure 7
Figure 7
Pharmacophore model. (A) Pharmacophoric features of 9b (pink) at the active site of the NS2B-NS3 protease. (B) Pharmacophoric features of 9b were selected at the active site of NS2B-NS3 protease. (C) Pharmacophore-based virtual screening from the ZINC and ChemDiv database. HBA (hydrogen bond acceptor-organ sphere). HP (hydrophobic areas-green sphere). AR (aromatic ring-violet sphere).
Figure 8
Figure 8
Redocking protocol and molecular dynamics of top three molecules virtual screened from ChemDiv and the ZINC database. HBA: hydrogen bond acceptor; HP: hydrophobic. Ar: aromatic ring.
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