Synthesis, Anti-Plasmodial Activities, and Mechanistic Insights of 4-Aminoquinoline-Triazolopyrimidine Hybrids.

pubs.acs.org/acsmedchemlett Letter Synthesis, Anti-Plasmodial Activities, and Mechanistic Insights of 4‑Aminoquinoline-Triazolopyrimidine Hybrids Shefali Chowdhary, Shalini, Joel Mosnier, Isabelle Fonta, Bruno Pradines, Nosipho Cele, Pule Seboletswe, Parvesh Singh, and Vipan Kumar* Cite This: ACS Med. Chem. Lett. 2022, 13, 1068−1076 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: In the search of new antiplasmodial agents, a multitargeted approach was used in the synthesis of triazolopyrimidine- and 4-aminoquinolines-based hybrids. In vitro antiplasmodial evaluation on chloroquine-sensitive (3D7) and -resistant (W2) P. falciparum strains identified triazolopyrimidine-4-aminoquinoline hybrids to be the most potent in the series, outperforming bistriazolopyrimidines. The active compounds were subjected to mechanistic studies with the plausible and expected targets including heme, PfCRT, and PfDHODH, that eventually validated the biological data. The active compound surpassed the antimalarial drug CQ by inhibiting the parasite’s cellular process (hemozoin formation) and parasitic enzymes (PfCRT and PfDHODH), as confirmed by UV−vis and molecular modeling studies. KEYWORDS: Triazolopyrimidine, 4-aminoquinoline, antiplasmodial, heme inhibition, PfCRT, PfDHODH M the quinoline nucleus. These moieties are designed to prevent drug efflux from the parasite’s digestive vacuole by interfering with the function of Pf CRT.12 The failure of existing therapies, however, has prompted the search for newer P. falciparum targets that interfere with the nucleotide biosynthesis pathway. The Plasmodium species lack pyrimidine salvaging enzymes and rely solely on the de novo pyrimidine synthesis pathway in their DNA and RNA synthesis. Since pyrimidine biosynthesis requires the catalytic enzyme P. falciparum dihydroorotate dehydrogenase (Pf DHODH), it has emerged as a viable target for antiplasmodial drug development.13 Triazolopyrimidines (TPs), a fused biaryl scaffold, is a subtype of the purine analogue. It possesses antimicrobial,14,15 antifungal,16 antibacterial,17 and antitumor18 activities and can act as a carbonic anhydrase inhibitor,19 an anti-Alzheimer agent,20 and a selective ATP site directed inhibitor of the EGF receptor protein tyrosine kinase, 21 making it riveting pharmacologically. Essramycin, (I; Figure 1), the first natural TP, was recently isolated and reported to possess antibacterial activity.22 The triazolopyrimidine scaffold has proven to be a highly effective chemical class for the identification of potent alaria remains the most endemic global infectious disease caused by female Anopheles mosquito. Over 241 million cases and 627 000 deaths due to malaria were estimated globally in 2020 with children under the age of five and pregnant women being the most susceptible.1 Among various parasitic species, Plasmodium falciparum, P. vivax, and P. ovale cause the majority of human malaria cases.2 For many decades, quinoline-containing compounds, including chloroquine (CQ), amodiaquine, and quinine have been commissioned in the treatment of malaria. However, in the late 1950s, CQ-resistant P. falciparum malaria emerged independently in Southeast Asia and in South America, and have expanded gradually throughout India and Africa.3,4 The World Health Organization (WHO) now recommends artemisinin-based combination therapy (ACT) that includes artemisinin- and quinoline-based drugs.5 However, artemisinin-resistant P. falciparum parasites have also spread in different regions during the past decade.6,7 CQ resistance has been linked to specific mutations in the polymorphism of P. falciparum chloroquine resistance transporter (Pf CRT) gene.8 Nonetheless, despite the development of resistance, CQ remains a compelling pharmacophore for chemical modification because of its excellent clinical efficacy; low host toxicity; and convenient, simple, and cost-effective synthesis.9,10 The antimalarial activity of the 7-chloro-4aminoquinoline core is due to its ability to inhibit hemozoin formation (parasite’s defense mechanism).11 Resistance to CQ can be reversed or avoided by making specific changes around © 2022 American Chemical Society Received: February 23, 2022 Accepted: June 16, 2022 Published: June 21, 2022 1068 https://doi.org/10.1021/acsmedchemlett.2c00078 ACS Med. Chem. Lett. 2022, 13, 1068−1076 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter approach used for the preparation of bis-triazolopyrimidines, 7a−e, by heating the precursor 4 with different diamines in ethanol. The nucleophilic substitution reaction of precursors 6a−f on 4 afforded the targeted triazolopyrimidine-4-aminoquinoline hybrids, 8a−f in 48−81% yield, Scheme 2ii. The structures of the synthesized compounds were determined using analytical and spectroscopic techniques. For example, the compound 8a, showed a peak at m/z 354.1172 [M + H]+ in its HRMS-ESI. Its 1H NMR spectrum showed the characteristic doublets at δ 8.39 (J = 5.4 Hz), 8.17 (J = 9.12 Hz), and 6.61 (J = 5.4 Hz) corresponding to quinoline ring protons along with singlets at δ 8.41 and 6.81 assigned to triazolopyrimidine protons. The absorptions peaks at δ 25.30 and δ 156.63 corresponding to methyl and triazolopyrimidine carbons along with aliphatic carbons at δ 69.01, 41.47 in 13C NMR spectrum further attested the assigned structure. The synthesized compounds were assayed for their in vitro antiplasmodial activities on both CQ-susceptible (3D7) and CQ-resistant (W2) strains of P. falciparum and the results are enlisted in Table 1. Among the synthesized triazolopyrimidines dimers, the compounds exhibited low antiplasmodial activities, IC50s ranging from 11.3 to 53.8 μM on the CQ-resistant strain and 11.6−52.2 μM on the CQ-susceptible strain, respectively. The structure−activity-relationship (SAR) studies among the dimers revealed poor activities at shorter (n = 1, 2) and longer (n = 7) alkyl chain lengths, while mild activities were observed at butyl and hexyl chain length as evident from 7c and 7d. The inclusion of quinoline core among these hybrids resulted in substantial enhancement in antiplasmodial activities, both on CQ-susceptible and CQ-resistant strains. The compound 8a with an ethyl linker displayed poor activity on both the tested strains. Increasing the spacer length substantially improved the activity as evidenced by hybrids 8b−e. The hybrid 8e with an octyl chain as spacer proved to be the most potent of synthesized series displaying IC50s of 0.17 and 0.20 μM on strains, respectively. In particular, compound 8e proved to be 3-fold more active than the standard drug CQ on the CQresistant strain. The compound 8f having non flexible piperazine linker displayed minor activity, 12.9 μM. In addition to this, active compounds 8b−e were also tested on Vero cell lines to evaluate cytotoxicity. The tested compounds were well tolerated on the normal cell and thus displayed good selectivity i (...truncated)