Discovery of new antimalarial agents: Second-generation dual inhibitors against FP-2 and PfDHFR via fragments assembely
Graphical abstract
Introduction
Malaria, a mosquito-borne disease caused by infection with Plasmodium parasites, is a devastating parasitic disease causing widespread mortality and morbidity across many parts of the developing world.1 Nearly half the world’s population are exposed in malaria endemic areas, and according to WHO 2016, malaria is responsible for an estimated 1.2 billion clinical cases of infection and 429,000 deaths in 2015 globally.2, 3 Children below five years of age and pregnant women living in poor countries are most vulnerable, it is considered that 303,000 malaria deaths happened in children aged under 5 years, which represented 70% of the global total deaths. Malaria remained a major killer of children, and it is estimated to take one child’s life every 2 min.4, 5 Mammalian infection is initiated by the bite of Plasmodium-infected female Anopheles mosquitoes.6, 7 Among the species of plasmodia, P. vivax and P. falciparum are responsible for the majority of malaria infections and recrudescent infection via activation of dormant liver-stage hypnozoites that re-establish the clinical blood-stage of infection.8, 9, 10, 11, 12 Natural products and their derivatives, including artemisinin and its derivatives, are identified as first-line antimalarial drugs used clinically. Although antimalarial drugs have successfully mitigated the epidemics in the past few decades, the control of malaria has been severely compromised in recent years, on account of the widespread resistance of P. falciparum to nearly all frontline therapeutics which were used for both prophylaxes and treatments.2, 13, 14, 15, 16, 17, 18 Consequently, the requirement for the development and discovery of new antimalarial drugs, which are structurally distinct from existing drugs and endowed with novel mechanisms of action, is greatly exigent.19, 20, 21, 22, 23, 24, 25
In P. falciparum, various proteases catalyze the degradation of human hemoglobin, and the amino acids derived from this process are incorporated into parasite proteins or utilized for energy metabolism.26 Cysteine protease falcipain-2 (FP-2) of P. falciparum is an indispensable protease involved in this metabolic process. FP-2, belonging to the family of cysteine proteases (papain-like enzymes known as clan CA), is expressed during the erythrocytic stage of the life cycle of the parasite.27, 28 In the past several years, various types of FP-2 inhibitors have been reported to be capable of inactivating the enzyme and a large number of studies have confirmed that inhibitors of FP-2 could block parasite hemoglobin hydrolysis, halt the development of culture parasites, and these inhibitors are effective against murine malaria.29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 FP-2 has emerged as a promising target for the development of novel antimalarial drugs40, 41, 42, 43
P. falciparum dihydrofolate reductase (PfDHFR) has received considerable attention for the prophylaxes and the treatments of P. falciparum infection.44, 45 The de novo synthesis of folate is required for DNA synthesis in Plasmodium species. The final step of folate synthesis is the enzymatic reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, which is catalyzed by the enzyme PfDHFR.46 Furthermore, PfDHFR is an essential metabolic enzyme that plays a critical role in one-carbon transfer reactions, including the biosynthetic pathways for deoxythymidine monophosphate (dTMP), purines, and several amino acids,47 which is necessary for the synthesis of DNA. Consequently, a more powerful pesticidal effect could be achieved by inhibiting FP-2 and PfDHFR simultaneously. Such dual inhibitors might show a good synergetic effect, and overcome the drug-resistance and be capable of providing “a combination therapy” in a single agent.48
In this study, based on the structures of uniform fragments of reported PfDHFR inhibitors and the first-generation dual inhibitors against FP-2 and PfDHFR identified by us previously,48 a novel series of second-generation dual inhibitors against FP-2 and PfDHFR had been designed through fragments assembly. In an attempt to gain novel structures with high potencies against both FP-2 and PfDHFR, we decided to explore the 2,4-diaminoquinazoline analogues (Structure D, Fig. 2) on the basis of the computational analysis of three compounds (compounds A–C, Table 2), we exhaustively evaluated whether the type of substituent (R, Fig. 2) at the terminal amide would improve the pharmacological activity. Finally, a potent dual inhibitor (24) effectively inhibited the proliferation of P. falciparum, furthermore, the in vivo assays indicated 24 had a more beneficial effect on the growth inhibition of P. berghei than artemisinin. Thus, 24 was identified as a unique lead compound for the development of antimalarial drugs.
Section snippets
Chemistry
As outlined in Scheme 1, tert-butyl(4-bromophenethyl)-carbamate (2) was prepared from commercially available 2-(4-bromophenyl)ethan-1-amine (1) in the presence of (Boc)2O in CH2Cl2 at 25 °C for 4 h in an yield of 84%. 2 was stirred with n-BuLi in THF at -78 °C under N2 for 1 h, then DMF was added into the solution for another 1 h to give tert-butyl (4-formylphenethyl)carbamate (3) in a yield of 70%. Further treatment of 3 with HCl-dioxane at 25 °C for 4 h gave 4-(2-aminoethyl)benzaldehyde (4)
Conclusion
In summary, we have identified a novel series of 2,4-diaminoquinazoline analogues (9–29) derived from 31 (2o) as FP-2 and PfDHFR dual inhibitors. On the basis of the structure of the lead compound 31, twenty-one completely novel 2,4-diaminoquinazoline analogues have been synthesized and tested in FP-2 and PfDHFR enzymatic inhibitory activities. Ten analogues, i.e., 10, 13, 16, 19, 21, 24 and 26–29, showed good inhibitory activities against FP-2 (IC50 = 4.9–10.0 µM) and eighteen analogues, i.e.,
Synthesis general
Reagents and solvents were purchased from Adamas-Beta, J & K, Energy Chemical, Target Molecule, TCI, Alfa Aesar, and Acros, and were used without further purification. Analytical thin-layer chromatography (TLC) was conducted on HSGF 254 plates (150–200 μm thickness; Yantai Huiyou Co., China) and spots were visualized with UV light. Yields were not optimized. Melting points were measured in capillary tubes on an SGW X-4 melting point apparatus without correction. Nuclear magnetic resonance (NMR)
Acknowledgments
Financial support for this research provided by the National Natural Science Foundation of China (Grants 21372001 and 21672064), the “Shu Guang” project supported by the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (Grant 14SG28), and the Fundamental Research Funds for the Central Universities are gratefully acknowledged.
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2021, European Journal of Medicinal ChemistryCitation Excerpt :In the continuation of the discovery of PfDHFR inhibitors, several authors explored different scaffolds to develop potent antimalarials. The design and synthesis of 2,4-diaminoquinazoline analogs as dual inhibitors active against falcipain-2 (FP-2) and PfDHFR were reported by Chen et al. [216] via fragments assembly method. During the study, authors discovered the best compound 18 (Fig. 14) through lead optimization which was reported with high potency against FP-2 (IC50 = 10.0 mM), PfDHFR (IC50 = 84.1 nM), P. falciparum 3D7 (IC50 = 53.1 nM), clinical isolated strains Fab9 (IC50 = 14.2 nM) and GB4 (IC50 = 23.4 nM) [217].
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These authors contributed equally to this work.