Elsevier

Bioorganic & Medicinal Chemistry

Volume 25, Issue 24, 15 December 2017, Pages 6467-6478
Bioorganic & Medicinal Chemistry

Discovery of new antimalarial agents: Second-generation dual inhibitors against FP-2 and PfDHFR via fragments assembely

https://doi.org/10.1016/j.bmc.2017.10.017Get rights and content

Abstract

Malaria parasites are a leading cause of worldwide mortality from infectious disease. Cysteine protease falcipain-2 (FP-2) and Plasmodium falciparum dihydrofolate reductase (PfDHFR) play vital roles, which are absolutely essential, in the parasite life cycle. 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, we identified a novel series of dual inhibitors through fragments assembly. Lead optimization led to the discovery of 24, which showed high potency against FP-2 (IC50 = 10.0 µM), 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). The in vivo inhibition assays against P. berghei in 10 days indicated 24 had a more beneficial effect on the growth inhibition of P. berghei than artemisinin and an identical effect with pyrimethamine. Additionally, 24 moderately inhibited the proliferation of chloroquine-resistant P. falciparum Dd2 strain. Collectively, these data revealed that 24 could be an excellent lead compound as FP-2 and PfDHFR dual inhibitor for the treatment of malaria.

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 AC, 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 (929) 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 2629, 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.

References (53)

  • A. Persidis

    Malaria

    Nat Biotechnol

    (2000)
  • T. Conroy et al.

    Synthesis of gallinamide analogues as potent falcipain inhibitors and antimalarials

    J Med Chem

    (2014)
  • P.J. Hotez et al.

    “Manifesto” for advancing the control and elimination of neglected tropical diseases

    PLoS Neglected Trop Dis

    (2010)
  • World Malaria Report 2016; World Health Organization: Geneva....
  • Y.K. Zhang et al.

    Benzoxaborole antimalarial agents. Part 5. Lead optimization of novel amide pyrazinyloxy benzoxaboroles and identification of a preclinical candidate

    J Med Chem

    (2017)
  • K. Singh et al.

    Antimalarial pyrido[1,2-a]benzimidazoles: Lead optimization, parasite life cycle stage profile, mechanistic evaluation, killing kinetics, and in vivo oral efficacy in a mouse model

    J Med Chem

    (2017)
  • T.F. de Koning-Ward et al.

    Plasmodium species: master renovators of their host cells

    Nat Rev Microbiol

    (2016)
  • K. Mendis et al.

    The neglected burden of Plasmodium vivax malaria

    Am J Trop Med Hyg

    (2001)
  • B. Autino et al.

    Pathogenesis of malaria in tissues and blood

    Mediterr J Hematol Infect Dis

    (2012)
  • R.T. Gazzinelli et al.

    Innate sensing of malaria parasites

    Nat Rev Immunol

    (2014)
  • M. Pieroni et al.

    Accepting the invitation to open innovation in malaria drug discovery: Synthesis, biological evaluation, and investigation on the structure-activity relationships of benzo[b]thiophene-2-carboxamides as antimalarial agents

    J Med Chem

    (2017)
  • A.M. Dondorp et al.

    Artemisinin resistance in Plasmodium falciparum malaria

    N Engl J Med

    (2009)
  • A.M. Dondorp et al.

    Artemisinin resistance: current status and scenarios for containment

    Nat Rev Microbiol

    (2010)
  • F. Ariey et al.

    A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    Nature

    (2014)
  • B. St. Laurent et al.

    Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa

    Nat Commun

    (2015)
  • Y.Q. Yang et al.

    Target elucidation by cocrystal structures of NADH-ubiquinone oxidoreductase of Plasmodium falciparum (PfNDH2) with small molecule to eliminate drug-resistant malaria

    J Med Chem

    (2017)
  • Cited by (14)

    • An insight into the recent development of the clinical candidates for the treatment of malaria and their target proteins

      2021, European Journal of Medicinal Chemistry
      Citation 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].

    • Falcipains as drug targets in antimalarial therapy

      2020, Antimalarial Agents: Design and Mechanism of Action
    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text