Elsevier

Antiviral Research

Volume 118, June 2015, Pages 103-109
Antiviral Research

Benzouracil–coumarin–arene conjugates as inhibiting agents for chikungunya virus

https://doi.org/10.1016/j.antiviral.2015.03.013Get rights and content

Highlights

  • New uracil–coumarin–arene triply conjugated compounds were designed and synthesized.

  • Five compounds exhibited inhibitory activity (EC50 = 10.2–19.1 μM) against chikungunya virus in Vero cells.

  • Compounds with a scaffold of benzouracil–SCH2–coumarin–OSO2–arene showed the most appealing antiviral results.

  • The potency was affected by the nature of uracil, the joint between the units, and the substituent on the arene.

Abstract

Chikungunya virus (CHIKV) is an arbovirus that was first recognized in an epidemic form in East Africa in 1952–1953. The virus is primarily transmitted through mosquitoes and the resulting disease, chikungunya fever, is found in nearly 40 countries. Neither an effective vaccine nor a specific antiviral drug exists for treatments of chikungunya fever. Thus 22 new conjugated compounds of uracil–coumarin–arene were designed and synthesized as potential inhibiting agents. Their chemical structures were determined unambiguously by spectroscopic methods, including single-crystal X-ray diffraction crystallography. The three units in these conjugates were connected by specially designed –SCH2– and –OSO2– joints. Five of these new conjugates were found to inhibit CHIKV in Vero cells with significant potency (EC50 = 10.2–19.1 μM) and showed low toxicity (CC50 = 75.2–178 μM). The selective index values were 8.8–11.5 for three conjugates. By analysis of the data from the anti-viral assays, the structure–activity relationship is derived on the basis of the nature of the uracil, the functional groups attached to the arene, and the joints between the ring units.

Introduction

Chikungunya virus (CHIKV) causes severe persistent arthralgia that has affected at least 41 countries with about one million suspected and 22,500 confirmed cases (Scholte et al., 2015). These cases are due to a recent massive outbreak of the Caribbean island in October 2013. Over the past half century, a number of CHIKV cases have emerged mainly in Africa and Asia (Gerardin et al., 2008) since it was first recognized in East Africa in 1952–1953 (Schwartz et al., 2012). The imported transmission happened to Italy in 2007 and to France in 2010 because of infected travelers (Albulescu et al., 2014). As the competent vectors exist in many regions, the CHIKV might continue to threat the Americans, including parts of the United States (Scholte et al., 2015). Presently, there is neither an effective vaccine nor a specific antiviral drug available for this disease (Cavrini et al., 2009). Hence the development of drugs with activity against CHIKV is becoming a global concern of the utmost importance.

CHIKV belongs to the genus Alphavirus and is an enveloped virus with a positive sense, single stranded RNA genome (Bassetto et al., 2013). With the growing available structural information about the CHIKV genome, the viral nonstructural proteins (particularly nsP2 acting as protease as well as helicase) and the viral envelope proteins are identified as the most promising targets for drug design through inhibitory binding of the targeted proteins (Rashad et al., 2014). Several chemical compounds have been reported to exhibit weak anti-CHIKV activity. They include flavones and their analogs (e.g., apigenin, chrysin, (5,7-dihydroxy)flavones, and silibinin), nucleoside derivatives (e.g., 6-azauridine and ribavirin), heterocyclic compounds (e.g., chloroquine, mycophenolic acid, and prothipendyl), interferon, etc. (Briolant et al., 2004, Ozden et al., 2008, Pohjala et al., 2011, Khan et al., 2011, Kaur et al., 2013). In 2011, arbidol and its two derived metabolites were tested in vitro on the CHIKV by use of two cell lines in pre- and post-infection conditions (Delogu et al., 2011). Arbidol is found to have an IC50 of 12.2 μM. D’Hooghe et al. reported that two purine-β-lactam hybrids possess anti-CHIKV activity with EC50 of 17.11 and 13.01 μM, respectively, and selectivity indices (SIs) of >5.75 and >4, respectively (D’hooghe et al., 2012). In 2013, Brancale et al. reported that benzylidene(cyclopropane)carbohydrazide inhibits the virus-induced cytopathic effect with an EC50 of 5.0 μM and SI of 14 (Bassetto et al., 2013).

In this study, we designed a new molecular structure 1 primarily composed of a “triply” conjugated skeleton of uracil–coumarin–arene for the development of new anti-CHIKV agents. The C-4′ and C-7′ positions of coumarins were deliberately linked to the uracil and arene units with a thiomethylene (SCH2) and a sulfonate ester (OSO2) (or replaced by an oxymethylene (OCH2)) joints to form triply conjugated compounds.

The uracil, thymine (i.e., 5-methyluracil), and benzouracil (i.e., quinazolinone) moieties exist in many biologically active compounds (Lagoja, 2005). Recently, Wang et al. (2012) revealed that the substituents on the C-5 and C-6 positions (i.e., R1 and R2, respectively, in 1) of the uracil unit are important to anti-viral activity. Abdel-Aal (2010) synthesized compounds with two hydrophobic residues at the C-5 and C-6 positions. These compounds exhibited potent activity against hepatitis B virus (HBV). Ordonez et al. (2012) found that the introduction of a substituent at the C-6 position of uracil resulted in elevated anti-HIV-1 activity. Benzouracil and its derivatives have been extensively studied because of their wide range of pharmacological activities. Alogliptin containing a benzouracil moiety is a potent selective inhibitor of serine protease (Feng et al., 2007). Moreover, the benzouracil unit is widely present in biologically active natural products (Horton et al., 2003).

Results from a recent review indicate the broad pharmacological activity of natural and synthetic coumarins (Riveiro et al., 2010). It has drawn much attention to drug discovery. The substituent pattern and structural feature within the coumarin family are vital to the improved activity. Kostova and co-workers found that coumarins and their derivatives have prominent inhibitory effect against chymotrypsin-like proteases of HIV (Kostova et al., 2005). Based on the structure–activity relationships of several 3-substituted-4-hydroxycoumarins, the substituents at positions 5 and 7 of the coumarin ring play an important role on the inhibitory potency of the HIV-1 protease (Kirkiacharian et al., 2002). Recently, coumarins conjugated with various benzimidazoles (Hwu et al., 2008, Tsay et al., 2013), benzothiazoles, or benzoxazoles (Neyts et al., 2009) have been synthesized by our research group. These conjugates exhibit appealing anti-HCV activity and become promising leads for further development. Replacement of the benzimidazole unit with nucleobases results in the related conjugates with enhanced anti-HCV activity (Hwu et al., 2011a, Hwu et al., 2011b). The data indicate the importance of the thiomethylene (–SCH2–) joint used for the connection of coumarin moieties with various heterocycles on the inhibitory potency of HCV (Hwu et al., 2011a, Hwu et al., 2011b).

Compounds containing a sulfo (R–OSO2–R′) joint have been identified as potent protease inhibitors (Shenai et al., 2003). The reaction of sulfonate compounds with the hydroxyl site of an active protease residue can form a stable enzyme derivative (Powers et al., 2002). Nitrophenyl esters of benzenesulfonic acid and phenylmethanesulfonic acid bearing various positively charged groups on the benzene ring act as inactivators of trypsin-like proteases (Powers et al., 2002). For example, p-nitrophenyl p-(amidinothiomethyl)benzenesulfonate inactivates thrombin. Recently, Udommaneethanakit et al. (2014) reported the inhibition of avian influenza A virus with sulfonate drugs, such as Oseltamivir, Peramivir–sulfonate, and Zanamivir.

Herein we report the scope of uracil–coumarin–arene conjugates as an unprecedented class of anti-CHIKV agents. In total, 22 compounds with the common skeleton 1 were synthesized, five of which exhibited significant inhibitory effect against CHIKV. Through analysis of their anti-CHIKV activities influenced by the types of joints and substituents on the uracil and the arene rings, their SAR guidelines were deduced accordingly.

Section snippets

Materials and methods

A detailed description of the materials and methods is available in the Supporting information file.

Results and discussion

For the synthesis of the triply conjugated compounds with scaffolds 1 (i.e., uracil–coumarin–arenes), three challenges had to be overcome. First, the labile sulfo group connecting the coumarin and arene moieties had to remain intact throughout the synthetic steps. Second, the conditions to form the S–CH2 single bond had to be sufficiently mild to maintain the bond intact. Third, the reagents could not induce an undesired Michael addition to the α,β-unsaturated ester group or ring-opening of the

Conclusions

For the development of new compounds with anti-CHIKV activity, 22 triply conjugated uracil–coumarin–arenes were designed and synthesized. Five of them (i.e., 10a, 10b, 10e, 12e, and 14e) were found to impede CHIKV replication at EC50 values of 19.1, 10.2, 17.2, 19.0, and 13.0 μM, respectively.

The coumarin moiety in the conjugated compounds was essential to their antiviral activity. This central moiety was attached to a pyrimidine unit at the C-4′ position through a SCH2 joint on one side and an

Acknowledgments

For financial support, we thank the Ministry of Science and Technology (Grant Nos. NSC 103-2923-I-008-001 and MOST 103-2113-M-007-018-MY3), Ministry of Education of R.O.C. (Grant Nos. 104N2011E1 and 104N2016E1), and National Central University, Taiwan (Grant No. 103G603-14). The work in Leuven is supported by the European Commission SILVER project within the 7th Framework Programme as Cooperation Project Grant Agreement (No. 260644).

References (37)

  • I.C. Albulescu

    An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors

    J. Gen. Virol.

    (2014)
  • F. Cavrini

    Chikungunya: an emerging and spreading arthropod-borne viral disease

    J. Infect. Dev. Ctries.

    (2009)
  • M. D’hooghe

    Design, synthesis, and antiviral evaluation of purine-β-lactam and purine-aminopropanol hybrids

    J. Med. Chem.

    (2012)
  • J. Feng

    Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV

    J. Med. Chem.

    (2007)
  • P. Gerardin

    Multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of la réunion

    PLOS Med.

    (2008)
  • A.K. Ghose

    A knowledge-based approach in designing combinatorial or to be medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases

    J. Comb. Chem.

    (1999)
  • M.M. Hann et al.

    Finding the sweet spot: the role of nature and nurture in medicinal chemistry

    Nat. Rev.

    (2012)
  • D.A. Horton

    The combinatorial synthesis of bicyclic privileged structures or privileged substructures

    Chem. Rev.

    (2003)
  • Cited by (35)

    • Synthesis and anti-cancer activity of naphthopyrone derivatives

      2021, Tetrahedron Letters
      Citation Excerpt :

      To date, lots of studies have indicated that coumarins possess a variety of pharmacological activities, such as: anti-inflammatory [9], antimicrobial [10], anti-viral [11], anti-oxidant [12], antinociceptive [13], anti-tumor [14], antiasthmatic, antidepressant [15], anti-HIV [16], antituberculosis [17], anti-Alzheimer [18], anti-influenza [19], antihyperlipidemic [20]. Thus, a large number of samples are required in the development of drug research and application.

    View all citing articles on Scopus
    View full text