Quinolone-N-acylhydrazone hybrids as potent Zika and Chikungunya virus inhibitors

Journal Pre-proofs Quinolone-N-Acylhydrazone Hybrids as Potent Zika and Chikungunya Virus Inhibitors Roberta K.F. Marra, Arthur E. Kümmerle, Guilherme P. Guedes, Caroline de S. Barros, Rafaela S.P. Gomes, Claudio C. Cirne-Santos, Izabel Christina N.P. Paixão, Amanda P. Neves PII: DOI: Reference:

S0960-894X(19)30859-5 https://doi.org/10.1016/j.bmcl.2019.126881 BMCL 126881

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

19 September 2019 4 November 2019 30 November 2019

Please cite this article as: Marra, R.K.F., Kümmerle, A.E., Guedes, G.P., Barros, C.d.S., Gomes, R.S.P., CirneSantos, C.C., Paixão, I.C.N.P., Neves, A.P., Quinolone-N-Acylhydrazone Hybrids as Potent Zika and Chikungunya Virus Inhibitors, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl. 2019.126881

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Quinolone-N-Acylhydrazone

Hybrids

as

Potent

Zika

and

Chikungunya Virus Inhibitors Roberta K. F. Marraa, Arthur E. Kümmerlea, Guilherme P. Guedesb, Caroline de S. Barrosc, Rafaela S. P. Gomesc, Claudio C. Cirne-Santosc, Izabel Christina N. P. Paixãoc, Amanda P. Nevesa. a

Instituto de Química, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ,

23897-000, Brasil. E-mail: [email protected] b

Instituto de Química, Universidade Federal Fluminense, Niterói, RJ, 24020-141,

Brasil. c

Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, 24020-141,

Brasil.

Abstract This work reports the synthesis of quinolone-N-acylhydrazone hybrids, namely 6-R-N'-(2-hydxoxybenzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (R = H: 5a, F: 5b, Cl: 5c and Br: 5d), which exhibited excellent activity against arbovirus Zika (ZIKV) and Chikungunya (CHIKV). The final compounds were characterized by spectroscopy techniques, mass spectrometry, and X-ray diffraction analysis for 5a. In vitro screening towards ZIKV and CHIKV inhibition revealed that all substances have significant antiviral activity, most of them being more potent than standard Ribavirin (5a-d: EC50 = 0.75-0.81 M, Ribavirin: EC50 = 3.95 M for ZIKV and 5a-d: 1.16-2.85 M, Ribavirin: EC50 = 2.42 M for CHIKV). The quinolone-N-acylhydrazone hybrids were non-toxic against Vero cells, in which compounds 5c and 5d showed the best selectivities (SI = 1410 and 630 against ZIKV and CHIKV, respectively). Antiviral activity was identified by inhibition of viral RNA production in a dose-dependent

manner. In the evaluation of the time of addition of the compounds, we observed that 5b and 5c remain with strong effect even in the addition for 12 h after infection. The above results indicate that quinolone-N-acylhydrazones represent a new and promising class to be further investigated as anti-ZIKV and anti-CHIKV agents. Keywords: Quinolone-N-acylhydrazones, Arbovirus, Zika, Chikungunya, Antiviral activity

Zika (ZIKV) and Chikungunya (CHIKV) are viruses transmitted by the bite of Aedes aegypit and Aedes albopictus female mosquitoes [1,2]. The viruses propagate rapidly and geographically affecting mainly in tropical areas, and there are cases of transmission in the Americas, Oceania and Africa [3]. A question of great concern is also that virus vectors can adapt in urban areas, survive under extreme conditions and be dispersed easily by humans [4]. Within South America, Brazil is one of the most affected countries, where ZIKV and CHIKV have been included in the National List of Compulsory Notification of Diseases, Injuries and Events of Public Health [4]. In 2019, there were registered 15.352 new probable cases of CHIKV, of which two resulted in death [5]. For ZIKV, in this same year, there were reported 2.493 probable cases, with the highest incidence rate in the North region [5]. ZIKV has been associated with neurological complications, especially in pregnant infected women, that can cause neurologic impairment in the newborns and fetuses, such as microcephaly and other congenital malformations. In addition,

there

are

reported

cases

of

Guillain-Barré

syndrome

(GBS),

meningoencephalitis, transverse myelitis (TM) and other neuropathies on teens and adults infected with ZIKV [4,5].

As these pathogens represent a serious public health problem, there has been a growing interest of the scientific community in the search for natural or synthetic substances for the treatment of ZIKV and CHIKV infections. For example, gemcitabine [6],

bithionol

[7],

chloroquine

[8],

hydroxychloroquine

[8],

7-Deaza-2’-C-

ethyladenosine [9], Merimepodib [10] and bis-naphthoquinones [11] have been reported as potent inhibitors of the ZIKV replication. Recently, a new series of 1,3-disubstituted (thio)urea derivatives were found to possess excellent activities against ZIKV [12]. For CHIKV, compounds that have proven broad-spectrum antiviral activities have been tested, such as Ribavirin [13], 6-azauridine [14], 4-hydroxycytidine (β-D-N4hydroxycytidine, NHC, 3) [15], Favipiravir (T-705) [16], Sofosbuvir [17], Arbidol [18] and Chloroquine [19]. Although many candidates are in active preclinical development, among which some are in phase I clinical trials [20], there is yet no vaccine available against these viruses or even effective antiviral therapies. For the treatment of CHIKV and ZIKV, only the symptoms are handled nowadays [1]. Quinolones represent a significant class in medicinal chemistry mostly owing to their recognized antibacterial properties [21]. For example, ciprofloxacin, ofloxacin and levofloxacin are second-choice drugs recommended by the WHO for the treatment of tuberculosis in cases of multiresistant bacilli [22]. Concerning their antiviral action, these privileged heterocycles are found to possess promising in vitro and in vivo anti‐HIV activities [23]. To the same mode, N-acylhydrazones comprise versatile molecules that exhibited significant antiviral activity against vaccinia virus (VV) and Influenza H1N1, with low selectivity indexes (SI) [24].

In view of the excellent activities of these two organic frameworks, we aimed to investigate the anti-ZIKV and anti-CHIKV potential of new synthetic quinolone-Nacylhydrazone hybrids 5a-d (Figure 1).

HO

O H

NH

N

N

O R

(5a-d)

R= H (a), F (b), Cl (c), Br (d)

Figure 1. Quinolone-N-acylhydrazone hybrids (5a-d) synthesized in this work. The choice of 6-halide-substituted-quinolones was inspired by the well-known success of fluoro-quinolones as antibiotics [21] and also due to the great anti-HIV-1 and HIV-2 activities of some fluoro- and chloro-quinolone derivatives [23]. The targeted compounds (5a-d) were prepared by a four-step synthetic route [25-27], outlined in scheme 1. Details of the synthesis, IR, 1H and

13C

spectroscopy and HRMS/ESI are given in the Supporting Information (SI).

H

NH2

N

i

R

CO2Et CO2Et

H ii

O (3a-d)

(2a-d) R (35-62%)

(1a-d)

R

O (3a-d)

iii

H

iv

H

HO NH

N

O R

(55-70%)

O NHNH2

N

CO2Et

N

N

O

(4a-d) (80-90%) R= H (a), F (b), Cl (c), Br (d)

R

(5a-d) (79-94%)

NMR

Scheme 1. Reactions conditions: (i) EMME, EtOH, reflux; (ii) Diphenyl ether, 250ºC; (iii) NH2NH2.H2O, 80%, EtOH; (iv) Salicylaldehyde, acetic acid, EtOH.

The crystal structure of 5a showed that the compound crystallized in a monoclinic P21/n space group and the asymmetric unit contains two crystallographic independent molecules and one lattice water molecule, as depicted in Figure 2. For the sake of discussion, the independent molecules were labeled as A and B. A summary of the crystal, data collection and refinement is listed in Table S1.

Figure. 2. Asymmetric unit of the compound 5a. Thermal ellipsoids are drawn at 50 % of probability. Dashed lines represent the intramolecular hydrogen bonds (see Table S3 for geometric parameters).

The molecular structure of 5a (molecules A and B) exhibits the average C–O, C=O, C–N and C=N bond lengths in typical values reported in the literature [28-30] (Table S2). Based on the structural features, it was possible to confirm that the quinolone-N-acyl-hydrazone hybrid 5a is in the keto form. Furthermore, one can observe that both molecules assumes E configuration related to the N2=C11 bond, as

predicted by 1H NMR results. The E isomer allowed the intramolecular hydrogen bond involving the phenol group and the iminic N2 atom, as well as between the quinolone carbonyl moiety, O2, and the hydrazone N1 atom (dashed lines in Figure. 2). The latter interaction was also observed for related hydrazone derivatives [30]. The quinolone ring connects to the N-acylhydrazone moiety in an approximately planar geometry with torsion angle of 1.6(3)º and -6.2(2)º in the fragments O2a–C10a–C2a–C1a and O2b– C10b–C2b–C1b. The same tendency was seen for the phenyl group and the iminic carbon atom, with torsion angles nearly to zero [-0.9(2) for O3a–C13a–C12a–C11a and 0.2(2)º for O3b–C13b–C12b–C11b)].

The in vitro cytotoxicity (CC50) of the compounds in Vero cells was assessed by MTT

[3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium

bromide]

(Sigma-

Aldrich), as previously described [31]. Further details are discussed in ESI. The results summarized in Table 1 show that the CC50 values of 5a-d ranged from 443 to 1113 µM, demonstrating a relatively low toxicity (Figure. 3), at least twofold lower than the control (Ribavirin CC50 = 297 M). Compound 5c (R = Cl) was the least toxic molecule (CC50 = 1113 M). Table 1. Cytotoxicity (CC50), anti-ZIKV and anti-CHIKV profiles (EC50) and selectivity index (SI) of the compounds. ZIKV Compound

CC50a (µM)

EC50b (µM)

CHIKV SIc

EC50b (µM)

SIc

5a

502 ± 4.38

0.76 ± 0.028

660.9

2.85 ± 0.12

176.2

5b

669 ± 4.33

0.75 ± 0.011

892.9

1.06 ± 0.077

631.7

5c

1113 ± 6.11

0.79 ± 0.005

1409.7

2.77 ± 0.18

402

5d

443± 5.1

0.81± 0.009

547.5

2.7 ± 0.13

164.2

Ribavirind

297 ± 4.95

3.95 ± 0.095

75.2

2.42 ± 0.49

122

Data represented as mean ± SD from three independent experiments. a Concentration that reduced the cytotoxic concentration in Vero cells by 50% when compared to untreated controls. b Concentration that reduced the CHIKV replication by 50% when compared to infected controls. c Selectivity index was defined as the ratio between CC and EC and represents the safety for 50 50 in vitro assays. d Ribavirin was used as a control.

To evaluate the potential of the compounds to inhibit ZIKV and CHIKV replication, Vero cells were infected in 96-well plates with 0.1 CHIKV MOI, incubated for 1 h for viral adsorption and, subsequently, treated with increasing concentrations of the compounds. The quinolone-N-acylhydrazone hybrids had a significant effect on the inhibition of replication of both viruses, with values ranging from 0.75-0.81 M for ZIKV and 1.16-2.85 M for CHIKV.

Fig. 3: Dose-dependent cell viability assessment ranging from 100 to 2000 micromolar to determine the CC50. Results were obtained by following the mean and standard deviation of 3 independent experiments.

It can be noted that the compounds exhibited slightly better activities against ZIKV than CHIKV, and were also more potent and selective than the reference drug (EC50 ~ 0.78 M for 5a-d vs 3.95 M for Ribavirin). Amongst the quinolone-Nacylhydrazone hybrids, compound 5c containing the -Cl as substituent presented the most remarkable result due to its highest selective index (SI =1410). The substituent in the phenyl ring (R = H, F, Cl and Br) seemed not to influence on ZIKV inhibition due to the similarity of the EC50 values, while the cytotoxicity against VERO cells was in part affected. For CHIKV, the fluoro substituted compound 5b was the most potent and selective inhibitor (EC50 = 1.06 M; SI = 631). On the other hand, 5a and 5c-d exhibited quite similar EC50 values compared to Ribavirin. When we evaluated the inhibition of viral RNA production, the compounds with the most significant EC50 and SI results exhibited a dose-dependent profile for both ZIKV and CHIKV (5c (Figure 4A) and 5b (Figure 4B), respectively), demonstrating an inhibitory profile not only in direct viral particle production and reduction of the cytopathic effect on Vero cells, but also in viral RNA production, directly influencing the assembly of the complete viral particle.

A

B

Figure 4A,B: Inhibition of the RNA production of ZIKV or CHIKV. A: Inhibition of ZIKV RNA production by 5c and Ribavirin. B: Inhibition of CHIKV RNA production by 5b and Ribavirin. Vero cells were infected with ZIKV or CHIKV (MOI 0.1) and treated at concentrations of 0.65, 1.25, 2.5, 5 and 10

µM. The results were evaluated from three independent experiments in triplicate. Data are presented as percentage of virus RNA titer, when compared with control cells and are expressed as the mean of three experiments ± standard error. Statistical analysis was performed using Tukey test in comparison of 5b and 5c compounds and ribavirin in each concentration: *p<0.05; **p<0.01; ***p<0.001.

In order to investigate the mechanism of action of the tested compounds, our group performed the addition time assay using compounds 5b and 5c in CHIKV and ZIKV infected Vero cells, respectively, using Ribavirin as the control. Figures 5A and 5B demonstrate a significant reduction in CHIKV and ZIKV titer when compounds were added at times 0 and 2, 6, 12 and 16 hpi time points. At 16 hpi, we observed that the antiviral activity against CHIKV of 5b decreased in the concentration used, however Ribavirin had its effect reduced before 12 hpi. In the treatment of ZIKV-infected cells with 5c, we observed reduction in viral titer up to 6 hpi, with loss of its effect by 12 hpi. Ribavirin control in the treatment of ZIKV infected cells showed a substantial reduction in the antiviral effect already at 6 hpi. These data indicate that the compounds 5b and 5c appear to act both in the early stages of the viral replication cycle and in some postinfection stages for CHIKV and ZIKV respectively. A

B

Figure 5. Drug addition time experiment with compounds 5b and 5c at 10 µM in inhibiting CHIKV and ZIKV respectively. Vero cells were infected with CHIKV (Fig 5A) and ZIKV (Fig 5B) with 106 PFU / mL at MOI of 0.1 after each treatment (0h; and after infection, + 1h, + 2h, + 6h, + 12h and + 16h), the results were evaluated by the plaque assay and the experiments were performed in triplicate. Ribavirin

(10 µM) was used as a positive control. Statistical analysis was performed using Tukey test in comparison of Compounds 5b and 5c and ribavirin in each concentration: * p <0.05; ** p <0.01; *** p <0.001. DMSO used as solvent remained at the final concentration of 1%, showing no activity in CHIKV replication.

In conclusion, a new series of quinolones-N-acylhydrazones derived from 2hydroxy-benzaldehyde has been obtained by simple and reproducible organic reactions, affording compounds with high purity and yields (5a-d). The molecules have been characterized by spectroscopic and spectrometric techniques, and also the crystalline structure of 5b was elucidated by X-Ray diffraction, confirming the expected structure. In vitro antiviral investigation revealed promising activity of the compounds against both ZIKV and CHIKV viruses, especially for ZIKV where 5a-d displayed remarkable potency in the nanomolar range. The substances were found to be more selective than the reference drug and may be added post infection while maintaining their antiviral effect, which implies that the quinolone-N-acylhydrazone hybrids can be considered excellent candidates for the development of anti-ZIKV and anti-CHIKV agents. Finally, expansion of the compounds series is underway to enable structure-activity relationship (SAR) study and mechanism of action investigation.

Acknowledgements The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support and for Productivity Fellowship to ICNPP (443930/2014-7). ICNPP (E-26/201.442/2014) also thank the FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) for the Cientista do Nosso Estado

Fellowship. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. We also thank LDRXUFF (www.ldrx.uff.br/) for the X-ray diffraction analysis and PPGQ-UFRRJ (http://cursos.ufrrj.br/posgraduacao/ppgq/) for the facilities.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Quinolone-N-acylhydrazone hybrids were tested against arbovirus Zika (ZIKV) and Chikungunya (CHIKV) The compounds exhibited excellent activities, with EC50 = 0.75-0.81 M for ZIKV and EC50 = 1.16-2.85 M for CHIKV Most of the compounds were more potent and selective than standard Ribavirin Antiviral activity was identified by inhibition of viral RNA production in a dosedependent manner The substances can be added post infection while maintaining their antiviral effect They can be considered excellent candidates for the development of anti-ZIKV and antiCHIKV agents