Accepted Manuscript Thiazolidone derivatives as inhibitors of chikungunya Virus Surender Singh Jadav, Barij Nayan Sinha, Rolf Hilgenfeld, Boris Pastorino, Xavier de Lamballerie, Venkatesan Jayaprakash PII:

S0223-5234(14)00969-6

DOI:

10.1016/j.ejmech.2014.10.042

Reference:

EJMECH 7449

To appear in:

European Journal of Medicinal Chemistry

Received Date: 1 March 2014 Revised Date:

13 October 2014

Accepted Date: 14 October 2014

Please cite this article as: S.S. Jadav, B.N. Sinha, R. Hilgenfeld, B. Pastorino, X. de Lamballerie, V. Jayaprakash, Thiazolidone derivatives as inhibitors of chikungunya Virus, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.10.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Graphical abstract: Thiazolidone derivatives as inhibitors of Chikungunya Virus Jadav et al.

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A series of twenty aralkylidene derivatives of thiazolidinone (1-20) were evaluated for anti-chikv activity. 5-[(2-methylphenyl)methylidene]-2sulfanylidene-1,3-thiazolidin-4-one (7) was found to have IC50 of 0.42 µM

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Thiazolidone derivatives as inhibitors of Chikungunya Virus Lamballerie3*, Venkatesan Jayaprakash1* 1

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Surender Singh Jadav1, Barij Nayan Sinha1, Rolf Hilgenfeld2, Boris Pastorino3, Xavier de

Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi-835215, Jharkhand, India 2

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of

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Lübeck, 23538 Lübeck, Germany 3

UMR_D 190 "Emergence des Pathologies Virales" (Aix-Marseille University, IRD French

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Institute of Research for Development, EHESP French School of Public Health), Marseille,

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France

Corresponding Author

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*Venkatesan Jayaprakash

E-mail: [email protected] Tel: +91-9470137264

*Xavier de Lamballerie

E-mail: [email protected] Tel: +33 49 132 4553

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Abstract A series of arylalkylidene derivatives of 1,3-thiazolidin-4-one (1-20) were synthesized and tested for their antiviral activity against chikungunya virus (LR2006_OPY1) in Vero cell culture by

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CPE reduction assay. Five compounds (7-9, 16 and 19) were identified to have anti-ChikV activity at lower micro molar concentration. The compounds 7, 8, 9, 16 and 19 inhibited the virus at 0.42, 4.2, 3.6, 40.1 and 6.8 µM concentrations respectively. Molecular docking simulation has been carried out using the available X-ray crystal structure of the ChikV nsp2 protease, in order

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to elucidate the possible mechanism of action. Interaction of ligands with ChikV nsp2 protease (PDB Code: 3TRK) suggested the possible mechanism of protease inhibition to act as potent

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anti-ChikV agents.

Keywords:

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Thiazolidinone; antiviral; chikungunya virus; nsp2 protease; molecular docking

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1. Introduction Chikungunya, an emerging arthropod-borne viral infection caused by the chikungunya virus (ChikV, an arbovirus) was first reported from Tanzania during 1952 [1]. A major outbreak

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has been reported more than 50 years later, during 2005-2007 in Africa and Asia [2, 3] that was followed by limited outbreaks in Europe [4] and the US [5].The emergence of a new clinical form of the virus [6] with vector adaptation (Aedes albopictus) [7] explains its geographical spread to developed nations. Threat due to this emerging virus is likely to be high in future if no

effective vaccine or chemotherapeutic agent available.

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means to prevent/treat the infection is developed and made available. To date,there is no

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Interferons [8] and their combination with Ribavirin [9] and Mercaptopurine [10] were reported to have antiviral activity against ChikV. Arbidol, an antiviral licensed for the treatment of influenza, was found to inhibit ChikV replication [11]. Extracts of a few plant materials have also been reported to exert anti-ChikV activity [12-18]. The current investigation presents the anti-ChikV activity of benzylidene rhodanine derivatives since rhodanine has been identified as a privileged scaffold [19] and reported with antiviral activity against HCV [20, 21] and HIV [22].

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Molecular docking simulation has been carried out with the recently deposited X-ray crystal structure of Chikv nsp2 protease (PDB Code: 3TRK) in order to understand the mechanism of action of the active molecules.

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2. Results and Discussion 2.1. Chemistry

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A series of twelve arylalkylidene derivatives of 1,3-thiazolidin-4-one (1-20) were synthesized following the reaction outlined in Scheme 1&2 [23]. Knoevenagel condensation of 2-sulfanylidene-1,3-thiazolidin-4-one (rhodanine) with aromatic/heteroaromatic aldehydes in the presence of acetic acid and ethanol provided compounds 1-12 (Scheme 1). Similarly, condensation

of

2-amino-4,5-dihydro-1,3-thiazole-4-one

(pseudothiohydantoin)

with

aromatic/heteroaromatic aldehydes in the presence of ammonium acetate and glacial acetic acid provided compounds 13-20 (Scheme 2). The resultant precipitates were recrystallized with ethanol to obtain pure final product. All the final compounds were found to have melting points

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closely matching with the available literature and further their structures were confirmed by their 1

H-NMR, 13CNMR and MS data. All the compounds were screened for their anti-ChikV and cytotoxic activity following

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the procedures discussed below.

2.2. Anti-Chikv & Cell viability assay

Anti-ChikV assay has been carried out with ChikV strain LR2006_OPY1 in Vero cell culture by CPE reduction assay. All the twenty (1-20) compounds have been evaluated for their

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antiviral activity and five compounds (7-9, 16 and 19) were found to have antiviral activity (Table 1 & 2, Graphs in Supplementary material). Three compounds (7-9), aralkylidene

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derivatives of 2-sulfanylidene-1,3-thiazolidin-4-one (1-12) were found to be active. Compound 7 with ortho-methyl substitution (IC50=0.1 µg/mL, Table 1) was found most potent amongst the three and was followed by compound 8 with para-methyl substitution (IC50=1.0 µg/mL, Table 1). Methyl substitution was favorable when it was at ortho-position (7) and was found to be ten times more potent than its para-counterpart (8). Compound 9 (IC50=1.0 µg/mL, Table 1) with 2napthyl ring inhibited virus replication at a concentration similar to that of compound 7. This

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clearly indicated that aralkylidene portion should be non-polar in nature to exert activity. This is further supported by the fact that compounds 1-6 with polar substitutions at para-position and compounds 10-12 with heteroaryl rings were found inactive at highest concentration studied (100 µg/mL). Two compounds (16 & 19, Table 2) from aralkylidene derivatives of 2-amino-4,5-

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dihydro-1,3-thiazole-4-one (13-20, Table 2) were found to be active. Compound 16 with orthonitro substitution and compound 19 with meta-methyl substitution were found active at concentrations IC50=10.0 µg/mL and IC50=1.5 µg/mL, respectively (Table 2). Compound 6 and

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16 differ in substitution at 2nd position of thiazolidine ring. A compound featuring sulfanylidene (6) was found inactive while the other with amino group (16) was found to exhibit activity. Similarly, compounds 7 and 8 differ from compound 18 and 20 at 2nd-position, but here the compounds having sulfanylidene group (7 & 8) were found to be active and compounds with amino group (18 & 20) were found inactive. Foregoing observation clearly indicates the influence of endo/exo nature of double bond involving 2nd position of thiazolidine and substitution in the phenyl ring of aralkylidene portions on the activity of the molecules. In the next section (2.3) interaction at molecular level will be discussed having simulations carried out

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with ChikV-nsp2 protease as a possible target. Active molecules did not show any cytotoxic effect at their active concentrations. Microscopic observation revealed no changes in the host cell morphology, which clearly indicates the antiviral property of the compounds analyzed (Potential cytotoxic/cytostatic effects of the compound were evaluated in uninfected cells by observing

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microscopically for any minor signs of virus-induced CPE or alterations to the cells caused by the compound).

2.3. Molecular docking simulation

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In an attempt to understand the possible mechanism of action of the active compounds, molecular docking simulation has been carried out with the X-ray crystal structure of Chikv nsp2

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protease (PDB Code: 3TRK). For compound 7, the aralkylidene portion of the molecule shows strong hydrophobic interaction with three amino-acid residues (TYR1047, TYR1049 and TRP1084) in S3 pocket (formed by TYR1047, TYR1049, TRP1084, MET1238, MET1242) while the thiazolidinone portion shows strong hydrophobic interaction with four amino-acid residues (CYS1013, TYR1047, TYR1049 and TRP1084) in the S2 pocket (formed by CYS1013, TRP1014, ALA1046, TYR1047, TYR1049, TRP1084). Also the carbonyl oxygen of

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thiazolidinone establishes H-bonding interaction with backbone-amide NH of TYR1047 (Fig 1 & 2). In compound 8, the aralkylidene portion is getting accommodated in S3 and exhibits two H-bonding interactions: (i) carbonyl oxygen of thiazolidinone with backbone-amide NH of TYR1047 and (ii) hydrogen of thiazolidinone ring nitrogen with backbone carbonyl oxygen of

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ASN1011. The second H-bonding interactions slightly bend the molecule away from the S2 pocket to make the thiazolidinone portion to interact with S1 pocket residue ASN1011. Internal

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strain, along with reduced interaction with the S2 pocket, may be the reason for the reduced potency of this compound (Figures in Supplementary Material). In case of compound 9, the aralkylidene portion is quite away from S3 and shows interaction with residues in S2 and SI. It does not exhibit any H-bonding interaction with TYR1047, but establishes two H-bonding interactions: (i) carbonyl oxygen of thiazolidinone with backbone-amide hydrogen of ALA1046 and (ii) hydrogen of thiazolidinone ring nitrogen with side-chain amino hydrogen of GLU1204. These two H-bonding interactions ensure partial interaction of the molecules with S2 and S3 pocket residues that may be the reason for its reduced potency (Figure in Supplementary Material). Compound 19 exhibits interactions quite similar to compound 8. It establishes three

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H-bonding interactions: (i) carbonyl oxygen of thiazolidinone with backbone-amide hydrogen of ALA1046, (ii) hydrogen of thiazolidinone ring nitrogen with backbone carbonyl oxygen of ASN1011 and (iii) hydrogen of thiazolidinone 2-amino nitrogen with side-chain carbonyl oxygen of ASN1011. The H-bonding interaction with ASN1011 slightly bends the molecule

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away from the S2 pocket, possibly leading to its reduced potency (Figure in Supplementary Material). Compound 16 interacts with the protein quite differently than the other four (7, 8, 9 and 19) discussed earlier. It exhibits two H-bonding interactions: (i) carbonyl oxygen of thiazolidinone with backbone-amide hydrogen of CYS1013 and (ii) hydrogen of thiazolidinone

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2-amino nitrogen with backbone carbonyl oxygen of ASN1082. The hydrophobic interaction is largely confined to S1 pocket leading to 7-fold decreased potency in comparison to compound 19

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(Figure in Supplementary Material). Based on the foregoing discussion, it can be concluded that the hydrophobic interaction with S2 & S3 pocket and H-bonding interaction with TYR1047 are crucial for activity and potency of the molecule.

3. Conclusion

Twenty compounds were synthesized and tested for their antiviral activity against ChikV strain

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LR2006_OPY1 by CPE reduction assay. Five compounds (7, 8, 9, 16 and 19) were found to have antiviral activity at low micromolar concentration. Compound 7 (IC50=0.42µM) was found to be most potent amongst the five. Molecular docking studies revealed that the inhibitors maypossibly

4. Experimental

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act by inhibiting ChikV protease.

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Materials and methods: Chemicals

and

solvents

were

of

reagent

grade

and

purchased

from

Sigma-

Aldrich/Merck/CDH/Rankem. Completion of reaction was monitored on TLC plates (Merck). Melting points were determined on a OPTIMELT automated system apparatus and are uncorrected melting point. Intermediates were characterized by their FT-IR spectra (FTIR8400S-Schimadzu). Final compounds were characterized by their 1H-NMR, 300 MHz (Varion), 1

H and 13CNR (400 MHz) , in DMSO-d6 as a solvent. Mass spectra were recorded by WATERS-

Q-T of Premier-HAB213 using the ESI-MS Electro spray Ionization technique. Proton NMR

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Spectra the coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to the solvent. 4. 1. General method for the synthesis of aralkylidene derivatives of 2-sulfanylidene-1,3thiazolidin-4-one (1-12)

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To a solution of rhodanine (0.13 mmol) in ethanol (10 mL) was added aromatic/heteroaromatic aldehydes (0.13 mmol) followed by few drops of glacial acetic acid. The resulting mixture was refluxed for about 24 h. Upon cooling to room temperature,the desired compounds (1-12) were obtained as yellow or yellowish-orange solid. It was then filtered, washed, dried and

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recrystallized from hot ethanol. Yield 61-85 %

4.1.1. 5-[(4-hydroxyphenyl)-methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (1)

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Yield: 65%; mp: 308 °C (310 °C) [24]; 1HNMR (400 MHz, DMSO-d6): δ (ppm) 7.59-7.70 (appeared as multiplet, 3H, Ar-H),7.80 (s, 1H, Ar-CH=), 7.48 ( d, J=7.2 Hz, 2H, Ar-H),8.07 (t, J=6.4 Hz, J=8 Hz, 2H, Ar-H), 8.21 (s, 1H, Ar-H), 13.95 (s, 1H, Ar-OH);

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CNMR (400 MHz,

DMSO-d6): δ (ppm) 126.217-133.82 (Ar-6C, C5, Ar-CH=C8), 169.85 (C=O C4), 196.16 (C2); ESI-MS: 236.16 (M)+; calcd for C10H7NO2S2: 236.99.

4.1.2. 5-[(4-methoxyphenyl)-methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (2)

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Yield: 80%; mp: 260 °C (261-261 °C) [25]; 1HNMR (400 MHz, DMSO-d6): δ (ppm) 3.83 (s, 3H, -OCH3), 7.09-7.56 (appeared as multiplet, 5H, -Ar-H), 9.51 (s, 1H, -NH);

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CNMR (400 MHz,

DMSO-d6): δ (ppm) 56.00 (Ar-OCH3), 115.52-133.09 (Ar-6C), 161.74 (Ar-CH= C8), 170.22 (C=O C4), 196.12 (C2); ESI-MS: 252.0 (M-1)+; calcd for C11H9NO2S2:251.01.

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4.1.3. 5-[(4-chlorophenyl)-methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (3) Yield: 69%; mp: 230 °C (229-231 °C) [26]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.60-7.63 13

CNMR (400

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(appeared as multiplet, 4H, Ar-H),7.65 (s, 1H, Ar-CH=), 13.80 (s, 1H, -NH);

MHz, DMSO-d6): δ (ppm) 126.70-132.48 (Ar-5C), 135.80 (Ar-CH=C8), 169.74 (C=O, C4), 195.81 (C2); ESI-MS: 254.66 (M)+; calcd for C10H6ClNOS2: 254.96. 4.1.4. 5-([4-(dimethylamino)-phenyl]-methylidene)-2-sulfanylidene-1,3-thiazolidin-4-one (4) Yield: 85%; mp: 289 °C (285-289 °C) [25, 27, 28]; 1HNMR (Varian 400 MHz, DMSO-d6) δ (ppm) 3.03 (appeared as singlet, 6H, Ar-N(CH3)2), 6.83 (d, J=8.8 Hz, 2H, Ar-H), 7.43 (d, J=9.2 Hz, 2H, -Ar-H), 7.51 (s, 1H, Ar-CH=), 13.57 (s, 1H, -NH); ); 13CNMR (400 MHz, DMSO-d6): δ (ppm) 39.96-40.03 (Ar-N(CH3)2, merged with DMSO peak), 112.63-133.51 (Ar-5C), 152.17 (ArCH= C8), 170.19 (C=O C4), 195.61 (C2); ESI-MS: 265 (M+1)+; calcd for C12H12N2OS2: 264.04.

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4.1.5. 5-[(4-cyanophenyl)-methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (5) Yield: 75%; mp: 286 °C (286 °C) [29]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.57 (d, J=2.8 Hz, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.71 (d, J=8.4 Hz, 1H, Ar-H), 8.85 (s, 1H, Ar-CH=), 13.9 (s, 1H, -NH);

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CNMR (400 MHz, DMSO-d6): δ (ppm) 124.76-129.60 (Ar-4C), 136.75 (Ar-CN),

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151.12 (Ar-CH= C8), 152.13 (C5), 169.82 (C=O C4), 195.65 (C2); ESI-MS: 245.09 (M)+; calcd for C11H6N2OS2: 245.99.

4.1.6. 5-[(2-nitrophenyl)-methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (6)

Yield: 78%; mp: 203 °C (204-205 °C) [30]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.73

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(appeared as multiplet, J=6.8 Hz, 2H, Ar-H), 7.88 (d, J=8.8 Hz, 2H, Ar-H), 8.21 (d, J=8 Hz, 1H, Ar-CH=), 13.98 (s, 1H, -NH); 13CNMR (400 MHz, DMSO-d6): δ (ppm) 125.97-131.69 (Ar-6C),

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135.05 (C5), 148.40 (Ar-CH=C8), 169.07 (C=O C4), 196.22 (C2); ESI-MS: 265.09 (M)+; calcd for C10H6N2O3S2: 265.98.

4.1.7. 5-[(2-methylphenyl)-methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (7) Yield: 61%; mp: 200 °C (195-199 °C) [31]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 2.41 (s, 3H), 7.34-7.41 (appeared as multiplet, 4H, Ar-H), 7.73 (s, 1H, Ar-CH=), 13.86 (s, 1H, -NH); 13

CNMR (400 MHz, DMSO-d6): δ (ppm) 19.84 (Ar-CH3), 127.24-131.49 (Ar-6C), 132.48 (C5),

C11H9NOS2: 235.01.

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139.51 (Ar-CH= C8), 169.78 (C=O C4), 196.65 (C2); ESI-MS: 234.1 (M-1)+; calcd for

4.1.8. 5-[(4-methylphenyl)-methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one (8) Yield: 79%; mp: 235 °C (233-234 °C) [32]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 2.36 (s, 3H,

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-CH3), 7.36 (appeared as multiplet, 2H, Ar-H), 7.50 (appeared as multiplet, 2H, Ar-H), 7.62 (s, 1H, Ar-CH=), 13.8 (s, 1H, -NH);

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CNMR (400 MHz, DMSO-d6): δ (ppm) 21.57 (Ar-CH3),

124.69-131.00 (Ar-4C), 132.22 (C5), 141.62 (Ar-CH= C8), 169.83 (C=O C4), 196.07 (C2); ESI-

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MS: 235.9 (M+1)+; calcd for C11H9NOS2: 235.01. 4.1.9. 5-(naphthalen-2-yl-methylidene)-2-sulfanylidene-1,3-thiazolidin-4-one (9) Yield: 84%; mp: 270 °C (269-270 °C) [26]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.62-8.19 (appeared as multiplet, 7H, Ar-H), 8.23 (s, 1H, Ar-CH=), 13.90 (s, 1H, -NH);

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CNMR (400

MHz, DMSO-d6): δ (ppm) 123.73-131.52 (Ar-10C), 133.73 (C5), 172.00 (Ar-CH= C8), 172.44 (C=O C4), 197.99 (C2); ESI-MS: 272.0 (M+1)+ 4.1.10. 5-(thiophen-2-yl-methylidene)-2-sulfanylidene-1,3-thiazolidin-4-one (10)

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Yield: 81%; mp: 230 °C (232-233 °C) [33]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.31 (t, J=4.4 Hz, 1H, Ar-H), 7.72 (d, J=2.8 Hz, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 8.09 (d, J=4.8 Hz, 1H, Ar-CH=), 13.80 (s, 1H, -NH);

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CNMR (400 MHz, DMSO-d6): δ (ppm)123.43-34.80 (Ar-4C),

for C8H5NOS3: 226.95.

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135.87 (C5), 137.89 (Ar-CH=C8), 169.49 (C=O C4), 195.00 (C2); ESI-MS: 226.09 (M)+; calcd

4.1.11. 5-(pyridin-2-yl-methylidene)-2-sulfanylidene-1,3-thiazolidin-4-one (11)

Yield: 64%; mp: 294 °C (291-295 °C) [29, 34]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.44 (t, J=4.8 Hz, J=6 Hz, 1H, Ar-H), 7.68 (s, 1H, Ar-H), 7.89-7.97 (appeared as multiplet, 2H, Ar-H), 13

CNMR (400 MHz, DMSO-d6): δ (ppm)

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8.79 s, J=4.8 Hz, 1H, Ar-CH=), 13.92 (s, 1H, -NH);

124.42-130.04 (Ar-4C), 138.04 (C4), 149.96-151.53 (Ar-C, Ar-CH=C8), 169.79 (C=O C4),

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202.44 (C2); ESI-MS: 222.05 (M+1)+; calcd for C9H6N2OS2: 221.99.

4.1.12 5-(pyridin-3-yl-methylidene)-2-sulfanylidene-1,3-thiazolidin-4-one (12) Yield: 68%; mp: 270 °C (269-272 °C) [34, 35]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.69 (s, 1H, Ar-CH=), 7.78 (d, J=8 Hz, 2H, Ar-H), 7.91(d, J=8 Hz, 2H, Ar-H);

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CNMR (400 MHz,

DMSO-d6): δ (ppm) 112.63-133.51 (Ar-5C), 137.80 (Ar-CH=C8), 169.69 (C=O), 195.69 (C2);

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ESI-MS: 222.05 (M+1)+; calcd for C9H6N2OS2: 221.99.

4.2. General method for the synthesis of 5-aralkylidine derivatives of 2-amino-4, 5-dihydro1, 3-thiazole-4-one derivatives (13-20)

To a solution of pseudothiohydantoin (1.0 mmol) in ethanol was added an equimolar

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aromatic/heteroaromatic aldehydes, ammonium acetate and glacial acetic acid (5 mL). The resulting mixture was refluxed for about 24 h. Upon cooling to room temperature provided

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desired compounds (13-20). It was then filtered, washed, dried and recrystallized from hot ethanol. Yield 49-76 %

4.2.1. 2-amino-5-[(3-hydroxyphenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (13) Yield: 52%; mp: 268 °C; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 6.91 (d, 2H, Ar-H), 7.45 (d, 2H, Ar-H), 7.70 (s, 1H, Ar-CH=), 10.32 (s, 1H, -NH), 12.47 (s, 1H, -OH); 13CNMR (400 MHz, DMSO-d6): δ (ppm) 116.56-129.92 (Ar-5C), 131.89 (C5), 159.41 (Ar-C10), 175.85 (C2), 181.12 (C=O C4);

ESI-MS: 219.5 (M-1)+; calcd for C10H8N2O2S: 220. 4.2.2. 2-amino-5-[(4-hydroxyphenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (14)

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Yield: 66%; mp: 294 °C (295-297°C) [36]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 6.80 (dd, 1H, Ar-H), 6.97 (s, 1H, Ar-H), 7.05 (d, 1H, Ar-H) 7.30-7.38 (t, 1H, Ar-H), 7.69 (s, 1H, ArCH=), 9.85 (s, 1H, -NH), 12.61 (s, 1H, -OH); 13CNMR (400 MHz, DMSO-d6): δ (ppm) 115.82-129.65 (Ar-6C), 130.61 (C5), 135.71 (Ar-CH= C8), 158.26 (C12), 176.03 (C2), 180.78 (C=O C4); ESI-MS: 219.5

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(M-1)+; calcd for C10H8N2O2S: 220.

4.2.3. 2-amino-5-[(2, 4-dihydroxyphenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (15) Yield: 57%; mp: 325 °C; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 6.45 (appeared as multiplet, 2H, Ar-H), 7.16 (d, 1H, Ar-H), 7.97 (s, 1H, Ar-CH=), 10.18 (s, 1H, -NH), 10.46 (s, 1H, -OH),

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12.35 (s, 1H, -OH);ESI-MS: 235.9 (M)+calcd for C10H8N2O3S: 236.

4.2.4. 2-amino-5-[(2-nitrophenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (16)

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Yield: 76%; mp: 289 °C (291-292 °C) [36]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.73 (t, J = 8.0 Hz, 2H, Ar-H), 7.90 (t, 2H, Ar-H), 8.01 (s, 1H, Ar-CH=),8.21 (d, J = 2.0 Hz, 1H, ArH),12.80 (s, 1H, -NH); 13CNMR (400 MHz, DMSO-d6): δ (ppm) 123.38-131.17 (Ar-5C), 132.77 (C5), +

136.34 (Ar-CH= C8), 148.69 (Ar-C13), 175.50 (C2), 180.32 (C=O C4); ESI-MS: 248.5 (M-1) ; calcd for

C10H7N3O3S: 249.

4.2.5. 2-amino-5-[(3-nitrophenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (17)

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Yield: 56%; mp: 262°C (264°C) [37]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 7.80 (t,1H, Ar-H), 7.96 (s, 1H, Ar-H), 8.01 (d, 1H, Ar-H),8.30 (dd, 1H, Ar-H), 8.45 (s, 1H, Ar-CH=), 12.79 (s, 1H, -NH); 13CNMR (400 MHz, DMSO-d6): δ (ppm) 125.85-129.67 (Ar-5C), 131.43 (C5), 134.92 (Ar-CH= C8) +

148.33 (Ar-C10), 167.16 (C2), 168.09 (C=O C4); ESI-MS: 248.5 (M-1) ; calcd for C10H7N3O3S: 249.

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4.2.6. 2-amino-5-[(2-methylphenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (18) Yield: 67%; mp: 274 °C; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 2.40 (s, 3H, -CH3), 7.34 – 7.43

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(m, 4H, Ar-H), 7.89 (s, 1H, Ar-CH=),12.64 (s, 1H, -NH); 13CNMR (400 MHz, DMSO-d6): δ (ppm) 19.81 (Ar-CH3), 125.58-131.38 (Ar-6C), 132.57 (C5), 139.08 (Ar-CH= C8), 167.53 (C2), 168.55 (C=O C4);

ESI-MS: 218.5 (M-1)+; calcd for C11H10N2OS:218. 4.2.7. 2-amino-5-[(3-methylphenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (19) Yield: 51%; mp: 279°C; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 2.40 (s, 3H, -CH3), 7.20 (s, 1H, Ar-H), 7.40 (m, 3H, Ar-H), 7.53 (s, 1H, Ar-CH=), 9.16 (s, 1H, -NH); 13CNMR (400 MHz, DMSOd6): δ (ppm) 21.41 (Ar-CH3), 126.99-130.64 (Ar-6C), 134.53 (C5), 138.81 (Ar-CH= C8), 175.92 (C2), 180.74 +

(C=O C4); ESI-MS: 219.0 (M+1) ; calcd for C11H10N2OS: 218).

4.2.8. 2-amino-5-[(4-methylphenyl)methylidene]-4,5-dihydro-1,3-thiazol-4-one (20)

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Yield: 49%; mp: 280 °C (281-283 °C) [36]; 1HNMR (400 MHz, DMSO-d6) δ (ppm) 2.40 (s, 3H, -CH3), 7.30 (d, 2H, Ar-H), 7.41 (d,2H, Ar-H), 7.53 (s, 1H, Ar-CH=),9.25 (s, 1H, -NH); 13CNMR (400 MHz, DMSO-d6): δ (ppm)21.42 (Ar-CH3), 128.76-130.22 (Ar-6C), 131.73 (C5), 139.94 (Ar-CH= C8), +

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175.89 (C2), 180.86 (C=O C4); ESI-MS: 219.0 (M+1) ; calcd for C11H10N2OS: 218.

4. 3. Anti-ChikV and cell viability assay

Cells and virus strain used: ChikV strain LR2006_OPY1 (Genbank DQ443544.2) belongs to the collection of viruses at the UMR 190, Marseille, France. The virus was propagated

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in African green monkey kidney cells [Vero cells (ATCC CCL-81)]. Verocells were maintained in cell growth medium composed of minimum essential medium (MEM, Gibco, Belgium)

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supplemented with 7% Foetal Bovine Serum (FBS, Integro, The Netherlands), 1% L-glutamine (Gibco), and 100U/mL penicillin, 100 µg/mL streptomycin sulfate (Gibco). Cell cultures were then maintained at 37°C in an atmosphere of 5% CO2 and 95-99% humidity. All compounds were dissolved in dimethyl sulfoxide (DMSO) to reach a final concentration of 20 mg/mL. CPE reduction assay: Vero cells were seeded in 96-well tissue culture plates (Becton Dickinson, Aalst, Belgium) at a density of 5 × 104 cells/well in 100 µL assay medium

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supplemented with 25 µL of the appropriate virus inoculums (0.01 MOI of ChikV) and 25 µL of a compound dilution series. Each assay was performed in duplicate in the same test. On day 3 post-infection (p.i.), the plates were processed using the cell Titer-Blue method as described by the manufacturer (Promega, The Netherlands) to estimate the % of cell surviving. The 50%

viral CPE

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inhibitory concentration (IC50), defined as the compound concentration that is required to inhibit by 50%,

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for

each

compound. Potential

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cytotoxic/cytostatic effects of the compound were evaluated in uninfected cells by checking microscopically for minor signs of virus-induced CPE or alterations to the cells caused by the compound.

Cell viability determination: CellTiter-Blue is a fluorescent assay used to measure cell

viability via non-specific redox enzyme activity. Vero cells (ATCC CCL-81) (100 µL, 1 × 105 cells/mL) were seeded into a 96-well flat-bottomed plate and incubated for 24 h at 37°C with 5% CO2. The medium was replaced with increasing concentrations of target compounds (4 to 4000 µg/mL) and cells were incubated for 72h. Cell supernatant was then removed and replaced with CellTiter-Blue (70 µL) reagents and the plate was incubated for 2 h protected from light

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before recording fluorescence intensity (excitation 560 nm, emission 590 nm). Both assays were measured on a Tecan M200 multimode plate reader (Tecan Austria GmbH, Grödig, Austria). CellTiter-Glo reagent was added to the wells (50 µL and 50 µL media) and incubated at room temperature for 10 min protected from light. The luminescence was recorded using the same

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multimode plate reader. Blank wells (with no reagents) were also measured for luminescence and deducted from the values in experimental wells. Values of viability of treated cells were expressed as a percentage of that from corresponding control cells. All experiments were

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repeated at least three times. All assay kits were purchased from Promega, Southampton, UK.

4. 4. Molecular docking

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Molecular docking: The X-ray crystal structure of ChikV nsp2 protease (PDB: 3TRK) was obtained from the Protein Data Bank (www.rcsb.org). Protein preparation was done using PDB2PQR Server (http://nbcr-222.ucsd.edu/pdb2pqr_1.8/), which adds hydrogens, missing nonhydrogen atoms, assigns protonation states for functional groups of amino-acid sidechains,assigns charge and optimizes H-bonding networks [38, 39]. Ligand structure was sketched and prepared for docking using PRODRG server (http://davapc1.bioch.dundee.ac.uk/cgi-

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bin/prodrg) [40]. MGLTools-1.5.6 rc3 (The Scripps Research Institute) was used to prepare all the parameter files required for performing molecular docking using Autodock-4.2. Grid maps were generated using autogrid-4 (keeping Cys1013 and His1083 as center of the grid with all the parameters at default level except grid dimension of 60x60x60) and docking was performed with

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Autodock-4 (with all the parameters at default level exceptno.of GA runs at 50) using Autodock Tools implemented in MGLTools-1.5.6.rc3 [41]. Autodock output file (.dlg) was then analyzed

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through Analysis option provided in MGLTools-1.5.6 rc3. Top-scoring molecules in the largest cluster were analyzed. Complex (in .pdbqt format) of the docked conformer of the ligand (compound 7) with the protein was manually prepared and converted to .pdb format through pdbqt_to_pdb.py script and a 2D-plot was generated using Ligplot+ [42].

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Acknowledgment Authors gratefully acknowledge the financial support given by the Department of Biotechnology (DBT), Govt. of India, and the German Ministry of Education and Research /BMBF) as New Indigo-Era net grant (BT/IN/NewIndigo/14/DV/2010 dt. 4th Feb 2011). Work at

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Lübeck and Marseille was partly supported by the European Commission through its SILVER project ("Small-molecule Lead Compounds versus Neglected and Emerging RNA Viruses", contract no. HEALTH-F3-2010-260644).We are also thankful to Institute of Life Sciences,

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Hyderabad, AP, India for providing spectral data.

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References

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[34] M. Sortino, P. Delgado, S. Juarez, J. Quiroga, R. Abonia, B. Insuasty, M. Nogueras, L. Rodero, F.M. Garibotto, R.D. Enriz, S.A. Zacchino, Synthesis and antifungal activity of (Z)-5-arylidenerhodanines, Bioorg. Med. Chem., 15 (2007) 484-494. [35] V. Opletalova, J. Dolezel, K. Kralova, M. Pesko, J. Kunes, J. Jampilek, Synthesis and characterization of (Z)-5-(arylmethylidene)rhodanine derivatives with photosynthesis-inhibiting properties, Molecules, 16 (2011) 5207-5227. [36] J.F. Zhou, X.J. Sun, F.X. Zhu, Y.L. Li, G.X. Gong, A Facile Synthesis of 5-Arylidene-2-imino-4thiazolidinones Under Microwave Irradiation, Synthetic Communications, 38 (2008) 4182-4187. [37] H. Taniyama, T. Yusa, T. Tabuchi, H. Uchida, Chemotherapeutics for Mycobacterium tuberculosis. XI. Syntheses and antibacterial activity of 4-thiazolidinone derivatives containing α,β-unsaturated ketone group. 1, Yakugaku Zasshi, 76 (1956) 154-157. [38] T.J. Dolinsky, P. Czodrowski, H. Li, J.E. Nielsen, J.H. Jensen, G. Klebe, N.A. Baker, PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations, Nucleic Acids Research, 35 (2007) W522-W525. [39] T.J. Dolinsky, J.E. Nielsen, J.A. McCammon, N.A. Baker, PDB2PQR: an automated pipeline for the setup of Poisson–Boltzmann electrostatics calculations, Nucleic Acids Research, 32 (2004) W665-W667. [40] A.W. Schuttelkopf, D.M. van Aalten, PRODRG: a tool for high-throughput crystallography of proteinligand complexes, Acta crystallographica. Section D, Biological crystallography, 60 (2004) 1355-1363. [41] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility, Journal of computational chemistry, 30 (2009) 2785-2791. [42] A.C. Wallace, R.A. Laskowski, J.M. Thornton, LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions, Protein engineering, 8 (1995) 127-134.

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Figure caption:

Figure 1a. Interaction of compound 7 with ChikV nsp2 protease (PDB Code; 3TRK), ligand and

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amino-acid residues are shown as tubes colored by atom type, H-bonding interaction shown as green dots, VdW radii shown as wired sphere (Figure generated in MGLTools-1.5.6 rc3)

Figure 1b. 2-Dplot showing interaction of compound 7 with ChikV nsp2 protease (PDB Code;

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3TRK), H-bonding interaction shown as green broken lines and hydrophobic interaction as red

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spikes (Figure generated in Ligplot+)

Scheme 1.Reagents and conditions: (a) R-CHO, EtOH, AcOH, reflux, 24 h

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Scheme 2. Reagents and conditions: (a) R-CHO, EtOH, AcOH, NH4Ac, reflux, 24 h

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7 8

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9 10 11 12

IC50 (µM) CC50 (µM) ND >100 ND >100 ND >100 ND >100 ND >100 ND >100 0.42 2-CH3-C6H4 >100 (0.1 µg/mL) 4.2 4-CH3-C6H4 >100 (1.0 µg/mL) 3.6 C10H7 (napth-2-yl) >100 (1.0 µg/mL) C4H3S (thiophen-2-yl) ND >100 C5H4N (pyridine-2-yl) ND >100 C5H4N (pyridine-3-yl) ND >100

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R 4-OH-C6H4 4-OCH3-C6H4 4-Cl-C6H4 4-N(CH3)2-C6H4 4-CN-C6H4 2-NO2-C6H4

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Code 1 2 3 4 5 6

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Table 1. Antiviral activity of Compounds 1-12 against ChikV

ND-Not Determined (Not showing any activity at the maximum concentration

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studiedie. 100 µM)

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Table 2. Antiviral activity of Compounds 13-20against ChickV

IC50 (µM) CC50 (µM) ND >100 ND >100 ND >100 40.1 >100 16 2-NO2-C6H4 (10.0 µg/mL) ND >100 17 3-NO2-C6H4 ND >100 18 2-CH3-C6H4 6.8 >100 19 3-CH3-C6H4 (1.5 µg/mL) ND >100 20 4-CH3-C6H4 ND-not determined (Not showing any activity at the maximum concentration

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Code R 13 3-OH-C6H4 14 4-OH-C6H4 15 2,4-diOH-C6H3

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Figure 1a

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Figure 1b

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Scheme 1.

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Scheme 2.

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8 9 10 11 12

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IC50 (µM) CC50 (µM) ND >100 ND >100 ND >100 ND >100 ND >100 ND >100 0.42 2-CH3-C6H4 >100 (0.1 µg/mL) 4.2 4-CH3-C6H4 >100 (1.0 µg/mL) 3.6 C10H7 (napth-2-yl) >100 (1.0 µg/mL) C4H3S (thiophen-2-yl) ND >100 C5H4N (pyridine-2-yl) ND >100 C5H4N (pyridine-3-yl) ND >100

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R 4-OH-C6H4 4-OCH3-C6H4 4-Cl-C6H4 4-N(CH3)2-C6H4 4-CN-C6H4 2-NO2-C6H4

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Code 1 2 3 4 5 6

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Table 1. Antiviral activity of Compounds 1-12 against ChikV

ND-Not Determined (Not showing any activity at the maximum concentration

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IC50 (µM) CC50 (µM) ND >100 ND >100 ND >100 40.1 >100 16 2-NO2-C6H4 (10.0 µg/mL) ND >100 17 3-NO2-C6H4 ND >100 18 2-CH3-C6H4 6.8 >100 19 3-CH3-C6H4 (1.5 µg/mL) ND >100 20 4-CH3-C6H4 ND-not determined (Not showing any activity at the maximum concentration

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Code R 3-OH-C 13 6H4 14 4-OH-C6H4 15 2,4-diOH-C6H3

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Table 2. Antiviral activity of Compounds 13-20against ChickV

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studiedie. 100 µg/mL)

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Figure caption:

Figure 1a. Interaction of compound 7 with ChikV nsp2 protease (PDB Code; 3TRK), ligand and amino-acid residues are shown as tubes colored by atom type, H-bonding

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interaction shown as green dots, VdW radii shown as wired sphere (Figure generated in MGLTools-1.5.6 rc3)

Figure 1b. 2-Dplot showing interaction of compound 7 with ChikV nsp2 protease

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(PDB Code; 3TRK), H-bonding interaction shown as green broken lines and hydrophobic interaction as red spikes (Figure generated in Ligplot+)

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Scheme 1.Reagents and conditions: (a) R-CHO, EtOH, AcOH, reflux, 24 h

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Scheme 2. Reagents and conditions: (a) R-CHO, EtOH, AcOH, NH4Ac, reflux, 24 h

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Figure 1a

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Figure 1b

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Scheme 1.

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Aralkylidene derivatives of thiazolidinone (1-20) were screened for their anti-chikv activity Five compounds (7, 8, 9, 16 & 19) were found to be active at lower µ M concentrations Compounds with polar substitution in (hetero)aryl portion were found to be inactive Docking simulation suggests the inhibition of chikv nsp2 protease inhibition

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HNMR spectra of compound 1

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CNMR Spectra of Compound 1

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HNMR spectra of compound 2

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CNMR of compound 2

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HNMR spectra of compound 3

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CNMR Spectra of Compound 3

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HNMR spectra of compound 4

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CNMR of compound 4

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HNMR spectra of compound 5

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CNMR of compound 5

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HNMR spectra of compound 6

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CNMR Spectra of Compound 6

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SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 7

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 7

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 8

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 8

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 9

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 9

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 10

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR Spectra of Compound 10

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 11

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR Spectra of Compound 11

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 12

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR Spectra of Compound 12

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 2

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 3

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 4

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 6

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 8

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 9

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 11

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 13

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 13

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 14

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 14

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 15

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 16

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 16

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 17

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 17

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 18

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 18

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 19

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 19

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

1

HNMR spectra of compound 20

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

13

CNMR of compound 20

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 13

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 14

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 15

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 16

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 19

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ESI Mass spectra of compound 20

ACCEPTED MANUSCRIPT

Antiviral activity Concentration Vs % of cell survival Graph CHIKV ANTIVIRAL EFFECT OF COMPOUND 7 100 90

RI PT

70 60 50 40 30

SC

% OF CELL SURVIVING

80

20

M AN U

10 0 0

1

2

3

4

5 6 7 8 9 10 11 COMPOUND 7 CONCENTRATION µg/ml

12

13

14

15

16

12

13

14

15

16

CHIKV ANTIVIRAL EFFECT OF COMPOUND 8 100

TE D

90 80

60

EP

50 40 30

AC C

% OF CELL SURVIVING

70

20 10 0

0

1

2

3

4

5

6

7

8

9

10

11

COMPOUND 8 CONCENTRATION µg/ml

ACCEPTED MANUSCRIPT

CHIKV ANTIVIRAL EFFECT OF COMPOUND 9 100 90

RI PT

80 % OF CELL SURVIVING

70 60 50

SC

40 30

M AN U

20 10 0 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

12

13

14

15

16

COMPOUND 9 CONCENTRATION µg/ml

CHIKV ANTIVIRAL EFFECT OF COMPOUND 19

TE D

100 90 80

EP

70

% OF CELL SURVIVING

60

AC C

50 40 30 20 10 0 0

1

2

3

4

5

6

7

8

9

10

11

COMPOUND 19 CONCENTRATION µg/ml

ACCEPTED MANUSCRIPT

CHIKV ANTIVIRAL EFFECT OF COMPOUND 16 60

RI PT

40

30

SC

20

0 0

1

2

3

M AN U

10

4

5

6

EP

TE D

COMPOUND 16 CONCENTRATION µg/ml

AC C

% OF CELL SURVIVING

50

7

8

9

10

ACCEPTED MANUSCRIPT

Molecular docking snapshots Ala1010

Asn1082

RI PT

Asn1082

Trp1014

OD1

S2

S2

Asn1011

CG CB

C3

ND2

Cys1013

C3

Leu1205

CA

N

S1

C

N

3.10

CD1

C2 CB

CG OH

0070

O N

C4

3.09

OH

N

C4

2.73

C5

CD2 CE2

C11 C5

CA

Tyr1047

CB

CG

Ala1046

CD2 CE2

0080

O

Tyr1047 CZ

C1

CZ

C2 C1

O

CD1

CE1

C11

SC

CE1

Ala1046

CA

C

C

C10

C6

Cys1013

S1

N

C8

O

C6

C9

C7

O

C9 C7

Glu1204

C8

C10

Trp1084

Trp1084

M AN U

Tyr1079

Ser1048

3trk_prep

A

Asn1082 O C2

C13

Leu1205

Asn1011

O

C3

Leu1205 Leu1205

EP

CE1 CE1

O

NN

CD

CB

AC C

3trk_prep_3

C4

His1083

Ala1046 O

C5

CB

C11 C5

CC

N

CA

C4

Ala1046 C

C6

OE1

C9 C10

C6 C7

C8 CD OE2

CB

C9 C7 C8

C

Glu1204 C

Glu1204

C10

O

CG

CA N

CA

Trp1084

Tyr1079

C

0080 0070

C11 C10 C1

2.73 3.09

OO

Glu1204

CG

C8

C11 CA CA

Tyr1047

2.90

S1

C2

Tyr1047

CD2 CD2 CE2 CE2

Cys1013 Cys1013

S1

C9 C7 C2 C1

C12 O O

CB CB

CG CG

CZ OH OH

OE1

Trp1084

N

3.10

C

OE2

C3

N

C

CD1 CD1

Tyr1047 CZ

2.62 S1 C6

C13 CA N

CB

His1083

S2

CB

ND2

Ala1046

CA

S2

Asn1202

C14 C5

CG

N

C11 C10

C3

S2

2.90

C8

N

C1

C4

OD1

Cys1013

C9 C7

C12

Tyr1047

0090

Trp1014

2.62 S1 C6

Asn1082 C2

S2

Asn1202

C14 C5

Asn1082 O

Lys1045

C3

C1

C4

Asn1011 Ala1010

N

TE D

0090

Ser1048

B

Asn1011

Lys1045

Tyr1079

N

Tyr1079

D

Figure 1. 2-D plot of compound 7 (A), 8 (B), 9 (C) and their overlap (D). H-bonding interactions were shown in green broken lines, hydrophobic interactions were shown as spikes over aminoacid residues and ligand atoms. Common interacting residues are highlighted in red circle.

O

ACCEPTED MANUSCRIPT

ND2

OD1

Asn1082

Asn1082 CG

2.81

RI PT

N

CB

NAB

S2

Asn1011 Leu1205

C2 CB

CG

2.95

0070

O

C4

3.09

N

OH

OAC

C10

C6

C8

CAH

CAG

CA

CE2

C

CAM

3.09

N

CD2

C11 C5

CA

CAD

CAN

CB

CZ

Ala1046

CD2 CE2

0191

C

C9 C7

Glu1204 Trp1084

His1083

CAE

CAA

CAF

Ser1048

A

M AN U

Trp1084

Tyr1079

Tyr1079

Trp1014

B

ND2

OD1

OD1 ND2 N CG

2.81 CB

TE D

Asn1082 CG

N

NAB

Asn1011

CA

Leu1205 O

NAI

2.95 CE1 CE1

CD1 CD1

CAB OAI

CAM C4

EP

CC

His1083

0070

CAH

CAG

CA CA

Tyr1047

3.09 3.09

N

Ala1046

C11 C5

0191

His1083

2.99 CAC CAG

0161

CA

CAE

C6

SAD

SG

CAK

CAA C10

C8

Cys1013

OO

CAN C OAP

CB

CAF C9 C7

AC C

NAF

2.87 NAA CAE

Val1012

CAD

C1

NN

O

S1

C2

CD2 CE2 CD2

Asn1082

C

Cys1013

N

CAN

OAC O

CB CB

CZ OH CZ

CE2

SAJ C3

CAO

Tyr1047 CG CG

CA CB

S2

CAL

C

Glu1204

CAM

CAH

Ala1010

CAJ

O

CAL NAO

Trp1084

OAQ

Tyr1079

CAK

Asn1011

Lys1009

Trp1014 Ser1048

C

Ala1046

CAK

O

O

OH

Cys1013

CAO

Tyr1047 CG

C1

CZ

Tyr1047

CD1

CE1

SC

OH

S1

CD1

SAJ

NAI

O

N

CE1

CAL

C

Cys1013

C3

CA

Lys1016

D

Figure 2. 2D-plot of compound 7 (A), 19 (B), their overlap (C) and 16. H-bonding interactions were shown in green broken lines, hydrophobic interactions were shown as spikes over aminoacid residues and ligand atoms. Common interacting residues are highlighted in red circle.