Quinolidene-rhodanine conjugates: Facile synthesis and biological evaluation

Accepted Manuscript Quinolidene-rhodanine conjugates: Facile synthesis and biological evaluation Dnyaneshwar D. Subhedar, Mubarak H. Shaikh, Bapurao B. Shingate, Laxman Nawale, Dhiman Sarkar, Vijay M. Khedkar, Firoz A. Kalam Khan, Jaiprakash N. Sangshetti PII:

S0223-5234(16)30795-4

DOI:

10.1016/j.ejmech.2016.09.059

Reference:

EJMECH 8925

To appear in:

European Journal of Medicinal Chemistry

Received Date: 11 March 2016 Revised Date:

17 September 2016

Accepted Date: 19 September 2016

Please cite this article as: D.D. Subhedar, M.H. Shaikh, B.B. Shingate, L. Nawale, D. Sarkar, V.M. Khedkar, F.A. Kalam Khan, J.N. Sangshetti, Quinolidene-rhodanine conjugates: Facile synthesis and biological evaluation, European Journal of Medicinal Chemistry (2016), doi: 10.1016/ j.ejmech.2016.09.059. 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.

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Graphical Abstract Quinolidene-Rhodanine

Conjugates:

Facile

Synthesis

and

Biological

Evaluation

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Dnyaneshwar D. Subhedar, Mubarak H. Shaikh, Bapurao B. Shingate,* Laxman Nawale,

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Dhiman Sarkar, Vijay M. Khedkar, Firoz A. Kalam Khan, Jaiprakash N. Sangshetti,

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Quinolidene-Rhodanine

Conjugates:

Facile

Synthesis

and

Biological

Evaluation Dnyaneshwar D. Subhedar,a Mubarak H. Shaikh,a Bapurao B. Shingate,*a Laxman Nawale,b Dhiman Sarkar,b Vijay M. Khedkar,c Firoz A. Kalam Khan,d Jaiprakash N. Sangshettid

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a

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Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, India b Combi-Chem BioResource Center, Organic Chemistry Division, CSIR-National Chemical Laboratory, Pune 411 008, India c School of Health Science, University of KwaZulu Natal, Westville Campus, Durban 4000, South Africa d Department of Pharmaceutical Chemistry, Y. B. Chavan College of Pharmacy, Aurangabad 431 001, India E-mail:* [email protected] (B. B. Shingate) *Corresponding author. Tel.: (91)-240-2403312; Fax: (91)-240-2403113 Abstract:

A series of rhodanine incorporated quinoline derivatives were efficiently synthesized using reusable DBU acetate as ionic liquid and evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Ra (MTB) (ATCC 25177) and Mycobacterium bovis

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BCG (ATCC 35743) both in active and dormant state. Compounds 3e, 3f, 3g, 3h and 3i exhibited very good antitubercular activity. The active compounds were studied for cytotoxicity against HUVEC, THP-1, macrophages, A549, PANC-1 and HeLa cell lines using modified MTT assay and were found to be noncytotoxic. Inactivity of all these compounds against Gram

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positive and Gram negative bacteria indicates their specificity towards the MTB. Further, the synthesized compounds have been screened for their in vitro antifungal activity. In addition, the

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molecular docking studies revealed the binding modes of these compounds in active site of Zmp1 enzyme, which in turn helped to establish a structural basis of inhibition of mycobacteria. The results of present study clearly indicate the identification of some novel, selective and specific inhibitors against MTB that can be explored further for potential antitubercular drug. Keywords: Antitubercular, Antibacterial activity, Antifungal, Cytotoxicity, Molecular docking study, [DBUH][OAc] ionic liquid.

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1.

Introduction

Bacterial infections are a major comprehensive health problem accounting for nearly 15 million deaths every year, numerous as of drug-resistant pathogenic agents, with a noteworthy figure of cases arising in emerging countries. The rebirth of tuberculosis (TB) has been convoyed by the

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rapid spread of multi-drug resistance TB (MDR-TB) resulting from MTB strains that were able to successfully resist the killing effect of all these TB drugs during treatment. Presently, less than 5% of nearby 0.5 million MDR-TB cases predictable universally are appropriately detected and treated since in part of the lengthy assay turnaround time accompanying with culture-based drug

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exposure analysis. It is reported that MTB exists in various physiological stages, such as replicating and non-replicating stage in human lung. It has been realized that multidrug

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combination is essential to target all MTB populations in demand to treatment of patients and reduce relapse rates. Presently, the drugs which are used for the first line treatment of drug sensitive to MTB are isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin [1]. Compounds bearing 2-thioxothiazolidin-4-one (rhodanine), as an imperious privileged scaffold, with a wide spectrum of biological activities such as antitubercular [2a-b], Zmp1 inhibitors [2c] (ZTB19, ZTB20, ZTB23, Figure 1), antifungal [3], anti-inflammatory [4], HIV

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inhibitors [5], anticancer [6], antibacterial [7], antidiabetic [8], inhibitors of histone acetyltransferases [9], Bcl-2 [10], PDE4 [11], JNK stimulating phosphatase-1 (JSP-1) [12], aldose reductase [13], ADAMTS-5 [14], trypanosoma brucei dolicholphosphate mannose synthase (DPMS) [15], photosynthesis [16], anthrax lethal factor protease [17], fungal protein

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mannosyl transferase-1 [18], protease [19], β-lactamase [20], HCV NS3 protease [21] inhibitor. Also, displays phosphate reductoisomerase (DXR) [22], staphylococcus aureus DNA gyrase

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[23], tau aggregation [24], dengue virus protease [25], chikungunya virus [26], MurD Ligase [27] and UDP-N-acetylmuramate/L-alanine ligase [28]. A significant number of compounds bearing a quinoline moiety have been reported in the literature with a variety of pharmacological activities, including anticancer [29], antimalarial [30], antibiotic [31], histamine H3 receptor antagonist [32], anti-inflammatory [33], anti-HIV [34], tyrosine kinase inhibitors [35] and antitubercular activity [36]. Some of the representive antitubercular compounds A, B and C bearing quinoline moiety as shown in Figure 1.

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(Insert Figure 1) The Knoevenagel condensation between aldehyde and rhodanine has been performed using pyridine-ethanol [37a], piperidine-ethanol-acetic acid [37b], CH3COONa-AcOH [37c],

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NH4OAc-AcOH [37d], NH4OAc [37e] and NH4OAc-toluene [37f]. Ionic liquids (ILs) have attracted interest as environmentally benign media for catalytic applications because of their unique chemical and physical properties [38]. Thus, ILs are considered to be a safer alternative to original organic solvents as they are cleaner and safer to use and reuse [39]. A variety of ionic

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liquids have been extensively applied in heterocyclic synthesis as solvent or catalysts [40]. The ionic liquid DBU acetate [DBUH][OAc] [41] have been mainly used for organic transformations including phospha-michael reaction [42a], diaryl thioethers [42b], synthesis of hydroxyl [42c],

1H-pyrazolo[1,2-b]phthalazine-5,10-diones

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naphthalene-1,4-diones

[42d],

triazolyl

spirocyclic oxindoles [42e], 3,4,5-trisubstituted oxazolones [42f], carbonylation of ophenylenediamines [42g] and dihydropyrano[3,2-c]quinolines via knoevenagel condensation reaction [42h].

In continuation of our work on the development of new synthetic methodologies using ionic liquids such as [Et3NH] [HSO4] and [HDBU] [HSO4] for the rhodanine-arylidene conjugates via

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knoevenagel condensation [43] and synthesis, bioevaluation of various compounds [44], herein we would like to report the synthesis of rhodanine incorporated quinoline derivatives via Knoevenagel condensation using DBU acetate as a catalyst under solvent free condition in high yields. In addition to this, the synthesized compounds were screened for antibacterial,

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antitubercular, antifungal and cytotoxic activity. Moreover, the molecular docking study against Zmp1 enzyme which revealed a significant correlation between the binding score and biological

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activity for this quinoline-rhodanine conjugates. 2. Results and Discussion 2.1. Chemistry

The rhodanine incorporated quinoline derivatives 3a-i, 4a-i and 5a-i were synthesized by DBU acetate catalyzed Knoevenagel condensation of various quinoline aldehydes 1a-i with rhodanines 2a-c in good yields. The required starting material, quinoline aldehydes 1a-i were prepared [45] from corresponding anilines by acylation followed by Vilsmeier-Haack formylation at 100 ºC for 16 h. The synthesis of rhodanines [46] 2a-c has been achieved by the reaction of respective

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amines with carbon disulfide in ammonia followed by sodium chloroacetate at 80 °C in good yield. In search of the best experimental reaction conditions for the preparation of (Z)-5-((2chloroquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one 3a, the Knoevenagel reaction between

model reaction (Scheme 1).

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(Insert Scheme 1)

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2-chloro-3-formylquinoline 1a and 2-thioxothiazolidin-4-one (rhodanine) 2a was selected as a

To evaluate the effect of solvent, model reaction was performed using DBU acetate in

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various solvents like polar protic (EtOH, MeOH, tert-BuOH and H2O), polar aprotic (THF, CH3CN and DMSO) and non polar (Toluene). Reactions in EtOH, MeOH and tert-BuOH resulted in moderate yields of product 3a (60, 67 and 69%, respectively, Table 1). H2O and THF produces very trace amount of product. Reaction in CH3CN, DMSO and toluene gave 48, 52 and 50% product, respectively. Considering the increasing importance of solvent-free reactions in

absence of solvent.

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organic synthesis, our attempt was to examine the catalytic efficiency of DBU acetate in the

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(Insert Table 1)

In the next, the effect of temperature was studied on the model reaction at different

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temperatures under solvent-free conditions (Table 1, entries 9-13) and the best results were obtained at 80 °C (Table 1, entry 11). To examine the catalyst efficiency, the model reaction was tested with 5, 10, 15, 20 and 25 mol% of the catalyst at 80 °C (Supporting information, Table S1). The maximum yield (90%) of the reaction was obtained in 30 min with 20 mol% of the catalyst. The DBU acetate was recovered and reused for four times without any appreciable decrease in the yield (Supporting information, Table S2). The structure of the synthesized compound 3a has been confirmed by 1H NMR,

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and HRMS spectral data. 1H NMR spectrum clearly indicates the formation of compound 3a as it shows a singlet at δ 7.75 and 12.57 ppm due to olefinic proton and -NH proton, respectively. 4

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While, in the 13C NMR spectrum of compound 3a, two characteristics carbon signals exhibited at δ 162.2 and 196.5 ppm due to the -N-C=O and -S-C=S groups, respectively, which confirms the presence of rhodanine ring. Further, the formation of compound 3a has been confirmed by HRMS spectrum. The calculated [M+Na]+ is 328.9688, and observed at 328.9632. The Z-

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configuration of the exocyclic C=C bond in compound 3a was assigned on the basis of chemical shift of vinylic proton, deshielded by the adjacent C=O, was detected at δ 7.75 ppm in 1H NMR spectra as observed for analogous 5-arylidene-2,4-thiazolidinediones [37c, 47a]. In E-isomers due to the lesser deshielding effect of 1-S, such a proton should resonate at lower chemical shift

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values [47b,c].

To explore the scope and efficiency of Knoevenagel condensation, the optimized reaction

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conditions, 20 mol% DBU acetate at 80 °C were used for the construction of a library of rhodanine incorporated quinoline derivatives 3a-i, 4a-i and 5a-i from wide range of group at the various position of 2-chloro-3-formyl quinoline 1a-i and rhodanine derivatives 2a-c (Scheme 2). The present reaction conditions are well suited for all the 2-chloro-3-formyl quinoline derivatives and resulted in good yields of product. Similarly, the synthesized rhodanine incorporated

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quinoline conjugates 3b-i, 4a-i and 5a-i were also confirmed by physical and spectral analysis.

(Insert Scheme 2)

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2.2. Biological activity

2.2.1. Antibacterial activity

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Specificity of all the synthesized compounds 3a-i, 4a-i and 5a-i for mycobacteria was evaluated for their in vitro activity against Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtillus). The preliminary antibacterial screening of these compounds was checked at the concentrations of 30, 10 and 3 µg/mL. However, all the compounds were found to show very poor or no activity up to 30 µg/mL, against all the tested bacteria except for 3f and 3h (Table 2). The results of antibacterial activity are summarized in Tables S3 and S4 of the supporting information. The compound 3f showed MIC value 7.86 µg/mL against S. aureus and 3h showed antibacterial activity against E. coli (MIC = 2.44 µg/mL), P. fluorescens (MIC = 2.8 µg/mL), S. aureus (MIC = 2.55 µg/mL) and 5

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B. subtillus (MIC = 9.33 µg/mL). As these compounds do not shown any significant antibacterial activities, we have extended our study for evaluation of antitubercular activity.

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(Insert Table 2)

2.2.2. Antitubercular activity

The synthesized rhodanine incorporated quinoline derivatives were screened against MTB

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H37Ra and M. bovis BCG using two-fold dilution technique, in order to determine the actual minimum inhibitory concentration (MIC) using an established XRMA antitubercular screening

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protocol [48]. Rifampicin was used as reference drug. In a preliminary screening, quinoline derivatives 3a-i, 4a-i and 5a-i were tested at concentrations of 30, 10 and 3 µg/mL. Compounds 3b, 3e, 3f, 3g, 3h, 3i and 4b exhibited higher antimycobacterial potency, inhibiting >90% of mycobacterial growth at 30 µg/mL, which strengthens the fact of the antimicrobial nature of quinoline derivatives (Supporting Information, Table S5-S9). Dose dependent tests of the most active conjugates 3b, 3e, 3f, 3g, 3h and 3i at concentrations from 100 to 0.78 µg/mL against

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dormant MTB, active MTB, dormant BCG and active BCG resulted in 3.5-18.0 µg/mL (dormant MTB, except 3e), 2.8-21.1 µg/mL (active MTB), 1.6-9.5 µg/mL (dormant BCG) and 0.9-6.5 µg/mL (active BCG) respectively.

The presence of a -OMe, -OEt, -Cl and -F group on the quinoline ring was found to

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substantially increase the anti-TB activity in dormant and active stage of BCG/MTB. Thioxothiazolidin-4-ones with free NH group (TTZ) exhibited higher potencies in comparison to

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the thioxothiazolidin-3-yl acetic acid (TTZAA) and thioxothiazolidin-3-yl propanoic acid (TTZPA), which are functionalized at the -NH group of thioxothiazolidin-4-one. In BCG and MTB, TTZAA, TTZPA compounds with -H, -Me, -OEt, -F and -Cl groups exhibited less potency. It was observed that the position and nature of the electron withdrawing substitution on the aromatic ring largely influenced the activity, after considering the results for the active compounds 3b, 3e, 3f, 3g, 3h and 3i.

(Insert Table 3)

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2.2.3. Structure Activity Relationship (SAR) According to the data, the presence of TTZ scaffold with quinoline displays significant antitubercular activity (Table 3). The hybrid molecules derived from TTZ and 2-chloro-3formyl-quinoline has been used as basic scaffold for the development of structural analogues,

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which produces the TTZ derivatives 3a-i, 4a-i and 5a-i. The results of the biological evaluation reveal that the activity was considerably affected by introducing a various substituents on quinoline ring and the homologation of -CH2 group in TTZ scaffold (Table 3). From the compounds 3a-i, compound 3b (R1 = -methyl and TTZ) showed good antitubercular activity with

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MIC value 12.9 µg/mL against dormant MTB H37Ra (Table 3). Compounds 3f, (R3 = -methoxy and TTZ) and 3g in which (R3 = -ethoxy and TTZ) showed prominent antitubercular activity other synthesized compounds.

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against dormant MTB H37Ra with MIC values 4.7 and 3.5 µg/mL, respectively as compared to

The compounds 3h (R3 = -chloro and TTZ) and 3i (R3 = -fluoro and TTZ) with MIC = 6.3 µg/mL and 18.0 µg/mL resp. showed promising antitubercular activity against dormant MTB H37Ra. The other compounds from the series 3a-i do not display significant antitubercular activity against dormant MTB H37Ra (MIC >30 µg/mL). The N-substitution of TTZ in

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compounds 4a-i (TTZAA) and 5a-i (TTZPA) decreased the antitubercular activity against dormant MTB H37Ra (MIC values >30 µg/mL, except 4b 19.9 µg/mL). Hence, among all the synthesized compounds 3a-i, 4a-i and 5a-i. The derivatives 3b, 3f, 3g, 3h, 3i and 4b showed excellent antitubercular activity against dormant MTB H37Ra (Table 3).

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From the series 3a-i, compound 3b (R1 = -methyl and TTZ) showed good antitubercular activity with MIC value 18.2 µg/mL against active MTB H37Ra (Table 3). Compound 3e (R2 =

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-methoxy and TTZ) showed moderate antitubercular activity against active MTB H37Ra with MIC value 21.1 µg/mL. Compound 3f (R3 = -methoxy and TTZ) shows better antitubercular activity against active MTB H37Ra with MIC value 14.3 µg/mL. Among all the synthesized compounds, 3g and 3i (R3 = -ethoxy, -fluoro and TTZ) showed excellent antitubercular activity with MIC values 2.8 against active MTB H37Ra. Compound 3h (R3 = -chloro and TTZ) shows good antitubercular activity against active MTB H37Ra with MIC value 3.8 µg/mL. The remaining compounds in the 3a-i series does not display significant antitubercular activity against active MTB H37Ra with MIC values >30 µg/mL. From the compounds 4a-i and 5a-i, having TTZAA and TTZPA moieties decreases the antitubercular activity against active MTB 7

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H37Ra with MIC values >30 µg/mL. Hence, among all the synthesized compounds the only 3g, 3h and 3i compounds showed excellent antitubercular activity against active MTB H37Ra (Table 3). From the series 3a-i, compound 3b (R1 = -methyl and TTZ), 3e (R2 = -methoxy and TTZ), 3f

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(R3 = -methoxy and TTZ), 3g (R3 = -ethoxy and TTZ) and 3h (R3 = -chloro and TTZ) showed good to excellent antitubercular activity with MIC values 9.5, 1.6, 6.8, 1.9 and 2.2 µg/mL, respectively against dormant M. bovis BCG strain. Compound 3i (R3 = -fluoro and TTZ) showed promising antitubercular activity against dormant M. bovis BCG with MIC value 5.2 µg/mL. The

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remaining compounds in the 3a-i series does not display significant antitubercular activity against dormant M. bovis BCG with MIC values >30 µg/mL. From the series 4a-i and 5a-i, with

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TTZAA and TTZPA, decreases the antitubercular activity against dormant M. bovis BCG with MIC values >30 µg/mL. Hence, among all the synthesized derivatives (3a-i, 4a-i and 5a-i), compounds 3b, 3e, 3f, 3g, 3h and 3i showed excellent antitubercular activity against dormant M. bovis BCG.

The synthesized compounds 3a-i, particularly 3b, 3e, 3f, 3g, 3h and 3i with MIC values 6.5, 2.9, 5.0, 0.9, 1.7 and 4.2 µg/mL, respectively, shows good antitubercular activity against active

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M. bovis BCG strain (Table 3). The remaining compounds of the series 3a-i, 4a-i and 5a-i do not show any significant antitubercular activity against the active M. bovis BCG strain (Table 3).

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2.2.4. Antifungal activity

The minimum inhibitory concentrations (MIC) were determined using the standard agar method

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[49]. Miconazole was used as a standard drug for the comparison of antifungal activity. Dimethyl sulfoxide was used as solvent control. MIC values of the tested compounds are presented in Table 2. Many of the synthesized compounds showed good to moderate antifungal activity. From the antifungal activity data, it is observed that, from the series 3a-i, compounds 3g and 3i are the most active among all the tested compounds. Compound 3g was found to be particularly active against the fungal strain A. niger with MIC value 25 µg/mL compared with standard drug miconazole. Similarly, compound 3i exhibited excellent antifungal activity against fungal strain F. oxysporum, A. niger, C. neoformans (MIC value 25 µg/mL) and A. flavus (MIC value 12.5 µg/mL). Again, from the series 4a-i, compound 4i found to be most potent antifungal agent. 8

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Compound 4i (MIC value 25 µg/mL) showed antifungal activity particularly against the fungal strain F. oxysporum, A. niger and C. neoformans. From the series 5a-i, compound 5c and 5i showed potent antifungal activity. Compound 5c (MIC value 25 µg/mL) found to be most potent

active against fungal strain A. flavus, A. niger and C. neoformans.

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against the fungal strain C. albicans. Similarly, compound 5i (MIC value 25 µg/mL) found to be

The results of the antifungal screening demonstrates that, the compounds 3g, 3i, 4i, 5c and 5i showed excellent activity against various fungal strains, which could be credited by the presence of ethoxy, fluoro- and methyl groups on the quinoline ring, which can serve as an

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important scaffold for the design and development of new antifungal agents.

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2.2.5. Cytotoxic activity

The synthesized quinoline-rhodanine derivatives 3b, 3e, 3f, 3g, 3h, 3i, 4b and 4i were further evaluated for their cytotoxic activity. The cytotoxic effect of lead compounds 3b, 3e, 3f, 3g, 3h, 3i, 4b and 4i were checked at the concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.7813 µg/mL to determine the GI50. Compounds 3b, 3e, 3f, 3g, 3h, 3i, 4b and 4i did not show any cytotoxicity up to 100 µg/mL on all the cells/cell lines used. The viability of HUVEC cells

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was not significantly affected (<30% inhibition) at 100 µg/mL (Table 4). Also, all compounds have very less (<35% inhibition) cytotoxic effect on A549, PANC-1 and HeLa cells (SI, Table S10). As an in vitro model to differentiate between antitubercular and side-effects, we have tested the cytotoxic effects on the viability of PMA (phorbol myristate acetate)-differentiated

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human THP-1 macrophages. Overall the GI50 (>100 µg/mL) values indicates that the compounds

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are potent and specific inhibitors against MTB.

(Insert Table 4)

2.2.6. Selectivity Index

The Table 4 observations interpret that, the screened compounds (3b, 3f, 3g, 3h, 3i, 4b) exhibited no mutagenic effect in the MTT cytotoxicity assay when HUVEC, THP-1, Macrophage, A549, PANC-1 and HeLa cells were exposed to the test compounds. Selective activities of these compounds towards human cell line against MTB are described via selectivity 9

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index. Selectivity index reflects the quantity of compound that exhibits good antimicrobial activity which can selectively kill microorganisms without being significantly toxic to host cells. Higher selectivity index signifies that compound can be used as therapeutic agent and can be a useful tool in evaluating the potential toxic effect of compounds in vitro. GI50 of all compounds

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against the tested cell lines was >100 µg/ml. It was found that compounds 3f, 3g and 3h have selectivity index of >15 at dormant state of MTB. The compounds 3b, 3e, 3f, 3g, 3h, 3i showed >10 SI index, which are actually good inhibitors of M. bovis BCG. According to Hartkoorn, antimycobacterial activity can be classified as significant if SI is >10 [50]. An improved in vitro

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efficacy (Table 3) and relatively high selectivity profile (Table 5) of compounds 3f, 3g and 3h indicates that these compounds may be useful as antitubercular agent and should be investigated

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further.

(Insert Table 5)

3.

Computational Study

3.1. Molecular docking

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Recently, the rhodanine derivatives have been reported to inhibit Zinc-dependent metalloprotease 1 (Zmp1) enzyme [2c]. Zmp1 a member of the M13 endopeptidase family [51a] has been proposed to play a key role during phagosome maturation and therefore, can be proposed as a potential therapeutic target against TB. Based on these reports, we have selected

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Zmp1 enzyme of MTB (PDB ID:3ZUK) [51b] as target for molecular docking study for the active compounds 3b, 3e, 3f, 3g, 3h, 3i, 4b and 4i to get insight into possible mechanism of

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action for antimycobacterial activity. In silico approaches like molecular docking have become very beneficial to identify the potential targets for different ligands and the associated thermodynamic interactions with the target enzyme governing the inhibition of the target microorganism especially in the absence of available resources to carry out the enzyme-based experimental studies.

Visual inspection of the ensuing docked structures show that all these rhodanine incorporated quinoline derivatives could snugly fit into the active site of Zmp1 enzyme with varying degree of affinities and their complexation was stabilized by formation of several steric and electrostatic interactions. Their docking scores ranged from -9.72 to -4.22 with a statistically 10

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significant harmony between the experimental antitubercular activity and the molecular docking scores wherein the active compounds showed higher docking scores while those with relatively low inhibition were also predicted to have a lower score. The more negative value of docking score indicates a good binding affinity of the ligand towards the target and vice versa. Even the

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minimum energy for the formation of complex between ligand and receptor (Glide energy) for each of these analogues was observed to be negative ranging from -42.06 to -26.20 kcal/mol, which as well suggests these molecules could serve as a pertinent starting point for the rational design of drugs targeting Zmp1. A detailed per-residue interaction analysis between the residue

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lining the active site of the enzyme and these quinoline derivatives was carried out to identify the most significantly interacting the residues and the type thermodynamic elements (bonded and

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non-bonded interactions) governing the binding of these molecules to the target. However for the sake of brevity we have elucidated this analysis only for one of the most active analog 3g and compared against the least active 4i. These results have been analyzed on the basis of glide score, glide energy, bonded and non-bonded interactions. The intermolecular interaction energy values obtained from the docking calculation were given in supporting information (Table S11). Results of the binding study have been analyzed and based on three main parameters-Glide score, Glide

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energy and the bonded and non-bonded interactions (van der Waals/steric and electrostatic). Considering these parameters the binding affinity of docked compounds towards Zmp1enzyme has been discussed.

The lowest energy docked conformation for the most active compound 3g into the active

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site of Zmp1 showed that the inhibitor binds at the same site as the native ligand with very

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favorable interactions (Figure 2).

(Insert Figure 2)

Its docking score was found to be -9.72 with an overall binding energy of -42.06 kcal/mol. The per-residue interaction analysis showed that the van der Waals interactions (-36.12 kcal/mol) surpassed the electrostatic contribution (-5.93 kcal/mol) in the overall binding affinity. The higher binding affinity demonstrated by 3g can be explained in terms of the specific bonded and non-bonded per residue interactions with the residues lining the active site of Zmp1. The 11

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compound is stabilized within the active site through an extensive network of significant van der Waals interactions with Arg628 (-1.66 kcal/mol), His622 (-6.54 kcal/mol), Asp620 (-3.52 kcal/mol), Asn452 (-1.42 kcal/mol) and Phe484 (-2.17 kcal/mol) through the rhodanine scaffold. Similarly, the 2-chloro-6-ethoxyquinolinine component is also engaged in favorable van der

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Waals interactions with Pro625 (-2.96 kcal/mol), Pro624 (-1.74 kcal/mol), Ser623 (-1.85 kcal/mol), Leu617 (-1.32 kcal/mol), Arg616 (-5.85 kcal/mol), Ala613 (-1.32 kcal/mol), Thr606 (1.67 kcal/mol) and 604 (-1.55 kcal/mol). The enhanced binding affinity of 3g can also be attributed to favorable electrostatic contacts observed with Arg628 (-1.37 kcal/mol), Asp620 (-

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3.24 kcal/mol), Asn452 (-1.42 kcal/mol), Ser623 (-1.84 kcal/mol) residues lining the active site. The enzyme-inhibitor complex was further stabilized by a close π-π stacking interactions

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observed between the quinoline ring and Pro625 residue. Furthermore, a crucial hydrogen bonding was also observed between the oxygen of rhodanine and Arg628 side chain. These type of hydrogen-bonding and the π-π stacking interactions serve as an "anchor", intensely determining the 3D orientation of the ligand in the active site. A similar binding mode and network of interactions was observed for the 3b, 3e, 3f, 3h and 3i (Figure 3 and SI, Table S11)

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as well but decreasing gradually with their observed anti-tubercular activity.

(Insert Figure 3)

The results obtained for the docking of the relatively less active analogues 4i and 4b showed that

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though they could as well occupy the same site as the native ligand but there binding orientation deviated marginally from other active analogues (Figure 4). This difference in binding

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orientation resulted in marked reduction their binding affinity to the target which could also be the reason for their relatively lower antitubercular activity. The overall binding energy of compound 4i (-26.20 kcal/mol) and 4b (-30.51 kcal/mol) was also found to be lower than 3g. This difference in binding affinity can be further explained in terms of the specific bonded and non-bonded per residue interactions observed with the residues in the active site.

(Insert Figure 4)

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Analysis of the best docked conformation of 4i (Figure 5) showed that it could as well form favorable van der Waals interactions with Pro625 (-1.01 kcal/mol), His622 (-1.11 kcal/mol), Arg616 (-1.05 kcal/mol), Thr606 (-1.01 kcal/mol) and Phe48 (-1.35 kcal/mol) through the rhodanine scaffold while substituted quinoline showed a similar type of interactions with Arg628

Ala453 (-1.17 kcal/mol) and Asn452 (-1.04 kcal/mol).

SC

(Insert Figure 5)

RI PT

(-1.00 kcal/mol), Trp604 (-1.06 kcal/mol), Glu494 (-1.13 kcal/mol), Val490 (-1.34 kcal/mol),

M AN U

It was further stabilized within the active site through a network of favorable electrostatic interactions observed with Arg628 (-1.04 kcal/mol), Pro625 (-1.10 kcal/mol), Arg616 (-1.29 kcal/mol), Thr606 (-1.42 kcal/mol), Asn452 (-1.01 kcal/mol) and Phe48 (-1.14 kcal/mol). A similar pattern of interactions were observed for 4b as well (Figure 6). The relatively weaker strength of these per-residue interactions rationalizes the lower binding affinity of 4i and 4b into

TE D

the active site of Zmp1 over 3g.

(Insert Figure 6)

EP

Furthermore, the hydrogen bonding interaction through Arg628 residue conserved in all the active analogues was also found to be missing in 4i and 4b. However, both these molecules did portray a hydrogen bonding interactions through Thr606 (2.050Å) and Arg616 (1.779Å),

AC C

respectively, which could assist in the anchoring of these compounds into the active site. Similarly, the π-π stacking interactions observed between the quinoline ring and Pro625 residue for the active analogues was replaced by His493 in case of 4i and 4b. Therefore considering binding pattern predicted by docking along with the detailed per-residue interaction analysis, compound 3g could be optimized using structure-based drug design approach to arrive at a potent antitubercular agent targeting Zmp1 enzyme.

13

ACCEPTED MANUSCRIPT

4. Conclusions In summary, we have developed a simple, efficient and environmentally benign method for the synthesis of rhodanine incorporated quinoline derivatives and evaluated for their in vitro antimycobacterial potency against MTB H37Ra and M. bovis BCG. Among all the tested

RI PT

compounds, 3b, 3e, 3f, 3g, 3h, 3i and 4b were identified as the most active compounds with MIC ranging from 3.5-19.9 µg/mL against MTB H37Ra. Compounds 3b, 3e, 3f, 3g, 3h and 3i exhibited significant activity against M. bovis BCG with MIC value 1.66-9.57 µg/mL. The synthesized compounds were evaluated for their cytotoxic effect against HUVEC, THP-1,

SC

Macrophage, A549, Panc-1 and HeLa cell lines and with the help of which, selectivity indexes for most active compounds 3b, 3e, 3f, 3g, 3h and 3i were calculated to determine their safety for

M AN U

further screening. Compounds 3i, 4i and 5i showed potential antifungal activity as compared to synthesized compound. Further, molecular docking study of the synthesized rhodanine incorporated quinoline derivatives has a high affinity towards the active site of Zinc-dependant metalloprotease 1 (Zmp1) enzyme of MTB provides a strong platform form for structure-based optimization of this scaffold.

TE D

5. Experimental section

All the reagents were purchased from Merck and Sigma Aldrich and used without further purification. Melting points of all the synthesized compounds were determined in open capillary tube and are uncorrected. 1H NMR and

13

C NMR spectra were recorded on a Bruker DRX-400

EP

MHz NMR spectrometer in DMSO-d6. High-resolution mass spectra (HRMS) were recorded on Agilent 6520 (QTOF) ESI-HRMS instrument and MALDI-TOF mass spectrometer. The purity

AC C

of each of the compound was checked by thin-layer chromatography (TLC) using silica-gel, (60F254) and visualization was accomplished by iodine/ultraviolet light.

5.1.

Typical

procedure

for

synthesis

of

(Z)-5-((2-chloroquinolin-3-yl)methylene)-2-

thioxothiazolidin-4-one (3a) A mixture of 2-chloro-3-formyl quinoline 1a (1 mmol), rhodanine 2a (1 mmol) and ionic liquid DBU acetate (20 mol %) was heated at 80 ºC. The progress of the reaction was monitored by thin layer chromatography (n-Hexane/EtOAc 9:1). After heating the reaction mass for 30 min, it was allowed to cool at room temperature. Then, ice cold water was added and whole reaction mass 14

ACCEPTED MANUSCRIPT

was stirred for 10 min. The obtained solid product was separated by filtration. The filtrate contains mixture of water and ionic liquid. The water was removed under reduced pressure (Temperature of bath: 50-55 °C, Vacuum: 1 torr) and then the trace amount of water was removed by keeping the flask under vacuum pump for 2-3 h (760 mmHg). The residual ionic

crude product 3a was recrystallized using the DMF-EtOH.

5.2. Spectral data for compounds (3a-i, 4a-i and 5a-i)

RI PT

liquid was reused to study its catalytic activity in subsequent runs for the synthesis of 3a. The

SC

5.2.1. (Z)-5-((2-chloroquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one

(3a)

[37c]:

The

compound 3a was obtained via Knoevenagel condensation reaction between 2-chloroquinoline3-carbaldehyde 1a and 2-thioxothiazolidin-4-one 2a in 30 min as yellow solid; Mp >300 °C;

M AN U

Yield: 90%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.57 (s, 1H, -NH), 8.84 (s, 1H, Ar-H), 7.88 (d, 1H, J = 8 Hz, Ar-H), 7.84 (d, 1H, J = 8 Hz, Ar-H), 7.75 (s, 1H), 7.54 (m, 1H, Ar-H), and 7.50 (m, 1H, Ar-H); 13C NMR (100 MHz, DMSO-d6, δ ppm): 196.5, 162.2, 148.2, 140.7, 134.6, 130.5, 129.2, 127.6, 126.0, 125.6, 121.0, 116.9 and 114.9; HRMS (ESI-qTOF): Calcd for

TE D

C13H7ClN2OS2Na [M+Na]+, 328.9624: found: 328.9632.

5.2.2. (Z)-5-((2-chloro-8-methylquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one (3b) [37c]: The compound 3b was obtained via Knoevenagel condensation reaction between 2-chloro-8methylquinoline-3-carbaldehyde 1b and 2-thioxothiazolidin-4-one 2a in 30 min as red solid; Mp

EP

>300 °C; Yield: 86%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.28 (s, 1H, -NH), 8.50 (s, 1H, Ar-H), 8.22 (s, 1H), 8.06 (d, 1H, J = 8 Hz, Ar-H), 8.03 (m, 1H, Ar-H), 7.25 (d, 1H, J = 8 Hz, Ar-

AC C

H) and 2.34 (s, CH3); 13C NMR (100 MHz, DMSO-d6, δ ppm): 185.5, 160.3, 149.7, 149.2, 143.8, 143.4, 129.6, 129.5, 129.4, 127.4, 125.7, 124.4, 124.1 and 22.4; HRMS (ESI-qTOF): Calcd for C14H10ClN2OS2N [M+H]+, 320.9845: found: 320.9851. 5.2.3. (Z)-5-((2-chloro-7-methylquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one (3c) [37c]: The compound 3c was obtained via Knoevenagel condensation reaction between 2-chloro-7methylquinoline-3-carbaldehyde 1c and 2-thioxothiazolidin-4-one 2a in 30 min as red solid; Mp >300 °C; Yield: 84%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.58 (s, 1H, -NH), 8.90 (s, 1H, Ar-H), 8.46 (s, 1H, Ar-H), 8.30 (s, 1H), 8.14 (d, 1H, J = 8 Hz, Ar-H), 7.43 (d, 1H, J = 8 Hz, Ar15

ACCEPTED MANUSCRIPT

H) and 2.68 (s, CH3); 13C NMR (100 MHz, DMSO-d6, δ ppm): 188.5, 172.0, 137.5, 135.9, 132.3, 129.0, 128.1, 126.4, 126.0, 125.9, 124.3, 122.2 and 21.7; HRMS (ESI-qTOF): Calcd for C14H9ClN2OS2N [M+Na]+, 342.9840: found: 342.9819.

RI PT

5.2.4. (Z)-5-((2-chloro-6-methylquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one (3d) [37c]: The compound 3d was obtained via Knoevenagel condensation reaction between 2-chloro-6methylquinoline-3-carbaldehyde 1d and 2-thioxothiazolidin-4-one 2a in 30 min as yellow solid; Mp >300 °C; Yield: 85%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.54 (s, 1H, -NH), 8.45 (s, Ar-H) and 2.70 (s, 3H, CH3);

13

SC

1H, Ar-H), 8.27 (s, 1H, Ar-H), 7.92 (s, 1H), 7.76 (d, 1H, J = 8 Hz, Ar-H), 7.69 (d, 1H, J = 8 Hz, C NMR (100 MHz, DMSO-d6, δ ppm): 198.1, 161.0, 145.6,

141.5, 139.7, 133.3, 132.2, 130.8, 129.0, 125.2, 124.2, 118.4, 114.3 and 20.3; HRMS (ESI-

M AN U

qTOF): Calcd for C14H10ClN2OS2N [M+H]+, 320.9807: found: 320.9812.

5.2.5. (Z)-5-((2-chloro-7-methoxyquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one (3e): The compound 3e was obtained via Knoevenagel condensation reaction between 2-chloro-7methoxyquinoline-3-carbaldehyde 1e and 2-thioxothiazolidin-4-one 2a in 30 min as yellow

TE D

solid; Mp >300 °C; Yield: 88%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 8.25 (s, 1H, Ar-H), 7.66 (s, 1H), 7.04-7.00 (m, 2H, Ar-H), 6.89 (s, 1H, Ar-H) and 3.92 (s, 3H, OCH3);

13

C NMR

(100 MHz, DMSO-d6, δ ppm): 199.4, 165.2, 161.6, 153.4, 144.5, 140.7, 132.9, 127.1, 125.9, 125.6, 119.8, 115.1, 105.8 and 56.6; HRMS (ESI-qTOF): Calcd for C14H10ClN2O2S2 [M+H]+,

EP

336.9736: found: 336.9741.

AC C

5.2.6. (Z)-5-((2-chloro-6-methoxyquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one (3f) [37c]: The compound 3f was obtained via Knoevenagel condensation reaction between 2-chloro-6methoxyquinoline-3-carbaldehyde 1f and 2-thioxothiazolidin-4-one 2a in 30 min as red solid; Mp >300 °C; Yield: 82%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.86 (s, 1H, -NH), 8.01 (s, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.50 (s, 1H), 7.30 (d, 1H, J = 8 Hz, Ar-H), 7.25 (d, 1H, J = 8 Hz, Ar-H) and 3.61 (s, 3H, OCH3);

C NMR (100 MHz, DMSO-d6, δ ppm): 197.8, 169.2, 162.6,

13

156.2, 137.0, 135.7, 132.6, 129.0, 126.07, 126.04, 124.0, 122.3, 121.8 and 55.2; HRMS (ESIqTOF): Calcd for C14H10ClN2O2S2 [M+H]+, 336.9754: found: 336.9760.

16

ACCEPTED MANUSCRIPT

5.2.7. (Z)-5-((2-chloro-6-ethoxyquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one (3g): The compound 3g was obtained via Knoevenagel condensation reaction between 2-chloro-6ethoxyquinoline-3-carbaldehyde 1g and 2-thioxothiazolidin-4-one 2a in 30 min as red solid; Mp >300 °C; Yield: 88%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 7.90 (s, 1H, Ar-H), 7.75-7.74 (d,

RI PT

1H, J = 4 Hz, Ar-H), 7.44-7.42 (d, 1H, J = 8 Hz, Ar-H), 7.20 (s, 1H), 6.90 (s, 1H, Ar-H), 4.05 (q, 2H, J = 8 Hz, OCH2) and 1.41 (t, 3H, J = 8 Hz, CH3); 13C NMR (100 MHz, DMSO-d6, δ ppm): 199.2, 170.0, 161.1, 156.2, 145.9, 141.9, 134.2, 128.8, 128.4, 127.6, 126.8, 126.5, 117.8, 61.3

5.2.8. (Z)-5-((2,

SC

and 16.4; HRMS (ESI-qTOF): Calcd for C15H12ClN2O2S2 [M+H]+, 350.9950: found: 350.9947. 6-dichloroquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one

(3h):

The

M AN U

compound 3h was obtained via Knoevenagel condensation reaction between 2,6dichloroquinoline-3-carbaldehyde 1h and 2-thioxothiazolidin-4-one 2a in 30 min as yellow solid; Mp >300 °C; Yield: 90%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.53 (s, 1H, -NH), 8.38 (s, 1H, Ar-H), 7.79 (s, 1H), 7.52-7.49 (m, 2H, Ar-H) and 7.15 (s, 1H, Ar-H); 13C NMR (100 MHz, DMSO-d6, δ ppm): 196.3, 168.6, 157.5, 143.0, 136.9, 133.6, 127.9, 126.4, 125.0, 123.2, 120.2, 117.6 and 111.8; HRMS (ESI-qTOF): Calcd for C13H7Cl2N2OS2 [M+H]+, 340.9256.: found:

TE D

340.9244.

5.2.9. (Z)-5-((2-chloro-6-fluoroquinolin-3-yl)methylene)-2-thioxothiazolidin-4-one

(3i):

The

compound 3i was obtained via Knoevenagel condensation reaction between 2-chloro-6-

EP

fluoroquinoline-3-carbaldehyde 1i and 2-thioxothiazolidin-4-one 2a in 30 min as yellow solid; Mp >300 °C; Yield: 80%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.41 (s, 1H, -NH), 8.68 (s, 1H, Ar-H), 7.84 (s, 1H), 7.75 (s, 1H, Ar-H) and 7.55-7.50 (m, 2H, Ar-H); 13C NMR (100 MHz,

AC C

DMSO-d6, δ ppm): 196.7, 161.7, 161.7, 160.2, 149.6, 149.1, 143.1, 139.6, 130.8, 130.7, 129.5, 129.3, 125.1, 124.0, 116.1, 115.9 and 115.5;

19

F NMR (471 MHz, DMSO-d6, δ ppm): -117.11;

HRMS (ESI-qTOF): Calcd for C13H7ClFN2OS2 [M+H]+, 324.9544: found: 324.9568. 5.2.10. (Z)-2-(5-((2-chloroquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic

acid

(4a) [37c]: The compound 4a was obtained via Knoevenagel condensation reaction between 2chloroquinoline-3-carbaldehyde 1a and 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid 2b in 30 min as yellow solid; Mp >300 °C; Yield: 90%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.20 (s, 17

ACCEPTED MANUSCRIPT

1H, COOH), 8.34 (s, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 7.75-7.69 (m, 2H, Ar-H), 7.14-7.09 (m, 2H, Ar-H) and 4.64 (s, 2H, CH2);

13

C NMR (100 MHz, DMSO-d6, δ ppm): 196.7, 166.7, 163.3,

155.1, 144.6, 139.3, 135.2, 133.5, 129.5, 126.0, 125.9, 124.2, 120.0, 111.0 and 45.4; HRMS

RI PT

(ESI-qTOF): Calcd for C15H10ClN2O3S2 [M+H]+, 364.9712: found: 364.9718.

5.2.11. (Z)-2-(5-((2-chloro-8-methylquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-

yl)acetic acid (4b) [37c]: The compound 4b was obtained via Knoevenagel condensation reaction

between

2-chloro-8-methylquinoline-3-carbaldehyde

1b

and

2-(4-oxo-2-

SC

thioxothiazolidin-3-yl)acetic acid 2b in 30 min as yellow solid; Mp >300 °C; Yield: 84%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.21 (s, 1H, -COOH), 8.41 (s, 1H), 7.78 (m, 1H, Ar-H), 7.71 (d, 1H, J = 8 Hz, Ar-H), 7.66 (d, 1H, J = 8 Hz, Ar-H), 7.36 (s, 1H), 4.37 (s, 2H, CH2) and 13

C NMR (100 MHz, DMSO-d6, δ ppm): 197.9, 168.8, 166.0, 160.7, 147.4,

M AN U

2.75 (s, 3H, CH3);

138.6, 133.5, 130.4, 128.4, 125.8, 123.2, 122.6, 121.9, 118.5, 45.9 and 17.9; HRMS (ESI-qTOF): Calcd for C16H12ClN2O3S2 [M+H]+, 378.9956: found: 378.9973.

5.2.12. (Z)-2-(5-((2-chloro-7-methylquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-

TE D

yl)acetic acid (4c): The compound 4c was obtained via Knoevenagel condensation reaction between 2-chloro-7-methylquinoline-3-carbaldehyde 1c and 2-(4-oxo-2-thioxothiazolidin-3yl)acetic acid 2b in 30 min as yellow solid; Mp >300 °C; Yield: 80%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 8.54 (s, 1H, Ar-H), 8.19 (d, 1H, J = 4 Hz, Ar-H), 7.94 (s, 1H), 7.79 (s, 1H, 13

C NMR (100

EP

Ar-H), 7.59 (d, 1H, J= 4 Hz, Ar-H), 4.54 (s, 2H, CH2) and 2.55 (s, 3H, CH3);

MHz, DMSO-d6, δ ppm): 196.4, 169.1, 165.4, 158.8, 137.2, 136.1, 132.2, 128.6, 125.7, 125.7, 124.5, 124.3, 123.6, 121.9, 45.7 and 18.7; MALDI-TOF (ms): Calcd for C16H12ClN2O3S2

AC C

[M+H]+, 378.9967: found: 378.9982.

5.2.13. (Z)-2-(5-((2-chloro-6-methylquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)acetic acid (4d) [37c]: The compound 4d was obtained via Knoevenagel condensation reaction

between

2-chloro-6-methylquinoline-3-carbaldehyde

1d

and

2-(4-oxo-2-

thioxothiazolidin-3-yl)acetic acid 2b in 30 min as yellow solid; Mp >300 °C; Yield: 80%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.50 (s, 1H, -COOH), 8.37 (s, 1H, Ar-H), 7.56 (s, 1H), 7.47 (d, 1H, J = 8 Hz, Ar-H), 7.35 (d, 1H, J = 8 Hz, Ar-H), 7.29 (s, 1H, Ar-H), 4.75 (s, 2H, CH2) 18

ACCEPTED MANUSCRIPT

and 2.72 (s, 3H, CH3);

13

C NMR (100 MHz, DMSO-d6, δ ppm): 198.0, 168.4, 167.6, 160.3,

146.7, 139.6, 137.0, 135.7, 130.1, 128.4, 127.7, 123.2, 120.2, 116.1, 47.2 and 19.7; HRMS (ESIqTOF): Calcd for C16H11ClN2O3S2 [M+Na]+, 400.2163: found: 400.2131.

RI PT

5.2.14. (Z)-2-(5-((2-chloro-7-methoxyquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)acetic acid (4e): The compound 4e was obtained via Knoevenagel condensation reaction between 2-chloro-7-methoxyquinoline-3-carbaldehyde 1e and 2-(4-oxo-2-thioxothiazolidin-3yl)acetic acid 2b in 30 min as red solid; Mp >300 °C; Yield: 90%; 1H NMR (400 MHz, DMSO-

SC

d6, δ ppm): 12.16 (s, 1H, -COOH), 8.35 (s, 1H, Ar-H), 7.75 (s, 1H), 7.72 (s, 1H, Ar-H), 6.91 (d, 1H, J = 8 Hz, Ar-H), 6.83 (d, 1H, J = 8 Hz, Ar-H), 4.72 (s, 2H, CH2) and 3.86 (s, 3H, OCH3); C NMR (100 MHz, DMSO-d6, δ ppm): 196.9, 169.0, 161.1, 155.7, 145.1, 136.7, 132.8, 126.1,

M AN U

13

124.6, 123.0, 117.8, 114.8, 107.8, 55.2 and 45.9; HRMS (ESI-qTOF): Calcd for C16H12ClN2O4S2 [M+H]+, 394.9849: found: 394.9861.

5.2.15. (Z)-2-(5-((2-chloro-6-methoxyquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)acetic acid (4f) [37c]: The compound 4f was obtained via Knoevenagel condensation reaction

TE D

between 2-chloro-6-methoxyquinoline-3-carbaldehyde 1f and 2-(4-oxo-2-thioxothiazolidin-3yl)acetic acid 2b in 30 min as red solid; Mp >300 °C; Yield: 88%; 1H NMR (400 MHz, DMSOd6, δ ppm): 12.60 (s, 1H, -COOH), 8.95 (s, 1H, Ar-H), 8.52 (s, 1H, Ar-H), 8.37 (s, 1H), 8.17 (d, 1H, J = 8 Hz, Ar-H), 7.44 (d, 1H, J = 8 Hz, Ar-H), 4.88 (s, 2H, -CH2) and 3.86 (s, 3H, -OCH3); C NMR (100 MHz, DMSO-d6, δ ppm): 188.7, 166.0, 162.2, 143.2, 139.6, 133.5, 130.6, 129.5,

EP

13

128.5, 125.4, 125.1, 124.5, 123.1, 55.0 and 48.8; MALDI-TOF (ms): Calcd for C16H12ClN2O4S2

AC C

[M+H]+, 394.9835: found: 394.9813.

5.2.16. (Z)-2-(5-((2-chloro-6-ethoxyquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)acetic acid (4g): The compound 4g was obtained via Knoevenagel condensation reaction between 2-chloro-6-ethoxyquinoline-3-carbaldehyde 1g and 2-(4-oxo-2-thioxothiazolidin-3yl)acetic acid 2b in 30 min as red solid; Mp. >300 °C; Yield: 85%; 1H NMR (400 MHz, DMSOd6, δ ppm): 12.50 (s, 1H, -COOH), 8.33 (s, 1H, Ar-H), 7.82 (s, 1H), 7.74 (d, 1H, J = 8 Hz, Ar-H), 7.39 (d, 1H, J = 8 Hz, Ar-H), 7.37 (s, 1H, Ar-H), 5.06 (q, 2H, J = 8 Hz, O-CH2), 4.17 (s, 2H, CH2), 1.41 (t, 3H, J = 8 Hz, CH3); 13C NMR (100 MHz, DMSO-d6, δ ppm): 198.9, 169.5, 164.6, 19

ACCEPTED MANUSCRIPT

160.1, 147.3, 139.9, 134.0, 133.2, 130.6, 128.0, 126.5, 122.0, 120.8, 115.7, 62.7, 46.6 and 15.4; HRMS (ESI-qTOF): Calcd for C17H14ClN2O4S2 [M+H]+, 409.0043: found: 409.2665. 5.2.17. (Z)-2-(5-((2,6-dichloroquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic

RI PT

acid (4h): The compound 4h was obtained via Knoevenagel condensation reaction between 2,6dichloroquinoline-3-carbaldehyde 1h and 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid 2b in 30 min as yellow solid; Mp >300 °C; Yield: 91%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.33 (s, 1H, -COOH), 8.06 (s, 1H, Ar-H), 7.33 (s, 1H), 7.28 (s, 1H, Ar-H), 7.16 (d, 1H, J = 4 Hz, Ar-H),

SC

7.14 (d, 1H, J = 4 Hz, Ar-H), 4.83 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6, δ ppm): 194.4, 165.9, 158.9, 153.3, 145.3, 135.6, 131.2, 129.7, 126.9, 126.5, 121.9, 120.9, 117.5, 111.1 and

M AN U

46.2; HRMS (ESI-qTOF): Calcd for C15H8Cl2N2O3S2Na [M+Na]+, 420.9368: found: 420.9383. 5.2.18. (Z)-2-(5-((2-chloro-6-fluoroquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)acetic acid (4i): The compound 4i was obtained via Knoevenagel condensation reaction between 2-chloro-6-fluoroquinoline-3-carbaldehyde 1i and 2-(4-oxo-2-thioxothiazolidin-3yl)acetic acid 2b in 30 min as red solid; Mp >300 °C; Yield: 80%; 1H NMR (400 MHz, DMSO-

TE D

d6, δ ppm): 12.47 (s, 1H, -COOH), 8.50 (s, 1H, Ar-H), 7.91 (s, 1H), 7.80 (d, 1H, J = 8 Hz, Ar-H), 7.29 (s, 1H, Ar-H), 7.21 (d, 1H, J = 8 Hz, Ar-H), 4.84 (s, 2H, CH2);

13

C NMR (100 MHz,

DMSO-d6, δ ppm): 194.4, 171.6, 161.8, 160.2, 150.7, 149.2, 147.6, 143.7, 143.3, 141.0, 129.4, 129.3, 127.3, 125.6, 124.3, 121.9 and 45.6;

19

F NMR (471 MHz, DMSO-d6, δ ppm): -116.12;

EP

HRMS (ESI-qTOF): Calcd for C15H9ClFN2O3S2 [M+H]+, 382.9649: found: 382.9639. 5.2.19. (Z)-3-(5-((2-chloroquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)propanoic

AC C

acid (5a): The compound 5a was obtained via Knoevenagel condensation reaction between 2chloroquinoline-3-carbaldehyde 1a and 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid 2c in 30 min as yellow solid; Mp >300 °C; Yield: 86%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 11.76 (s, 1H, -COOH), 7.99 (s, 1H, Ar-H), 7.63 (m, 1H, Ar-H) 7.54 (s, 1H), 7.43 (m, 1H, Ar-H), 6.986.96 (m, 2H, Ar-H), 4.87 (t, 2H, J = 8 Hz, CH2) and 2.23 (t, 2H, J = 8 Hz, CH2); 13C NMR (100 MHz, DMSO-d6, δ ppm): 195.2, 170.0, 162.8, 150.6, 149.0, 144.6, 140.9, 133.5, 131.3, 129.1, 127.4, 125.2, 122.1, 118.2, 45.3 and 31.9; HRMS (ESI-qTOF): Calcd for C16H11ClN2O3S2 [M+K]+, 416.2179: found: 416.2147.

20

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5.2.20. (Z)-3-(5-((2-chloro-8-methylquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)propanoic acid (5b): The compound 5b was obtained via Knoevenagel condensation reaction between 2-chloro-8-methylquinoline-3-carbaldehyde 1b and 3-(4-oxo-2-thioxothiazolidin-3yl)propanoic acid 2c in 30 min as yellow solid; Mp >300 °C; Yield: 86%; 1H NMR (400 MHz,

RI PT

DMSO-d6, δ ppm): 12.21 (s, 1H, -COOH), 8.35 (s, 1H, Ar-H), 7.97 (s, 1H, Ar-H), 7.77 (s, 1H), 7.72 (d, 1H, J = 8 Hz, Ar-H), 7.16-7.11 (m, 1H, Ar-H), 4.64 (t, 2H, J = 8 Hz, -CH2), 2.62 (t, 2H, J = 8 Hz, -CH2) and 2.42 (s, -CH3); 13C NMR (100 MHz, DMSO-d6, δ ppm): 186.4, 172.7, 169.0, 145.2, 141.0, 136.8, 132.9, 129.2, 127.8, 126.5, 125.8, 123.8, 122.4, 121.6, 46.0, 33.6 and 20.0;

SC

MALDI-TOF (ms): Calcd for C17H13ClN2O3S2Na [M+Na]+, 415.0072: found: 415.0191.

M AN U

5.2.21. (Z)-3-(5-((2-chloro-7-methylquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)propanoic acid (5c): The compound 5c was obtained via Knoevenagel condensation reaction between 2-chloro-7-methylquinoline-3-carbaldehyde 1c and 3-(4-oxo-2-thioxothiazolidin-3yl)propanoic acid 2c in 30 min as yellow solid; Mp >300 °C; Yield: 84%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 8.52 (s, 1H, Ar-H), 8.18 (d, 1H, J = 4 Hz, Ar-H), 7.92 (s, 1H, Ar-H), 7.77 (s, 1H), 7.57 (d, 2H, J = 8 Hz, Ar-H), 4.52 (t, 2H, J = 4 Hz, -CH2), 2.62 (t, 2H, J = 8 Hz, -CH2) and

TE D

2.48 (s, 3H, -CH3); 13C NMR (100 MHz, DMSO-d6, δ ppm): 187.8, 174.8, 168.4, 146.0, 140.9, 137.0, 136.5, 132.3, 130.9, 130.8, 127.4, 126.6, 124.9, 48.0, 34.0 and 21.0; MALDI-TOF (ms): Calcd for C17H13ClN2O3S2K [M+K]+, 431.0067: found: 431.0972.

EP

5.2.22. (Z)-3-(5-((2-chloro-6-methylquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)propanoic acid (5d): The compound 5d was obtained via Knoevenagel condensation reaction between 2-chloro-6-methylquinoline-3-carbaldehyde 1d and 3-(4-oxo-2-thioxothiazolidin-3-

AC C

yl)propanoic acid 2c in 30 min as yellow solid; Mp; >300 °C; Yield: 82%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.35 (s, 1H, -COOH), 8.28 (s, 1H, Ar-H), 7.89 (d, 1H, J = 8 Hz, Ar-H), 7.81 (s, 2H), 7.17 (d, 1H, J = 8 Hz, Ar-H), 6.72 (s, 1H, Ar-H), 4.39 (t, 2H, J = 8 Hz CH2), 2.74 (t, 2H, J = 8 Hz CH2), 2.40 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6, δ ppm): 192.1, 175.0, 168.9, 151.2, 145.8, 141.5, 139.7, 132.2, 130.2, 129.0, 127.1, 125.2, 124.2, 123.4, 45.3, 32.8 and 20.3; HRMS (ESI-qTOF): Calcd for C17H13ClN2O3S2Na [M+Na]+, 415.0075: found: 415.0059. 5.2.23. (Z)-3-(5-((2-chloro-7-methoxyquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)propanoic acid (5e): The compound 5e was obtained via Knoevenagel condensation reaction 21

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between 2-chloro-7-methoxyquinoline-3-carbaldehyde 1e and 3-(4-oxo-2-thioxothiazolidin-3yl)propanoic acid 2c in 30 min as yellow solid; Mp >300 °C; Yield: 87%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 11.97 (s, 1H, -COOH), 8.61 (s, 1H, Ar-H), 8.27 (s, 1H, Ar-H), 8.21 (s, 1H), 7.95 (d, 1H, J = 8 Hz, Ar-H), 7.78 (d, 1H, J = 8 Hz, Ar-H), 4.81 (t, 2H, J = 8 Hz, -CH2), 3.85 (s,

RI PT

3H, -OCH3) and 2.73 (t, 2H, J = 8 Hz, -CH2); 13C NMR (100 MHz, DMSO-d6, δ ppm): 188.4, 173.7, 168.4, 146.2, 140.9, 137.0, 135.7, 132.6, 129.0, 126.1, 126.0, 124.0, 122.3, 121.8, 55.4, 47.3 and 32.7; HRMS (ESI-qTOF): Calcd for C17H13ClN2O4S2 [M+Na]+, 431.0073: found:

SC

431.0986.

5.2.24. (Z)-3-(5-((2-chloro-6-methoxyquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)propanoic acid (5f): The compound 5f was obtained via Knoevenagel condensation reaction

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between 2-chloro-6-methoxyquinoline-3-carbaldehyde 1f and 3-(4-oxo-2-thioxothiazolidin-3yl)propanoic acid 2c in 30 min as yellow solid; Mp >300 °C; Yield: 89%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.33 (s, 1H, -COOH), 7.42 (s, 1H, Ar-H), 6.95 (s, 1H), 6.70 (d, 1H, J = 8 Hz, Ar-H), 6.61 (d, 1H, J = 8 Hz, Ar-H), 6.58 (s, 1H, Ar-H), 4.51 (t, 2H, J = 8 Hz, CH2), 3.58 (s, 3H, OCH3) and 2.35 (t, 2H, J = 8 Hz, CH2);

13

C NMR (100 MHz, DMSO-d6, δ ppm): 193.9,

174.5, 168.9, 153.5, 145.3, 139.2, 133.2, 132.2, 130.9, 129.0, 125.2, 124.2, 123.4, 116.8, 55.8,

409.2383.

TE D

45.5 and 32.7; HRMS (ESI-qTOF): Calcd for C17H14ClN2O4S2 [M+H]+, 409.3528: found:

5.2.25. (Z)-3-(5-((2-chloro-6-ethoxyquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-

EP

yl)propanoic acid (5g): The compound 5g was obtained via Knoevenagel condensation reaction between 2-chloro-6-ethoxyquinoline-3-carbaldehyde 1g and 3-(4-oxo-2-thioxothiazolidin-3-

AC C

yl)propanoic acid 2c in 30 min as red solid: Mp >300 °C; Yield: 88%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 13.71 (s, 1H, -COOH), 8.66 (s, 1H, Ar-H), 8.46 (d, 1H, J = 8 Hz, Ar-H), 8.28 (d, 1H, J = 8 Hz, Ar-H), 8.14 (s, 1H, Ar-H), 7.61 (s, 1H), 4.95 (t, 2H, J = 8 Hz, CH2), 4.23 (q, 2H, J = 8 Hz, -OCH2), 2.66 (t, 2H, J = 8 Hz, CH2) and 1.38 (t, 3H, J = 8 Hz, CH3);

13

C NMR

(100 MHz, DMSO-d6, δ ppm): 194.1, 168.1, 157.2, 149.6, 148.8, 139.9, 138.9, 137.0, 129.7, 128.8, 127.6, 125.0, 123.4, 120.2, 65.8, 44.5, 29.7 and 16.9; HRMS (ESI-qTOF): Calcd for C18H16ClN2O4S2 [M+H]+, 423.0154: found: 423.0168.

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5.2.26. (Z)-3-(5-((2,6-dichloroquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3yl)propanoic acid (5h): The compound 5h was obtained via Knoevenagel condensation reaction between

2,6-dichloroquinoline-3-carbaldehyde

1h

and

3-(4-oxo-2-thioxothiazolidin-3-

yl)propanoic acid 2c in 30 min as yellow solid; Mp >300 °C; Yield: 90%; 1H NMR (400 MHz,

RI PT

DMSO-d6, δ ppm): 8.57 (s, 1H, Ar-H), 8.26 (d, 1H, J = 8 Hz, Ar-H), 8.17 (s, 1H, Ar-H), 7.95 (s, 1H), 7.77 (d, 1H, J = 8 Hz, Ar-H), 4.75 (t, 2H, J = 8 Hz, -CH2) and 2.74 (t, 2H, J = 8 Hz, -CH2); C NMR (100 MHz, DMSO-d6, δ ppm): 187.0, 172.4, 165.7, 140.3, 137.8, 133.5, 132.7, 131.5,

13

C16H12Cl2N2O3S2 [M+H]+, 412.9548: found: 412.9570.

SC

129.4, 129.1, 127.8, 127.5, 127.1, 126.2, 49.2 and 34.7; HRMS (ESI-qTOF): Calcd for

5.2.27. (Z)-3-(5-((2-chloro-6-fluoroquinolin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-

M AN U

yl)propanoic acid (5i): The compound 5i was obtained via Knoevenagel condensation reaction between 2-chloro-6-fluoroquinoline-3-carbaldehyde 1i and 3-(4-oxo-2-thioxothiazolidin-3yl)propanoic acid 2c in 30 min as red solid; Mp >300 °C; Yield: 80%; 1H NMR (400 MHz, DMSO-d6, δ ppm): 12.51 (s, 1H, -COOH), 8.06 (s, 1H, Ar-H), 7.49 (d, 1H, J = 8 Hz, Ar-H), 7.28 (s, 1H), 7.20 (d, 1H, J = 8 Hz, Ar-H), 7.09 (s, 1H, Ar-H), 4.59 (t, 2H, J = 8 Hz, CH2) and 2.61 (t, 2H, J = 8 Hz, CH2); 13C NMR (100 MHz, DMSO-d6, δ ppm): 194.2, 163.7, 162.1, 161.3, 160.3,

TE D

149.1, 148.9, 143.3, 136.0, 129.4, 125.3, 124.2, 115.7, 45.8 and 31.1;

19

F NMR (471 MHz,

DMSO-d6, δ ppm): -115.23; HRMS (ESI-qTOF): Calcd for C16H10ClFN2O3S2 [M+Na]+,

6. Pharmacology

EP

418.9825: found: 418.9842.

6.1. Antibacterial activity

AC C

All bacterial cultures were first grown in Lysogeny Broth (LB) media at 37 °C at 180 RPM. Once the culture reaches 1 O.D, it is used for antibacterial assay. Bacterial strains E. coli (NCIM2688), Pseudomonas fluorescens (NCIM-2036) as gram-negative and B. subtilus (NCIM-2079), S. aureus (NCIM-2010) as gram-positive were obtained from NCIM (NCL, Pune) and were grown in Luria Burtony medium from Himedia, India. The assay was performed in 96 well plates after 8 and 12 h. for gram negative and gram positive bacteria respectively. 0.1 % of 1 OD culture at 620 nm was used for screening [52]. 0.1 % inoculated culture was added in to each well of 96 well plates containing the compounds to be tested. Optical density for each plate was

23

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measured at 620 nm after 8 h for gram negative bacteria and after 12 h for gram positive bacteria.

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6.2. Antitubercular assay All the chemicals such as sodium salt XTT, DMSO, sulfanilic acid, sodium nitrate, HCl, NEED and rifampicin were purchased from Sigma-Aldrich, USA. Dubos medium was purchased from DIFCO, USA. Compounds were dissolved in DMSO and it was used as stock solution for further antimycobacterial testing. Microbial strains such as MTB H37Ra (ATCC 25177) and M. bovis

SC

BCG (ATCC 35734) were obtained from AstraZeneca, India. The stock culture was maintained at -80 ºC and sub cultured once in a liquid medium before inoculation into an experimental

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culture. Cultures were grown in Dubos media (enrichment media). Mycobacterium pheli medium (minimal essential medium) was used for antimycobacterial assay. It contains 0.5 g KH2PO4, 0.25 g trisodium citrate, 60 mg MgSO4, 0.5 g aspargine and 2 ml glycerol in distilled water (100 ml) followed by pH adjustment to 6.6. All the newly synthesized compounds were screened in vitro against two Mycobacterium species such as MTB H37Ra and M. bovis BCG. Both species of Mycobacterium were grown in Mycobacterium pheli medium. Screening of MTB H37Ra was

TE D

done by using XTT reduction menadione assay (XRMA) and M. Bovis BCG screening was done by using NR (Nitrate reductase) assay, both of them were developed [48]. Briefly 2.5 µl of these inhibitor solutions were added in a total volume of 250 µl of Mycobacterium pheli medium consisting of bacilli. The incubation was terminated on the 8th day for Active and 12th days for

EP

Dormant MTB culture. The XRMA and NR was then carried out to estimate viable cells present in different wells of the assay plate. The optical density was read on a micro plate reader

AC C

(Spectramax plus384 plate reader, Molecular Devices Inc) at 470 nm filter for XTT and at 540 nm filter for NR against a blank prepared from cell-free wells. Absorbance given by cells treated with the vehicle alone was taken as 100% cell growth. Initially primary screening was done at 30, 10 and 3 µg/ml. Compounds showing 90% inhibition of bacilli at or lowers than 30 µg/ml were selected for further dose response curve. All experiments were performed in triplicates and the quantitative value was expressed as the average ± standard deviation. MIC and IC50 values of selected compound were calculated from their dose response curves by using Origin 6 software. % Inhibition was calculated by using following formula: % Inhibition = [(absorbance of

24

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compound − absorbance of Test)/(absorbance of Control – absorbance of Blank )] × 100, where control is the medium with bacilli along with vehicle and blank is cell free medium.

6.3. Antifungal activity

RI PT

All the compounds 3a-i, 4a-i and 5a-i were tested for in vitro (Table 2) against the five human pathogenic fungal strains, as Candida albicans (NCIM-3471), Fusarium oxysporum (NCIM1332), Aspergillus Flavus (NCIM-539), Aspergillus niger (NCIM-1196) and Cryptococcus neoformons (NCIM-576) and miconazole as a standard reference drug. The minimum inhibitory

SC

concentration (MIC) values were determined using the standard agar method [49].

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6.4. Antiproliferative activity against HUVEC, Macrophages, THP-1, A549, PANC-1 and HeLa cell lines using the MTT assay (Cytotoxic activity)

The effect of compounds 3b, 3e, 3f, 3g, 3h, 3i, 4b and 4i on cell growth was determined in primary human umbilical vein endothelial cells HUVEC (ATCCCRL-1730) macrophages and in a panel of human tumor cells including lung adenocarcinoma (A549), cervix adenocarcinoma (HeLa), acute monocytic leukemia cell line (THP-1) and pancreatic adenocarcinoma (PANC-1)

TE D

obtained from the European Collection of Cell Cultures (ECCC, Salisbury, UK). The HUVEC were maintained in M200500 Media supplemented with 50X LVES (Gibco, Invitrogen) and THP-1 were maintained in RPMI 1640 without phenol red, A549 and PANC-1 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM), while HeLa cells were maintained

EP

in Minimum Essential Medium (MEM). THP-1 macrophages were differentiated by PMA (phorbol myristate acetate) treatment for 24 h on THP-1 cells.

AC C

In vitro cytotoxicity of compounds 3b, 3e, 3f, 3g, 3h, 3i, 4b and 4i against above mentioned cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT cell proliferation) assay [53] with 48 h exposure time of the tested compounds and paclitaxel was used as positive control. Each concentration was tested in triplicate in a single experiment. The viability and growth in the presence of test material is calculated by using the following formula: % cytotoxicity = [(control-test)/(control-blank)] ×100; where control is the culture medium with cells and DMSO and blank is the culture medium without cells. IC50 and MIC values were calculated by plotting the percentage survival versus concentrations, using Origin Pro software.

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6.5. Selectivity index The selectivity index (SI) was calculated by dividing the 50% growth inhibition concentration (GI50) for cell lines (HUVEC, Macrophages, THP-1, A549, PANC-1 and HeLa) by the MIC for

7.

RI PT

in vitro activity against active/dormant MTB and BCG [54].

Computational study

7.1. Molecular docking

Molecular docking study was performed using the Glide (Grid-Based Ligand Docking with

SC

Energetics) program [55] incorporated in the Schrodinger molecular modeling package (Schrodinger, LLC, New York, NY, 2015). With this purpose, the crystal structure of Zinc-

M AN U

dependent metalloprotease 1 (Zmp1) enzyme of MTB (PDB ID:3ZUK) in complex with its inhibitor was retrieved from the RCSB Protein Data Bank (PDB) (www.rcsb. org). The protein preparation wizard tool was used to refine the protein-inhibitor complex which involved eliminating the crystallographically observed water molecules (since no water molecule was observed to be conserved) and addition of hydrogen to the structure corresponding to pH 7.0 considering the appropriate ionization states for both the acidic and basic amino acid residues.

TE D

Following the assignment of appropriate charge and protonation state, the prepared complex structure was subjected to restrained energy minimization, using the force field OPLS2005 (RMSD of the atom displacement for terminating the minimization 0.3 Å), in order to relieve the steric clashes among the residues caused due to addition of hydrogen atoms. The 3D structures of

EP

the molecules to be docked were sketched with the build panel in Maestro and optimized using the LigPrep module which generates a number of low energy 3D structures, with various

AC C

ionization states, tautomers, stereochemistries, and ring conformations, for each input molecule. Partial atomic charges were assigned for each these structures using the OPLS-2005 force-field and the possible ionization states were generated corresponding to pH 7.0. The ligand structures thus obtained were then subjected to energy minimization until a gradient of 0.01 kcal/mol/Å was reached. Next, the shape and properties of the active site of Zmp1 enzyme was characterized and setup for the docking study using the receptor grid generation panel in Glide. It generates two cubical boxes having a common centroid for organizing the calculations: a larger enclosing and a smaller binding box. With the non-covalently bound native ligand, the receptor grid was defined by a box that has dimensions of 10×10×10Å centered on the centroid of native ligand in 26

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the crystal complex to allow the ligands to explore a sufficiently large region of the enzyme structure. The native ligand is used as the reference coordinate as it signifies the binding sites of a molecule with respect to the target. The optimized ligand structures were docked flexibly into the enzyme binding site using with extra precision (GlideXP) scoring function to estimate their

RI PT

binding affinities. The output files in the form of the docking poses were visualized and analyzed for the key elements of interaction with the enzyme through the Maestro’s Pose Viewer utility.

Acknowledgments

SC

The authors D.D.S. and M.H.S. are very much grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi for the award of senior research fellowship. Authors are also

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thankful to the University Grants Commission, Department of Science and Technology, New

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EP

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Delhi for financial support under UGC-SAP and DST-FIST Schemes.

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33

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Tables Captions Table 1. Screening of solvent.a

RI PT

Table 2. Antibacterial and antifungal activity of synthesized compounds 3a-i, 4a-i and 5a-i. Table 3. In vitro antitubercular activity against MTB H37Ra and M. bovis BCG strains of compounds 3a-i, 4a-i and 5a-i.

Table 4. Cytotoxic effect of compounds on primary and human cancer cells.

SC

Table 5. Selectivity index on human cell lines against dormant MTB H37Ra and M. bovis BCG.

Scheme 1. Standard model reaction.

M AN U

Schemes Captions

Scheme 2. Synthesis of rhodanine incorporated quinoline derivatives 3a-i, 4a-i and 5a-i.

Figure Captions

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Figure 1. Rhodanine and quinoline based antitubercular agents. Figure 2. Binding mode of 3g into the active site of Zmp1. π-π stacking interactions are represented by green lines while the dotted lines signify the hydrogen bonding interactions.

EP

Figure 3. Binding mode of 3b, 3e, 3f, 3h and 3i into the active site of Zmp1. π-π stacking interactions are represented by green lines while the dotted lines signify the hydrogen bonding interactions.

AC C

Figure 4. Superimposition of the binding pose of the most active compound 3g (orange backbone) versus the less active 4i (green backbone). Figure 5. Binding mode of 4i into the active site of Zmp1. π-π stacking interactions are represented by green lines while the dotted lines signify the hydrogen bonding interactions. Figure 6. Binding mode of 4b the active site of Zmp1. π-π stacking interactions are represented by green lines while the dotted lines signify the hydrogen bonding interactions.

34

ACCEPTED MANUSCRIPT

Tables

Yieldb (%)

Solvent

Temp (°C)

1

EtOH

Reflux

2

MeOH

Reflux

3

Tert-BuOH

Reflux

69

4

H2O

Reflux

Trace

5

CH3CN

6

THF

7

DMSO

8

Toluene

12

AC C

13

M AN U

67

48

Reflux

Trace

95

52

Reflux

50

solvent-free

50

65

solvent-free

60

75

solvent free

80

90

solvent-free

100

90

solvent-free

110

86

TE D

11

60

Reflux

EP

10

SC

Entry

9

a

RI PT

(Table 1)

Reaction conditions: 1a (1 mmol), 2a (1 mmol) and [DBUH][OAc] (20 mol%) for 30 min. Isolated yield.

b

1

ACCEPTED MANUSCRIPT

(Table 2)

Antibacterial activity MIC (µg/mL)

Antifungal activity MIC (µg/mL)

Entry SA

BS

CA

FO

3a

>30

>30

>30

>30

150

100

3b

>30

>30

>30

>30

200

200

3c

>30

>30

>30

>30

100

100

3d

>30

>30

>30

>30

175

3e

>30

>30

>30

>30

50

3f

>30

>30

7.86

3g

>30

>30

>30

3h

2.44

2.8

2.55

3i

>30

>30

22.64

4a

>30

>30

>30

4b

>30

>30

4c

>30

>30

4d

>30

>30

4e

>30

4f

>30

AF

AN

RI PT

PF

CN

150

100

50

100

150

100

50

75

50

SC

EC

125

125

150

50

100

75

50

M AN U

100

150

125

100

150

100

28.38

125

150

50

25

50

9.33

100

100

125

150

100

>30

50

25

12.5

25

25

>30

125

125

*

100

100

TE D

>30

>30

*

175

100

200

150

>30

>30

125

125

100

75

150

>30

>30

125

150

150

175

125

EP

>30

>30

>30

50

75

125

150

100

>30

>30

>30

150

125

200

*

150

AC C

>30

>30

>30

>30

>30

*

150

100

125

*

>30

>30

>30

>30

125

150

150

100

125

>30

>30

>30

>30

50

25

100

25

25

5a

>30

>30

>30

>30

100

125

50

75

50

5b

>30

>30

>30

>30

50

100

150

75

100

5c

>30

>30

>30

>30

25

100

150

150

150

4g 4h 4i

2

ACCEPTED MANUSCRIPT

>30

>30

>30

>30

50

100

150

100

175

5e

>30

>30

>30

>30

150

*

100

200

150

5f

>30

>30

>30

>30

125

175

100

175

150

5g

>30

>30

>30

>30

200

125

100

175

175

5h

>30

>30

>30

>30

200

*

5i

>30

>30

>30

>30

50

50

AMP

0.41

2.83

0.12

5.89

NT

NT

KAN

0.67

0.012

16

0.25

NT

MA

NT

NT

NT

NT

25

100

200

150

25

25

25

NT

NT

NT

SC

RI PT

5d

NT

NT

NT

25

12.5

25

25

M AN U

NT

AC C

EP

TE D

AMP: Ampicillin; KAN: Kanamycin; MA, Miconazole; EC, Escherichia. coli; PF, Pseudomonas fluorescens; SA, Staphylococcus aureus; BS, Bacillus subtillus; CA, Candida albicans; FO, Fusarium oxysporum; AF, Aspergillus flavus; AN, Aspergillus niger and CN, Cryptococcus neoformans; NT, Not Tested; Note:* No activity up to 200 µg/mL.

3

ACCEPTED MANUSCRIPT

MTB H37Ra (µg/mL) R1

R2

Dormant

R3

M. bovis BCG (µg/mL)

Active

Dormant

IC50

IC90

IC50

IC90

H

H

H

>30

>30

>30

>30

3b

Me

H

H

7.6

12.9

4.8

18.2

3c

H

Me

H

>30

>30

>30

3d

H

H

Me

>30

>30

3e

H

OMe

H

10

>30

3f

H

H

OMe

3.3

4.7

3g

H

H

OEt

2.3

3h

H

H

Cl

3i

H

H

F

4a

H

H

4b

Me

H

4c

H

Me

H

IC90

IC50

IC90

>30

>30

>30

>30

5.1

9.5

3.7

6.5

>30

>30

>30

>30

>30

>30

>30

>30

>30

>30

>30

5.7

21.1

4.4

1.6

1.7

2.9

6.9

14.3

2.9

6.8

3.7

5.0

3.5

1.9

2.8

1.1

1.9

1.4

0.9

2.2

6.3

1.9

3.8

1.3

2.2

1.0

1.7

5.1

18.0

5.4

2.8

3.9

5.2

2.4

4.2

H

>30

>30

>30

>30

>30

>30

>30

>30

H

10.3

19.9

21.2

27.3

>30

>30

>30

>30

H

>30

>30

>30

>30

>30

>30

>30

>30

H

Me

>30

>30

>30

>30

>30

>30

>30

>30

H

OMe

H

>30

>30

>30

>30

>30

>30

>30

>30

H

H

OMe

>30

>30

>30

>30

>30

>30

>30

>30

4g

H

H

OEt

>30

>30

>30

>30

>30

>30

>30

>30

4h

H

H

Cl

>30

>30

>30

>30

>30

>30

>30

>30

4e 4f

TE D

EP

AC C

4d

M AN U

3a

IC50

Active

SC

Entry

RI PT

(Table 3)

4

ACCEPTED MANUSCRIPT

H

H

F

>30

>30

>30

>30

>30

>30

>30

>30

5a

H

H

H

>30

>30

>30

>30

>30

>30

>30

>30

5b

Me

H

H

>30

>30

>30

>30

>30

>30

>30

>30

5c

H

Me

H

>30

>30

>30

>30

>30

>30

>30

>30

5d

H

H

Me

>30

>30

>30

>30

>30

5e

H

OMe

H

>30

>30

>30

>30

>30

5f

H

H

OMe

>30

>30

>30

>30

>30

5g

H

H

OEt

>30

>30

>30

>30

5h

H

H

Cl

>30

>30

>30

>30

5i

H

H

F

>30

>30

RP

-

-

-

0.0019±

0.020±

0.00022

0.0021

>30

>30

>30

>30

>30

>30

>30

>30

>30

SC >30

>30

>30

>30

>30

>30

>30

M AN U

>30

>30

>30

>30

>30

>30

>30

0.0021±

0.028±

0.0043±

0.0173±

0.0040±

0.0151±

0.00015

0.0025

0.00028

0.039

0.00043

0.0058

AC C

EP

TE D

RP, Rifampicin.

RI PT

4i

5

ACCEPTED MANUSCRIPT

(Table 4) *

HUVEC

THP-1

GI50 (µg/mL)

GI50 (µg/mL)

3b

>100

3e

**

Macrophage

A549

PANC-1

HeLa

GI50 (µg/mL)

GI50 (µg/mL)

GI50 (µg/mL)

GI50 (µg/mL)

>100

>100

>100

>100

>100

>100

>100

3f

>100

>100

>100

>100

3g

>100

>100

>100

3h

>100

>100

>100

3i

>100

>100

4b

>100

>100

4i

>100

>100

Paclitaxel

>10

5.7± 0.3

RI PT

Entry

>100

>100

>100

>100

>100

SC

>100

>100

>100

>100

>100

>100

M AN U

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

0.04± 0.003

5.8± 0.8

0.08± 0.007

AC C

EP

TE D

GI50 indicates concentration to inhibit 50% growth of cells.

6

ACCEPTED MANUSCRIPT

(Table 5)

Entry

MIC (µg/mL)

HUVEC

THP-1

Macrophage

A549

PANC-1

HeLa

3b

12.9 ± 0.4

7.8

7.8

7.8

7.8

7.8

7.8

3f

4.8 ± 0.4

20.8

20.8

20.8

20.8

20.8

20.8

3g

3.5 ± 0.6

28.6

28.6

28.6

28.6

28.6

28.6

3h

6.4 ± 0.8

15.6

15.6

15.6

15.6

15.6

15.6

3i

18.0 ± 0.7

5.6

5.6

4b

19.9 ± 0.5

5.0

5.0

Rifampicin

0.04 ± 0.03

2500.0

2500.0

3b

9.57

10.4

3e

1.66

3f

5.6

5.6

5.6

5.6

5.0

5.0

5.0

5.0

2500.0

2500.0

2500.0

2500.0

10.4

10.4

10.4

10.4

10.4

60.2

60.2

60.2

60.2

60.2

60.2

6.81

14.7

14.7

14.7

14.7

14.7

14.7

3g

1.96

50.0

50.0

50.0

50.0

50.0

50.0

3h

2.29

43.7

43.7

43.7

43.7

43.7

43.7

3i

5.29

18.9

18.9

18.9

18.9

18.9

18.9

Rifampicin

0.04 ± 0.03

2500.0

2500.0

2500.0

2500.0

2500.0

2500.0

EP AC C

BCG

TE D

M. bovis

M AN U

H37Ra

SC

MTB

RI PT

Strain

7

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

Figures

AC C

EP

TE D

Figure 1.

1

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

(Figure 2)

RI PT

3g

2

ACCEPTED MANUSCRIPT

RI PT

3b

M AN U

SC

3e

AC C

3h

EP

TE D

3f

3

ACCEPTED MANUSCRIPT

AC C

EP

TE D

SC

M AN U

(Figure 3)

RI PT

3i

4

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

(Figure 4)

5

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

(Figure 5)

SC

RI PT

4i

6

ACCEPTED MANUSCRIPT

AC C

EP

TE D

SC

M AN U

(Figure 6)

RI PT

4b

7

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

(Scheme 1)

RI PT

Schemes

(Scheme 2)

1

ACCEPTED MANUSCRIPT

Highlights A series of quinolidene-rhodanine conjugates were synthesized.

•

In vitro antitubercular activity against MTB H37Ra and M. bovis BCG strains.

•

Structure activity relationship of quinolidene-rhodanine conjugates.

•

Docking study against the active site of Zmp1 enzyme.

•

Most active quinolidene-rhodanine conjugates are nontoxic against the HUVEC,

RI PT

•

AC C

EP

TE D

M AN U

SC

THP-1, macrophages, A549, PANC-1 and HeLa cell lines.

1