Accepted Manuscript Synthesis, cytotoxicity and antimicrobial of thiourea derivatives incorporating 3(trifluoromethyl)phenyl moiety Anna Bielenica, Ph.D., Joanna Stefańska, Karolina Stępień, Agnieszka Napiórkowska, Ewa Augustynowicz-Kopeć, Giuseppina Sanna, Silvia Madeddu, Stefano Boi, Gabriele Giliberti, Małgorzata Wrzosek, Marta Struga PII:

S0223-5234(15)30102-1

DOI:

10.1016/j.ejmech.2015.06.027

Reference:

EJMECH 7954

To appear in:

European Journal of Medicinal Chemistry

Received Date: 22 August 2014 Revised Date:

9 June 2015

Accepted Date: 9 June 2015

Please cite this article as: A. Bielenica, J. Stefańska, K. Stępień, A. Napiórkowska, E. AugustynowiczKopeć, G. Sanna, S. Madeddu, S. Boi, G. Giliberti, M. Wrzosek, M. Struga, Synthesis, cytotoxicity and antimicrobial of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.027. 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

CF3

Cl

CF3 F

S NH

NH

Cl

S NH

5

CF3

Cl

Br S NH NH 15

NH 6

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SC

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MIC for S. aureus (MRSA): 0.25 ‒ 2 µg/ml MIC for S. epiermidis (MRSE): 0.25 ‒ 4 µg/ml IC50 for S. epidermidis biofilm formation: 0.97-5 µg/ml Cytotoxicity in MT-4 cells: 8 ‒ 9.2 µM

ACCEPTED MANUSCRIPT Submitted to: European Journal of Medicinal Chemistry Corresponding author: Anna Bielenica, Ph.D. Chair and Department of Biochemistry Medical University of Warsaw

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02-097 Warszawa, Poland E-mail address: [email protected] (A. Bielenica)

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Phone: +48-22 5720693; Fax: +48-22 5720679

Synthesis, cytotoxicity and antimicrobial of thiourea derivatives incorporating 3-

Anna Bielenica

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(trifluoromethyl)phenyl moiety a,*

, Joanna Stefańska b, Karolina Stępień b, Agnieszka Napiórkowska c, Ewa

Augustynowicz-Kopeć c, Giuseppina Sanna d, Silvia Madeddu d, Stefano Boi d, Gabriele Giliberti d, Małgorzata Wrzosek e, Marta Struga e

Chair and Department of Biochemistry, Medical University, 02-097 Warszawa, Poland

b

Department of Pharmaceutical Microbiology, Medical University, 02-007 Warszawa,

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a

Poland

Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute,

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c

01-138 Warszawa, Poland e

Department of Biomedical Science, University of Cagliari, 09042 Monserrato (CA), Italy

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d

Department of Pharmacogenomics, Faculty of Pharmacy, Medical University, 02-097

Warszawa, Poland

Keywords: Antimicrobial activity, Biofilm, Cytotoxicity, Thiourea derivatives, 3(Trifluoromethyl)aniline

1

ACCEPTED MANUSCRIPT Abstract:

A

total

of

31

of

thiourea

derivatives

was

prepared

reacting

3-

(trifluoromethyl)aniline and commercial aliphatic and aromatic isothiocyanates. The yields varied from 35% to 82%. All compounds were evaluated in vitro for antimicrobial activity. Derivatives 3, 5, 6, 9, 15, 24 and 27 showed the highest inhibition against Gram-positive cocci (standard and hospital strains). The observed MIC values were in the range of 0.25-16

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µg/ml. Inhibitory activity of thioureas 5 and 15 against topoisomerase IV isolated from S. aureus was studied. Products 5 and 15 effectively inhibited the formation of biofilms of methicillin-resistant and standard strains of S. epidermidis. Moreover, all obtained thioureas were evaluated for cytotoxicity and antiviral activity against a large panel of DNA and RNA

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viruses. Compounds 5, 6, 8-12, 15 resulted cytotoxic against MT-4 cells (CC50 ≤ 10 µM).

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1. Introduction

The emergence of resistance to the major classes of antibacterial agents is recognized as a serious health problem. Particularly, in recent years much attention has been focused on the multi-drug resistant bacteria and fungi resulting from the widespread use and misuse of classical antimicrobial drugs. Some of these resistant strains, such as methicilin-resistant

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Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are capable of surviving the effect of most of the currently used antibiotics [1,2]. On the other hand, many chronic infections, such as otitis media, tonsillitis, adenoiditis, as well as device-related infections are caused by biofilm-forming mucosal

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pathogens of Staphylococcus aureus and Staphylococcus epidermidis [3,4]. Indeed, biofilms now account for over 85% of implants associated infections [5]. Biofilm formation constitutes an alternative lifestyle, in which bacterial cells adopt a multicellular behavior that facilitates

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and prolongs survival in diverse environmental conditions. One of the most important features of these sessile aggregates is their extreme resistance to conventional antibiotics, antiseptics and the host immune system [6-8]. Currently available antibiotics admittedly inhibit metabolically active bacteria growing on the biofilm surface, but do not influence the cells in its core [5,9]. Therefore the development of new therapeutic agents, active not only against planktonic microorganisms, but also on biofilm, is one of the fundamental challenges in medicinal chemistry. Up to now only a few thioureas with antibiofilm properties have been reported [10,11]. Thioureas, both symmetrical and unsymmetrical, have attracted much attention as antimicrobial drug candidates. While unsubstituted 1,3-diphenylthiourea exerts no relevant 2

ACCEPTED MANUSCRIPT antimicrobial activity [12], its various structural modifications improve the biological effectiveness of a compound [13]. Literature survey reveals that incorporation of halogen atom(s) within the molecule is one of the most effective strategies to enhance its biopotency, bioavailability and lipophilicity. Suresha et al. proved that fluoro-containing arylthiourea compounds show better activity as compared to other analogues [14], however fluoro-methyl,

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methyl or metoxy substituents on the benzene ring also improve antimicrobial potency. According to other authors findings [15-18], the antibacterial and antifungal efficiency depends on the presence of such electron-withdrawing substituent at C-2 and C-4 position of the phenyl ring. On the other hand, modification of isoxyl, the symmetrical diphenylthiourea

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derivative, produced the library of compounds with antimycobacterial activity [19]. The introduction of aliphatic para functionalities to either or both positions increased the potency of the inhibitor. Also the presence of oxygen in a side chain improved the effectivity of a

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thiourea derivative against M. tuberculosis. Potent Gram-positive antibacterial activity of several analogs of thiourea, urea [20] and thiosemicabazide derivatives [21] is explained by an inhibition of the catalytic site of bacterial type II topoisomerases, in particular DNA gyrase and topoisomerase IV.

Whereas naphtylthiourea, phenylthiourea and 1,3-diphenylthiourea were found to be

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highly cytotoxic in rat hepatocytes, introducing of short-chain alkyl substituents to phenylthiourea core reduced its toxic influence. Similarly, the methylene unit insertion between phenyl and thiourea remarkably reduced the cytotoxic activity [22]. Many organic connections of thiourea were found to be cytotoxic against different cell lines derived from

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human tumors [23-26]. Thiourea-derived compounds have also been reported as inhibitors of herpes virus family [27-29], as well as they are effectively used in the antiretroviral therapy [30,31].

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As a part of our extensive research program to rapidly assemble novel bioactive

compounds under mild condition [32,33], and to investigate the role of substitutions by functional groups attached to the thiourea bridge, we synthesized new disubstituted thioureas containing 3-(trifluoromethyl)phenyl moiety. The compounds were evaluated for their antimicrobial, cytotoxic and antiviral activities, as well as further investigated as potential inhibitors on biofilm formation of Gram-positive pathogens. The mechanism of their action through topoisomerase IV inhibition was proved.

2. Results and discussion 2.1. Chemistry 3

ACCEPTED MANUSCRIPT Our aim was to obtain a small library of 3-(trifluoromethyl)phenylthiourea derivatives by condensation of 3-(trifluoromethyl)aniline with appropriate isothiocyanates (Fig.1., Table 1). Among 31 presented derivatives, 25 have not been published previously. In order to assure structural variability, different aryl (compounds 1‒20) and alkyl substituents (21‒31) at thiourea moiety were introduced. In the class of N-aryl substituents, electron-withdrawing (2-

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12, 14, 15, 19, 20) and electron-donating groups (13, 16, 17, 18) were inserted. On the other hand, proposed N-alkyl functionalities are of different sizes, bearing cyclic, heterocyclic, aromatic or carbonyl terminal substituents. The effect of this modifications was evaluated on biological activity of thiourea derivatives. Our investigations involved antimicrobial,

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cytotoxic and antiviral profile, as well as biofilm formation inhibition of synthesized thiourea series. The antiproliferative properties of selected derivatives were established. [Figure 1]

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[Table 1]

2.2. Characterization

Spectral data (NMR, MS, IR) of all compounds were in full agreement with the proposed structures. The 1H NMR spectrum exhibited singlets at δ 12.6 – 7.9 ppm, which were assigned to the N-H protons. 13C NMR revealed peaks at δ 182.7 ‒ 176.7 ppm for C=S (thiourea) and

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quartets at 124.8 ‒ 122 ppm with high coupling constants (272.8 – 270.0 Hz) for CF3 group. The second quartet at 131.5 – 128.6 ppm with low coupling constant (33.6 − 30.7 Hz) proved the presence of the carbon attached to CF3 (C3). Quartets of neighboring carbons (C2 and C4)

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were also visible, both at 125.7 – 118.2 ppm (J ranged from 4.2 to 3.7 Hz). Signals at δ 140.9 – 134.2 ppm reflected the presence of the aniline carbon bound to the NH group. Other

13

C

NMR signals were considered a singlet if the multiplicity was not assigned. The IR spectra

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display absorption bands for the C=S group between 689.0 and 720.0 cm-1.

2.3. Biological studies

2.3.1. Antimicrobial study

All obtained compounds were tested in vitro against a number of bacteria, including Gram-positive cocci, Gram-negative-rods and fungi. Microorganisms used in this study have common applications in the antimicrobial tests for many substances like antibiotics, antiseptic drugs and in the search for new antimicrobial agents [34,35].

4

ACCEPTED MANUSCRIPT The procedure started with a preliminary screening by the disc diffusion method [36,37], in order to select derivatives with significant antimicrobial properties. Once detected, the most interesting (1-6, 8-10, 12, 15, 19, 21, 22, 24, 27-29, 31) were examined for their minimal inhibitory concentration (MIC) by the twofold serial microdilution method [38,39]. The observed data are summarized in Tables 2 and 3.

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[Table 2] [Table 3]

According to data generated from this study, over 60% of the synthesized compounds presented variable activity against wide range of standard strains of Gram-positive bacteria.

Ciprofloxacin.

The

most

active

of

the

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Compounds 5, 6, 15 (Table 3) exhibited antibacterial activity comparable to the standard series,

1-(3,4-dichlorophenyl)-3-[3-

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(trifluoromethyl)phenyl]thiourea (6) revealed the inhibitory effect against Gram-positive bacteria at the level of 0.5 µg/ml. Derivatives 1-4, 8, 9, 19, 22, 24, 27, 31 were found to be potent to moderate antibacterial agents (MIC values from 4 to 32 µg/ml). Several Narylthiourea derivatives (1, 2, 4, 5, 15) showed also some inhibition activity against Proteus vulgaris. Interestingly, the observed MIC values of the derivatives investigated in this research are higher than that reported for other thiourea derivatives [32], due to the presence

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of aniline moiety substituted with an electron-withdrawing CF3 group. Compounds 7, 11, 13, 14, 16, 18, 20, 23, 25, 26, 30 exerted no antimicrobial activity. Gram-negative strains such as Escherichia coli, Pseudomonas aeruginosa, Bordetella bronchiseptica and yeasts (Candida albicans, Candida parapsilosis) were completely unsusceptible for the presence of tested

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thiourea conjugates. The latter may be explained by the differences between structures of bacterial and fungal cells [40,41].

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The next step was to evaluate the activity of the previously assigned most active antibacterial thioureas (3, 5, 6, 9, 15, 24, 27) against hospital methicillin-resistant strains of Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). The observed results have confirmed high activity of 3-(trifluoromethyl)phenylthiourea derivatives against Gram-positive bacteria (Table 4). MIC values varied from 0.25 to 16 µg/ml, however for the most active compounds 5 and 6 they ranged from 0.25 to 2 µg/ml. The level of activity of disubstituted

halogen

derivatives

(trifluoromethyl)phenyl]thiourea),

6

5

(1-(3-chloro-4-fluorophenyl)-3-[3-

(1-(3,4-dichlorophenyl)-3-[3-(trifluoromethyl)-

phenyl]thiourea), as well as 3-bromophenyl derivative 15 against standard strains of S. aureus and S. epidermidis is comparable to Ciprofloxacin. However, the tested thioureas are even 5

ACCEPTED MANUSCRIPT 128-256 times more active against selected hospital bacterial strains than the reference drug. In case of several compounds (e.g. 8-10, 12, 17, 19) no evident correlation between the zones of inhibition and MIC values was observed. This is due the fact that the zone of inhibition depends on both the diffusion of a compound into the agar medium and the solubility of a compound. When the solubility is low, the diffusion is limited, which results in the small

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zone, even for highly active derivatives presenting low MICs. On the other hand high inhibition zone correlated with relatively high MIC could be explained by precipitation of a compound in the liquid medium and as the result changing its real concentration.

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[Table 4]

In addition, in vitro antimycobacterial activity of compounds 1-10, 12, 15, 17-22, 24, 27-

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31 was investigated against the drug-resistant M. tuberculosis H37Rv strain and two “wildtype” strains isolated from tuberculosis patients: one (Spec. 210) resistant to p-aminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), etambutol (ETB) and rifampicin (RMP) and another (Spec. 192) fully sensitive to the administrated tuberculostatics. The tested derivatives showed weak tuberculostatic activity (Table 1S, Supplementary material). According to the obtained results, N-arylthiourea derivatives showed better antibacterial

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activity than those with alkyl substituents at the thiourea moiety. The introduction of variable functionalities to the phenyl ring induced antimicrobial activity of a compound compared to the initial 1,3-diphenylthiourea, and allowed to reach MIC values above 512 µg/ml. When the effect of the substituent at thiourea nitrogen was evaluated, it was found that the

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functionalities could be arranged in order of their decreasing influence as follows: 3-chloro-4fluorophenyl >3-bromophenyl > 3,4-dichlorophenyl > 3-fluorophenyl > phenylethyl > benzyl

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> 4-chlorophenyl. Furthermore, it was observed that for N-aryl derivatives (3, 5, 6, 9, 15) optimum CLogP values ensuring antimicrobial activity ranged from 4.15 to 5.26, and the higher CLogP value was attributed, the stronger activity observed. Moreover, substituent groups on different positions of the phenyl ring resulted in various degrees of effect. Derivatives 3, 5, 6, 9 and 15 possessing weakly deactivating halogen substituents at metaand/or para- position of the benzene ring were found as the most active. For most of Grampositive bacteria, disubstituted derivatives (5, 6) were more active than monosubstituted halogen compounds, because of stronger electronegativity effect produced. That phenomenon was also noticed for 3-bromo- (15) and 3-fluorophenyl (3) derivatives. The presence of halogen atoms at ortho- position, as well as the introducing of electron-donating substituents 6

ACCEPTED MANUSCRIPT on aromatic ring has reduced antibacterial activity. On the other hand, among Nalkylthioureas, only these with the methylene groups linked the thiourea branch to the phenyl ring (24, 27) have reached good antibacterial activity. For these derivatives CLogP factors varied from 3.37 to 4.19. Bulky aliphatic or alicyclic functionalities at the phenyl ring corresponded with very high CLogP values (5-8.3) that probably resulted in poor absorption

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and permeation, and caused loss of activity (compounds 13, 16, 18). Also the class of thiourea derivatives with non aromatic N-substituents (23, 25, 30), characterized by low lipophilicity (CLogP from 2 to 3.9), was devoid of antimicrobial properties.

To sum up, due to the presence of 3-(trifluoromethyl)phenyl core, the antibacterial profile

connections of thiourea [32,42].

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2.3.2. Topoisomerase IV inhibition assay

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of the presented compounds was found to be higher than the activity of heterocyclic

Topoisomerase IV is a bacterial type II topoisomerase that is essential for proper chromosome segregation. It is the primary target of second-generation fluoroquinolones, such as Ciprofloxacin and Levofloxacin [43], that stimulate topoisomerase IV-mediated DNA

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cleavage both by increasing rates of DNA scission and by inhibiting religation of cleaved DNA. As a result, quinolones inhibit the overall catalytic activity of topoisomerase IV primarily by interfering with enzyme-ATP interactions [44]. It is supposed that in Grampositive bacterial species, topoisomerase IV rather than DNA gyrase, another example of type

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II topoisomerase, appears to be the primary target of most quinolone-based antibiotics. Considering the results of the in vitro antibacterial assay, we have investigated the inhibitory effect on topoisomerase IV of compounds 5 and 15, which showed highest therapeutic

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potential against strains of S. aureus and S. epidermidis, including clinically relevant resistant isolates.

In decatenation assay both tested thiourea derivatives were found as potent

topoisomerase IV inhibitors (Fig.2.). Synthesized compounds applied at concentration 32 µg/ml are equally active as Ciprofloxacin. At lower concentrations (4 µg/ml and 1 µg/ml) their inhibitory potency was stronger than the control drug. This enzymatic inhibition corresponded well with the antibacterial activity of thioureas observed for Gram-positive cocci.

[Figure 2] 7

ACCEPTED MANUSCRIPT Presented preliminary results showed that thiourea-derived compounds incorporating 3-(trifluoromethyl)phenyl were able to inhibit the activity of bacterial topoisomerases, such as topoisomerase IV from S. aureus.

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2.3.3. Inhibition of S. epidermidis biofilm formation

Preliminary antibacterial studies revealed that 1-(3-chloro-4-fluorophenyl)-3-[3(trifluoromethyl)phenyl]thiourea

(5)

and

1-(3-bromophenyl)-3-[3-(trifluoromethyl)-

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phenyl]thiourea (15) have shown the most promising activity against free-swimming (planktonic) forms of staphylococcal species. In general MIC values for standard and

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medically relevant methicillin-resistant strains of S. aureus and S. epidermidis ranged from 0.25 to 2 µg/ml. These thiourea conjugates were further studied for their ability to inhibit the formation of biofilms of eight methicillin-resistant strains of S. epidermidis (MRSE) and two standard strains of S. epidermidis (ATCC 12228, ATCC 35984).

The compounds were tested at concentrations ranging from 0.125 to 4 µg/ml (derivative 5) and 0.25 to 8 µg/ml (15). Both thiourea connections exhibited good biofilm

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inhibitory activity against the aforesaid standard and hospital bacterial strains, regardless of the level on which biofilm was formed. IC50 values, calculated for each strain from the dose (concentrations)-response curve for both derivatives ranged from 1xMIC to 2xMIC estimated against planktonic forms (Table 5). The compound 5 was found to be more promising with

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IC50 values of 0.97-2.85 µg/ml. For most of the tested strains the highest concentrations of compounds (for 5 - 4 µg/ml, for 15 - 8 µg/ml) inhibited biofilm formation of hospital strains

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by above 70% (Figures 3 and 4; Table 2S, Supplementary material), as compared to the control. The stronger inhibitor, compound 5, prevented the formation of biofilm of standard S. epidermidis strains by 84-95%. To compare, the reference compound Ciprofloxacin used at the same concentration, blocked biofilm formation by 5-45% (only for S. epidermidis 62/04 by 60%; Table 2S). In its highest concentration (8 µg/ml) it inhibited biofilm formation of four tested strains of S. epidermidis by approximately 40% (Fig.5.), whereas inhibitory potency of the compound 15 remained the same as observed at 4 µg/ml. For the one and only hospital strain (S. epidermidis 62/04) the highest concentration of the standard drug blocked biofilm formation by 80%. Observed values of IC50 for Ciprofloxacin were much higher than for both tested thiourea derivatives (Table 5.). 8

ACCEPTED MANUSCRIPT [Table 5] [Figure 3] [Figure 4]

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[Figure 5] Mature biofilm forms when critical bacterial cell density is achieved. The process involves also the arrangement of multiple bacterial cells into a complex tertiary structure, an extracellular matrix. In many bacterial strains the biofilm growth is controlled by the cell-cell

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signaling system termed quorum sensing (QS) [45]. The exact mechanism of action of small molecule biofilm inhibitors is still unknown, but different hypotheses can be proposed. Compounds can modulate biofilm formation through non-microbicidal or biocidal mechanism

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[46]. The latter is even more promising, since it reduced the probability of bacterial resistance. The non-microbicidal strategies include biofilm weakening by degradation of extracellular matrix and targeting of extracellular and intracellular signaling molecules. The other agents act through biofilm disruption in mechanical or biological (enzymatic) ways. Moreover, antibiotic, biocides and ion coatings interfere with the attachment and expansion of immature biofilm. It was estimated that small cationic peptides are able to infect swimming and

biofilm degradation.

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swarming motilities of bacterial cells and stimulate twitching motility [47], that results in

Many compounds inhibiting the biofilm growth contain a halogen atom in their

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structure [46]. The presence of multiple amino or guanidine group, as well as the charge of the compounds, could be important contributors to their inhibitory activity [8]. Natural products, such as the plant auxin 3-indolylacetonitrile, were found to modulate the

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development of Gram-negative bacteria biofilm via a QS-dependent mechanism [45]. Compounds of that group could inhibit and disperse biofilm through a zinc-dependent mechanism that differs significantly between Gram-negative and Gram-positive strains [48]. On the other hand, phytoalexin resveratrol and its derivatives act through the inhibition of the LuxR-type QS receptors [45]. In addition to QS, bacteria use other ways to recognize and replay to various external signals and stimuli [7]. The signaling pathways are additionally mediated by two-component regulatory system [TCSs]. Walkmycin, the natural compound produced by Streptomyces strains, found as TCS inhibitor, disrupts the interbacterial communication and bacteria’s ability to analyze the external environment [49]. 9

ACCEPTED MANUSCRIPT Bactericidal profile of activity against P. aeruginosa biofilm was confirmed for several antibiotics, e.g. amikacin, levofloxacin and tetracycline [50]. The link between biofilm inhibitory properties of thiourea-derived compounds and the possible mechanism of action is still undocumented.

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2.3.4. Cytotoxicity and antiviral study

With the aim to evaluate more widely the biological properties of synthesized compounds and on the basis of reported anti-HIV activities of thiourea derivatives,

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compounds were tested in cell-based assay against the human immunodeficiency virus type-1 (HIV-1), using Efavirenz as reference inhibitor. The cytotoxicity against the MT-4 cells was evaluated in parallel with the antiviral activity (Table 7). None of the tested compounds

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showed selective anti-HIV-1 activity; however compounds 5, 6, 8-12, 15, 24 turned out cytotoxic for exponentially growing MT-4 cells in the low micromolar range (CC50 ≤ 10 µM). That study revealed that compounds containing phenylethyl, dihalogen, halogen, nitro and cyano groups at C-3 and/or C-4 position of benzene ring showed significant biological activity.

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The results were also analyzed from the point of view of the relationship between cytotoxicity and activity against Gram-positive cocci. To compare, the high cytotoxicity is not evidently correlated with antimicrobial potency. Derivatives 5, 6, 9, 15 and 24 presented both biological profiles. Compounds 3 and 27, which displayed well-marked activity against

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Gram-positive pathogens showed moderate cytotoxicity, similar to the reference Efavirenz. However cytotoxic 8, 10, 11 and 12 are only moderate to weak antibacterial agents. What is more, except of compound 24, cytotoxic properties were observed only for the group of N-

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arylthioureas. On the contrary, bacterial strains were susceptible to both aryl and alkyl derivatives.

All obtained compounds were further tested in cell-based assays against representative

members of several RNA and DNA viruses, causing infectious diseases; their cytotoxicity was evaluated in parallel assays with uninfected MDBK, BHK and Vero-76 cell lines. No relevant activities were found; interestingly, compounds turned out as cytotoxic against MT-4 cells showed a similar level of cytotoxicity against at least one other used cell line, with the exception of compound 24. [Table 6] 10

ACCEPTED MANUSCRIPT Compounds 5, 6, 8-12, 15 were also evaluated for their antiproliferative activity against different cell lines derived from human haematological (Table 3S, Supplementary material). Observed CC50 values were comparable to that obtained with MT-4 cells. None of them showed CC50 values comparable with Doxorubicin, used as reference compound, but activities could be considered for a better future evaluation of the anticancer potential of this

derivatives. 2.3.5. Cytotoxic activity in HaCaT cells

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class of compounds, offering interesting indications to design and develop more potent

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Cytotoxic effect of the most active derivatives 5 and 15 was measured in cell viability assessment and cell mortality assay in human immortal keratinocyte cell line from adult

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human skin (HaCaT). Compounds were tested in concentration 16 µM, which was two times higher than their cytotoxic concentration against the MT-4 cells. According to obtained results 24-hour incubation of HaCaT cells together with tested derivatives caused approximately 10% decrease in cell viability, as compared to the control (not treated cells) (Table 7). In the LDH (lactate dehydrogenase release) assay the observed cells mortality in the presence of the compound 5 was below LD10 value (the concentration that causes mortality in 10% of treated

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cells). The compound 15 had no influence on the increase of the mortality of tested cells. This study demonstrates that tested thiourea derivatives decreased viability of HaCaT cells by 10% and slightly increased (below 5%) their mortality. Therefore short-term exposure

[Table 7]

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3. Conclusion

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of HaCaT cells on synthesized compounds should not cause irreversible cytotoxic effect.

In summary, we have presented a synthetic approach to rapidly access thiourea

conjugates of 3-(trifluoromethyl)aniline. A set of 31 analogues was evaluated for in vitro antimicrobial, cytotoxic and antiviral activities. Most of derivatives exhibited potent to moderate antibacterial activity. Compounds 5, 6 and 15 presented the strongest potency against standard and methicillin-resistant S. aureus and S. epidermidis strains, with MIC values varied from 0.25 to 2 µg/ml. N-arylthioureas 5 and 15 inhibited the formation of S. epidermidis biofilm in above 70% in the low micromolar range. Compounds 6, 5, 8-12 and 15 turned out as cytotoxic against human leukaemia/lymphoma cells at a similar level as 11

ACCEPTED MANUSCRIPT against MT-4 cells. Derivatives 5 and 15 decreased viability of HaCaT cells by 10% and only in 5% increased their mortality. For N-aryl derivatives a relationship between the high ClogP values and their biological activity was observed. The proposed scaffolds of the most active halogenophenyl derivatives 5 and 15 offer the possibility of convenient further modifications that could give rise to structures with improved antimicrobial and

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topoisomerase inhibitory activities.

4. Experimental

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4.1. Chemistry 4.1.1. General procedure

3-(Trifluoromethyl)aniline was supplied from Alfa Aesar. Isothiocyanates were

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purchased from Alfa Aesar or Sigma Aldrich. Acetonitrile, chloroform and methanol were supplied from POCh (Polskie Odczynniki Chemiczne). All chemicals were of analytical grade and were used without any further purification. Prior usage, dry acetonitrile was kept in crown cap bottles over anhydrous phosphorus pentoxide (Carl Roth). The IR spectra were obtained on Perkin Elmer Spectrum 1000 spectrometer in KBr pellets. The NMR spectra

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were recorded on Varian VNMRS 300 Oxford NMR spectrometer, operating at 300 MHz (1H NMR, relax. delay 1.000 sec, pulse 30.0 degrees) and 75.4 MHz (13C NMR, relax. delay 3.700 sec, pulse 45.0 degrees, proton-decoupled: Waltz-16 modulated). Chemical shifts (δ) were expressed in parts per million relative to tetramethylsilane used as the internal

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reference. Mass spectral ESI measurements were carried out on Waters ZQ Micro-mass instruments with quadruple mass analyzer. The spectra were performed in the negative ion

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mode at a declustering potential of 40-60 V. The sample was previously separated on a UPLC column (C18) using UPLC ACQUITYTM system by Waters connected with DPA detector. Flash chromatography was performed on Merck silica gel 60 (200-400 mesh) using chloroform eluent. Analytical TLC was carried out on silica gel F254 (Merck) plates (0.25 mm thickness).

4.1.2. General procedure for the preparation of N-aryl-[3-(trifluoromethyl)phenyl]thiourea derivatives (1‒20) and N-alkyl-[3-(trifluoromethyl)phenyl]thiourea derivatives (21‒31) A solution of commercially available 3-(trifluoromethyl)aniline (0.0031 mol, 0.50 g) in anhydrous acetonitrile (10 mL) was treated with appropriate isothiocyanate (0.0031 mol) and

12

ACCEPTED MANUSCRIPT the mixture was stirred at room temperature for 12 h. Then solvent was removed on rotary evaporator. The residue was crystallized from acetonitrile or purified by column chromatography (chloroform: methanol; 9.5:0.5 vol.).

4.1.2.1. 1-Phenyl-3-[3-(trifluoromethyl)phenyl]thiourea (1)

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1-Phenyl-3-[3-(trifluoromethyl)phenyl]thiourea (1) has been synthesized as described previously [51].

Yield 75%, white powder, m.p. 82-84.5 °C. FT-IR (KBr, cm-1): 3214.1, 3189.7 (N-Hamide stretching);

3032.1 (C-Haromatic stretching); 1597.5 (N-Hamide bending); 1493.8, 1449.2 (ring stretching);

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1332.5, 1174.5, 1112.5 (C-Haromatic bending); 1069.8; 696.0 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.03 (s, 1H, NH), 9.99 (s, 1H, NH), 7.97 (m, 1H, H-2), 7.76 (d, J = 8.1 Hz, 1H, H-4), 7.55 (t, J = 7.95 Hz, 1H, H-5), 7.50-7.44 (m, 3H, H-6, H-2’, H-6’), 7.38-7.30

M AN U

(m, 2H, H-3’, H-5’), 7.16 (t, J = 7.35 Hz, 1H, H-4’). 13C NMR (75.4 MHz, DMSO) δ: 179.64 (C=S), 138.26 (C1’), 136.2 (C1), 131.52 (q, J = 33.6 Hz, C3), 130.20 (C5), 129.60 (C4’), 128.40 (C3’, C5’), 127.85 (C6), 125.47 (C2’, C6’), 123.61 (q, J = 272 Hz, C3a), 123.23 (q, J = 3.8 Hz, C2), 121.73 (q, J = 3.8 Hz, C4). HRMS (ESI) calc. for C14H11F3N2S [M]• ‒: 296.0595, found: 296.0607.

TE D

4.1.2.2. 1-(4-Fluorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (2) Yield 70%, white powder, m.p. 112.5-114.5 °C. FT-IR (KBr, cm-1): 3216.2 (N-Hamide stretching); 3014.6 (C-Haromatic stretching); 1602.2 (N-Hamide bending); 1507.2, 1452.1 (ring stretching); 1331.1, 1177.4, 1118.1 (C-Haromatic

bending);

1071.4, 716.5 (C=Sthioamide

stretching).

1

H NMR (300 MHz,

EP

DMSO) δ: 9.98 (s, 2H, NH), 7.95 (s, 1H, H-2), 7.75 (d, J = 8.1 Hz, 1H, H-4), 7.56 (t, J = 7.95 Hz, 1H, H-5), 7.50-7.44 (m, 3H, H-6, H-3’, H-5’), 7.23-7.14 (m, 2H, H-2’, H-6’).

13

C NMR

AC C

(75.4 MHz, DMSO) δ: 180.20 (C=S), 159.36 (d, J = 241.9 Hz, C4’), 140.41 (C1), 135.35 (d, J = 2.8 Hz, C1’), 129.45 (C5), 128.79 (q, J = 30.7 Hz, C3), 127.33 (C6), 126.42 (C2’, C6’), 126.31 (C3’, C5’), 124.05 (q, J = 272.7 Hz, C3a), 120.58 (q, J = 3.9 Hz, C2), 119.84 (q, J = 3.9 Hz, C4). HRMS (ESI) calc. for C14H9F4N2S [M ‒ H] ‒: 313.0423, found: 313.0411. 4.1.2.3. 1-(3-Fluorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (3) Yield 70%, pale orange powder, m.p. 120-122 °C. FT-IR (KBr, cm-1): 3230.5, 3202.9 (NHamide

stretching);

3044.5 (C-Haromatic

stretching);

1606.8 (N-Hamide

bending);

1485.4, 1452.5 (ring

stretching); 1330.4 1161.5, 1120.6 (C-Haromatic bending); 1069.9, 703.8 (C=Sthioamde stretching). 1H NMR (300 MHz, CD3OD) δ: 8.08 (s, 1H, NH), 7.89 (s, 1H, NH), 7.69-7.62 (m, 2H, H-2, H5’), 7.56-7.51 (m, 2H, H-4, H-4’), 7.45-7.37 (m, 1H, H-5), 7.18-7.11 (m, 2H, H-6, H-2’), 13

ACCEPTED MANUSCRIPT 7.06-7.00 (m, 1H, H-6’). 13C NMR (75.4 MHz, DMSO) δ: 179.76 (C=S), 161.60 (d, J = 242 Hz, C3’), 140.98 (d, J = 10.8 Hz, C1’), 140.22 (C1), 130.16 (d, J = 9.4 Hz, C5’), 129.53 (C5), 129.00 (q, J = 32 Hz, C3), 127.43 (C6), 124.03 (q, J = 272 Hz, C3a), 120.82 (q, J = 3.9 Hz, C2), 119.94 (q, J = 3.9 Hz, C4), 119.17 (d, J = 2.8 Hz, C6’), 111.10 (d, J = 21.3 Hz, C2’), 110.21 (d, J = 24.9 Hz, C4’). HRMS (ESI) calc. for C14H9F4N2S [M ‒ H] ‒: 313.0423, found:

RI PT

313.0433. 4.1.2.4.

1-(2-Fluorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (4)

Yield 74%, white powder, m.p. 119-120 °C. FT-IR (KBr, cm-1): 3196.5 (N-Hamide

stretching);

3019.9 (C-Haromatic stretching); 1599.5 (N-Hamide bending); 1491.5, 1454.5 (ring stretching); 1328.5, 1

H NMR (300 MHz,

SC

1169.8, 1123.0 (C-Haromatic bending); 1070.4, 720.0 (C=Sthioamide stretching).

DMSO) δ: 10.15 (s, 1H, NH), 9.72 (s, 1H, NH), 7.99 (s, 1H, H-2), 7.78 (d, J = 8.1 Hz, 1H,

5’, H-6’).

13

M AN U

H-4), 7.61-7.54 (m, 2H, H-5, H-3’), 7.47 (d, J = 7.8 Hz, 1H, H-6), 7.30-7.16 (m, 3H, H-4’, HC NMR (75.4 MHz, DMSO) δ: 180.80 (C=S), 156.40 (d, J = 245.3 Hz, C2’),

140.30 (C1), 129.53 (C5), 128.96 (q, J = 32 Hz, C3), 128.63 (C1’), 127.64 (d, J = 7.8 Hz, C6’), 127.28 (C6), 126.63 (d, J = 11.8 Hz, C4’), 124.25 (d, J = 3.7 Hz, C5’), 122.02 (q, J = 271.5 Hz, C3a), 120.73 (q, J = 4.1 Hz, C2), 119.72 (q, J = 4.1 Hz, C4), 116.00 (d, J = 31.2 Hz, C3’). HRMS (ESI) calc. for C14H9F4N2S [M ‒ H] ‒: 313.0423, found: 313.0415.

TE D

4.1.2.5. 1-(3-Chloro-4-fluorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (5) Yield 70%, white powder, m.p. 127-128.5 °C. FT-IR (KBr, cm-1): 3229.4, 3189.3 (N-Hamide stretching);

3041.4 (C-Haromatic stretching); 1598.5 (N-Hamide bending); 1497.7, 1452.5 (ring stretching);

EP

1326.6, 1180.5, 1120.2 (C-Haromatic bending); 1066.2, 701.6 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.08 (br.s, 2H, NH), 7.92 (s, 1H, H-2), 7.76-7.73 (m, 2H, H-4, H-5’), 7.57 (t, J = 7.95 Hz, 1H, H-5), 7.48 (d, J = 7.8 Hz, 1H, H-6), 7.42-7.39 (m, 2H, H-2’, H-6’).

13

C

AC C

NMR (75.4 MHz, DMSO) δ: 180.22 (C=S), 154.30 (d, J = 242.6 Hz, C4’), 140.15 (C1), 136.28 (d, J = 3.3 Hz, C1’), 129.56 (C5), 129.01 (q, J = 31.7 Hz, C3), 127.54 (C6), 126.07 (C2’), 124.85 (d, J = 7.2 Hz, C6’), 124.01 (q, J = 272 Hz, C3a), 120.88 (q, J = 3.7 Hz, C2), 120.03 (q, J = 4.2 Hz, C4), 118.83 (d, J = 18.6 Hz, C3’), 116.58 (d, J = 22 Hz, C5’). HRMS (ESI) calc. for C14H8ClF4N2S [M ‒ H] ‒: 347.0033, found: 347.0019. 4.1.2.6.

1-(3,4-Dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (6)

1-(3,4-Dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (6) has been synthesized as described previously [52].

14

ACCEPTED MANUSCRIPT Yield 71%, white powder, m.p. 156.5-158.5 °C. FT-IR (KBr, cm-1): 3221.1, 3178.3 (N-Hamide stretching);

3036.5 (C-Haromatic stretching); 1589.3 (N-Hamide bending); 1472.3, 1452.6 (ring stretching);

1323.5, 1183.9, 1117.9 (C-Haromatic bending); 1068.5, 717.1 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.18 (br.s, 2H, NH), 7.92 (s, 1H, H-2), 7.86 (d, 1H, J =2.4 Hz, H-4), 7.74 (d, J = 8.1 Hz, 1H, H-5’), 7.61-7.55 (m, 2H, H-5, H-2’), 7.50-7.42 (m, 2H, H-6, H-6’).

13

C

RI PT

NMR (75.4 MHz, DMSO) δ: 179.91 (C=S), 140.09 (C1), 139.36 (C1’), 130.58 (C2’), 130.30 (C3’), 129.59 (C5), 129.06 (q, J = 31.8 Hz, C3), 127.53 (C6), 126.37 (C4’), 125.03 (C6’), 124.00 (q, J = 272.1 Hz, C3a), 123.76 (C5’), 120.95 (q, J = 3.8 Hz, C2), 120.02 (q, J = 4.0 Hz, C4). HRMS (ESI) calc. for C14H8Cl2F3N2S [M ‒ H] ‒: 362.9737, found: 362.9729. 1-(3-Chloro-4-methylphenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (7)

SC

4.1.2.7.

Yield 71%, white powder, m.p. 144.5-146 °C. FT-IR (KBr, cm-1): 3297.4 (N-Hamide stretching);

M AN U

3076.1 (C-Haromatic stretching); 1637.6 (N-Hamide bending), 1492.6, 1445.8 (ring stretching); 1428.5, 1385.5 (C-Halifatic rocking); 1338.3, 1160.2, 1117.7 (C-Haromatic bending); 1068.5, 700.5 (C=Sthioamide stretching).

1

H NMR (300 MHz, DMSO) δ: 10.04 (s, 2H, NH), 7.93 (s, 1H, H-2), 7.75 (d, 1H, J

= 8.1 Hz, H-4), 7.61-7.54 (m, 1H, H-5, H-5’), 7.46 (d, J = 7.5 Hz, 1H, H-6), 7.34-7.27 (m, 2H, H-2’, H-6’), 2.30 (s, 3H, H-4’a). 13C NMR (75.4 MHz, DMSO) δ: 179.87 (C=S), 140.41 (C1), 138.53 (C1’), 133.10 (C3’), 132.65 (C4’), 131.15 (C5’), 129.87 (C5), 129.50 (q, J =

TE D

31.8 Hz, C3), 128.59 (C2’), 127.40 (C6), 123.96 (q, J = 270.1 Hz, C3a), 126.93 (C6’), 119.92 (q, J = 4.1 Hz, C2), 118.20 (q, J = 3.8 Hz, C4), 18.99 (C4’a). HRMS (ESI) calc. for C15H11ClF3N2S [M ‒ H] ‒: 343.0284, found: 343.0273. 1-(3-Chlorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (8)

EP

4.1.2.8.

Yield 82%, white powder, m.p. 106-108 °C. FT-IR (KBr, cm-1): 3227.6, 3187.5 (N-Hamide stretching);

3048.4 (C-Haromatic stretching); 1594.5 (N-Hamide bending); 1475.3, 1450.6 (ring stretching);

AC C

1330.0, 1173.5, 1116.4 (C-Haromatic bending); 1072.7, 689.0 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.14 (s, 2H, NH), 7.93 (s, 1H, H-2), 7.75 (d, 1H, J = 8.4 Hz, H-4), 7.667.65 (m, 1H, H-5’), 7.57 (t, 1H, J = 7.95 Hz, H-5), 7.48 (d, J = 7.5 Hz, 1H, H-6), 7.42-7.34 (m, 2H, H-2’, H-4’), 7.22-7.19 (m, 1H, H-6’). 13C NMR (75.4 MHz, DMSO) δ: 180.37 (C=S), 141.18 (C1’), 140.72 (C1), 133.14 (C3’), 130.62 (C5’), 129.74 (C5), 129.00 (q, J = 32 Hz, C3), 127.91 (C6), 124.71 (C6’), 123.81 (q, J = 272 Hz, C3a), 123.12 (C2’), 122.52 (C4’), 120.84 (q, J = 3.9 Hz, C2), 119.90 (q, J = 4.0 Hz, C4). HRMS (ESI) calc. for C14H9ClF3N2S [M ‒ H] ‒: 329.0127, found: 329.0116. 4.1.2.9.

1-(4-Chlorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (9) 15

ACCEPTED MANUSCRIPT Yield 74%, white powder, m.p. 167-169 °C. FT-IR (KBr, cm-1): 3210.7, 3181.4 (N-Hamide stretching);

3030.4 (C-Haromatic stretching); 1594.7 (N-Hamide bending); 1488.5, 1450.7 (ring stretching);

1327.4, 1178.5, 1116.9 (C-Haromatic bending); 1089.0, 715.5 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.07 (s, 2H, NH), 7.94 (s, 1H, H-2), 7.75 (d, J = 8.4 Hz, 1H, H-4), 7.597.39 (m, 6H, H-5, H-6, H-2’, H-3’, H-5’, H-6’).

13

C NMR (75.4 MHz, DMSO) δ: 179.88

RI PT

(C=S), 140.30 (C1), 138.06 (C1’), 129.50 (C5), 128.90 (q, J = 31.3 Hz, C3), 128.63 (C4’), 128.44 (C2’, C6’), 127.33 (C6), 125.41 (C3’, C5’), 124.03 (q, J = 272.7 Hz, C3a), 120.69 (q, J = 3.8 Hz, C2), 119.83 (q, J = 3.8 Hz, C4). HRMS (ESI) calc. for C14H9ClF3N2S [M ‒ H] ‒: 329.0127, found: 329.0136.

1-(4-Cyanophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (10)

SC

4.1.2.10.

Yield 64%, white powder, m.p. 91-92.5 °C. FT-IR (KBr, cm-1): 3241.5, 3204.7 (N-Hamide 3046.3 (C-Haromatic stretching); 2222.4 (C≡Naromatic nitrile stretching); 1594.1 (N-Hamide bending);

M AN U

stretching);

1508.6, 1452.8 (ring stretching); 1326.3, 1178.4, 1118.4 (C-Haromatic 1

bending);

1069.6, 698.6

(C=Sthioamide stretching). H NMR (300 MHz, DMSO) δ: 10.37 (s, 2H, NH), 7.95 (s, 1H, H-2), 7.81-7.73 (m, 5H, H-4, H-2’, H-3’, H-5’, H-6’), 7.56 (t, 1H, J = 7.8 Hz, H-5), 7.50 (d, 1H, J = 7.8 Hz, H-6).

13

C NMR (75.4 MHz, DMSO) δ: 179.65 (C=S), 143.77 (C1’), 140.02 (C1),

132.81 (C3’, C5’), 129.66 (C5), 129.10 (q, J = 31.7 Hz, C3), 127.41 (C6), 124.00 (q, J =

TE D

272.8 Hz, C3a), 122.58 (C2’, C6’), 121.07 (br. q, C2), 119.94 (C4), 118.97 (C4’a), 105.70 (C4’). HRMS (ESI) calc. for C15H9F3N3S [M ‒ H] ‒: 320.0469, found: 320.0470. 4.1.2.11.

1-(4-Iodophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (11)

EP

1-(4-Iodophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (11) has been synthesized as described previously [53].

Yield 55%, white powder, m.p. 166-168 °C. FT-IR (KBr, cm-1): 3185.4, 3142.6 (N-Hamide 3014.5 (C-Haromatic stretching); 1591.7 (N-Hamide bending); 1482.5, 1449.5 (ring stretching);

AC C

stretching);

1324.6, 1173.1, 1119.6 (C-Haromatic bending); 1067.2, 706.3 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.06 (s, 2H, NH), 7.94 (s, 1H, H-2), 7.75 (d, J = 8.4 Hz, 1H, H-4), 7.717.66 (m, 2H, H-3’, H-5’), 7.56 (t, J = 7.95 Hz, 1H, H-5), 7.48-7.45 (m, 1H, H-6), 7.34-7.29 (m, 2H, H-2’, H-6’).

13

C NMR (75.4 MHz, DMSO) δ: 179.67 (C=S), 140.29 (C1), 138.96

(C1’), 137.22 (C3’, C5’), 129.49 (C5), 128.94 (q, J = 32.1 Hz, C3), 127.33 (C6), 125.80 (C2’, C6’), 123.92 (q, J = 270 Hz, C3a), 120.65 (C2), 119.83 (q, J = 3.9 Hz, C4), 89.00 (C4’). HRMS (ESI) calc. for C14H9IF3N2S [M ‒ H] ‒: 420.9483, found: 420.9470. 4.1.2.12.

1-(4-Nitrophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (12) 16

ACCEPTED MANUSCRIPT Yield 75%, yellow powder, m.p. 193-195 °C. FT-IR (KBr, cm-1): 3256.2, 3224.1 (N-Hamide stretching);

3070.5 (C-Haromatic

stretching);

1594.3 (N-Hamide

bending);

1557.8 (aromatic-NO2

stretching); 1511.4, 1453.6 (ring stretching); 1328.4 (aromatic-NO2 stretching); 1184.5, 1117.4 (C-Haromatic bending); 1071.6, 700.6 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.56 (s, 1H, NH), 10.45 (s, 1H, NH), 8.25-8.20 (m, 2H, H-3’, H-5’), 7.96 (br s, 1H, H-2),

RI PT

7.85-7.77 (m, 3H, H-4, H-2’, H-6’), 7.61 (t, J = 7.95 Hz, 1H, H-5), 7.52 (d, J = 7.8 Hz, 1H, H6). 13C NMR (75.4 MHz, DMSO) δ: 179.66 (C=S), 145.84 (C1’), 142.58 (C4’), 139.93 (C1), 129.72 (C5), 129.12 (q, J = 32.1 Hz, C3), 127.44 (C6), 124.44 (C2’, C6’), 123.99 (q, J = 272.1 Hz, C3a), 121.88 (C3’, C5’), 121.19 (q, J = 3.9 Hz, C2), 119.91 (q, J = 3.9 Hz, C4).

4.1.2.13.

SC

HRMS (ESI) calc. for C14H9F3N3O2S [M ‒ H] ‒: 340.0368, found: 340.0357.

1-[4- Trans-(4-butylcyclohexyl)phenyl]-3-[3-(trifluoromethyl)phenyl]thiourea (13)

stretching);

3054.3 (C-Haromatic

stretching);

M AN U

Yield 63%, white powder, m.p. 116-118 °C. FT-IR (KBr, cm-1): 3255.5, 3201.7 (N-Hamide 2919.3, 2847.3 (C-Halifatic

stretching);

1605.2 (N-Hamide

bending);

1449.5 (ring stretching); 1255.6, 1219.3 (C-Califatic stretching); 1181.3, 1122.4 (C-Haromatic

bending);

1072.3, 703.6 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 9.94 (s, 1H, NH),

9.93 (s, 1H, NH), 7.96 (br.s, 1H, H-2), 7.76 (d, J = 8.4 Hz, 1H, H-4), 7.54 (t, J = 7.8 Hz, 1H, H-5), 7.44 (d, J = 7.8 Hz, 1H, H-6), 7.34 (d, 2H, J = 8.4 Hz, H-3’, H-5’) 7.20 (d, 2H, J = 8.4

TE D

Hz, H-2’, H-6’), 2.45-2.41 (m, 1H, H-4’a), 1.82-1.79 (m, 4H, H-4’b, H-4’f), 1.48-1.35 (m, 2H, H-4’c), 1.29-1.22 (m, 7H, H-4’e, H-4’d, H-4’da, H-4’db), 1.08-0.97 (m, 2H, H-4’dc), 0.90-0.86 (t, J = 7.2 Hz, 3H, H-4’dd). 13C NMR (75.4 MHz, DMSO) δ: 179.64 (C=S), 144.01 (C4’), 140.55 (C1), 136.68 (C1’), 129.37 (C5), 128.87 (q, J = 31.4 Hz, C3), 127.19 (C6),

EP

126.78 (C3’, C5’), 124.03 (q, J = 272.5 Hz, C3a), 123.81 (C2’, C6’), 120.42 (br. q, C2), 119.65 (q, J = 3.9 Hz, C4), 43.31 (C4’a), 36.58 (C4’b, C4’f), 33.81 (C4’c, C4’e), 33.06

AC C

(C4’d), 28.62 (C4’da), 22.42 (C4’db), 20.15 (C4’dc), 13.98 (C4’dd). HRMS (ESI) calc. for C24H28F3N2S [M ‒ H] ‒: 433.1925, found: 433.1927. 4.1.2.14.

1-(2-Bromophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (14)

Yield 72%, white powder, m.p. 167-169 °C. FT-IR (KBr, cm-1): 3292.5 (N-Hamide

stretching);

3185.2 (C-Haromatic stretching); 1591.8 (N-Hamide bending); 1507.6, 1446.4 (ring stretching); 1333.5, 1196.0, 1123.1 (C-Haromatic

bending);

1075.2, 707.0 (C=Sthioamide

stretching).

1

H NMR (300 MHz,

DMSO) δ: 10.17 (s, 1H, NH), 9.67 (s, 1H, NH), 8.03 (m, 1H, H-2), 7.80 (d, 1H, J = 8.1 Hz, H-4), 7.71-7.68 (m, 1H, H-3’), 7.60-7.46 (m, 3H, H-5, H-6, H-5’), 7.43-7.38 (m, 1H, H-6’), 7.25-7.19 (m, 1H, H-4’). 13C NMR (75.4 MHz, DMSO) δ: 180.52 (C=S), 140.20 (C1), 137.50 (C1’), 132.67 (C4’), 130.25 (C5), 129.54 (C3’), 128.95 (q, J = 31.9 Hz, C3), 128.27 (C6’), 17

ACCEPTED MANUSCRIPT 127.94 (C5’), 127.29 (C6), 124.03 (q, J = 272.4 Hz, C3a), 121.43 (C2’), 120.76 (q, J = 4.0 Hz, C2), 119.78 (q, J = 4.0 Hz, C4). HRMS (ESI) calc. for C14H9BrF3N2S [M ‒ H] ‒: 372.9622, found: 372.9608. 4.1.2.15.

1-(3-Bromophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (15)

Yield 72%, white powder, m.p. 99-101 °C. FT-IR (KBr, cm-1): 3203.8 (N-Hamide

stretching);

RI PT

3034.2, (C-Haromatic stretching); 1588.0 (N-Hamide bending); 1477.0, 1452.5 (ring stretching); 1328.9, 1182.4, 1117.8 (C-Haromatic bending), 1067.3, 711.3 (C=Sthioamide stretching).

1

H NMR (300 MHz,

DMSO) δ: 10.34 (s, 1H, NH), 10.31 (s, 1H, NH), 7.97 (s, 1H, H-2), 7.83 (m, 1H, H-4’), 7.77 (d, 1H, J = 8.1 Hz, H-4), 7.57 (t, J = 7.95 Hz, 1H, H-5), 7.52-7.43 (m, 2H, H-6, H-5’), 7.3713

C NMR (75.4 MHz, DMSO) δ: 180.36 (C=S), 141.06 (C1’),

SC

7.28 (m, 2H, H-2’, H-6’).

140.42 (C1), 130.87 (C5’), 130.26 (C4’), 130.02 (C5), 129.44 (q, J = 31.8 Hz, C3), 127.98

M AN U

(C6), 127.82 (C2’), 126.56 (C6’), 123.09 (C3’), 124.65 (q, J = 270.0 Hz, C3a), 121.38 (C2), 120.48 (C4). HRMS (ESI) calc. for C14H9BrF3N2S [M ‒ H] ‒: 372.9622, found: 372.9620. 4.1.2.16. 1-[4- Trans-(4-ethylcyclohexyl)phenyl]-3-[3-(trifluoromethyl)phenyl]thiourea (16) Yield 76%, white powder, m.p. 135-139 °C. FT-IR (KBr, cm-1): 3249.8 (N-Hamide

stretching);

3106.7 (C-Haromatic stretching); 2964.7, 2850.6 (C-Halifatic stretching); 1594.1 (N-Hamide bending); 1496.5, 1445.3 (ring stretching); 1224.8, 1200.4 (C-Califatic stretching); 1173.0, 1119.4 (C-Haromatic bending);

TE D

1073.4, 710.8 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 9.94 (s, 1H, NH), 9.92 (s, 1H, NH), 7.96 (br.s, 1H, H-2), 7.76 (d, J = 8.1 Hz, 1H, H-4), 7.54 (t, J = 7.95 Hz, 1H, H-5), 7.44 (d, J = 7.8 Hz, 1H, H-6), 7.33 (d, 2H, J = 8.4 Hz, H-3’, H-5’) 7.20 (d, 2H, J = 8.4 Hz, H-

EP

2’, H-6’), 2.46-2.41 (m, 1H, H-4’a), 1.84-1.79 (m, 4H, H-4’b, H-4’f), 1.49-1.36 (m, 2H, H4’c), 1.29-1.16 (m, 3H, H-4’e, H-4’d), 1.08-0.97 (m, 2H, H-4’da), 0.89 (t, J = 7.2 Hz, 3H, H4’db).

13

C NMR (75.4 MHz, DMSO) δ: 179.63 (C=S), 143.99 (C4’), 140.57 (C1), 136.70

AC C

(C1’), 129.35 (C5), 128.88 (q, J = 31.6 Hz, C3), 127.15 (C6), 126.77 (C3’, C5’), 124.07 (q, J = 271.8 Hz, C3a), 123.79 (C2’, C6’), 120.36 (q, J = 3.6 Hz, C2), 119.64 (q, J = 4.0 Hz, C4), 43.33 (C4’a), 38.37 (C4’b), 33.80 (C4’b, C4’f), 32.63 (C4’c, C4’e), 29.46 (C4’da), 11.34 (C4’db). HRMS (ESI) calc. for C22H24F3N2S [M ‒ H] ‒: 405.1612, found: 405.1608. 4.1.2.17.

1-(3-Methoxyphenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (17)

Yield 35%, grey crystals, m.p. 112-114 °C. FT-IR (KBr, cm-1): 3192.8 (N-Hamide

stretching);

3039.4 (C-Haromatic stretching); 1603.9 (N-Hamide bending); 1491.1, 1454.2 (ring stretching); 1211.7 (C-O-Cether stretching); 1163.8, 1122.1 (C-Haromatic bending); 1066.2, 710.6 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.04 (s, 1H, NH), 10.01 (s, 1H, NH), 7.96-7.93 (m, 1H, H-2), 18

ACCEPTED MANUSCRIPT 7.75 (d, J = 8.1 Hz, 1H, H-4), 7.61-7.50 (m, 1H, H-5), 7.45 (d, J = 7.8 Hz, 1H, H-6), 7.25 (t, J = 8.1 Hz, 1H, H-5’), 7.15-7.14 (m, 1H, H-4’), 7.03-7.00 (m, 1H, H-2’), 6.74 (dd, J1 = 2.1 Hz, J2 = 1.8 Hz, 1H, H-6’), 3.74-3.73 (m, 3H, H-3’a).

13

C NMR (75.4 MHz, DMSO) δ: 179.84

(C=S), 159.63 (C3’), 140.70 (C1’), 140.40 (C1), 129.71 (C5’), 129.63 (C5), 129.18 (q, J = 32 Hz, C3), 127.60 (C6), 124.29 (q, J = 272 Hz, C3a), 120.80 (q, J = 3.8 Hz, C2), 120.10 (q, J =

RI PT

4.0 Hz, C4), 115.96 (C6’), 110.41 (C4’), 109.60 (C2’), 55.35 (C3’a). HRMS (ESI) calc. for C15H12F3N2OS [M ‒ H] ‒: 325.0622, found: 325.0618. 4.1.2.18.

1-(4-Butyl-2-methylphenyl)-3-[3-(trifluoromethyl)phenyl]thiourea (18)

Yield 58%, white powder, m.p. 106-108 °C. FT-IR (KBr, cm-1): 3250.4 (N-Hamide

stretching);

SC

3042.5 (C-Haromatic stretching); 2954.6, 2855.5 (C-Halifatic stretching); 1602.7 (N-Hamide bending); 1486.2, 1452.4 (ring stretching); 1326.5, 1171.2, 1119.7 (C-Haromatic bending); 1070.4; 704.1 (C=Sthioamide 1

H NMR (300 MHz, DMSO) δ: 9.78 (s, 1H, NH), 9.51 (s, 1H, NH), 7.96 (s, 1H, H-

M AN U

stretching).

2), 7.77 (d, J = 8.1 Hz, 1H,H-4), 7.54 (t, J = 7.8 Hz, 1H, H-5), 7.43 (d, J = 7.5 Hz, 1H, H-6), 7.15-7.08 (m, 2H, H-3’, H-5’), 7.03-7.00 (m, 1H, H-6’), 2.57-2.49 (m, 2H, H-4’a), 2.22 (s, 3H, H-2’a), 1.60-1.50 (m, 2H, H-4’b), 1.38-1.25 (m, 2H, H-4’c), 0.90 (t, J = 7.2Hz, 3H, H4’d).

13

C NMR (75.4 MHz, DMSO) δ: 180.53 (C=S), 140.81 (C4’), 140.61 (C1), 134.88

(C2’), 134.48 (C1’), 130.30 (C3’), 129.30 (C5), 128.83 (q, J = 31.7 Hz, C3), 127.68 (C6),

TE D

127.29 (C5’), 126.09 (C6’), 124.07 (q, J = 272.1 Hz, C3a), 120.36 (q, J = 3.8 Hz, C2), 119.74 (q, J = 4.2 Hz, C4), 34.36 (C4’a), 33.05 (C4’b), 21.77 (C4’c), 17.77 (C2’a), 13.74 (C4’d). HRMS (ESI) calc. for C19H20F3N2S [M ‒ H] ‒: 365.1299, found: 365.1292. Methyl 2-({[3-(trifluoromethyl)phenyl]carbamothioyl}amino)benzoate (19)

EP

4.1.2.19.

Yield 40%, white powder, m.p. 164-165 °C. FT-IR (KBr, cm-1): 3175.5 (N-Hamide 3020.2 (C-Haromatic

stretching);

1704.6 (C=Ocarbamate

stretching);

1614.6 (N-Hamide

bending);

stretching);

1487.5,

AC C

1446.4 (ring stretching); 1329.4, 1172.6, 1129.3 (C-Haromatic bending); 1073.5, 697.1 (C=Sthioamide stretching).

1

H NMR (300 MHz, DMSO) δ: 10.61 (s, 1H, NH), 10.29 (s, 1H, NH), 8.01-7.86 (m,

2H, H-2, H-3’), 7.83-7.70 (m, 3H, H-4, H-5’, H-6’), 7.66-7.57 (m, 1H, H-5), 7.48 (t, J = 9.0 Hz, 1H, H-4’), 7.39-7.26 (m, 1H, H-6), 3.83 (s, 3H, H-2’b). 13C NMR (75.4 MHz, DMSO) δ: 176.75 (C=S), 161.25 (C2’a), 140.80 (C1’), 140.48 (C1), 137.19 (C5’), 134.36 (C3’), 131.46 (C5), 131.08 (q, J = 31.8 Hz, C3), 128.60 (C6), 126.67 (C4’), 125.93 (C6’), 125.93 (C2’), 124.77 (q, J = 270.7 Hz, C3a), 117.00 (C2), 116.70 (C4), 49.72 (C2’b). HRMS (ESI) calc. for C16H12F3N2O2S [M ‒ H] ‒: 353.0577, found: 353.0572. 4.1.2.20.

Ethyl 4-({[3-(trifluoromethyl)phenyl]carbamothioyl}amino)benzoate (20) 19

ACCEPTED MANUSCRIPT Yield 40%, white powder, m.p. 130-132 °C. FT-IR (KBr, cm-1): 3242.4, 3194.1 (N-Hamide stretching);

3081.3 (C-Haromatic

stretching);

2992.5, 2900.7 (C-Halifatic

stretching);

1674.4 (C=Ocarbamate

stretching);

1602.8 (N-Hamide bending); 1486.5, 1453.4 (ring stretching); 1328.5, 1177.5, 1121.1 (C-

Haromatic bending); 1074.5, 694.2 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.33 (s, 1H, NH), 10.24 (s, 1H, NH), 7.96-7.94 (m, 2H, H-3’, H-5’), 7.91 (s, 1H, H-2), 7.76 (d, J =

RI PT

8.1 Hz, 1H, H-4), 7.68 (d, J = 8.7 Hz, 2H, H-2’, H-6’), 7.58 (t, J = 7.95 Hz, 1H, H-5), 7.48 (d, J = 7.8 Hz, 1H, H-6), 4.30 (dd, J1 = 6.9 Hz, J2 = 7.2 Hz, 2H, H-4’b), 1.31 (t, J = 7.0 Hz, 3H, H-4’c). 13C NMR (75.4 MHz, DMSO) δ: 179.61 (C=S), 165.27 (C4’a), 143.72 (C1’), 140.15 (C1), 129.78 (C3’, C5’), 129.58 (C5), 129.41 (q, J = 31.7 Hz, C3), 127.34 (C6), 125.11 (C4’), 123.60 (q, J = 272 Hz, C3a), 122.12 (C2’, C6’), 120.87 (C2), 119.83 (C4), 60.48 (C4’b),

1-(Furan-2-ylmethyl)-3-[3-(trifluoromethyl)phenyl]thiourea (21)

M AN U

4.1.2.21.

SC

14.16 (C4’c). HRMS (ESI) calc. for C17H14F3N2O2S [M ‒ H] ‒: 367.0728, found: 367.0712.

Yield 57%, yellow crystals, m.p. 107-108 °C. FT-IR (KBr, cm-1): 3173.6 (N-Hamide stretching); 3022.3 (C-Haromatic stretching); 1596.1 (N-Hamide bending); 1450.7 (ring stretching); 1332.3, 1165.7, 1120.0 (C-Haromatic bending); 1068.5, 699.5 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 9.81 (s, 1H, NH), 8.34 (br.s, 1H, NH), 8.01 (s, 1H, H-2), 7.68 (d, J = 7.8 Hz, 1H, H-4), 7.63-7.62 (m, 1H, H-5’), 7.54 (t, J = 7.95 Hz, 1H, H-5), 7.43 (d, J = 7.8 Hz, 1H, H-6), 6.43

H-1’).

13

TE D

(dd, J1=J2 = 1.8 Hz, 1H, H-4’), 6.34 (dd, J1=J2 = 0.9 Hz, 1H, H-3’), 4.73 (d, J= 5.1 Hz, 2H, C NMR (75.4 MHz, DMSO) δ: 180.72 (C=S), 151.28 (C2’), 142.31 (C5’), 140.37

(C1), 129.56 (C5), 128.99 (q, J = 31.7 Hz, C3), 126.41 (C6), 124.06 (q, J = 272.05 Hz, C3a), 120.20 (br. q, C2), 118.88 (C4), 110.52 (C4’), 107.58 (C3’), 40.51 (C1’). HRMS (ESI) calc.

4.1.2.22.

EP

for C13H10F3N2OS [M ‒ H] ‒: 299.0466, found: 299.0460. 1-[3-(Methylsulfanyl)propyl]-3-[3-(trifluoromethyl)phenyl]thiourea (22)

AC C

Yield 45%, white powder, m.p. 56-57.5 °C. FT-IR (KBr, cm-1): 3135.3 (N-Hamide

stretching);

2982.3 (C-Haromatic stretching); 2955.6, 2845.0 (C-Halifatic stretching); 1590.0 (N-Hamide bending); 1484.1, 1441.1 (ring stretching); 1389.2 (C-Halifatic rocking); 1331.7, 1163.5, 1124.1 (C-Haromatic bending); 1065.8, 697.1 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 9.71 (s, 1H, NH), 8.04 (s, 1H, NH), 7.97 (s, 1H, H-2), 7.67 (d, J = 8.1 Hz, 1H, H-4), 7.53 (t, J = 7.95 Hz, 1H, H-5), 7.41 (m, J = 7.8 Hz, 1H, H-6), 3.57-3.55 (m, 2H, H-1’), 2.54-2.50 (m, 2H, H-3’), 2.07 (s, 3H, H-4’), 1.88-1.79 (m, 2H, H-2’). 13C NMR (75.4 MHz, DMSO) δ: 180.58 (C=S), 140.47 (C1), 129.53 (C5), 128.95 (q, J = 31.9 Hz, C3), 126.12 (C6), 124.10 (q, J =272 Hz, C3a), 119.94 (C2), 118.65 (C4), 42.77 (C1’), 30.62 (C3’), 27.70 (C2’), 14.57 (C4’). HRMS (ESI) calc. for C12H14F3N2S2 [M ‒ H] ‒: 307.0551, found: 307.0552. 20

ACCEPTED MANUSCRIPT 4.1.2.23.

1-[2-(Morpholin-4-yl)ethyl]-3-[3-(trifluoromethyl)phenyl]thiourea (23)

Yield 60%, pale yellow oil. FT-IR (KBr, cm-1): 3277.2 (N-Hamide stretching); 3053.6 (C-Haromatic stretching);

2955.3, 2856.8 (C-Halifatic stretching); 1600.3 (N-Hamide bending); 1453.2 (ring stretching);

1332.4 (C-Haromatic bending); 1168.0 (C-Haromatic bending, C-O-Cstretching); 1119.1 (C-Haromatic bending); 1068.8, 700.4 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 9.95 (s, 1H, NH), 8.00 (s,

RI PT

1H, NH), 7.84 (s, 1H, H-2), 7.70 (d, J = 8.1 Hz, 1H, H-4), 7.54 (t, J = 7.95 Hz, 1H, H-5), 7.41 (d, J = 7.5 Hz, 1H, H-6), 3.59 (t, J = 4.5 Hz, 6H, H-1’, H-4’, H-6’), 2.52-2.48 (m, 4H, H13

3’, H-5’), 2.43-2.41 (m, 2H, H-2’).

C NMR (75.4 MHz, DMSO) δ: 180.23 (C=S), 140.45

(C1), 129.61 (C5), 129.10 (q, J = 32.1 Hz, C3), 125.87 (C6), 124.06 (q, J = 272.3 Hz, C3a), 119.91 (br. q, C2), 118.44 (C4), 66.15 (C4’, C5’), 56.39 (C2’), 53.13 (C3’, C6’), 40.67 (C1’).

SC

HRMS (ESI) calc. for C14H17F3N3OS [M ‒ H] ‒: 332.1044, found: 332.1049.

M AN U

4.1.2.24. 1-(2-Phenylethyl)-3-[3-(trifluoromethyl)phenyl]thiourea (24)

Yield 65%, white powder, m.p. 98-100 °C. FT-IR (KBr, cm-1): 3151.3 (N-Hamide 3015.1 (C-Haromatic

stretching);

2986.5 (C-Halifatic

stretching); 1399.0 (C-Halifatic 1170.8, 1137.3 (C-Haromatic

rocking);

bending);

stretching);

stretching);

1594.0 (N-Hamide bending); 1449.5 (ring

1327.7 (C-Haromatic

bending);

1069.8, 697.1 (C=Sthioamide

1260.5 (C-Califatic

stretching).

1

stretching);

H NMR (300 MHz,

DMSO) δ: 9.79 (s, 1H, NH), 7.95 (s, 2H, NH, H-2), 7.63 (d, J = 8.1 Hz, 1H, H-4), 7.51 (t,

TE D

1H, J = 7.8 Hz, H-5), 7.40-7.35 (d, J = 7.8 Hz, 1H, H-6), 7.33-7.20 (m, 5H, H-4’, H-5’, H-6’, H-7’, H-8’), 3.72 (m, 2H, H-1’), 2.89 (t, J = 7.35 Hz, 2H, H-2’).

13

C NMR (75.4 MHz,

DMSO) δ: 180.47 (C=S), 140.36 (C3’), 139.16 (C1), 129.55 (C5), 128.62 (C5’, C7’), 128.61 (q, J = 30.7 Hz, C3), 128.38 (C4’, C8’), 126.74 (C6’), 126.17 (C6), 124.04 (q, J = 272.5 Hz,

EP

C3a), 120.01 (C2), 118.63 (C4), 45.18 (C1’), 34.25 (C2’). HRMS (ESI) calc. for C16H14F3N2S [M ‒ H] ‒: 323.0830, found: 323.0834. N-{[3-(trifluoromethyl)phenyl]carbamothioyl}acetamide (25)

AC C

4.1.2.25.

N-{[3-(trifluoromethyl)phenyl]carbamothioyl}acetamide (25) has been synthesized as

described previously [54].

Yield 40%, dark yellow crystals, m.p. 207-209 °C. FT-IR (KBr, cm-1): 3182.5 (N-Hamide stretching);

3076.1 (C-Haromatic

stretching);

1597.8 (N-Hamide

stretching);

bending);

3020.5, 2943.7 (C-Halifatic

stretching);

1764.3 (C=Ocarbamate

1495.1, 1449.7 (ring stretching); 1415.8 (C-Halifatic

rocking);

1333.2, 1184.2, 1134.7 (C-Haromatic bending); 1066.5, 700.5 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 10.49 (s, 2H, NH), 7.83-7.72 (m, 3H, H-2, H-4, H-5), 7.65-7.62 (d, J = 7.8 Hz, 1H, H-6), 4.29 (s, 3H, H-2’).

13

C NMR (75.4 MHz, DMSO) δ: 182.95 (C=S), 172.24

(C1’), 134.50 (C1), 133.40 (C5), 130.19 (C6), 129.60 (q, J = 32.3 Hz, C3), 125.98 (q, J = 4.0 21

ACCEPTED MANUSCRIPT Hz, C2), 125.52 (q, J = 3.8 Hz, C4), 124.04 (q, J = 272.4 Hz, C3a), 49.54 (C2’). HRMS (ESI) calc. for C11H10F3N2O2S [M ‒ H] ‒: 261.0315, found: 261.0320. 4.1.2.26. Exo-1-bicyclo[2.2.1]hept-2-yl-3-[3-(trifluoromethyl)phenyl]thiourea (26) Yield 65%, yellow crystals, m.p. 155-157 °C. FT-IR (KBr, cm-1): 3247.6 (N-Hamide stretching); 3046.6 (C-Haromatic stretching); 2954.7, 2871.7 (C-Halifatic stretching); 1606.4 (N-Hamide bending); 1484.3, stretching).

1

RI PT

1456.7 (ring stretching); 1332.6, 1176.3, 1114.4 (C-Haromatic bending); 1068.5; 711.7 (C=Sthioamide H NMR (300 MHz, DMSO) δ: 9.48 (s, 1H, NH), 8.10 (s, 1H, NH), 7.92 (m, 1H, H-

2), 7.67 (d, J = 8.1 Hz, 1H, H-4), 7.51 (t, J = 7.95 Hz, 1H, H-5), 7.37 (d, J = 7.8 Hz, 1H, H-6), 4.11-3.99 (m, 1H, H-1’), 2.30-2.26 (m, 2H, H-7’), 1.77-1.70 (m, 1H, H-2’), 1.54-1.30 (m, 4H, 13

C NMR (75.4 MHz, DMSO) δ: 179.55 (C=S),

SC

H-3’, H-6’), 1.22-1.08 (m, 3H, H-4’, H-5’).

140.76 (C1), 129.40 (C5), 128.98 (q, J = 31.2 Hz, C3), 125.56 (C6), 124.1 (q, J = 272.1 Hz,

M AN U

C3a), 119.60 (C2), 118.07 (C4), 56.75 (C1’), 41.58 (C2’), 35.23 (C6’), 27.84 (C5’, C7’), 26.02 (C3’, C4’). HRMS (ESI) calc. for C15H16F3N2S [M ‒ H] ‒: 313.0986, found: 313.0977. 4.1.2.27.

1-Benzyl-3-[3-(trifluoromethyl)phenyl]thiourea (27)

Yield 50%, white powder, m.p. 114-115.5 °C. FT-IR (KBr, cm-1): 3265.0, 3169.2 (N-Hamide stretching); bending);

3031.4 (C-Haromatic

stretching);

2926.5, 2874.0 (C-Halifatic

stretching);

1601.6 (N-Hamide

1493.5, 1454.4 (ring stretching); 1332.5, 1165.2, 1116.2 (C-Haromatic

bending);

1070.7,

TE D

699.5 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 9.85 (s, 1H, NH), 8.41 (s, 1H, NH), 8.00 (s, 1H, H-2), 7.71 (d, 1H, J = 8.1 Hz, H-4), 7.54 (t, 1H, J = 7.95 Hz, H-5), 7.42 (d, 1H, J = 7.8 Hz, H-6), 7.36-7.32 (m, 4H, H-3’, H-4’, H-5’, H-6’), 7.29-7.27 (m, 1H, H-7’),

EP

4.75 (d, 2H, J = 5.4 Hz, H-1’). 13C NMR (75.4 MHz, DMSO) δ: 180.90 (C=S), 140.42 (C1), 138.60 (C2’), 129.57 (C5), 129.00 (q, J = 31.6 Hz, C3), 128.29 (C4’, C6’), 127.46 (C6), 126.96 (C3’, C7’), 126.39 (C5’), 124.10 (q, J = 272.1 Hz, C3a), 120.14 (C2), 118.84 (C4),

AC C

47.09 (C1’). HRMS (ESI) calc. for C15H12F3N2S [M ‒ H] ‒: 309.0673, found: 309.0673. 4.1.2.28.

1-Ethyl-3-[3-(trifluoromethyl)phenyl]thiourea (28)

Yield 55%, white powder, m.p. 91-92.5 °C. FT-IR (KBr, cm-1): 3261.1, 3238.6 (N-Hamide stretching); bending);

3072.6 (C-Haromatic

stretching);

2983.2, 2880.6 (C-Halifatic

stretching);

1614.7 (N-Hamide

1496.6, 1448.5 (ring stretching); 1337.2 (C-Haromatic bending); 1224.5 (C-Califatic stretching);

1169.4, 1114.5 (C-Haromatic

bending),

1073.4, 700.5 (C=Sthioamide

stretching).

1

H NMR (300 MHz,

DMSO) δ: 9.67 (s, 1H, NH), 7.97 (s, 2H, NH, H-2), 7.66 (d, J = 7.8 Hz, 1H, H-4), 7.52 (t, J = 7.95 Hz, 1H, H-5), 7.40 (d, J = 7.8 Hz, 1H, H-6), 3.51-3.45 (m, 2H, H-1’), 1.13 (t, J = 7.2 Hz, 3H, H-2’).

13

C NMR (75 MHz, DMSO) δ: 180.23 (C=S), 140.53 (C1), 129.50 (C5), 128.97 22

ACCEPTED MANUSCRIPT (q, J = 31 Hz, C3), 126.10 (C6), 122.27 (q, J = 271.8 Hz, C3a), 119.85 (C2), 118.67 (C4), 38.59 (C1’), 13.97 (C2’). HRMS (ESI) calc. for C10H10F3N2S [M ‒ H] ‒: 247.0517, found: 247.0510. 4.1.2.29.

1-(2-Methylprop-2-en-1-yl)-3-[3-(trifluoromethyl)phenyl]thiourea (29)

Yield 68%, pale yellow powder, m.p. 83-84.5 °C. FT-IR (KBr, cm-1): 3207.9 (N-Hamide 3049.5 (C-Haromatic bending); 1656.3 (C=Califatic stretching); 1596.2 (N-Hamide bending ); 1451.0

(ring stretching); 1332.6, 1167.8, 1119.2 (C-Haromatic stretching).

1

bending);

RI PT

stretching);

1072.6, 704.5 (C=Sthioamide

H NMR (300 MHz, DMSO) δ: 9.80 (br.s, 1H, NH), 8.09 (br.s, 1H, NH), 8.03 (s,

1H, H-2), 7.70 (d, J = 8.4 Hz, 1H, H-4), 7.54 (t, J = 7.8 Hz, 1H, H-5), 7.41 (d, J= 7.8 Hz, 1H,

SC

H-6), 4.85-4.84 (m, 2H, H-3’), 4.10 (m, 2H, H-1’), 1.73 (s, 3H, H-4’). 13C NMR (75.4 MHz, DMSO) δ: 181.85 (C=S), 142.37 (C2’), 140.91 (C1), 130.49 (C5), 129.78 (q, J = 31.5 Hz,

M AN U

C3), 127.63 (C6), 124.7 (q, J = 272.7 Hz, C3a), 121.49 (C2), 120.25 (C4), 111.25 (C3’), 49.74 (C1’), 20.99 (C4’). HRMS (ESI) calc. for C12H12F3N2S [M ‒ H] ‒: 273.0673, found: 273.0662. 4.1.2.30.

1-Cyclohexyl-3-[3-(trifluoromethyl)phenyl]thiourea (30)

1-Cyclohexyl-3-[3-(trifluoromethyl)phenyl]thiourea

(30)

has

been

synthesized

according to the method described previously [55].

TE D

Yield 60%, white powder, m.p. 135-136.5 °C. FT-IR (KBr, cm-1): 3231.1 (N-Hamide stretching); 3076.3 (C-Haromatic stretching); 2932.7, 2853.2 (C-Halifatic stretching); 1602.4 (N-Hamide bending); 1485.1, 1459.4 (ring stretching); 1327.3 (C-Haromatic bending); 1273.3, 1212.5 (C-Califatic stretching); 1164.2,

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1116.8 (C-Haromatic bending); 1073.2, 700.1 (C=Sthioamide stretching). 1H NMR (300 MHz, DMSO) δ: 9.57 (s, 1H, NH), 8.05 (s, 1H, NH), 7.90 (m, 1H, H-2), 7.66 (d, J = 8.1 Hz, 1H, H-4), 7.51 (t, J = 7.95 Hz, 1H, H-5), 7.38 (d, J = 7.8 Hz, 1H, H-6), 4.09 (br.s, 1H, H-1’), 1.93-1.90 (m, 2H, 13

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H-2’), 1.71-1.67 (m, 2H, H-6’), 1.59-1.55 (m, 1H, H-4’), 1.38-1.15 (m, 5H, H-3’, H-4’, H-5’). C NMR (75.4 MHz, DMSO) δ: 179.20 (C=S), 140.65 (C1), 129.42 (C5), 128.66 (q, J =31.1

Hz, C3), 125.90 (C6), 124.07 (q, J = 272.5 Hz, C3a), 119.67 (C2), 118.35 (C4), 52.08 (C1’), 31.71 (C2’, C6’), 25.10 (C4’), 24.42 (C3’, C5’). HRMS (ESI) calc. for C14H17F3N2S [M]• ‒: 302.1065; found 302.1088.

4.1.2.31.

N-{[3-(Trifluoromethyl)phenyl]carbamothioyl}benzamide (31)

N-{[3-(Trifluoromethyl)phenyl]carbamothioyl}benzamide (31) has been synthesized as described previously [56].

23

ACCEPTED MANUSCRIPT Yield 75%, pale yellow crystals, m.p. 111-113 °C. FT-IR (KBr, cm-1): 3283.1 (NHamide

stretching);

bending); 1

2998.9 (C-Haromatic

stretching);

1673.2 (C=Ocarbamate

stretching);

1599.3 (N-Hamide

1474.5 (ring stretching); 1345.1, 1148.3 (C-Haromatic bending); 718.2 (C=Sthioamide stretching).

H NMR (300 MHz, DMSO) δ: 12.65 (s, 1H, NH), 11.70 (s, 1H, NH), 8.21 (s, 1H, H-2),

8.00-7.98 (m, 2H, H-3’, H-7’), 7.92 (d, J = 7.2 Hz, 1H, H-4), 7.70-7.62 (m, 3H, H-5, H-4’, H13

C NMR (75.4 MHz, DMSO) δ: 180.02 (C=S), 169.23

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6’), 7.58-7.53 (m, 2H, H-6, H-5’).

(C1’), 139.37 (C1), 134.33 (C2’), 132.59 (C5’), 130.89 (C5), 130.20 (q, J = 32.0 Hz, C3), 129.57 (C4’, C6’), 129.24 (C3’, C7’), 128.22 (C6), 124.62 (J = 271.8 Hz, C3a), 123.96 (q, J = 3.8 Hz, C2), 122.03 (q, J = 4.2 Hz, C4). HRMS (ESI) calc. for C15H10F3N2OS [M ‒ H] ‒:

4.2. Biological assays

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4.2.1. In vitro evaluation of antimicrobial activity

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323.0466, found: 323.0454.

Microorganisms used in this study were as follows: Gram-positive bacteria: Staphylococcus aureus NCTC 4163, Staphylococcus aureus ATCC 25923, Staphylococcus aureus

ATCC

6538,

Staphylococcus

aureus

ATCC

29213,

Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis ATCC 35984,

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Enterococcus hirae ATCC 10541, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Micrococcus luteus ATCC 9341, Micrococcus luteus ATCC 10240; Gram-negative rods: Escherichia coli ATCC 10538, Escherichia coli ATCC 25922, Escherichia coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseudomonas

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aeruginosa ATCC 15442, Pseudomonas aeruginosa NCTC 6749, Pseudomonas aeruginosa ATCC 27863, Bordetella bronchiseptica ATCC 4617 and yeasts: Candida albicans ATCC 10231, Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019.

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Other microorganisms used were obtained from the collection of the Department of

Pharmaceutical Microbiology, Medical University of Warsaw, Poland.

4.2.2. Media, growth conditions and antimicrobial activity assays

Antibacterial activity was examined by the disc-diffusion method under standard conditions using Mueller-Hinton II agar medium (Becton Dickinson) according to CLSI (previously NCCLS) guidelines [36]. Antifungal activities were assessed using MuellerHinton agar + 2% glucose and 0.5µg/mL Methylene Blue Dye Medium [37].

24

ACCEPTED MANUSCRIPT Sterile filter paper discs (9mm diameter, Whatman No 3 chromatography paper) were dripped with tested compound solutions (in DMSO) to load 400µg of a given compound per disc. Dry discs were placed on the surface of appropriate agar medium. The results (diameter of the growth inhibition zone) were read after 18 h of incubation at 35°C. Minimal Inhibitory Concentration (MIC) was tested by the twofold serial

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microdilution method (in 96-well microtiter plates) using Mueller-Hinton Broth medium (Beckton Dickinson) for bacteria or RPMI-1640 medium for Candida species according to CLSI guidelines [38,39]. The stock solution of tested agent was prepared in DMSO and diluted in sterile water. Concentrations of tested agents ranged from 0.125 to 512 µg/mL-1.

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The final inoculum of all studied microorganisms was 105 CFU/ mL-1 (colony forming units per ml). Minimal inhibitory concentrations (the lowest concentration of an tested agent that prevents visible growth of a microorganism) were read after 18h (bacteria) or 24h (yeasts) of

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incubation at 35 °C.

4.2.2. Inhibition of bacterial topoisomerase IV. Decatenation assay

The assay was performed using S. aureus topoisomerase IV decantation kit

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(Inspiralis). Kinetoplast DNA (kDNA) was the substrate for topoisomerase IV. 1 U of topoisomerase IV decatenated 200 ng of kDNA, in the dedicated decantation assay buffer supplied by the manufacturer. Enzyme activity was detected by incubation for 30 min at 37oC in a total reaction volume of 30 µL and in the presence of compounds 5, 15 and the reference

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Ciprofloxacin at concentrations 1 µg/ml, 4 µg/ml and 32 µg/ml. The reactions were terminated by adding of an equal volume of STEB buffer (40% sucrose, 100 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mg/ml bromophenol blue), followed by extraction with 1 volume of

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chloroform/isoamyl alcohol (24: 1). Then, 20 µL of the aqueous phase of each sample was loaded onto a 1% agarose gel.

Electrophoresis was conducted in Tris-acetate-EDTA buffer for 1 h at 120 V. Gels were stained with ethidium bromide and visualized under UV light in a transilluminator (ChemiDoc MP, Bio Rad).

4.2.3. Biofilm inhibitory assay

Inhibition of bacterial biofilm formation was screened using modified method, described previously [57]. Eight hospital isolates of methicillin-resistant and two standard 25

ACCEPTED MANUSCRIPT strains of Staphylococcus epidermidis (ATCC 12228, ATCC 35984) were cultured overnight in Tryptone Soy Broth supplemented with 0.5% glucose. The solution of tested compounds in TSB-glucose medium were mixed (1:1) with the bacterial inoculums (107 CFU/mL) in sterile 96-well polystyrene microtiter plates (Karell - Medlab, Italy) and incubated at 37ºC for 24 h. The final concentrations of tested compounds ranged from 0.125 to 4 µg/ml (compound 5)

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and 0.25 to 8 µg/ml (15). The negative control was TSB-glucose medium, the positive control (biofilm formation) was bacterial culture in TSB-glucose. After incubation, medium was removed from wells and washed with sterile phosphate buffered saline (PBS) to remove the non-

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adherent bacteria. Alive bacterial cells in each well of the microtiter plate were stained with 3(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium bromide (MTT; 0.5% in PBS) for 2 hours at 37°C. Adherent bacterial cells, which usually formed biofilm on wells, were

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uniformly stained with MTT. After incubation, the solution was removed and bacterial biofilm was solubilized by DMSO with glycine buffer and mixed 15 minutes at room temperature. The absorbance (A554) was measured at 554 nm using spectrophotometer (PowerWave XS, BioTek).

The biofilm-inhibition results were interpreted from dose (concentrations)-response

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curve. IC50 value is defined as the concentrations of tested compounds are required to inhibit 50% of biofilm formation under the assay conditions. All the experiments were carried out in quadruplicates.

S. epidermidis strain ATCC 12228 and S. epidermidis ATCC 35984 were used in

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assays as a negative (low biofilm-producing) and positive (high biofilm-producing) control, respectively. Ciprofloxacin was used as the reference antimicrobial compound; its final

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concentration ranged from 0.125 to 8 µg/ml.

4.2.4. Cells and Viruses

Cell lines were purchased from American Type Culture Collection (ATCC). The

absence of mycoplasma contamination was checked periodically by the Hoechst staining method. Cell lines supporting the multiplication of RNA and DNA viruses were the following: CD4+ human T-cells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK) [ATCC CCL 22 (NBL-1) Bos Taurus]; Baby Hamster Kidney (BHK-21) [ATCC CCL 10 (C-13) Mesocricetus auratus] and Monkey kidney (Vero 76) [ATCC CRL 1587 Cercopithecus Aethiops]. 26

ACCEPTED MANUSCRIPT Viruses were purchased from American Type Culture Collection (ATCC), with the exception of Yellow Fever Virus (YFV), and Human Immunodeficiency Virus type-1 (HIV1). Viruses representative of positive-sense, single-stranded RNAs (ssRNA+) were: i) Retroviridae: the IIIB laboratory strain of HIV-1, obtained from the supernatant of the persistently infected H9/IIIB cells (NIH 1983); ii) Flaviviridae: yellow fever virus (YFV)

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[strain 17-D vaccine (Stamaril Pasteur J07B01)] and bovine viral diarrhoea virus (BVDV) [strain NADL (ATCC VR-534)]; iii) Picornaviridae: human enterovirus B [coxsackie type B5 (CVB-5), strain Ohio-1 (ATCC VR-29)], and human enterovirus C [poliovirus type-1 (Sb-1), Sabin strain Chat (ATCC VR-1562)]. Viruses representative of negative-sense, single-

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stranded RNAs (ssRNA-) were: iv) Rhabdoviridae: vesicular stomatitis virus (VSV) [lab strain Indiana (ATCC VR 1540)]. The virus representative of double-stranded RNAs (dsRNA) was reovirus type-1 (Reo-1) [simian virus 12, strain 3651 (ATCC VR-214)],

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Reoviridae family. DNA virus representatives were: v) Poxviridae: vaccinia virus (VV) [vaccine strain Elstree-Lister (ATCC VR-1549)]; vi) Herpesviridae: human herpes 1 (HSV-1) [strain KOS (ATCC VR-1493)].

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4.2.5. Cytotoxicity Assays

Cytotoxicity assays were run in parallel with antiviral assays. Cytotoxicity against MT-4 cells was determined as described in Stefanska et al. [32]. As far as stationary monolayers (analogous to those which support the replication of

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the other RNA and DNA viruses) are concerned, MDBK and BHK cells were seeded in 24well plates at an initial density of 6x105 and 1x106 cells/mL, respectively, in Minimum Essential Medium with Earle’s salts (MEM-E), L-glutamine, 1mM sodium pyruvate and

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25mg/L kanamycin, supplemented with 10% horse serum (MDBK) or 10% foetal bovine serum (FBS) (BHK). Cell viability was determined after 48-96 hrs at 37°C by the MTT method [58]. Vero-76 cells were seeded in 24-well plates at an initial density of 4x105 cells/mL, in Dulbecco’s Modified Eagle Medium (D-MEM) with L-glutamine and 25mg/L kanamycin, supplemented with 10% FBS. Cell viability was determined after 48-96 hrs at 37 °C by the crystal violet staining method. All cell cultures were then incubated at 37 °C in a humidified, 5% CO2 atmosphere, in the absence or presence of serial dilutions of test compounds in culture medium. Before dilutions, compounds were dissolved in DMSO at 100 mM.

27

ACCEPTED MANUSCRIPT 4.2.7. Antiviral assays

Compound's activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity in exponentially growing MT-4 cell, determined by the MTT method, as described in Stefanska et al. [32].

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Compound's activity against YFV and Reo-1 was based on inhibition of virus-induced cytopathogenicity in BHK-21 cells acutely infected with a m.o.i. of 0.01. Compound's activity against BVDV was based on inhibition of virus-induced cytopathogenicity in MDBK cells acutely infected with a m.o.i. of 0.01. Briefly, BHK and MDBK cells were seeded in 96-well

SC

plates at a density of 5x104 and 3x104 cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified CO2 (5%) atmosphere. Cell monolayers were then infected with 50 µL of a proper virus

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dilution in maintenance medium [MEM-Earl with L-glutamine, 1mM sodium pyruvate and 0.025g/L kanamycin, supplemented with 0.5% inactivated FBS] to give an m.o.i of 0.01. After 1 hr, 50 µL of maintenance medium, without or with serial dilutions of test compounds, were added. After a 3-/4-day incubation at 37°C, cell viability was determined by the MTT method.

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Compound's activity against CVB-5, Sb-1, VV and HSV-1 was determined by plaque reduction assays in infected cell monolayers. To this end, Vero 76-cells were seeded in 24well plates at a density of 2x105 cells/well and were allowed to form confluent monolayers by incubating overnight in growth medium [Dulbecco's Modified Eagle Medium (D-MEM) with

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L-glutamine and 4500 mg/L D-glucose and 0.025g/L kanamycin, supplemented with 10% FBS] at 37°C in a humidified CO2 (5%) atmosphere. Then, monolayers were infected for 2 hrs with 250 µL of proper virus dilutions to give 50-100 PFU/well. Following removal of

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unadsorbed virus, 500 µL of maintenance medium [D-MEM with L-glutamine and 4500 mg/L D-glucose, supplemented with 1% inactivated FBS] containing 0.75% methyl-cellulose, without or with serial dilutions of test compounds, were added. Cultures were incubated at 37°C for 2 (Sb-1 and VSV) or 3 days (CVB-5, VV and HSV-1) and then fixed with PBS containing 50% ethanol and 0.8% crystal violet, washed and air-dried. Plaques were then counted. The extent of cell growth/viability and viral multiplication, at each drug concentration tested, were expressed as percentage of untreated controls. Concentrations resulting in 50% inhibition (CC50 or EC50) were determined by linear regression analysis.

28

ACCEPTED MANUSCRIPT Efavirenz, 2’-C-methylguanosine, 2’-C-methylcytidine, 6-azauridine, Mycophenolic Acid and Acycloguanosine were used as reference inhibitors.

4.2.8. Cytotoxic activity in HaCaT cells

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4.2.8.1. Cell Culture: Conditions and Treatments

Human immortal keratinocyte cell line from adult human skin (HaCaT) was purchased from American Type Culture Collection (Rockville, USA), and cultured in Dulbecco's

SC

Modified Eagle's Medium (DMEM), supplemented with antibiotics (penicillin and streptomycin), 10% heat-inactivated FBS-fetal bovine serum (Gibco Life Technologies, USA) at 37ºC and 5% CO2 atmosphere. Cells were passaged using trypsin-EDTA (Gibco Life

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Technologies, USA) and cultured in 24-well plates (2.5 x 104 cells per well). Experiments were conducted in DMEM with 2% FBS.

4.2.8.2. Cell Viability Assessment (Mitochondrial Function Assessment)

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The cell viability assay was assessed by determination of MTT salt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich Chemie, Germany) conversion by mitochondrial dehydrogenase. The cells were incubated for 24 h in 24-well plates with tested compounds (5 and 15) at concentration of 16 µM, and subsequently for

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another 2 h with 0.5 mg/ml of MTT solution, which was converted in living cells under the effect of mitochondrial dehydrogenase into insoluble formazan. Then the converted dye was solubilized with the use of 0.04 M HCl in absolute isopropanol. Absorbance of solubilized

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formazan was measured spectrophotometrically at 570 nm (using Epoch microplate reader, BioTek Inc., USA, equipped with Gen5 software (BioTech Instruments,Inc., Biokom). Cell viability was presented as a percent of MTT in the treated cells versus the control

(cells incubated in serum-free DMEM without extracts). The relative MTT level (%) was calculated as [A]/[B] × 100, where [A] is the absorbance of the test sample and [B] is the absorbance of control sample containing the untreated cells. Decreased relative MTT level indicates decreased cell viability. 4.2.8.3. Lactate Dehydrogenase Release Assay (Cellular Membrane Integrity Assessment)

29

ACCEPTED MANUSCRIPT Release of lactate dehydrogenase (LDH) from the cytosol to culture medium is a marker of cell death. The assay was performed after 24 h incubation of HaCaT cells in 24well plates with compounds 5 and 15 (concentration of 16 µM). The activity of LDH released from cytosol of damaged cells to the supernatant was measured using the protocol of the cytotoxicity detection kit LDH test described by the manufacturer (Roche Diagnostics,

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Germany). Absorbance was measured at 490 nm using a microplate reader (using Epoch microplate reader, BioTek Inc., USA) equipped with Gen5 software (BioTech Instruments,Inc., Biokom)

Compounds mediated cytotoxicity expressed as the LDH release (%) was determined

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by the following equation: [(A test sample − A low control)/(A high control − A low control)] × 100% (A-absorbance); where “low control” were cells in DMEM with 2% FBS without

Triton X-100 (100% LDH release).

Acknowledgments

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tested compounds, and “high control” were cells incubated in DMEM with 2% FBS with 10%

The authors thank to Sylwester Krukowski from Department of Inorganic and Analytical

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Chemistry, Faculty of Pharmacy, Medical University of Warsaw, for recording IR spectra.

Appendix A. Supplementary data

The following is the supplementary data related to this article:

References

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[56] M.K. Rauf, A. Talib, A. Badshah, S. Zaib, K. Shoaib, M. Shahid, U. Flörke, Imtiaz-udDin, J. Iqbal, Eur. J. Med. Chem. 70 (2013) 487–496.

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[57] P. Nagender, G. Malla Reddy, R. Naresh Kumar, Y. Poornachandra, C. Ganesh Kumar, B. Narsaiah, Bioorg. Med. Chem. Lett. 224 (2014) 2905-2908. [58] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, E.

AC C

De Clercq, J. Virol. Methods 20 (1988) 309−321.

FIGURE CAPTIONS LIST

Fig.1. Synthetic procedure for thiourea derivatives 1-31.

33

ACCEPTED MANUSCRIPT Fig.2. The effect of studied compounds on topoisomerase IV in decatenation assays. The reactions were performed in the presence of compounds 5 and 15 at concentrations 32 µg/ml (lane I), 4 µg/ml (lane II) and 1 µg/ml (lane III). Ciprofloxacin (CFX) was used as the control compound at the same concentrations. The first lane, labelled A, contains a control assay without enzyme. The control (lane B) were performed in the absence of compounds. To

RI PT

checked the effect of DMSO on the reaction, tubes +/- DMSO was included (lane C). Fig. 3. Biofilm inhibitory activity of compound 5 – the percentage of S. epidermidis biofilm formed in the presence of the tested compound. (compared to drug-free control). All presented

SC

results are mean from experiments performed in quadruplicate ± S.D.

Fig. 4. Biofilm inhibitory activity of compound 15 – the percentage of S. epidermidis biofilm formed in the presence of the tested compound. (compared to drug-free control). All presented

M AN U

results are mean from experiments performed in quadruplicate ± S.D.

Fig. 5. Biofilm inhibitory activity of Ciprofloxacin – the percentage of S. epidermidis biofilm formed in its presence. All presented results are mean from experiments performed in

AC C

EP

TE D

quadruplicate ± S.D.

34

ACCEPTED MANUSCRIPT

Table 1. Structures, yields and calculated CLogP values of aryl- (1-20) and alkylthiourea (21-31) derivatives of 3-(trifluoromethyl)aniline.

S NH

Compound

R

Yields, %

CLogP*

R NH

Compound

R

RI PT

CF3

Yields, %

CLogP*

75

1

4.05

17

SC

OCH 3

35

3.98

58

6.06

40

3.96

40

4.37

57

2.94

45

3.35

60

2.01

65

4.19

2

F

70

4.15

M AN U

H3C

18

C 4H 9

O

F

H3CO

3

70

4.15

74

4.15

Cl

5

F

70

6

Cl

Cl

7

CH3

Cl

8

71

20

O C OC2H5

21

O

5.26

22

AC C

Cl

4.75

EP

4

19

TE D

F

C

71

5.00

23

82

4.65

24

CH2 CH2

S CH2

N

CH3

O

ACCEPTED MANUSCRIPT

O

9

74

Cl

4.65

25

40

C

2.39

CN

64

3.88

26

11

I

55

4.48

27

12

NO2

75

3.63

28

63

8.30

29

CH2

65

4.60

50

3.76

55

2.74

68

3.62

60

3.89

75

3.84

CH2

C

M AN U

C 4H 9

13

-CH2CH3

SC

10

RI PT

CH3

CH3

Br

14

72

4.77

72

4.77

76

7.25

Br

O

C2H5

AC C

EP

* Calculated by online program OSIRIS Property Explorer

31

TE D

15

16

30

ACCEPTED MANUSCRIPT

Table 2. Antibacterial activity of the synthesized thiourea derivatives (zone of inhibition in mm). 3

4

5

6

8

9

10 12 15 17 19 21 22 24 27 28 29 31 Ref.*

24 23 21 23 23 23 21 22 25 15 22 24 11

27 23 25 25 26 25 25 23 30 20 28 28 14

20 22 20 21 19 19 20 18 25 14 21 23 ‒

18 19 21 19 20 20 20 18 ‒ 13 23 21 11

30 27 30 30 30 27 30 25 34 20 30 32 14

15 14 14 15 13 13 11 13 15 12 16 15 ‒

29 27 29 27 28 26 26 23 ‒ 20 27 30 ‒

12 12 13 12 ‒ ‒ 12 12 ‒ ‒ ‒ ‒ ‒

20 19 22 18 18 20 19 19 ‒ ‒ 16 18 ‒

20 20 23 22 22 21 20 19 12 13 19 24 ‒

25 24 25 23 26 24 20 19 11 14 24 25 ‒

14 14 14 19 12 13 15 13 13 0 13 13 ‒

19 18 18 18 20 16 18 19 ‒ 13 16 20 ‒

SC

28 28 29 29 29 27 26 24 13 18 30 33 12

M AN U

16 20 15 16 13 12 15 15 13 ‒ 13 13 ‒

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2

TE D

S. aureus NCTC 4163 S. aureus ATCC 25923 S. aureus ATCC 6538 S. aureus ATCC 29213 S. epidermidis ATCC 12228 S. epidermidis ATCC 35984 B. subtilis ATCC 6633 B. cereus ATCC 11778 E. hirae ATCC 10541 E. faecalis ATCC 29212 M. luteus ATCC 10240 M. luteus ATCC 9341 P. vulgaris NCTC 4635 ‒ lack of activity * Ref. – Ciprofloxacin

1

15 15 15 15 14 17 16 16 17 ‒ 15 18 ‒

18 19 15 16 17 13 16 15 ‒ ‒ 15 19 ‒

14 15 14 19 14 13 15 15 13 ‒ 13 13 ‒

20 18 18 17 17 13 16 16 ‒ ‒ 16 20 ‒

17 17 17 17 17 16 17 14 ‒ 12 19 17 ‒

26 26 28 22 30 32 40 30 18 15 22 24 36

Table 3. Antibacterial activity of the synthesized thiourea derivatives (MIC values in µg/ml). 3 4

5 6

16 16 16 16 32 16

8 8 16 16 16 16

4 4 4 8 8 8

1 1 1 1 2 2

16 16 16 16 16 32

8

9 10

12

15 17 19 21

22 24 27 28

29 31 Ref.*

8 8 8 8 8 4

128 128 64 64 64 64

1 1 1 1 2 2

32 32 32 32 32 64

64 64 64 64 64 64

EP

2

0.5 0.5 0.5 0.5 2 1

32 32 32 32 32 64

AC C

S. aureus NCTC 4163 S. aureus ATCC 25923 S. aureus ATCC 6538 S. aureus ATCC 29213 S. epidermidis ATCC 12228 S. epidermidis ATCC 35984 * Ref. – Ciprofloxacin

1

128 64 64 64 64 64

32 32 32 32 32 32

32 32 32 32 64 64

256 256 256 256 256 512

8 8 8 8 16 8

16 16 8 8 32 16

256 128 256 256 256 512

32 32 32 32 64 64

0.25 0.5 0.25 0.5 0.25 0.25

ACCEPTED MANUSCRIPT

15

24 27 Ref.*

4 8 4 8 8 8 8 8 8 8 8 8 8 16 16 16

1 1 1 1 0.25 0.25 0.5 0.5 1 1 1 1 0.5 0.5 0.25 2

2 2 2 2 2 1 1 1 2 1 1 1 1 2 1 1

1 0.5 1 0.5 0.5 1 0.5 0.25 2 2 4 2 1 1 1 2

8 8 8 16 8 16 16 16 8 8 16 16 8 8 8 16

64 16 128 64 16 8 128 64 32 2 64 64 0.125 4 0.125 4

M AN U

8 8 8 16 16 8 16 16 16 16 16 16 16 16 8 16

TE D

16 16 16 16 16 16 16 8 16 16 16 16 16 16 8 8

SC

6 9

EP

* Ref. – Ciprofloxacin

5

AC C

S. aureus MRSA 53/05 S. aureus MRSA 54/05 S. aureus MRSA 57/05 S. aureus MRSA 79/05 S. aureus MRSA 80/05 S. aureus MRSA 81/05 S. aureus MRSA 522/11 S. aureus MRSA 573/11 S. epidermidis MRSE 16/04 S. epidermidis MRSE 23/04 S. epidermidis MRSE 24/04 S. epidermidis MRSE 31/04 S. epidermidis MRSE 62/04 S. epidermidis MRSE 63/04 S. epidermidis MRSE 76/04 S. epidermidis MRSE 151/04

3

RI PT

Table 4. Activity of compounds against hospital methicillin-resistant strains of Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) – minimal inhibitory concentrations (MIC, µg/ml).

ACCEPTED MANUSCRIPT

Ref.*

IC50 7.00 11.59 6.81 42.76 12.95 22.13 4.81 7.14 7.93 13.10

SC

15 IC50 2.80 3.84 3.91 3.13 3.01 3.76 1.98 5.00 2.98 2.34

M AN U

S. epidermidis ATCC 12228 S. epidermidis ATCC 35984 S. epidermidis 16/04 S. epidermidis 23/04 S. epidermidis 24/04 S. epidermidis 31/04 S. epidermidis 62/04 S. epidermidis 63/04 S. epidermidis 76/04 S. epidermidis 151/04

5 IC50a 1.62 1.37 1.35 2.01 1.59 1.18 0.97 2.39 2.85 −

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Table 5. Biofilm inhibitory activity of compounds 5 and 15 against standard and hospital methicillin-resistant strains of S. epidermidis - IC50 values (µg/ml).

AC C

EP

TE D

− no possibility of IC50 calculation a concentration (µg/ml) of tested compound required to inhibit 50% of biofilm formation under the assay conditions. All the experiments were carried out in quadruplicates. * Ref. – Ciprofloxacin

ACCEPTED MANUSCRIPT

Table 6. Cytotoxicity and antiviral activity of compounds 1-31 against representatives of ssRNA+ (HIV-1, BVDV, YFV, CBV-5, Sb-1), ssRNA- (RSV, VSV), dsRNA (Reo-1) and dsDNA (VV, HSV-1) viruses°.

1.1

YFV EC50f >41 >26 >9.4 >54 >10 >9.5 >10 >9.5 >10 >9.5 >9.4 >9.5 >9.5 >59 >10 >10 >12 >9.0 >100 >50 >48 >100 >100 >29 >100 >9.6 >45 >100 >100 >14 >100

>10 >100

Reo-1 EC50g >41 >26 >9.4 >54 >10 >9.5 >10 >9.5 >10 >9.5 >9.4 >9.5 >9.5 >59 >10 >10 >12 >9.0 >100 >50 >48 >100 >100 >29 >100 >9.6 >45 >100 >100 >14 >100

Vero-76 CC50h 39 40 20 64 20 16 20 20 11 10 9.5 10 17 50 14 10 20 18 >100 71 50 >100 >100 46 >100 20 35 >100 >100 60 >100

CVB-5 EC50i >39 >40 >20 >64 >20 >16 >20 >20 >11 >10 >9.5 >10 >17 >50 >14 >10 >20 >18 >100 >71 >50 >100 >100 >46 >100 >20 >35 >100 >100 >60 >100

Sb-1 EC50j >39 >40 >20 >64 >20 >16 >20 >20 >11 >10 >9.5 >10 >17 >50 >14 >10 >20 >18 >100 >71 >50 >100 >100 >46 >100 >20 >35 >100 >100 >60 >100

16

>100

18

7.3

RI PT

>10

BHK CC50e 41 26 9.4 54 10 9.5 10 9.5 10 9.5 9.4 9.5 9.5 59 10 10 12 9.0 >100 50 48 >100 >100 29 >100 9.6 45 >100 >100 14 >100

SC

BVDV EC50d >49 >41 >10 >100 >11 >10 >10 >19 >17 >10 >9.5 >10 >10 >100 >10 >9.0 >52 >10 >100 >64 >100 >100 >100 >48 >100 >10 >40 >100 >100 >48 >100

M AN U

MDBK CC50c 49 41 10 >100 11 10 10 19 17 10 9.5 10 10 >100 10 9.0 52 10 >100 64 >100 >100 >100 48 >100 10 40 >100 >100 48 >100

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HIV-1 EC50b >39 >31 >20 >47 >8.0 >9.0 >20 >10.0 >6.8 >7.0 >10.5 >9.2 >22 >36 >9.2 >39 >26 >43 >100 >23 >43 >43 >100 >8.8 >100 >42 >45 >100 >43 >45 >100 0.002

EP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Efavirenz 2’-C-methyl-guanosine 2'-C-methyl-cytidine

MT-4 CC50a 39 31 20 47 8.0 9.0 20 10 6.8 7.0 10 9.2 22 36 9.2 39 26 43 >100 23 43 43 >100 8.8 >100 42 45 >100 43 45 >100 40

AC C

Compound

1.9

RSV EC50k >39 >40 >20 >64 >20 >16 >20 >20 >11 >10 >9.5 >10 >17 >50 >14 >10 >20 >18 >100 >71 >50 >100 >100 >46 >100 >20 >35 >100 >100 >60 >100

VSV EC50l >39 >40 >20 >64 >20 >16 >20 >20 >11 >10 >9.5 >10 >17 >50 >14 >10 >20 >18 >100 >71 >50 >100 >100 >46 >100 >20 >35 >100 >100 >60 >100

VV EC50m >39 >40 >20 >64 >20 >16 >20 >20 >11 >10 >9.5 >10 >17 >50 >14 >10 >20 >18 >100 >71 >50 >100 >100 >46 >100 >20 >35 >100 >100 >60 >100

HSV-1 EC50n >39 >40 >20 >64 >20 >16 >20 >20 >11 >10 >9.5 >10 >17 >50 >14 >10 >20 >18 >100 >71 >50 >100 >100 >46 >100 >20 >35 >100 >100 >60 >100

ACCEPTED MANUSCRIPT

SC

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1.4 6-Aza-uridine 12 Mycophenolic acid 13 1.5 Acycloguanosine >100 3.0 °Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%. a Compound concentration (µM) required to reduce the proliferation of mock-infected MT-4 cells by 50%. bCompound concentration (µM) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity.cCompound concentration (µM) required to reduce the viability of mock-infected MDBK cells by 50%. dCompound concentration (µM) required to achieve 50% protection of MDBK cells from BVDV-induced cytopathogenicity. eCompound concentration (µM) required to reduce the viability of mock-infected BHK cells by 50%. f-gCompound concentration (µM) required to achieve 50% protection of BHK cells from YFV-induced(f) and Reo-1-induced(g) cytopathogenicity. hCompound concentration (µM) required to reduce the viability of mock-infected VERO-76 cells by 50%. i-nCompound concentration (µM) required to reduce the plaque number of CVB-5(i), Sb-1(j), RSV(k), VSV(l), VV(m), HSV-1(n) by 50% in VERO-76 monolayers.

EP

TE D

MTT reduction (% of control) 89.85 ± 6.22 91.04 ± 6.87

AC C

Compound 5 15

M AN U

Table 7. Cells viability assessed by MTT mitochondrial conversion and LDH release as a marker of cell death in HaCaT cells. Data are expressed as means ± SD from three independent experiments performed in triplicate.

LDH release (%) 8 ± 0.40 0

ACCEPTED MANUSCRIPT

CF3 + R-NCS NH2

MeCN 12h, 20°C

S R

RI PT

CF3

NH

NH

R = aryl, alkyl

SC

1-31

AC C

EP

TE D

M AN U

Fig.1. Synthetic procedure for thiourea derivatives 1-31.

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig.2. The effect of studied compounds on topoisomerase IV in decatenation assays. The reactions were performed in the presence of compounds

EP

5 and 15 at concentrations 32 µg/ml (lane I), 4 µg/ml (lane II) and 1 µg/ml (lane III). Ciprofloxacin (CFX) was used as the control compound at

AC C

the same concentrations. The first lane, labelled A, contains a control assay without enzyme. The control (lane B) were performed in the absence of compounds. To checked the effect of DMSO on the reaction, tubes +/- DMSO was included (lane C).

ACCEPTED MANUSCRIPT

100 90

RI PT

80

ATCC 12228 16/04 23/04

SC

70

31/04

M AN U

60 50 40

62/04

TE D

30 20 10 0 0.25

AC C

0.125

EP

% of biofilm formed

ATCC 35984

0.5

1

2

4

Concentration of compound 5 (µg/ml)

Fig. 3. Biofilm inhibitory activity of compound 5 – the percentage of S. epidermidis biofilm formed in the presence of the tested compound. (compared to drug-free control). All presented results are mean from experiments performed in quadruplicate ± S.D.

ACCEPTED MANUSCRIPT

100

RI PT

90 80

SC M AN U

60 50 40 30

TE D

% of biofilm formed

70

ATCC 35984 ATCC 12228 16/04 23/04 31/04 62/04

20

0 0.25

0.5

AC C

EP

10

1

2

4

8

Concentration of compound 15 (µg/ml) Fig. 4. Biofilm inhibitory activity of compound 15 – the percentage of S. epidermidis biofilm formed in the presence of the tested compound. (compared to drug-free control). All presented results are mean from experiments performed in quadruplicate ± S.D.

ACCEPTED MANUSCRIPT

100

S. epidermidis

RI PT

90

16/04

SC

70

ATCC 12228

23/04 31/04

M AN U

60 50 40 30

62/04

TE D

% of biofilm formed

80

ATCC 35984

20

0 0.125

0.25

AC C

EP

10

0.5

1

2

4

8

Concentration of Ciprofloxacin (µg/ml)

Fig. 5. Biofilm inhibitory activity of Ciprofloxacin – the percentage of S. epidermidis biofilm formed in its presence. All presented results are mean from experiments performed in quadruplicate ± S.D.

ACCEPTED MANUSCRIPT • New thiourea derivatives were tested for their antimicrobial and cytotoxic activity. • Electron-withdrawing substituent in benzene ring promoted antibacterial activity. • Two compounds effectively inhibited biofilm formation of S. epidermidis strains.

AC C

EP

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M AN U

SC

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• New derivatives are inhibitors of topoisomerase IV isolated from S. aureus.

ACCEPTED MANUSCRIPT

Supplementary material

RI PT

Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety Anna Bielenica a,*, Joanna Stefanska b, Karolina Stępień b, Agnieszka Napiórkowska c, Ewa Augustynowicz-Kopeć c, Giuseppina Sanna d, Silvia

SC

Madeddu d, Stefano Boi d, Gabriele Giliberti d, Małgorzata Wrzosek e, Marta Struga e

Chair and Department of Biochemistry, Medical University, 02-097 Warszawa, Poland

b

Department of Pharmaceutical Microbiology, Medical University, 02-007 Warszawa, Poland

c

M AN U

a

Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, 01-138 Warszawa, Poland

d

Department of Biomedical Science, University of Cagliari, 09042 Monserrato (CA), Italy

e

AC C

EP

TE D

Department of Pharmacogenomics, Faculty of Pharmacy, Medical University, 02-097 Warszawa, Poland

S1

ACCEPTED MANUSCRIPT

I. Tuberculostatic activity inhibitory concentrations (MIC, µg/ml). MIC (µg/ml)

SC M AN U

50 25 >100 100 50 100 >100 100 >100 >100 >100 >100 >100 100 >100 >100 >100 100 100 25 >100 100 >100 >100 12.5

TE D

50 25 25 100 50 50 >100 100 >100 >100 50 100 >100 50 >100 >100 >100 50 50 25 >100 >100 >100 >100 6.25

EP

1 2 3 4 5 6 7 8 9 10 12 15 17 18 19 20 21 22 24 27 28 29 30 31 Ref.*

M. tuberculosis Spec. M. tuberculosis Spec. 192 210

AC C

Compounds

M. tuberculosis H37Rv ATCC 25618 50 25 25 100 50 50 >100 100 >100 >100 50 100 >100 50 >100 >100 >100 50 50 25 >100 >100 >100 >100 6.25

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Table 1S. Activity of compounds against the M. tuberculosis H37Rv strain and two “wild-type” strains isolated from tuberculosis patients – minimal

S2

ACCEPTED MANUSCRIPT

* Ref. – Isonicotinic acid hydrazide (INH)

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The tuberculostatic activity of compounds was tested against the M. tuberculosis H37Rv strain (ATCC 25618) and two “wild-type” strains isolated from tuberculosis patients (Spec. 192, Spec. 210). All strains were obtained from the collection of the Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland.

SC

The synthesized compounds were examined in vitro for their tuberculostatic activity. Investigations were performed by a classical testtube method of successive dilution in Youmans’ modification of the Proskauer and Beck liquid medium containing 10% of bovine serum [1S,

M AN U

2S]. Bacterial suspensions were prepared from 14 days old cultures of slowly growing strains. Solutions of compounds in DMSO were tested. Stock solutions contained 10 mg of compounds in 1 milliliter. Dilutions (in geometric progression) were prepared in Youmans’ medium. The medium containing no investigated substances and containing isoniazid (INH) as reference drugs were used for comparison. Incubation was performed at a temperature of 37 °C. The MIC values were determined as minimum concentration inhibiting the growth of tested tuberculous

AC C

EP

6.2, 12.5, 25, 50 and 100 µg/ml, were evaluated.

TE D

strains in relation to the probe with no tested compound. The influence of the compound on the growth of bacteria at a certain concentration, 3.1,

S3

ACCEPTED MANUSCRIPT

II. Inhibition of S. epidermidis biofilm formation

Compound 15 % 86,99 46,83 61,71 71,57 70,06 84,80

Ref. * % 21,30 45,43 41,96 5,71 18,66 59,89

SC

S. epidermidis ATCC 12228 S. epidermidis ATCC 35984 S. epidermidis 16/04 S. epidermidis 23/04 S. epidermidis 31/04 S. epidermidis 62/04 * Ciprofloxacin

Compound 5 % 95,20 84,03 71,97 77,90 77,06 89,74

M AN U

Bacterial strain

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Table 2S. The percentage of inhibition of biofilm formation by compounds 5 and 15 for selected S. epidermidis strains (concentration of compounds 4 µg/ml).

TE D

III. Antiproliferative assays

Table 3S. Cytotoxicity of derivatives 5, 6, 8-12, 15 against human leukaemia/lymphoma cell lines°. b

5

CCRF-CEM CC50a 7.0

EP

Compounds

c

WIL-2NS CC50a 9.0

d

CCRF-SB CC50a 9.5

8.0

8.0

8.8

8

9.1

11

9.6

9

12

13

11

10

8.7

9.2

8.3

11

8.0

8.0

9.1

12

8.7

9.4

8.2

15

9.3

9.9

9

AC C

6

S4

ACCEPTED MANUSCRIPT

Doxorubicin

0.02

0.02

0.03

RI PT

°Data represent mean values for three independent determinations. a

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Compound concentration (µM) required to reduce cell proliferation by 50%, under conditions allowing untreated controls to undergo at least three consecutive rounds of multiplication. b CD4+ human acute T-lymphoblastic leukaemia. cHuman splenic B-lymphoblastoid cells. dHuman acute B-lymphoblastic leukaemia.

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Cell lines derived from human haematological tumours [CD4+ human T-cells containing an integrated HTLV-1 genome (MT-4); CD4+ human acute T-lymphoblastic leukaemia (CCRF-CEM), human splenic B-lymphoblastoid cells (WIL-2NS), human acute B-lymphoblastic leukaemia (CCRF-SB)] were seeded at an initial density of 1×105 cells/ml in 96 well plates in RPMI-1640 medium supplemented with 10% foetal calf serum (FCS), 100 units/ml penicillin G and 100 µg/ml streptomycin. Cell cultures were then incubated at 37 °C in a humidified, 5% CO2 method [3S].

EP

[1S] G.P. Youmans, Am. Rev. Tuberc. 56 (1947) 376

TE D

atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 96 hrs at 37 °C by the MTT

[2S] G.P. Youmans, A.S. Youmans, J. Bacteriol. 58 (1949) 247

AC C

[3S] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, E. De Clercq, J. Virol. Methods 20 (1988) 309−321.

S5

ACCEPTED MANUSCRIPT

IV. The 1H NMR, 13C NMR spectra of compounds 1-31. MS spectra of compounds 2, 6, 15, 24, 27.

RI PT

Spectrum 1. 1H NMR of compd 1 (300 MHz, DMSO-d6). Spectrum 2. 13C NMR of compd 1 (75.4 MHz, DMSO-d6). Spectrum 3. 1H NMR of compd 2 (300 MHz, DMSO-d6).

SC

Spectrum 4. 13C NMR of compd 2 (75.4 MHz, DMSO-d6).

Spectrum 6. 1H NMR of compd 3 (300 MHz, DMSO-d6). Spectrum 7. 13C NMR of compd 3 (75.4 MHz, DMSO-d6). Spectrum 8. 1H NMR of compd 4 (300 MHz, DMSO-d6).

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Spectrum 9. 13C NMR of compd 4 (75.4 MHz, DMSO-d6).

M AN U

Spectrum 5. MS of compd 2.

Spectrum 10. 1H NMR of compd 5 (300 MHz, DMSO-d6).

EP

Spectrum 11. 13C NMR of compd 5 (75.4 MHz, DMSO-d6). Spectrum 12. 1H NMR of compd 6 (300 MHz, DMSO-d6).

Spectrum 14. MS of compd 6.

AC C

Spectrum 13. 13C NMR of compd 6 (75.4 MHz, DMSO-d6).

Spectrum 15. 1H NMR of compd 7 (300 MHz, DMSO-d6). Spectrum 16. 13C NMR of compd 7 (75.4 MHz, DMSO-d6). Spectrum 17. 1H NMR of compd 8 (300 MHz, DMSO-d6). S6

ACCEPTED MANUSCRIPT

Spectrum 18. 13C NMR of compd 8 (75.4 MHz, DMSO-d6).

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Spectrum 19. 1H NMR of compd 9 (300 MHz, DMSO-d6). Spectrum 20. 13C NMR of compd 9 (75.4 MHz, DMSO-d6). Spectrum 21. 1H NMR of compd 10 (300 MHz, DMSO-d6).

SC

Spectrum 22. 13C NMR of compd 10 (75.4 MHz, DMSO-d6).

Spectrum 24. 13C NMR of compd 11 (75.4 MHz, DMSO-d6). Spectrum 25. 1H NMR of compd 12 (300 MHz, DMSO-d6). Spectrum 26. 13C NMR of compd 12 (75.4 MHz, DMSO-d6).

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Spectrum 27. 1H NMR of compd 13 (300 MHz, DMSO-d6).

M AN U

Spectrum 23. 1H NMR of compd 11 (300 MHz, DMSO-d6).

Spectrum 28. 13C NMR of compd 13 (75.4 MHz, DMSO-d6).

EP

Spectrum 29. 1H NMR of compd 14 (300 MHz, DMSO-d6).

Spectrum 30. 13C NMR of compd 14 (75.4 MHz, DMSO-d6).

AC C

Spectrum 31. 1H NMR of compd 15 (300 MHz, DMSO-d6).

Spectrum 32. 13C NMR of compd 15 (75.4 MHz, DMSO-d6). Spectrum 33. MS of compd 15.

Spectrum 34. 1H NMR of compd 16 (300 MHz, DMSO-d6). Spectrum 35. 13C NMR of compd 16 (75.4 MHz, DMSO-d6). S7

ACCEPTED MANUSCRIPT

Spectrum 36. 1H NMR of compd 17 (300 MHz, DMSO-d6).

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Spectrum 37. 13C NMR of compd 17 (75.4 MHz, DMSO-d6). Spectrum 38. 1H NMR of compd 18 (300 MHz, DMSO-d6). Spectrum 39. 13C NMR of compd 18 (75.4 MHz, DMSO-d6).

SC

Spectrum 40. 1H NMR of compd 19 (300 MHz, DMSO-d6).

Spectrum 42. 1H NMR of compd 20 (300 MHz, DMSO-d6). Spectrum 43. 13C NMR of compd 20 (75.4 MHz, DMSO-d6). Spectrum 44. 1H NMR of compd 21 (300 MHz, DMSO-d6).

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Spectrum 45. 13C NMR of compd 21 (75.4 MHz, DMSO-d6).

M AN U

Spectrum 41. 13C NMR of compd 19 (75.4 MHz, DMSO-d6).

Spectrum 46. 1H NMR of compd 22 (300 MHz, DMSO-d6).

EP

Spectrum 47. 13C NMR of compd 22 (75.4 MHz, DMSO-d6). Spectrum 48. 1H NMR of compd 23 (300 MHz, DMSO-d6).

AC C

Spectrum 49. 13C NMR of compd 23 (75.4 MHz, DMSO-d6). Spectrum 50. 1H NMR of compd 24 (300 MHz, DMSO-d6). Spectrum 51. 13C NMR of compd 24 (75.4 MHz, DMSO-d6). Spectrum 52. MS of compd 24. Spectrum 53. 1H NMR of compd 25 (300 MHz, DMSO-d6).

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Spectrum 54. 13C NMR of compd 25 (75.4 MHz, DMSO-d6).

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Spectrum 55. 1H NMR of compd 26 (300 MHz, DMSO-d6). Spectrum 56. 13C NMR of compd 26 (75.4 MHz, DMSO-d6). Spectrum 57. 1H NMR of compd 27 (300 MHz, DMSO-d6).

SC

Spectrum 58. 13C NMR of compd 27 (75.4 MHz, DMSO-d6).

Spectrum 60. 1H NMR of compd 28 (300 MHz, DMSO-d6). Spectrum 61. 13C NMR of compd 28 (75.4 MHz, DMSO-d6). Spectrum 62. 1H NMR of compd 29 (300 MHz, DMSO-d6).

TE D

Spectrum 63. 13C NMR of compd 29 (75.4 MHz, DMSO-d6).

M AN U

Spectrum 59. MS of compd 27.

Spectrum 64. 1H NMR of compd 30 (300 MHz, DMSO-d6).

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Spectrum 65. 13C NMR of compd 30 (75.4 MHz, DMSO-d6). Spectrum 66. 1H NMR of compd 31 (300 MHz, DMSO-d6).

AC C

Spectrum 67. 13C NMR of compd 31 (75.4 MHz, DMSO-d6).

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Spectrum 1. 1H NMR of compd 1 (300 MHz, DMSO-d6). S10

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Spectrum 2. 13C NMR of compd 1 (75.4 MHz, DMSO-d6).

S11

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Spectrum 3. 1H NMR of compd 2 (300 MHz, DMSO-d6).

S12

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Spectrum 4. 13C NMR of compd 2 (75.4 MHz, DMSO-d6). S13

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Spectrum 5. 1H NMR of compd 3 (300 MHz, DMSO-d6). S14

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Spectrum 6. 13C NMR of compd 3 (75.4 MHz, DMSO-d6).

S15

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Spectrum 7. 1H NMR of compd 4 (300 MHz, DMSO-d6).

S16

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Spectrum 8. 13C NMR of compd 4 (75.4 MHz, DMSO-d6). S17

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Spectrum 9. 1H NMR of compd 5 (300 MHz, DMSO-d6). S18

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Spectrum 10. 13C NMR of compd 5 (75.4 MHz, DMSO-d6). S19

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Spectrum 11. MS of compd 5. S20

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Spectrum 12. 1H NMR of compd 6 (300 MHz, DMSO-d6).

S21

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Spectrum 13. 13C NMR of compd 6 (75.4 MHz, DMSO-d6). S22

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Spectrum 14. MS of compd 6. S23

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Spectrum 15. 1H NMR of compd 7 (300 MHz, DMSO-d6).

S24

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Spectrum 16. 13C NMR of compd 7 (75.4 MHz, DMSO-d6). S25

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Spectrum 17. 1H NMR of compd 8 (300 MHz, DMSO-d6). S26

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Spectrum 18. 13C NMR of compd 8 (75.4 MHz, DMSO-d6). S27

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Spectrum 19. 1H NMR of compd 9 (300 MHz, DMSO-d6).

S28

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Spectrum 20. 13C NMR of compd 9 (75.4 MHz, DMSO-d6). S29

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Spectrum 21. 1H NMR of compd 10 (300 MHz, DMSO-d6). S30

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Spectrum 22. 13C NMR of compd 10 (75.4 MHz, DMSO-d6). S31

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Spectrum 23. 1H NMR of compd 11 (300 MHz, DMSO-d6). S32

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Spectrum 24. 13C NMR of compd 11 (75.4 MHz, DMSO-d6). S33

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Spectrum 25. 1H NMR of compd 12 (300 MHz, DMSO-d6).

S34

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Spectrum 26. 13C NMR of compd 12 (75.4 MHz, DMSO-d6).

S35

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Spectrum 27. 1H NMR of compd 13 (300 MHz, DMSO-d6). S36

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Spectrum 28. 13C NMR of compd 13 (75.4 MHz, DMSO-d6). S37

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Spectrum 29. 1H NMR of compd 14 (300 MHz, DMSO-d6).

S38

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Spectrum 30. 13C NMR of compd 14 (75.4 MHz, DMSO-d6). S39

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Spectrum 31. 1H NMR of compd 15 (300 MHz, DMSO-d6). S40

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Spectrum 32. 13C NMR of compd 15 (75.4 MHz, DMSO-d6). S41

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Spectrum 33. MS of compd 15.

S42

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Spectrum 34. 1H NMR of compd 16 (300 MHz, DMSO-d6). S43

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Spectrum 35. 13C NMR of compd 16 (75.4 MHz, DMSO-d6). S44

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Spectrum 36. 1H NMR of compd 17 (300 MHz, DMSO-d6). S45

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Spectrum 37. 13C NMR of compd 17 (75.4 MHz, DMSO-d6). S46

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Spectrum 38. 1H NMR of compd 18 (300 MHz, DMSO-d6). S47

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Spectrum 39. 13C NMR of compd 18 (75.4 MHz, DMSO-d6).

S48

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Spectrum 40. 1H NMR of compd 19 (300 MHz, DMSO-d6). S49

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Spectrum 41. 13C NMR of compd 19 (75.4 MHz, DMSO-d6). S50

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Spectrum 42. 1H NMR of compd 20 (300 MHz, DMSO-d6).

S51

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Spectrum 43. 13C NMR of compd 20 (75.4 MHz, DMSO-d6). S52

AC C

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Spectrum 44. 1H NMR of compd 21 (300 MHz, DMSO-d6).

S53

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Spectrum 45. 13C NMR of compd 21 (75.4 MHz, DMSO-d6).

S54

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Spectrum 46. 1H NMR of compd 22 (300 MHz, DMSO-d6).

S55

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Spectrum 47. 13C NMR of compd 22 (75.4 MHz, DMSO-d6). S56

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Spectrum 48. 1H NMR of compd 23 (300 MHz, DMSO-d6).

S57

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Spectrum 49. 13C NMR of compd 23 (75.4 MHz, DMSO-d6). S58

AC C

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Spectrum 50. 1H NMR of compd 24 (300 MHz, DMSO-d6). S59

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Spectrum 51. 13C NMR of compd 24 (75.4 MHz, DMSO-d6).

S60

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Spectrum 52. MS of compd 24.

S61

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Spectrum 53. 1H NMR of compd 25 (300 MHz, DMSO-d6).

S62

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Spectrum 54. 13C NMR of compd 25 (75.4 MHz, DMSO-d6). S63

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Spectrum 55. 1H NMR of compd 26 (300 MHz, DMSO-d6). S64

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Spectrum 56. 13C NMR of compd 26 (75.4 MHz, DMSO-d6). S65

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Spectrum 57. 1H NMR of compd 27 (300 MHz, DMSO-d6).

S66

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Spectrum 58. 13C NMR of compd 27 (75.4 MHz, DMSO-d6). S67

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Spectrum 59. MS of compd 27. S68

AC C

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Spectrum 60. 1H NMR of compd 28 (300 MHz, DMSO-d6).

S69

AC C

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Spectrum 61. 13C NMR of compd 28 (75.4 MHz, DMSO-d6).

S70

AC C

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Spectrum 62. 1H NMR of compd 29 (300 MHz, DMSO-d6). S71

AC C

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Spectrum 63. 13C NMR of compd 29 (75.4 MHz, DMSO-d6). S72

AC C

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Spectrum 64. 1H NMR of compd 30 (300 MHz, DMSO-d6). S73

AC C

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Spectrum 65. 13C NMR of compd 30 (75.4 MHz, DMSO-d6).

S74

AC C

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Spectrum 66. 1H NMR of compd 31 (300 MHz, DMSO-d6).

S75

AC C

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Spectrum 67. 13C NMR of compd 31 (75.4 MHz, DMSO-d6). S76

Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety.

A total of 31 of thiourea derivatives was prepared reacting 3-(trifluoromethyl)aniline and commercial aliphatic and aromatic isothiocyanates. The yiel...
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