Vol. 63, No. 3225 Chem. Pharm. Bull. 63, 225–236 (2015)

Regular Article

Antimicrobial and Anti-biofilm Activity of Thiourea Derivatives Incorporating a 2-Aminothiazole Scaffold Joanna Stefanska,a Grażyna Nowicka,b Marta Struga,*,b Daniel Szulczyk,c Anna Eugenia Koziol,d Ewa Augustynowicz-Kopec,e Agnieszka Napiorkowska,e Anna Bielenica, f Wojciech Filipowski,g Anna Filipowska,g,h Aleksandra Drzewiecka,i Gabriele Giliberti, j Silvia Madeddu, j Stefano Boi, j Paolo La Colla, j and Giuseppina Sanna j a

 Department of Pharmaceutical Microbiology, Medical University; Warszawa 02–007, Poland: b Department of Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw; Warszawa 02–097, Poland: c Department of Medical Chemistry, Medical University; Warszawa 02–007, Poland: d Faculty of Chemistry, Maria Curie-Sklodowska University; Lublin 20–031, Poland: e Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute; Warszawa 01–138, Poland: f Chair and Department of Biochemistry, Medical University of Warsaw; Warszawa 02–097, Poland: g Silesian University of Technology, Faculty of Automatic Control, Electronics and Computer Science, Institute of Electronics; Gliwice 44–102, Poland: h Silesian University of Technology, Faculty of Biomedical Engineering, Department of Biosensors and Processing of Biomedical Signals; Gliwice 44–100, Poland: i  Institute of Physics, Polish Academy of Sciences; Warszawa 02–668, Poland: and j Department of Biomedical Science, University of Cagliari; Monserrato (CA) 09042, Italy. Received December 9, 2014; accepted January 6, 2015 A series of new thiourea derivatives of 1,3-thiazole have been synthesized. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. Compounds were also tested for their in vitro tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain, as well as two ‘wild’ strains isolated from tuberculosis patients. Compounds 3 and 9 showed significant inhibition against Gram-positive cocci (standard strains and hospital strain). The range of MIC values is 2–32 µg/mL. Products 3 and 9 effectively inhibited the biofilm formation of both methicillin-resistant and standard strains of S. epidermidis. The halogen atom, especially at the 3rd position of the phenyl group, is significantly important for this antimicrobial activity. Moreover, all obtained compounds resulted in cytotoxicity and antiviral activity on a large set of DNA and RNA viruses, including Human Immunodeficiency Virus type 1 (HIV-1) and other several important human pathogens. Compound 4 showed activity against HIV-1 and Coxsackievirus type B5. Seven compounds resulted in cytotoxicity against MT-4 cells (CC50512 − >512 − 16 (17) 32 (15) 8 (20) 32 (12) 512 (20) 512 (10) 512 (11)

32 (13) 32 (14) 32 (14) 32 (13) 4 (13) 8 (18) 64 (11) 64 − 16 (17) 4 (14) 8 (23) 32 (13) − − − − − −

32 (12) 16 (14) 32 (12) 16 (11) 16 (13) 16 (16) >512 − 512 − 16 (17) 16 (16) 4 (20) 32 (13) 256 (20) 128 (11) 128 (15)

16 (13) 16 (13) 16 (13) 16 (13) 8 (16) 8 (15) >512 − >512 − 16 (16) 8 (14) 8 (18) 16 (12) 512 (14) 512 (11) 512 (11)

0.25 (26) 0.5 (26) 0.25 (28) 0.5 (22) 0.25 (30) 0.25 (32) 1 (18) 1 (15) 2 (22) 1 (24) 100 µM). Interestingly, seven compounds (2, 3, 8–10, 16, 17) turned out cytotoxic for exponentially growing MT4 in a low micromolar range (CC50=7.8–9.0 µM). As indicated in Table 5, selected compounds showed high level of cytotoxicity, also against other cell monolayers used to support the multiplication of different viruses (MDBK, BHK, Vero-76) in stationary growth. Thiourea derivatives of 1,3-thiazole were also tested in cell-based assays against representative members of several virus families. Among ssRNA+ viruses, were: bovine viral diarrhoea virus [BVDV] and yellow fever virus [YFV] (Flaviviridae), two Picornaviridae, human enterovirus B [coxsackie virus B5, CVB-5] and human enterovirus C [polio virus type-1, Sb-1]. Among ssRNA-viruses, we tested vesicular stomatitis virus [VSV] (Rhabdoviridae). Among dsRNA viruses, we tested reovirus type-1 [Reo-1] (Reoviridae). Finally, representatives of two DNA virus families were also included: vaccinia virus [VV] (Poxviridae) and human herpes virus 1 [herpes simplex type-1, HSV-1] (Herpesviridae). In order to be able to establish whether test compounds were endowed with selective antiviral activity, their cytotoxicity was evaluated in parallel assays with uninfected cell lines. 2′-C-Methyl-guanosine, 2′-C-methyl-cytidine, 2′-C-ethynylcytidine, mycophenolic acid and acyclo-guanosine (acyclovir) were used as reference inhibitors. Three compounds (4, 19, 22) resulted not cytotoxic against all cell lines used (MT4, MDBK, BHK, Vero-76), while other

Detection of Slime Production on Congo Red Agar

(A) Black colonies of slime-positive (high biofilm-producer) S. epidermidis ATCC 35984, (B) Red colonies of slime-negative (low biofilm-producer) S. epidermidis ATCC 12228.

Chem. Pharm. Bull. Vol. 63, No. 3 (2015)229

Fig. 3.

Inhibitory Effect of the Compound 3 for Biofilm Formation by Standard and Selected Hospital Methicillin-Resistant Strains S. epidermidis

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

Fig. 4.

Inhibitory Effect of the Compound 9 for Biofilm Formation by Standard and Selected Hospital Methicillin-Resistant Strains S. epidermidis

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

Chem. Pharm. Bull.

230

Fig. 5. Inhibitory Effect of the Ciprofloxacin for Biofilm Formation by Standard and Selected Hospital Methicillin-Resistant Strains S. epidermidis All presented results are mean from experiments performed in quadruplicate±S.D.

Table 4. Antibiofilm Activity of Compounds 3 and 9 against Standard and Hospital Methicillin-Resistant Strains of S. epidermidis Test compounds Bacterial strain

S. S. S. S. S. S. S. S. S. S.

epidermidis epidermidis epidermidis epidermidis epidermidis epidermidis epidermidis epidermidis epidermidis epidermidis

ATCC 12228 ATCC 35984 517/12 519/12 523/12 526/12 528/12 531/12 532/12 533/12

Compd. 3 IC50 values (µg/mL)

Compd. 9 IC50 values (µg/mL)

Ciproflox. IC50 values (µg/mL)

3.71 2.55 7.32 2.52 2.53 1.61 2.50 2.83 3.49 1.96

0.35 — 0.66 2.50 3.14 4.95 1.10 1.13 2.21 2.88

11.89 18.55 — 1.65 — 9.28 5.00 6.59 6.73 1.90

compounds showed a different degree of cytotoxicity. Very interestingly compound 4, in addition to anti-HIV-1 activity already described, showed selective activity also against CVB-5 virus, with EC50 value of 14 µM, and lack of cytotoxicity for BHK cell lines. None of other tested compounds showed antiviral activity with the exception of compound 19, resulted weakly active against CVB-5. As presented in Tables 3 and 4, compounds with electronwithdrawing substituents, such as fluoro or chloro-fluoro (3, 6, 9, 10) were found as the most potent antibacterial agents. For most of Gram-positive bacteria, it is reasonable that disubstituted compounds (3, 9) are more potent than monosubstituted compounds, because the first group produce stronger electronegativity effect. However, substituent groups on different positions definitely resulted in various degrees of effects. The halogen atom at meta position of phenyl ring improved potency against hospital S. epidermidis as the electronegativity increases (compounds 6 and 2). On the other hand all parasubstituted compounds showed similar activity, even with the electron donating methoxy group (compounds 15, 13, 16, 17, 12). The latter suggests the activity against S. epidermidis is not sensitive to 4-substituted groups. For M. tuberculosis, the 3-halogen atoms did not show different effects (compounds 6, 2), but 4-halogen atoms decreased activity as electronegativity increases (compounds 17>15, 13).

Vol. 63, No. 3 (2015)

The structure–activity relationship was also observed when the positional weighed electronic effect of substituent is analyzed (Table 6). Subsequently, some physicochemical parameters as molar weight (M), volume (V), surface area (SA), surface area grid (gSA), logarithm of the partition coefficient (log P), the difference between higest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels (HLG), the HOMO, the LUMO, hardness (η) obtained from the equation η=(HOMO−LUMO)/2, Mullkien electronegativity (χ) obtained from the equation χ=−(HOMO+LUMO)/2, total energy (ET), binding energy (EB), isolated atomic energy (EIA), electronic energy (EE), core–core interaction (IC–C), heat of formation (HF), refractivity (Rf), polarizability (α) were studied (Table 6). However, there was no close correlation between such important parameters as log P, HOMO, LUMO. On the other hand, the relationship between ET (total energy), EIA (isolated atomic energy) and antibacterial activity was observed. As seen in Table 6, the most active compounds (3, 9, 10) were characterized by lower value of ET and EIA parameters in comparison with inactive derivatives.

Conclusion

On the basis of predicted biological activities, reported literature and results of our previous studies, we designed, synthesized and tested 22 thiourea derivatives of 1,3-thiazole, with the aim of developing new therapeutic agents with antimicrobial activity. The antimicrobial screening for the most of tested thiourea derivatives exhibited significant antibacterial effect. On the basis of structural activity relationship (SAR) it was established that compounds with electron withdrawing halogens, such as fluorine and chlorine (3, 6, 9) displayed strong antibacterial potency. The presence of 3-chloro-4-fluorophenyl (9) moiety resulted in the highest activity against standard and hospital Gram-positive cocci among all investigated compounds. Two compounds (3, 9), the most active against planktonic forms of staphylococcal species, significantly affected the tested staphylococcal biofilm, in most of cases at concentrations next to the MIC observed against the planktonic form. The effect on tuberculostatic activity was especially apparent for the phenyl (11), 4-iodophenyl (17), as well as 3, 9, 10 thiourea derivatives. We explore their biological activities also in antiviral assays, against HIV-1 and other RNA and DNA viruses causing infectious diseases. When tested against representatives of ssRNA+, ssRNA−, dsRNA and DNA virus families, compound 4 have proved to be active against HIV-1 and interestingly also against CVB-5, a member of Enterovirus genus (Picornaviridae family) that are important human pathogens that cause both acute and chronic diseases in infants, young children and immunocompromised individuals.26)

Experimental

Chemistry The NMR spectra were recorded on a Bruker AVANCE DMX400 spectrometer, operating at 300 MHz (1H-NMR) and 75 MHz (13C-NMR). The chemical shift values are expressed in ppm relative to TMS as an internal standard. Mass spectral ESI measurements were carried out on Waters

Chem. Pharm. Bull. Vol. 63, No. 3 (2015)231 Table 5. Cytotoxicity and Antiviral Activity of Thiourea Derivatives of 1,3-Thiazole against Representatives of ssRNA+ (HIV-1, BVDV, YFV, CBV-5, Sb-1), ssRNA− (VSV), dsRNA (Reo-1) and dsDNA (VV, HSV-1) Viruses* Compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Efavirenz 2′-C-Methylguanosine 2′-C-Methylcytidine 2′-C-Ethynylcytidine Mycophenolic acid Acycloguanosine

MT-4 CC50a)

HIV-1 EC50b)

MDBK CC50c)

BVDV EC50d)

BHK CC50e)

YFV EC50f)

>100 9.0 9.0 >100 38.5 10.8 20.8 8.5 7.8 9.0 50 >100 >100 >100 10.8 9.0 9.0 >100 >100 40.0 43.8 >100 40

>100 >9.0 >9.0 36 >38.5 >10.8 >20.8 >8.5 >7.8 >9.0 >50 >100 >100 >100 >10.8 >9.0 >9.0 >100 >100 >40.0 >43.8 >100 0.002

>100 52 12 >100 >100 46 >100 26 12 12 >100 >100 65 ≥100 46 32 79 ≥100 >100 >100 >100 >100

85.4 >52 >12 >100 >100 >46 78 >26 >12 >12 >100 >100 >65 38 >46 >32 >79 >100 >100 >100 >100 >100

14 9.4 7.0 >100 51 9.2 35 9.5 12 7.0 32 63 9.0 >38 >46 8.0 6.0 42 >100 21 17 >100

>14 >9.4 >7.0 >100 >51 >9.2 >35 >9.5 >12 >7.0 >32 >63 >9.0 >38 9.2 >8.0 >6.0 >42 >100 >21 >17 >100

>10

1.1

>10 >100

Reo-1 VERO 76 CVB-5 EC50g) CC50h) EC50i)

Sb-1 EC50j)

VSV EC50k)

VV EC50l)

HSV-1 EC50m)

44 20 13 >100 >100 49 ≥100 33 15 13 45 >100 12 >47 >9.2 20 50 >100 >100 ≥100 ≥100 >100

>44 >20 >13 14 >100 >49 >100 >33 >15 >13 >45 >100 >12 >47 49 >20 >50 >100 40 >100 >100 >100

>44 >20 >13 >100 >100 >49 >100 >33 >15 >13 >45 >100 >12 >47 >49 >20 >50 >100 >100 >100 >100 >100

>44 >20 >13 >100 >100 >49 >100 >33 >15 >13 >45 >100 >12 >47 >49 >20 >50 >100 >100 >100 >100 >100

>44 >20 >13 >100 >100 >49 >100 >33 >15 >13 >45 >100 >12 >47 >49 >20 >50 >100 >100 >100 >100 >100

>44 >20 >13 >100 >100 >49 >100 >33 >15 >13 >45 >100 >12 >47 >49 >20 >50 >100 >100 >100 >100 >100

27

23

>14 >9.4 >7.0 >100 >51 >9.2 >35 >9.5 >12 >7.0 >32 >63 >9.0 47 >9.2 >8.0 >6.0 >42 >100 >21 >17 >100

1.9 16 >100 ≥12.5 >100

1.5 3

* 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%, as determined by the MTT method. b) Compound concentration (µM) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by the MTT method. c) Compound concentration (µM) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method. d) Compound concentration (µM) required to achieve 50% protection of MDBK cells from BVDV-induced cytopathogenicity, as determined by the MTT method. e) Compound concentration (µM) required to reduce the viability of mock-infected BHK cells by 50%, as determined by the MTT method. f, g) Compound concentration (µM) required to achieve 50% protection of BHK cells from YFV-induced(f) and Reo-1-induced(g) cytopathogenicity, as determined by the MTT method. h) Compound concentration (µM) required to reduce the viability of mock-infected VERO-76 cells by 50%. as determined by the MTT method. i–m) Compound concentration (µM) required to reduce the plaque number of CVB-5(i), Sb-1(j), VSV(k), VV(l), HSV-1(m) by 50% in VERO-76 monolayers.

ZQ Micro-mass instruments with quadrupol mass analyzer. The spectra were performed in the positive ion mode at a declustering potential of 40–60 V. The sample was previously separated on a UPLC column (C18) using UPLC ACQUITY™ system by Waters connected with DPA detector. Flash chromatography was performed on Merck silica gel 60 (200–400 mesh) using chloroform–methanol (19 : 1, v/v) mixture as eluent. Analytical TLC was carried out on silica gel F254 (Merck) plates (0.25 mm thickness). The intensity measurements of diffraction reflections were carried out at 200 K with a Xcalibur CCD diffractometer, using graphite monochromated CuKα radiation (λ=1.54178 Å) and ω scan mode. Crystal structure wase solved by the SHELXS-97 program and refined by full-matrix least squares on F2 using the SHELXL-97 program.27) All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were positioned geometrically and allowed to ride on their parent atoms, with Uiso(H)=1.2Ueq(C, N). Thiourea Derivatives of 1,3-Thiazol-2-amine General procedure: A solution of 1,3-thiazol-2-amine (0.0069 mol,

0.69 g) in acetonitrile (25 mL) was treated with appropriate isothiocyanate (0.0075 mol) and the mixture was refluxed for 8 h. Then solvent was removed on rotary evaporator. The residue was purified by column chromatography (chloroform– methanol; 9.5 : 0.5, v/v). The compound was crystallized from acetonitrile. 1-(2-Bromophenyl)-3-(1,3-thiazol-2-yl)thiourea (1) Yield 81%. mp: 163–165°C. 1H-NMR (DMSO-d6) δ (ppm): 7.08 (br s, 1H, CHarom.); 7.16–7.21 (t, 1H, CHarom., J=7.5 Hz); 7.38–7.45 (m, 2H, CHarom.); 7.67–7.70 (d, 1H, CHarom., J=7.8 Hz); 7.76–7.79 (d, 1H, CHarom., J=6.9 Hz); 12.31 (br s, 1H, NH). 13 C-NMR (DMSO-d6) δ (ppm): 111.45, 119.64, 127.69, 127.77 (2C), 129.01, 132.51 (2C), 137.44, 180.12. Electrospray ionization (ESI)-MS: m/z=338.0 [M+Na+H]+ (100%). 1-(3-Bromophenyl)-3-(1,3-thiazol-2-yl)thiourea (2) Yield 90%. mp: 160–161°C. 1H-NMR (DMSO-d6) δ (ppm): 6.99 (br s, 1H, CHarom.); 7.22–7.27 (m, 2H, CHarom.); 7.46–7.48 (d, 1H, CHarom., J=4.5 Hz); 7.82 (br s, 1H, CHarom.); 7.98 (s, 1H, CHarom.); 10.18 (br s, 1H, NH); 12.97 (br s, 1H, NH). 13 C-NMR (DMSO-d6) δ (ppm): 110.11, 120.47, 121.13 (2C),

Chem. Pharm. Bull.

232 Table 6.

SAR Study of Compounds 1–22

Compd. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Vol. 63, No. 3 (2015)

M

SA

log P

HOMO

η

ET

EIA

IC–C

Rf

V

g

SA

HLG

LUMO

χ

EB

EE

HF

α

362.87 441.17 384.31 455.11 407.18 464.01 360.18 451.11 339.35 423.74 352.03 424.83 354.08 429.89 412.97 466.09 381.77 446.71 381.99 462.55 340.44 414.73 399.64 463.53 375.68 444.73 384.09 450.71 352.46 425.68 384.83 450.14 396.00 457.56 366.90 455.83 330.12 440.15 375.17 441.57 390.00 453.32 343.64 405.65

4.54 −7.269 4.54 −7.269 4.78 −7.168 3.51 −7.395 3.89 −7.265 3.89 −7.268 4.26 −7.239 4.73 −7.205 4.40 −7.225 4.63 −8.663 3.75 −7.239 3.49 −7.087 4.26 −7.163 4.21 −7.175 3.89 −7.229 4.54 −7.250 5.00 −7.132 3.70 −7.406 5.45 −7.080 4.26 −7.265 4.73 −7.202 2.31 −7.536

−8.752 −1.483 −8.835 −1.566 −8.779 −1.611 −8.692 −1.297 −8.786 −1.521 −8.855 −1.587 −8.745 −1.506 −8.718 −1.513 −8.870 −1.645 −8.663 −1.506 −8.697 −1.458 −8.503 −1.416 −8.711 −1.548 −8.610 −1.435 −8.808 −1.579 −8.829 −1.579 −8.679 −1.547 −8.719 −1.313 −8.704 −1.624 −8.806 −1.541 −8.726 −1.524 −9.178 −1.642

3.635 5.118 3.635 5.201 3.584 5.195 3.698 4.995 3.633 5.154 3.634 5.221 3.620 5.126 3.603 5.116 3.613 5.258 3.579 5.085 3.620 5.078 3.544 4.960 3.582 5.130 3.588 5.023 3.615 5.194 3.625 5.204 3.566 5.113 3.703 5.016 3.540 5.164 3.633 5.174 3.601 5.125 3.768 5.410

−58072 −2514 −58074 −2516 −64178 −2514 −52451 −2913 −60075 −2558 −60076 −2559 −57228 −2531 −60681 −2816 −67026 −2541 −83144 −2882 −50278 −2549 −60492 −2922 −57229 −2532 −53731 −2833 −60077 −2559 −58073 −2515 −56578 −2501 −53727 −2829 −59783 −2816 −57229 −2532 −60679 −2815 −55641 −2262

−55558 −342363 −55558 −335941 −61664 −374979 −49538 −340912 −57518 −346844 −57518 −340740 −54697 −342990 −57865 −373931 −64485 −379595 −80262 −474922 −47730 −299463 −57570 −374310 −54697 −334594 −50898 −333631 −57518 −339181 −55558 −334383 −54077 −331966 −50898 −338045 −56967 −376258 −54697 −336222 −57865 −380319 −53379 −319919

284291 110.17 277867 108.63 310801 88.90 288461 49.24 286769 58.79 280663 57.57 285762 95.25 313250 85.40 312569 52.40 391778 −57.16 249185 100.83 313818 62.52 277365 94.25 279900 91.32 279104 57.51 276309 108.78 275388 122.41 284318 95.53 316475 64.35 278993 94.32 319639 86.92 264277 −6.07

75.29 30.04 75.29 30.04 77.28 31.27 68.61 28.00 67.88 27.33 67.88 27.33 72.47 29.35 77.51 31.18 72.69 29.26 73.64 28.98 67.67 27.42 74.13 29.89 72.47 29.35 72.71 29.25 67.88 27.33 75.29 30.04 80.08 32.45 72.21 29.25 69.44 29.34 72.47 29.35 77.51 31.18 58.64 23.99

314.22 711.27 314.22 722.90 304.21 740.53 241.37 715.94 253.31 667.58 253.31 670.34 269.77 691.27 283.79 749.51 287.76 711.68 303.32 736.17 235.32 661.34 265.35 739.07 269.77 702.90 249.35 712.64 253.31 669.83 314.22 722.01 361.22 738.70 249.35 721.31 263.33 706.94 269.77 702.99 283.79 740.67 231.29 639.58

Surfaces and refractivity in Å−2, volumes and polarizability in Å−2, energies in kcal/mol.

123.61, 125.60, 130.23 (2C), 141.61, 179.79. ESI-MS: m/z=338.0 [M+Na+H]+ (100%). 1-(3,4-Dichlorophenyl)-3-(1,3-thiazol-2-yl)thiourea (3) Yield 81%. mp: 163–165°C. 1H-NMR (DMSO-d6) δ (ppm): 6.99–7.01 (d, 1H, CHarom., J=4.5 Hz); 7.48–7.51 (t, 2H, CHarom., J=4.5 Hz); 7.75–7.78 (d, 1H, CHarom., J=6.6 Hz); 8.11–8.12 (d, 1H, CHarom., J=4.5 Hz); 10.29 (br s, 1H, NH); 13.02 (br s, 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 110.05, 121.16, 122.20, 124.09, 129.99 (2C), 130.55 (2C), 140.21, 182.25. ESI-MS: m/z=325.9 [M+Na]+ (100%). 1-Cyclohexyl-3-(1,3-thiazol-2-yl)thiourea (4) Yield 92%. mp: 154–156°C. 1H-NMR (DMSO-d6) δ (ppm): 1.28–1.41 (m,

5H, CH2cyclo.); 1.53–1.67 (m, 3H, CH2cyclo.); 1.90–1.93 (m, 2H, CH2cyclo.); 4.08–4.11 (m, 1H, CHcyclo.); 7.09–7.11 (d, 1H, CHarom., J=3.6 Hz); 7.38–7.40 (d, 1H, CHarom., J=6.0 Hz); 9.70 (br s, 1H, NH); 11.42 (s, 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 24.00 (2C), 25.03, 31.42 (2C), 52.28, 112.02, 137.17, 161.79, 176.35. ESI-MS: m/z=264.1 [M+Na]+ (100%). 1-(2-Fluorophenyl)-3-(1,3-thiazol-2-yl)thiourea (5) (PubChem Compound ID: 36777148; CCA 002249—Sigma-Aldrich) Yield 87%. mp: 167–169°C. 1H-NMR (DMSO-d6) δ (ppm): 7.04 (br s, 1H, CHarom.); 7.19–7.28 (m, 3H, CHarom.); 7.42 (s, 1H, CHarom.); 7.78 (br s, 1H, CHarom.); 12.31 (br s, 1H, NH); 12.39 (s, 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 115.88,

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124.12 (2C), 126.93, 127.43, 128.51 (2C), 154.63, 157.89, 181.51. ESI-MS: m/z=276.0 [M+Na]+ (100%). 1-(3-Fluorophenyl)-3-(1,3-thiazol-2-yl)thiourea (6) have been synthesized as described previously.28) 1-(2-Chlorophenyl)-3-(1,3-thiazol-2-yl)thiourea (7) Yield 91%. mp: 160–161°C. 1H-NMR (DMSO-d6) δ (ppm): 7.10 (br s, 1H, CHarom.); 7.23–7.28 (t, 1H, CHarom., J=6.6 Hz); 7.34–7.38 (t, 1H, CHarom., J=7.5 Hz); 7.44–7.54 (m, 2H, CHarom.); 7.82–7.84 (d, 1H, CHarom., J=7.8 Hz); 10.39 (br s, 1H, NH); 12.35 (br s, 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 111.54, 127.31 (3C), 128.65 (2C), 129.48 (2C), 136.10, 180.21. ESI-MS: m/z=292.0 [M+Na+H]+ (100%). 1-(3-Chloro-4-methylphenyl)-3-(1,3-thiazol-2-yl)thiourea (8) Yield 79%. mp: 158–159°C. 1H-NMR (DMSO-d6) δ (ppm): 2.28 (s, 3H, CH3); 6.98–6.99 (d, 1H, CHarom., J=3.9 Hz); 7.23–7.26 (d, 1H, CHarom., J=8.4 Hz); 7.45–7.56 (d, 1H, CHarom., J=4.2 Hz); 7.56–7.59 (d, 1H, CHarom., J=8.1 Hz); 7.84 (s, 1H, CHarom.); 10.31 (br s, 1H, NH); 12.70 (br s, 1H, NH). 13 C-NMR (DMSO-d6) δ (ppm): 18.96, 110.22, 120.59, 121.77, 122.56 (2C), 129.46, 130.72, 132.62, 139.07, 180.81. ESI-MS: m/z=306.0 [M+Na]+ (100%). 1-(3-Chloro-4-fluorophenyl)-3-(1,3-thiazol-2-yl)thiourea (9) (PubChem Compound ID: 21008657) Yield 83%. mp: 171–172°C. 1H-NMR (DMSO-d6) δ (ppm): 6.99–7.01 (d, 1H, CHarom., J=4.2 Hz); 7.35–7.38 (d, 1H, CHarom., J=7.5 Hz); 7.47–7.49 (d, 2H, CHarom., J=4.5 Hz); 8.06 (s, 1H, CHarom.); 10.42 (br s, 1H, NH); 12.81 (br s, 1H, NH). 13C-NMR (DMSOd6) δ (ppm): 110.21, 117.67, 119.34, 125.39, 127.50, 129.48 (2C), 130.74, 140.74, 182.26. ESI-MS: m/z=310.1 [M+Na]+ (100%). Crystal data for 9: monoclinic space group P21/c, unit cell dimensions a=10.091(2) Å, b=3.949(1) Å, c=28.384(6) Å, β=95.45(3)°, V=1126.0(4) Å3, Z=4, dcalc=1.697 g/cm3, μ= 6.430 mm, F(000)=584. Crystal size 0.15×0.03×0.02 mm3; reflections collected/independent/observed 3754/2025/1393; Goodness-of-fit on F2=1.031; final R indices [I>2σ(I)]R1=0.0913, wR2=0.2286; Δρ max/min 0.99/−0.67 e Å−3. The experimental details and final atomic parameters have been deposited with the Cambridge Crystallographic Data Centre as supplementary material (CCDC No. 994080). Copies of the data can be obtained free of charge by emailing [email protected] or on request via www.ccdc. cam.ac.uk/data_request/cif. 1-(1,3-Thiazol-2-yl)-3-[3-(trifluoromethyl)phenyl]thiourea (10) Yield 81%. mp: 1741–1775°C. 1H-NMR (DMSO-d6) δ (ppm): 6.98–6.99 (d, 1H, CHarom., J=4.2 Hz); 7.29–7.35 (t, 1H, CHarom., J=9.3 Hz); 7.47–7.48 (d, 1H, CHarom., J=4.2 Hz); 7.66–7.71 (m, 2H, CHarom.); 7.99–8.02 (dd, 1H, CHarom., J=4.5 Hz); 10.29 (br s, 1H, NH); 12.90 (br s, 1H, NH). 13C-NMR (DMSO-d6) δ (ppm): 110.25, 116.27 (2C), 116.56 (2C), 118.70, 118.95, 122.14, 123.23, 137.34, 182.17. ESIMS: m/z=326.0 [M+Na]+ (100%). 1-Phenyl-3-(1,3-thiazol-2-yl)thiourea (11),29) 1-(4-methoxyphenyl)-3-(1,3-thiazol-2-yl)thiourea (12), 1-(4-chlorophenyl)-3(1,3-thiazol-2-yl)thiourea (13), 1-(4-methylphenyl)-3-(1,3thiazol-2-yl)thiourea (14), 1-(4-fluorophenyl)-3-(1,3-thiazol-2yl)thiourea (15), 1-(4-bromophenyl)-3-(1,3-thiazol-2-yl)thiourea (16),30) 1-(4-iodophenyl)-3-(1,3-thiazol-2-yl)thiourea (17),31) 1-(4-benzylophenyl)-3-(1,3-thiazol-2-yl)thiourea (18),32) N-(1,3thiazol-2-ylcarbamothioyl)benzamide (19),33) 1-(3-chlorophenyl)-3(1,3-thiazol-2-yl)thiourea (20),34) 1-(3-chloro-6-methylphenyl)3-(1,3-thiazol-2-yl)thiourea (21)35) and ethyl(1,3-thiazol-2-

ylcarbamothioyl)carbamate (22)36) have been synthesized as described previously. Biology In Vitro Evaluation of Antimicrobial Activity The antibacterial activity of compounds was tested against a series of Gram-positive bacteria: Staphylococcus aureus ATC C 4163, Staphylococcus aureus ATC C 25923, Staphylococcus aureus ATC C 29213, Staphylococcus aureus ATC C 6538, Staphylococcus epidermidis ATC C 12228, Bacillus subtilis ATC C 6633, Bacillus cereus ATC C 11778, Enterococcus hirae ATC C 10541, Micrococcus luteus ATC C 9341, Micrococcus luteus ATC C 10240, and Gram-negative rods: Escherichia coli ATC C 10538, Escherichia coli ATC C 25922, Escherichia coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseudomonas aeruginosa ATC C 15442, Pseudomonas aeruginosa NCTC 6749, Pseudomonas aeruginosa ATC C 27853, Bordetella bronchiseptica ATC C 4617. Antifungal activity was tested against yeasts: Candida albicans ATC C 10231, Candida albicans ATC C 90028, Candida parapsilosis ATC C 220191. Microorganisms used in this study were obtained from the collection of the Department of Pharmaceutical Microbiology, Medical University of Warsaw, Poland. The tuberculostatic activity of compounds was tested against the M. tuberculosis H37Rv strain (ATC C 25618) and two ‘wild’ 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. Media, Growth Conditions and Antimicrobial Activity Assays Antimicrobial activity was examined by the disc diffusion and MIC method under standard conditions, using Mueller–Hinton II agar medium (Becton Dickinson) for bacteria and RPMI agar with 2% glucose (Sigma) for yeasts, according to CLSI (previously NCCLS) guidelines.22) Solutions containing the tested agents were prepared in methanol or dimethyl sulfoxide (DMSO). For the disc diffusion method, sterile paper discs (9 mm diameter, Whatman No. 3 chromatography filter paper) were dripped with the compound solutions tested to obtain 400 µg of substance per disc. Dry discs were placed on the surface of an 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) were examined by the twofold serial agar dilution technique.23) Concentrations of the tested compounds in solid medium ranged from 3.125 to 400 µg/mL. The final inoculum of studied organisms was 104 (colony forming units per milliliter (CFU/mL)), except the final inoculum for E. hirae ATC C 10541, which was 105 CFU/mL. MICs were read off after 18 h) of incubation at 35°C. The synthesized compounds were examined in vitro for their tuberculostatic activity. Investigations were performed by a classical test-tube method of successive dilution in Youmans’ modification of the Proskauer and Beck liquid medium containing 10% of bovine serum.37,38) Bacterial suspensions were prepared from 14 d old cultures of slowly growing strains. Solutions of compounds in DMSO were tested. Stock solutions contained 10 mg of compounds in 1 mililitre. 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 37°C. The MIC

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values were determined as minimum concentration inhibiting the growth of tested tuberculous strains. The influence of the compound on the growth of bacteria at the certain concentration, 3.1, 6.2, 12.5, 25, 50 and 100 µg/mL, were evaluated. Biofilm Inhibitory Assay Inhibition of bacterial biofilm formation was screened using method, described previously,39) with some modification. Eight hospital isolates of methicillinresistant and two standard strains of Staphylococcus epidermidis (ATC C 12228, ATC C 35984) were cultured overnight in Tryptone Soy Broth 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 37°C for 24 h. The final concentrations of tested compounds ranged from 1 to 16 µg/mL. The positive control—biofilm formation, was bacterial culture in TSB-glucose, negative control was TSB-glucose medium. After incubation, medium was removed from wells and washed with sterile phosphate buffered saline (PBS) to remove the non-adherent bacteria. Alive bacterial cells in each well were stained with 0.5% MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) for 2 h at 37°C. Adherent bacterial cells, which usually formed biofilm on wells, were uniformly stained with MTT. After incubation, the solution was removed and bacterial biofilm was solubilized by DMSO with glycine buffer and mixed 15 min at room temperature. The absorbance (A554) was recorded at 554 nm using spectrophotometer (PowerWave XS, BioTek). The biofilm-inhibition results were interpreted from concentrations-response curve. IC50 value is defined as the concentrations of tested compounds required to inhibit 50% of biofilm formation under the assay conditions. All the experiments were performed in four replicates. Statistical analysis was performed using program Statistica 5.0 PL. S. epidermidis strain ATC C 12228 and S. epidermidis ATC C 35984 were used in assays as a negative (low biofilmproducing) and positive (hgh biofilm-producing) control, respectively. Ciprofloxacin was used as reference antibacterial compound. All strains of S. epidermidis (hospital and standard) were also tested for slime production on Congo Red Agar (CRA) supplemented with 0.8 g/L of Congo red (Sigma) and 50 g/L sucrose (Sigma). Plates were incubated for 24 h at 37°C and for 24 h at room temperature, in the dark. Cell-Based Assays Cells and Viruses Cell lines were purchased from American Type Culture Collection (ATC C). 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 Tcells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK) [ATC C CCL 22 (NBL-1) Bos Taurus]; Baby Hamster Kidney (BHK-21) [ATC C CCL 10 (C-13) Mesocricetus auratus] and Monkey kidney (Vero 76) [ATC C CRL 1587 Cercopithecus Aethiops]. Viruses were purchased from American Type Culture Collection (ATC C), with the exception of Yellow Fever Virus (YFV), and Human Immunodeficiency Virus type-1 (HIV-1). 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: yel-

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low fever virus (YFV) [strain 17-D vaccine (Stamaril Pasteur J07B01)] and bovine viral diarrhoea virus (BVDV) [strain NADL (ATC C VR-534)]; (iii) Picornaviridae: human enterovirus B [coxsackie type B5 (CVB-5), strain Ohio-1 (ATC C VR-29)], and human enterovirus C [poliovirus type-1 (Sb-1), Sabin strain Chat (ATC C VR-1562)]. Viruses representative of negative-sense, single-stranded RNAs (ssRNA−) were: (iv) Rhabdoviridae: vesicular stomatitis virus (VSV) [lab strain Indiana (ATC C VR 1540)]. The virus representative of double-stranded RNAs (dsRNA) was reovirus type-1 (Reo-1) [simian virus 12, strain 3651 (ATC C VR-214)], Reoviridae family. DNA virus representatives were: (v) Poxviridae: vaccinia virus (VV) [vaccine strain Elstree-Lister (ATC C VR-1549)]; vi) Herpesviridae: human herpes 1 (HSV-1) [strain KOS (ATC C VR-1493)]. Cytotoxicity Assays Exponentially growing MT-4 cells were seeded at an initial density of 1×105 cells/mL in 96-well plates in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin G and 100 µg/mL streptomycin. Cell viability was determined after 96 h at 37°C by the MTT method.40) As far as stationary monolayers (analogous to those which support the replication of the other RNA and DNA viruses) are concerned, MDBK and BHK cells were seeded in 24well plates at an initial density of 6×105 and 1×106 cells/mL, respectively, in minimum essential medium with Earle’s salts (MEM-E), L-glutamine, 1 m M sodium pyruvate and 25 mg/L kanamycin, supplemented with 10% horse serum (MDBK) or 10% foetal bovine serum (FBS) (BHK). Cell viability was determined after 48–96 h at 37°C by the MTT method. Vero-76 cells were seeded in 24-well plates at an initial density of 4×105 cells/mL, in Dulbecco’s modified Eagle’s medium (DMEM) with L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell viability was determined after 48–96 h 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 dimethyl sulfaxide (DMSO) at 100 m M. Antiviral Assays Compound’s activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity in exponentially growing MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 µL of RPMI containing 1×104 MT-4 cells were added to each well of flatbottom microtitre trays, containing 50 µL of RPMI without or with serial dilutions of test compounds. Then, 20 µL of a HIV-1 suspension containing 100 CCID50 were added. After a 4-d incubation at 37°C, cell viability was determined by the MTT method. 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 plates at a density of 5×104 and 3×104 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 dilution

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in maintenance medium [MEM-Earl with L-glutamine, 1 m M sodium pyruvate and 0.025 g/L kanamycin, supplemented with 0.5% inactivated FBS] to give an m.o.i of 0.01. After 1 h, 50 µL of maintenance medium, without or with serial dilutions of test compounds, were added. After a 3-/4-d incubation at 37°C, cell viability was determined by the MTT method. 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 plates at a density of 5×104 and 3×104 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 dilution in maintenance medium (MEM-E with L-glutamine, supplemented with 0.5% inactivated FBS, 1 m M sodium pyruvate and 0.025 g/L kanamycin) to give an m.o.i of 0.01. After 1 h, 50 µL of maintenance medium, without or with serial dilutions of test compounds, were added. After a 3–4 d incubation at 37°C, cell viability was determined by the MTT method. 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 24-well plates at a density of 2×105 cells/well and were allowed to form confluent monolayers by incubating overnight in growth medium (DMEM with L-glutamine and 4.5 g/L D -glucose and 0.025 g/L kanamycin, supplemented with 10% FBS) at 37°C in a humidified CO2 (5%) atmosphere. Then, monolayers were infected for 2 h with 250 µL of proper virus dilutions to give 50–100 PFU/well. Following removal of unadsorbed virus, 500 µL of maintenance medium (DMEM with L-glutamine and 4.5 g/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 d (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. Efavirenz, 2′-β-methylguanosine, 2′-ethynyl-D -citidine, acycloguanosine and mycophenolic acid were used as reference inhibitors of ssRNA+, ssRNA− and DNA viruses, respectively. Linear Regression Analysis 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. Computational Details Conformational search and physicochemical parameters were calculated using HyperChem Release 8.0.7.41) Extensive conformational search was performed at molecular mechanics level with OPLS force field. The most stable structures obtained were subsequently optimized to the closest local minimum at the semiempirical level using PM3 parametrizations. Converegence criteria were set to 0.1 and 0.01 kcal·mol−1·Å−1 for OPLS and PM3 calculations, respectively. Some physicochemical parameters as molar weight (M), volume (V), surface area (SA), surface area grid (gSA), logarithm of the partition coefficient (log P), the difference

between HOMO and LUMO energy levels (HLG), the higest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), hardness (η) obtained from the equation η=(HOMO−LUMO)/2, Mullkien electronegativity (χ) obtained from the equation χ=−(HOMO+LUMO)/2, total energy (ET), binding energy (EB), isolated atomic energy (EIA), electronic energy (EE), core–core interaction (IC–C), heat of formation (HF), refractivity (Rf), polarizability (α) were collected in Table 6. Acknowledgments We gratefully acknowledge the Sardinia Regional Government for the financial support of Silvia Madeddu through his Ph.D. scholarship (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous Region of Sardinia, European Social Found 2007–2013). Conflict of Interest interest.

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