Antimicrobial and Anti-biofilm Activity of Thiourea Derivatives Incorporating a 2-Aminothiazole Scaffold

Abstract

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 (CC₅₀<10 µM).

1,3-Thiazoles are an important group of compounds due to their wide range of application as pharmaceutical agents. Especially 2-amino-1,3-thiazole scaffold (fused and non-fused) would serve as a privileged structure due to their prevalence in antibacterial agents and other biologically active molecules.^1–5) Database of biologically active substances⁶⁾ reveals the extensive use of the 2-aminothiazole structural motif in numerous applied drugs as well as in preclinical and clinical candidates (e.g., Sulfatizole, Cetraxone, Aztreonam, Riluzole).

Moreover, the literature surrey shows that 2-aminothiazole-based compounds serve as muscarinic and serotonergic ligands.^7–9) Therefore 2-amino-1,3-thiazoles are perspective scaffolds for designing new families of compounds with therapeutic importance.

The structure of 2-amino-1,3-thiazole can be considered as a compound having thiourea system built into the five membered ring. In our previous studies,^10,11) we reported the synthesis and biological activity of thiourea derivatives. In the recent literature, the thioureas are described as most useful class of agents with large number of activities including antiviral,^12,13) antibacterial¹⁴⁾ and high density lipoprotein (HDL)-elevating properties.¹⁵⁾

Isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea)—another example of the thiourea pharmacofore is known to be an effective anti-tuberculosis drug clinically used against a range of multidrug-resistant strains of Mycobacterium tuberculosis.¹⁶⁾ Isoxyl inhibits M. bovis with 6 h of exposure, which is similar to isoniazid and ethionamide, two other prominent anti-tuberculosis drugs.¹⁶⁾

Isoniazid, ethionamide and isoxyl inhibit biosynthesis of mycolic acids (fatty acids). Isoniazid and ethionamide stimulate biosynthesis of short chain fatty acids, while isoxyl inhibits their synthesis.¹⁷⁾

Analogues of isoxyl, with symmetrical and unsymmetrical modifications of the molecule, have significantly enhanced activity against both Mycobacterium tuberculosis and M. bovis BCG.¹⁸⁾

Although the twentieth century has been characterized by a drastic reduction in the mortality caused by infectious diseases and a rise in the control of neoplastic pathologies, the treatment of cancer and of infectious diseases caused by viruses still remains an important and challenging problem. As a matter of fact cancer remains the leading cause of death in economically developed countries and AIDS is the major health problem with 34 million people were living with Human immunodeficiency virus (HIV)¹⁹⁾; therefore, despite the considerable progress made, there is continued interest in developing therapeutic agents.²⁰⁾

On the basis of the biological properties previously described, we have accomplished the synthesis of 22, 10 of them new, thiourea derivatives of 1,3-thiazole and the evaluation of their antibacterial and antiviral activities.

Chemistry

The preparation of 10 new thiourea derivatives is described. 1,3-Thiazol-2-amine was subjected to the reaction with isothiocyanates in order to be transformed into the corresponding thiourea derivatives (Chart 1). Obtained compounds were purified by a flash chromatography. MS, ¹H- and ¹³C-NMR spectra confirmed the identity of the products. In ¹H-NMR spectra labile proton of NH group of thiourea branch was not observed for compound 1. The molecular structure of 9 (Fig. 1) was determined by an X-ray crystal structure analysis.

Chart 1. Synthesis of Studied Compounds

R=2-bromophenyl (1), 3-bromophenyl (2), 3,4-dichlorophenyl (3), cyclohexyl (4), 2-fluorophenyl (5), 3-fluorophenyl (6), 2-chlorophenyl (7), 3-chloro-4-methylphenyl (8), 3-chloro-4-fluorophenyl (9), 3-trifluorophenyl (10), phenyl (11), 4-metoxyphenyl (12), 4-chlorophenyl (13), 4-methylphenyl (14), 4-fluorophenyl (15), 4-bromophenyl (16), 4-iodophenyl (17), benzyl (18), benzoyl (19), 3-chlorophenyl (20), 5-chloro-2-methylphenyl (21), ethoxycarbonyl (22).

Fig. 1. View of the Molecule 9

Results and Discussion

All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. Microorganisms used in this study have common application in the antimicrobial tests for many substances like antibiotics, disinfectants and antiseptic drugs or in research on new antimicrobial agents.²¹⁾ All compounds were screened for their antimicrobial activity by a disc diffusion method.²²⁾ Compounds showing significant activity in the test were next examined for their minimal inhibitory concentration (MIC).²³⁾ The results of activity for these compounds are summarized in Table 1.

Table 1. Activities of Obtained Compounds against Gram-Positive Bacteria and C. albicans—Minimal Inhibitory Concentations (MIC, µg/mL) and Diameter of Growth Inhibitory Zone (GIZ, mm, Applied 400 µg per Disc)

	2	3	5	6	8	9	10	11	12	15	16	Ref.a)	Ref.b)
S. aureus NCTC 4163	32	16	512	32	32	4	32	32	32	32	16	0.25	−
	(13)	(15)	(11)	(13)	(12)	(26)	(14)	(13)	(13)	(12)	(13)	(26)	−
S. aureus ATCC 25923	32	16	512	16	16	4	32	32	32	16	16	0.5	−
	(12)	(20)	(13)	(15)	(15)	(27)	(15)	(13)	(14)	(14)	(13)	(26)	−
S. aureus ATCC 6538	32	16	256	64	32	8	32	32	32	32	16	0.25	−
	(11)	(17)	(12)	(15)	(15)	(26)	(14)	(11)	(14)	(12)	(13)	(28)	−
S. aureus ATCC 29213	16	4	256	64	16	8	32	32	32	16	16	0.5	−
	(15)	(18)	(11)	(18)	(19)	(25)	(16)	(12)	(13)	(11)	(13)	(22)	−
S. epidermidis ATCC 12228	16	8	516	16	2	4	16	32	4	16	8	0.25	−
	(15)	(17)	(12)	(16)	(14)	(26)	(14)	(13)	(13)	(13)	(16)	(30)	−
S. epidermidis ATCC 35984	16	4	516	16	4	4	16	16	8	16	8	0.25	−
	(15)	(21)	(12)	(19)	(20)	(30)	(20)	(14)	(18)	(16)	(15)	(32)	−
E. hirae ATCC 10541	>512	32	256	512	32	16	128	>512	64	>512	>512	1	−
	(11)	(17)	−	−	(11)	(23)	(13)	−	(11)	−	−	(18)	−
E. faecalis ATCC 29212	>512	32	516	512	32	16	128	>512	64	512	>512	1	−
	−	−	−	−	−	(20)	−	−	−	−	−	(15)	−
M. luteus ATCC 10541	16	16	516	16	8	4	32	16	16	16	16	2	−
	(18)	(24)	(17)	(18)	(19)	(29)	(19)	(17)	(17)	(17)	(16)	(22)	−
M. luteus ATCC 9341	16	16	128	16	4	8	16	32	4	16	8	1	−
	(13)	(20)	(30)	(20)	(17)	(31)	(14)	(15)	(14)	(16)	(14)	(24)	−
B. subtilis ATCC 6633	2	8	128	16	4	2	4	8	8	4	8	<0.12	−
	(21)	(24)	(17)	(24)	(24)	(36)	(25)	(20)	(23)	(20)	(18)	(38)	−
B. cereus ATCC 11778	32	16	256	16	16	4	16	32	32	32	16	0.25	−
	(11)	(14)	(17)	(24)	(13)	(22)	(13)	(12)	(13)	(13)	(12)	(26)	−
C. albicans ATCC 10231	−	−	256	−	−	128	−	512	−	256	512	−	0.5
	−	−	(16)	−	−	(11)	−	(20)	−	(20)	(14)	−	(20)
C. albicans ATCC 90028	−	−	256	−	−	64	−	512	−	128	512	−	0.5
	−	−	(19)	−	−	(12)	−	(10)	−	(11)	(11)	−	(29)
C. albicans ATCC 22019	−	−	256	−	−	64	−	512	−	128	512	−	0.5
	−	−	(20)	−	−	(18)	−	(11)	−	(15)	(11)	−	(25)

a) Ref.—ciprofloxacin (GIZ—5 µg/9 mm disc). b) Ref.—Fluconazole (GIZ—25 µg/9 mm disc). − Lack of the growth inhibition area.

Preliminary test by a disc-diffusion method showed antimicrobial activity against standard Gram-positive cocci, therefore the next step was evaluation of compounds’ MIC values for standard and hospital strains. The research was carried out over 20 standard strains and 15 hospital strains of Staphylococcus epidermidis used for routine antimicrobial media susceptibility testing. Hospital strains were isolated from different biological materials of patients hospitalized in Warsaw Medical University Hospitals.

MIC values of hospital S. epidermidis strain were in the range 128—2 µg/mL (Table 2). For the most active compounds 3, 6 and 9 the MICs were in the range 16—2 µg/mL.

Table 2. Activity of Compounds against Hospital Strains of S. epidermidis—Minimal Inhibitory Concentrations (MIC, µg/mL)

	2	3	5	6	8	9	10	11	12	15	16	Cipa)
S. epidermidis 517/12	32	4	128	8	32	8	64	64	32	32	32	32
S. epidermidis 518/12	32	4	128	8	32	8	64	64	32	32	32	32
S. epidermidis 519/12	16	2	128	4	8	4	32	16	16	16	8	0.25
S. epidermidis 520/12	128	4	256	16	32	8	64	64	128	32	32	16
S. epidermidis 521/12	16	2	128	8	8	4	32	32	32	16	16	32
S. epidermidis 523/12	32	8	128	8	32	4	64	64	32	32	32	64
S. epidermidis 524/12	32	4	128	4	8	4	32	32	32	16	16	8
S. epidermidis 526/12	32	4	128	4	16	4	32	32	32	32	32	4
S. epidermidis 527/12	32	4	128	16	32	8	64	64	64	32	32	0.25
S. epidermidis 528/12	32	8	128	8	32	8	64	64	128	32	32	32
S. epidermidis 529/12	32	4	128	4	8	4	32	32	32	16	16	4
S. epidermidis 530/12	32	4	128	8	32	4	64	64	32	32	32	32
S. epidermidis 531/12	32	4	128	8	8	4	32	32	32	32	32	8
S. epidermidis 532/12	16	4	128	8	8	4	32	16	8	16	8	0.5
S. epidermidis 533/12	16	4	128	8	8	4	32	16	32	16	32	0.25

a) Cip—ciprofloxacin.

The biological activity depends on the structure of compounds, viz. on an aryl substituent. Based on Table 2, methoxy (12) and trifluoromethyl (10) derivatives are less potent than Ciprofloxacin, while compounds 3 and 9 are more active than the reference drug. It led to the conclusion that activity depends on the type of the substituent on phenyl ring. Furthermore, the position of the substitution is also important. For example, compound 5 is the least active compound, suggesting that halogen on meta position of benzene ring does not favor activity against S. epidermidis.

Generally, the phenyl group substituted with the fluorine, bromine or chlorine atoms, and methoxy or trifluoromethyl groups increases the antimicrobial activity in comparison to the aliphatic substituents. The most active compound was 9 which is the 3-chloro-4-fluorophenyl substituted derivative.

Preliminary test by disc-diffusion method showed antimicrobial activity against standard strain of fungi C. albicans for five compounds, therefore the next step was evaluation of compounds’ MIC values for standard and hospital strains. The MIC values for these compounds were higher than 64 µg/mL.

Compounds were also tested for their in vitro tuberculostatic activity against the M. tuberculosis H₃₇Rv strain and two ‘wild’ strains isolated from tuberculosis patients: one (Spec. 210) resistant to p-aminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB) and rifampicin (RMP), and the other (Spec. 192) fully sensitive to the administrated tuberculostatics. The MIC values were determined as the minimum concentration inhibiting the growth of tested tuberculous strains. INH was used as the reference drugs. The results of tuberculostatic activity for these compounds are summarized in Table 3.

Table 3. In Vitro Tuberculostatic Activity of Compounds 1–9 and 11–21

Compdb)	MICa) [µg/mL]
	M. tuberculosisc)
	H37Rv	Spec. 192	Spec. 210
1	50	50	50
2	25	25	12.5
3	25	25	25
4	100	100	100
5	25	25	25
6	50	25	25
7	50	25	25
8	50	25	50
9	25	25	12.5
11	12.5	12.5	12.5
12	25	25	12.5
13	50	25	50
14	25	25	25
15	50	50	50
16	nt	nt	nt
17	12.5	25	12.5
18	50	50	50
19	100	100	100
20	50	50	50
21	50	50	50

a) Minimum inhibitory concentrations for mycobacterial strains were determined by two-fold classical test-tube method of successive dilution. b) INH, isoniazid; nt, no tested. c) Mycobacterium tuberculosis H₃₇Rv (ATCC 25618), Spec. 192, Spec. 210.

The most active compounds were 11 and 17 which are the phenyl and the 4-iodophenyl substituted derivatives. The activity against three strains of M. tuberculosis is similar for all compounds, indicating that these compounds could be promising drug candidates for multidrug-resistant tuberculosis treatment.

Antimicrobial activity can be related to cytotoxicity of compounds (e.g., 2, 3, 16). However it is not a rule because compounds 1 and 5 that significantly inhibit the growth of Gram-positive cocci, turned out cytotoxic in the high micromolar range (CC₅₀ above 100 µM and 38.5 µM).

Preliminary antibacterial studies revealed that halogen derivatives of thiourea 3 and 9 have shown the most promising activity against free-swimming (planktonic) forms of staphylococcal species. In general MIC values for standard and medically relevant methicillin-resistant strains of S. aureus and S. epidermidis ranged from 16 to 4 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 (ATC C 12228, ATC C 35984).

Based on their absorbance, the tested strains of S. epidermidis were divided into two groups: the first that form biofilm on low level (A₅₅₄ <0.7) and high level biofilm forming (A₅₅₄ <1.9). Strains that produced slime, formed black colonies with a dry crystalline consistency on Congo Red agar (CRA), whereas slime-negative isolates were pinkish-red with darkening at the centre (Fig. 2). In MTT test slime-positive strains were high biofilm-producers, slime-negative—low biofilm producers.

Fig. 2. 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.

The compounds were tested at concentrations ranging from 1 to 16 µg/mL. Both thiourea connections exhibited good biofilm inhibitory activity against the aforesaid standard and hospital bacterial strains, regardless of the level on which biofilm was formed.

The compounds 3 and 9 were found to be more promising with IC₅₀ values of 0.35–7.32 µg/mL. For most of tested strains the highest concentrations of compounds (for 3—4 µg/mL, for 9—8 µg/mL) inhibited biofilm formation in above 70% (Figs. 3, 4), as compared to the control.

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.

To compare, the reference compound, ciprofloxacin, in its highest concentration (16 µg/mL) inhibited biofilm formation of four tested strains of S. epidermidis in approximately 40% (Fig. 5). For the two and only hospital strain (S. epidermidis 519/12 and 533/12) the highest concentration of the standard drug blocked biofilm formation in 80%. Observed values of IC₅₀ for ciprofloxacin were much higher than for both tested thiourea derivatives (Table 4).

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

Bacterial strain	Test compounds
	Compd. 3 IC50 values (µg/mL)	Compd. 9 IC50 values (µg/mL)	Ciproflox. IC50 values (µg/mL)
S. epidermidis ATCC 12228	3.71	0.35	11.89
S. epidermidis ATCC 35984	2.55	—	18.55
S. epidermidis 517/12	7.32	0.66	—
S. epidermidis 519/12	2.52	2.50	1.65
S. epidermidis 523/12	2.53	3.14	—
S. epidermidis 526/12	1.61	4.95	9.28
S. epidermidis 528/12	2.50	1.10	5.00
S. epidermidis 531/12	2.83	1.13	6.59
S. epidermidis 532/12	3.49	2.21	6.73
S. epidermidis 533/12	1.96	2.88	1.90

Due to the previously reported anti-HIV activities of thiourea derivatives,^24,25) title compounds were tested in the cell-based assay against the human immunodeficiency virus type-1 (HIV-1), using Efavirenz as reference inhibitor. The cytotoxicity against the MT4 cells was evaluated in parallel with the antiviral activity (Table 5).

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	MT-4 CC50a)	HIV-1 EC50b)	MDBK CC50c)	BVDV EC50d)	BHK CC50e)	YFV EC50f)	Reo-1 EC50g)	VERO 76 CC50h)	CVB-5 EC50i)	Sb-1 EC50j)	VSV EC50k)	VV EC50l)	HSV-1 EC50m)
1	>100	>100	>100	85.4	14	>14	>14	44	>44	>44	>44	>44	>44
2	9.0	>9.0	52	>52	9.4	>9.4	>9.4	20	>20	>20	>20	>20	>20
3	9.0	>9.0	12	>12	7.0	>7.0	>7.0	13	>13	>13	>13	>13	>13
4	>100	36	>100	>100	>100	>100	>100	>100	14	>100	>100	>100	>100
5	38.5	>38.5	>100	>100	51	>51	>51	>100	>100	>100	>100	>100	>100
6	10.8	>10.8	46	>46	9.2	>9.2	>9.2	49	>49	>49	>49	>49	>49
7	20.8	>20.8	>100	78	35	>35	>35	≥100	>100	>100	>100	>100	>100
8	8.5	>8.5	26	>26	9.5	>9.5	>9.5	33	>33	>33	>33	>33	>33
9	7.8	>7.8	12	>12	12	>12	>12	15	>15	>15	>15	>15	>15
10	9.0	>9.0	12	>12	7.0	>7.0	>7.0	13	>13	>13	>13	>13	>13
11	50	>50	>100	>100	32	>32	>32	45	>45	>45	>45	>45	>45
12	>100	>100	>100	>100	63	>63	>63	>100	>100	>100	>100	>100	>100
13	>100	>100	65	>65	9.0	>9.0	>9.0	12	>12	>12	>12	>12	>12
14	>100	>100	≥100	38	>38	>38	47	>47	>47	>47	>47	>47	>47
15	10.8	>10.8	46	>46	>46	9.2	>9.2	>9.2	49	>49	>49	>49	>49
16	9.0	>9.0	32	>32	8.0	>8.0	>8.0	20	>20	>20	>20	>20	>20
17	9.0	>9.0	79	>79	6.0	>6.0	>6.0	50	>50	>50	>50	>50	>50
18	>100	>100	≥100	>100	42	>42	>42	>100	>100	>100	>100	>100	>100
19	>100	>100	>100	>100	>100	>100	>100	>100	40	>100	>100	>100	>100
20	40.0	>40.0	>100	>100	21	>21	>21	≥100	>100	>100	>100	>100	>100
21	43.8	>43.8	>100	>100	17	>17	>17	≥100	>100	>100	>100	>100	>100
22	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100	>100
Efavirenz	40	0.002
2′-C-Methyl-guanosine			>10	1.1	>10	1.9
2′-C-Methyl-cytidine					>100		16
2′-C-Ethynyl-cytidine								>100	27	23
Mycophenolic acid								≥12.5				1.5
Acycloguanosine								>100					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.

Only compound 4 showed anti-HIV activity, although not very potent (EC₅₀=36 µM), associated with lack of cytotoxicity (CC₅₀>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-ethynyl-cytidine, 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 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 electron-withdrawing 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 para-substituted 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).

The structure–activity relationship was also observed when the positional weighed electronic effect of substituent is analyzed (Table 6).

Table 6. SAR Study of Compounds 1–22

Compd.	M	SA	log P	HOMO	η	ET	EIA	IC–C	Rf
	V	gSA	HLG	LUMO	χ	EB	EE	HF	α
1	314.22	362.87	4.54	−8.752	3.635	−58072	−55558	284291	75.29
	711.27	441.17	−7.269	−1.483	5.118	−2514	−342363	110.17	30.04
2	314.22	384.31	4.54	−8.835	3.635	−58074	−55558	277867	75.29
	722.90	455.11	−7.269	−1.566	5.201	−2516	−335941	108.63	30.04
3	304.21	407.18	4.78	−8.779	3.584	−64178	−61664	310801	77.28
	740.53	464.01	−7.168	−1.611	5.195	−2514	−374979	88.90	31.27
4	241.37	360.18	3.51	−8.692	3.698	−52451	−49538	288461	68.61
	715.94	451.11	−7.395	−1.297	4.995	−2913	−340912	49.24	28.00
5	253.31	339.35	3.89	−8.786	3.633	−60075	−57518	286769	67.88
	667.58	423.74	−7.265	−1.521	5.154	−2558	−346844	58.79	27.33
6	253.31	352.03	3.89	−8.855	3.634	−60076	−57518	280663	67.88
	670.34	424.83	−7.268	−1.587	5.221	−2559	−340740	57.57	27.33
7	269.77	354.08	4.26	−8.745	3.620	−57228	−54697	285762	72.47
	691.27	429.89	−7.239	−1.506	5.126	−2531	−342990	95.25	29.35
8	283.79	412.97	4.73	−8.718	3.603	−60681	−57865	313250	77.51
	749.51	466.09	−7.205	−1.513	5.116	−2816	−373931	85.40	31.18
9	287.76	381.77	4.40	−8.870	3.613	−67026	−64485	312569	72.69
	711.68	446.71	−7.225	−1.645	5.258	−2541	−379595	52.40	29.26
10	303.32	381.99	4.63	−8.663	3.579	−83144	−80262	391778	73.64
	736.17	462.55	−8.663	−1.506	5.085	−2882	−474922	−57.16	28.98
11	235.32	340.44	3.75	−8.697	3.620	−50278	−47730	249185	67.67
	661.34	414.73	−7.239	−1.458	5.078	−2549	−299463	100.83	27.42
12	265.35	399.64	3.49	−8.503	3.544	−60492	−57570	313818	74.13
	739.07	463.53	−7.087	−1.416	4.960	−2922	−374310	62.52	29.89
13	269.77	375.68	4.26	−8.711	3.582	−57229	−54697	277365	72.47
	702.90	444.73	−7.163	−1.548	5.130	−2532	−334594	94.25	29.35
14	249.35	384.09	4.21	−8.610	3.588	−53731	−50898	279900	72.71
	712.64	450.71	−7.175	−1.435	5.023	−2833	−333631	91.32	29.25
15	253.31	352.46	3.89	−8.808	3.615	−60077	−57518	279104	67.88
	669.83	425.68	−7.229	−1.579	5.194	−2559	−339181	57.51	27.33
16	314.22	384.83	4.54	−8.829	3.625	−58073	−55558	276309	75.29
	722.01	450.14	−7.250	−1.579	5.204	−2515	−334383	108.78	30.04
17	361.22	396.00	5.00	−8.679	3.566	−56578	−54077	275388	80.08
	738.70	457.56	−7.132	−1.547	5.113	−2501	−331966	122.41	32.45
18	249.35	366.90	3.70	−8.719	3.703	−53727	−50898	284318	72.21
	721.31	455.83	−7.406	−1.313	5.016	−2829	−338045	95.53	29.25
19	263.33	330.12	5.45	−8.704	3.540	−59783	−56967	316475	69.44
	706.94	440.15	−7.080	−1.624	5.164	−2816	−376258	64.35	29.34
20	269.77	375.17	4.26	−8.806	3.633	−57229	−54697	278993	72.47
	702.99	441.57	−7.265	−1.541	5.174	−2532	−336222	94.32	29.35
21	283.79	390.00	4.73	−8.726	3.601	−60679	−57865	319639	77.51
	740.67	453.32	−7.202	−1.524	5.125	−2815	−380319	86.92	31.18
22	231.29	343.64	2.31	−9.178	3.768	−55641	−53379	264277	58.64
	639.58	405.65	−7.536	−1.642	5.410	−2262	−319919	−6.07	23.99

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

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 (E_T), binding energy (E_B), isolated atomic energy (E_IA), electronic energy (E_E), core–core interaction (I_C–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 E_T (total energy), E_IA (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 E_T and E_IA 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 immuno-compromised 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 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 U_iso(H)=1.2U_eq(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). ¹³C-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). ¹³C-NMR (DMSO-d₆) δ (ppm): 110.11, 120.47, 121.13 (2C), 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, 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). ¹³C-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 (DMSO-d₆) 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) ų, Z=4, d_calc=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 [e-mail] 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. ESI-MS: 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-(1,3-thiazol-2-yl)thiourea (14), 1-(4-fluorophenyl)-3-(1,3-thiazol-2-yl)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-(1,3-thiazol-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 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 methicillin-resistant 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 biofilm-producing) 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 T-cells 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 III_B laboratory strain of HIV-1, obtained from the supernatant of the persistently infected H9/III_B cells (NIH 1983); (ii) Flaviviridae: yellow 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 24-well 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 mM 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 mM.

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 flat-bottom 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 in maintenance medium [MEM-Earl with L-glutamine, 1 mM 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 mM 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 (E_T), binding energy (E_B), isolated atomic energy (E_IA), electronic energy (E_E), core–core interaction (I_C–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

The authors declare no conflict of interest.

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