Pyrimidine-derived disulfides as potential antimicrobial agents: Synthesis and evaluation in vitro

Article abstract of DOI:10.1080/17415993.2015.1024679

Antibiotic resistance is a worldwide problem. The synthesis and evaluation of new antimicrobial compounds, without cytotoxicity against human cells, are highly desired. In this paper, the preparation of a class of pyrimidine-derived disulfides is describe

Full text of DOI:10.1080/17415993.2015.1024679

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Pyrimidine-derived disulfides as
potential antimicrobial agents:
synthesis and evaluation in vitro
Paola Bonaccorsia, Anna Barattuccia, Teresa Papaliab, Giuseppe
Criseoc, Caterina Faggioc & Orazio Romeoc
a Dipartimento di Scienze Chimiche, Università degli Studi di
Messina, viale F. Stagno d'Alcontres 31, vill. S. Agata, 98166
Messina, Italy
b Dipartimento di Scienze del Farmaco e dei Prodotti per la
Salute, Università degli Studi di Messina, Polo Universitario, viale
SS. Annunziata, 98168 Messina, Italy
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c Dipartimento di Scienze Biologiche e Ambientali, Università
degli Studi di Messina, viale F. Stagno d'Alcontres 31, vill. S.
Agata, 98166 Messina, Italy
Published online: 09 Apr 2015.
To cite this article: Paola Bonaccorsi, Anna Barattucci, Teresa Papalia, Giuseppe Criseo,
Caterina Faggio & Orazio Romeo (2015) Pyrimidine-derived disulfides as potential antimicrobial
agents: synthesis and evaluation in vitro, Journal of Sulfur Chemistry, 36:3, 317-325, DOI:
10.1080/17415993.2015.1024679
To link to this article: http://dx.doi.org/10.1080/17415993.2015.1024679
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Journal of Sulfur Chemistry, 2015
Vol. 36, No. 3, 317–325, http://dx.doi.org/10.1080/17415993.2015.1024679
Pyrimidine-derived disulfides as potential antimicrobial agents:
synthesis and evaluation in vitro
Paola Bonaccorsia∗, Anna Barattuccia, Teresa Papaliab, Giuseppe Criseoc, Caterina Faggioc and
Orazio Romeoc,
aDipartimento di Scienze Chimiche, Università degli Studi di Messina, viale F. Stagno d’Alcontres 31, vill.
S. Agata, 98166 Messina, Italy; bDipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Univer-
sità degli Studi di Messina, Polo Universitario, viale SS. Annunziata, 98168 Messina, Italy; cDipartimento
di Scienze Biologiche e Ambientali, Università degli Studi di Messina, viale F. Stagno d’Alcontres 31, vill.
S. Agata, 98166 Messina, Italy
(Received 3 February 2015; accepted 26 February 2015)
Antibiotic resistance is a worldwide problem. The synthesis and evaluation of new antimicrobial com-
pounds, without cytotoxicity against human cells, are highly desired. In this paper, the preparation of a
class of pyrimidine-derived disulfides is described. Sulfenic acids were generated from suitable precursors
and used as reactive intermediates that condensed with the thiol function of thiocytosine. An introductory
study on the antimicrobial activity of this disulfide family is reported. Three family components, that
revealed no cytotoxicity against human erythrocytes, exhibited inhibitory activity against Gram-positive
Staphylococcus aureus.
Keywords: antibacterial activity; cytotoxicity; disulfides; sulfenic acids; thiocytosine
1. Introduction
Several derivatives of pyrimidine have been used in the synthesis of compounds with
notable antimicrobial activity. Both the antibacterial, 5-[(3,4,5-trimethoxyphenyl)methyl]-
*Corresponding author. Email: pbonaccorsi@unime.it
© 2015 Taylor & Francis

318 P. Bonaccorsi et al.
2,4-pyrimidinediamine, better known as trimethoprim, and the antimalarial pyrimethamine
[5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine] are inhibitors of dihydrofolate reductase,
causing folate deficiency in some patients.[1] A variety of cephalosporins, possessing
C(3)-aminopyrimidinyl substituents, exhibited excellent antibacterial activities [2] in vitro. The
synthesis of a family of carbapenem derivatives bearing an aminopyrimidinylthioether moiety
at the C-5 position of the pyrrolidine ring was described, together with their potent antibacte-
rial activities.[3] The bacteriostatic and fungistatic activities of pyrimidinophanes, macrocycles
with pyrimidine fragments, possessing in their skeleton thiocytosine and uracil moieties, were
estimated.[4] The reported data show that some of these macrocycles exhibit a remarkable
selectivity toward Gram-positive bacteria, in dependence of their size and substituent nature.
Finally, amino-substituted pyrimidines used as capping for gold nanoparticles induced bacterial
resistance and appeared harmless to human cells.[5]
On the other hand, the existing collection of antimicrobial agents seems to be insufficient to
protect the human species from infectious diseases, due to the resistance that many clinically rel-
evant pathogens have developed against some of the well-known classes of antibacterial agents,
such as the classical β-lactam antibiotic penicillins and cephalosporins. There is a real need
of new compounds endowed with antimicrobial activity that show, possibly, new mechanisms of
action. Staphylococcus aureus is perhaps the pathogen of greatest concern because of its intrinsic
virulence and its ability to develop antibiotic resistance.[6]
In continuing our study directed to the development of the sulfenic acid chemistry for the
preparation of new biologically active compounds,[7–10] we had the idea of connecting a pyrim-
idine residue to a planar structural skeleton by disulfide bond, as spacer, and prepare a small
library of mono, di, and tri-pyrimidine-derived disulfides 2–8 (Figure 1) with different structural
characteristics. In this paper we present the synthetic strategy, the hemolytic impact on red blood
cells (RBCs), and an introductory study of the potential antibacterial and antifungal activities of
compounds 2–8.
2. Results and discussion
The sulfenic acid/thiol condensation was used to prepare the pyrimidine-derived family of disul-
fides shown in Figure 1. This is a well-assessed synthetic process (Scheme 1) that allowed us
to obtain unsymmetrical disulfides in good yields and in three steps, starting from the suitable
thiols.[11]
The syntheses of compounds 2 [11] and 8 [12,13] were previously reported. Compounds 4
and 5 have been analogously prepared from the commercially available thiophenol and 1,4-
benzenedimethanethiol, respectively. Disulfides 6 and 7 have been obtained from thiols 9 and 10
(Figure 2), that can be easily prepared from the corresponding 1,3,5-tris(bromomethyl)benzenes,
through the formation of the related thiouronium salts.[14] Finally, disulfide 3 has been prepared
in quantitative yield by base-catalyzed deprotection of compound 2 with aqueous ammonia.
The procedure for the synthesis of 2ꢀ-[1,4-phenylenebis(methylenedithio)]bis-4-pyrimidin-
amine (5) is outlined in Scheme 2, as an example of the sulfenic acid chemistry used for the
preparation of the molecules under study. The synthesis started with the preparation of the
sulfenic acid precursors, such as racemic bis-sulfoxide 13, that was obtained from bis-thiol 11
in two steps: (i) a conjugated addition in basic media of the corresponding thiolate to methyl
acrylate to give 12, obtained in high yields after column chromatography, and (ii) the subsequent
controlled and quantitative oxidation of sulfide 12–13, by means of mCPBA.[8] Thermolysis of
13 was first conducted in 1,2-dichloroethane (DCE) at 83 °C, but compound 5 was obtained in
very poor yields. Side-products were isolated from the top of the chromatographic column, due

Journal of Sulfur Chemistry 319
Figure 1. Thiocytosine (1) and derived disulfides 2–8.
Scheme 1. The general procedure for the synthesis of disulfides 2–8.
to the nucleophilic attack of the sulfur atom of thiocytosine (1) to electron-poor carbon atoms of
DCE. In order to avoid this side-reaction, several other solvents were tested, such as acetonitrile,
THF or 1,4-dioxane (bp 101 °C) and the last one resulted in the most efficient in term of yield
and work-up of the thermolysis crude. Thermolysis of bis-sulfoxide 13, in refluxing 1,4-dioxane,
allowed the stepwise generation of the transient sulfenic functions in 14 and 15, not isolated,[15]
that in turn condensed with an excess of commercially available thiocytosine (1), giving rise to

320 P. Bonaccorsi et al.
Figure 2. Starting products for the synthesis of 2,2ꢀ,2ꢀꢀ-[1,3,5-benzenetriyltris(methylenedithio)]tris-4-pyrimidinamines
6 and 7.
Scheme 2. The synthetic pathway to 2ꢀ-[1,4-phenylenebis(methylenedithio)]bis-4-pyrimidinamine (5).
product 5 in 88% yield. All the thiocytosine-derived disulfides 2, 4–8 were obtained under the
same reaction conditions and in good yields, separated from the excess of thiocytosine (1) by
simply washing the crude with acetone and, finally, purified by column chromatography.
The in vitro antibacterial and antifungal activity of the family of pyrimidine derived disul-
fides 2–8 was investigated against representative Gram-negative (Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853), Gram-positive (Bacillus cereus ATCC 11778, S. aureus
ATCC 29213 including three our clinical isolates MRSA: CI26428, CI25967, CI26348) and yeast
(Candida albicans ATCC 10231, Cryptococcus neoformans ATCC MYA-565) strains. Thiocyto-
sine glucosides 2 and 3 are part of a project in which we intended to prepare small glucosides to
be tested as antimicrobial and/or anticancer therapeutics.[16] However, they are inactive against
all the bacteria and fungi tested in this study, together with thiocytosine (1). In compounds 4–8

Journal of Sulfur Chemistry 321
Table 1. Percentage of hemolysis by compounds 4, 5,
and 8 (50 µg/mL).
Osmolarity (mOsm/Kg)
300
200
150
100
0
Control
0
0
0
0
0.3
0.1
0.2
0.2
34
22
36
31
100
98
99
100
100
100
100
Compound 4
Compound 5
Compound 8
98
(Figure 1) pyrimidine residues are connected, by disulfide bonds, to phenyl or benzylic skele-
tons. The monosubstituted benzene derivative 4 and the elongated benzenedimethanedisulfide 5
were synthesized taking into account the previously obtained results with similarly shaped bio-
logically active molecules.[7,8] In vitro susceptibility data showed that E. coli, P. aeruginosa,
and B. cereus strains grew well in all tubes containing these compounds and, therefore, were
considered to be resistant. Only S. aureus isolates showed inhibition of the growth. A total inhi-
bition of the four examined S. aureus strains was observed at a concentration of 50 µg/mL using
compound 5, whereas minimal inhibitory concentration (MIC) values obtained for compound 4
were lower (25 µg/mL). Molecules possessing a C3 symmetry, such as the multi-armed benzene
derivatives 6–8 (Figure 1), have been tested as potential antibacterials on the basis of literature
data.[17] Noteworthy, whereas molecules 6 and 7 were inactive against all the bacteria studied,
disulfide 8, a hexafunctionalized 1,3,5(R1)3–2,4,6-(R2)3-benzene (R1 = ethyl) that adopts the
most thermodynamically stable tripodal conformation,[12] with an alternate geometrical orien-
tation of the ring substituents above and below the benzene plane, exhibited inhibitory activity
against Gram-positive S. aureus. MIC values were comparable with those of compound 5. In
addition, no antifungal effect was observed for the two pathogenic fungi included in this study
indicating that compounds 5, 8 and, in particular, 4 can be opportunely modified to enhance a
species-specific inhibitory activity of these pyrimidine derivatives against the important human
pathogen S. aureus. Finally, the potential toxicity of the biologically active compounds 4, 5, and 8
against human erythrocytes was evaluated (Table 1). The trypan blue assay showed a viability of
92–100% in all experiments (data not shown). Hemolytic activity was determined by hemoglobin
release in the plasma after exposure to the molecules. No cytotoxic activity was observed. There
were no statistical differences in the viability at different concentrations of the molecules.
3. Conclusions
In conclusion, we have described the synthesis of a small family of pyrimidine derivatives, disul-
fides 2–8, with chemical moieties in common but distinct structural and spatial characteristics.
Three of these compounds, disulfides 4, 5, and 8, that reveal no cytotoxicity activity against
human erythrocytes, are promising candidates for the development of new antibacterial agents.
4. Experimental
Solvents were purified according to standard procedures. All reactions were monitored by thin
layer chromatography (TLC) on commercially available precoated plates (Aldrich silica gel 60
F254) and the products were visualized with vanillin [1 g dissolved in MeOH (60 mL) and conc.
H2SO4 (0.6 mL)]. Silica gel used for column chromatography was Aldrich 60. 1H and 13C NMR

322 P. Bonaccorsi et al.
spectra were recorded with a Varian 500 spectrometer at 500 and 125 MHz, respectively; J are
given in Hz; the attributions are supported by Heteronuclear Single Quantum Coherence and
correlation spectroscopy experiments. Proton and carbon nuclei identified by apex’ pertain to
thiocytosine residues (for numbering see Figure 1).
1-(4-Amino-2-pyrimidinyldithio)-1-deoxy-β-d-glucopyranose (3)
To a solution of disulfide 2 [11] (60 mg, 0.12 mmol) in MeOH/THF (16 mL, 5:5) aqueous
ammonia (30%, 5 mL) was added under stirring, and stirring was maintained for 16 h, at r.t. The
reaction was monitored by TLC (EtOAc/MeOH 9.5:0.5) and 1H NMR. The solvent was evapo-
rated and the crude was washed with MeOH (3 × 5 mL), to remove acetamide. Yield 92%. TLC:
1
Rf 0.20 (EtOAc/MeOH 9.5:0.5). White solid. M.p. 198–202 °C. H NMR (DMSO-d6): δ 7.96
(d, 1H, J5 ,6 5.9, H-6ꢀ), 7.36 (br s, 2H, NH2), 6.29 (d, 1H, H-5ꢀ), 6.03 (d, 1H, Jvic 3.4, 2-OH),
5.04 (d, 1H, Jvic 4.9, 4-OH), 4.99 (d, 1H, Jvic 5.4, 3-OH), 4.56 (t, 1H, Jvic 5.6, 6-OH), 4.41
(d, 1H, J1,2 8.8, H-1), 3.66 and 3.40 (split AB system, 2H, H2-6), 3.29–3.12 (m, 2H, H-3,5),
3.12 (m, 1H, H-2), 3.02 (m, 1H, H-4). 13C NMR (DMSO-d6): δ 168.9 (C-2ꢀ), 163.2 (C-4ꢀ),
154.7 (C-6ꢀ), 102.9 (C-5ꢀ), 86.9 (C-1), 81.7 and 76.3 (C-3,5), 70.8 (C-2), 69.8 (C-4), 61.0 (C-6).
Anal. Calcd for C10H15N3O5S2 (321.37): C, 37.37; H, 4.70; N, 13.08. Found: C, 37.24; H, 4.71;
N, 13.12.
ꢀ
ꢀ
2-(Phenyldithio)-4-pyrimidinamine (4)
3-(phenylsulfinyl)propanenitrile [18] (200 mg, 1.12 mmol) was dissolved in 1,4-dioxane (6 mL)
and thiocytosine (1) (426 mg, 3.35 mmol) was added, forming a suspension that was heated up
to the reflux (101 °C) and maintained for 2 h at this temperature. During this time, the reaction
was monitored via TLC (DCM/EtOAc 8:2). The excess of thiocytosine (1) was filtered. The solid
residue was washed with acetone (3 × 5 mL) and the solution was recovered. The solvent was
removed and the crude was purified by column chromatography (DCM/EtOAc 9.5:0.5). Yield
80%. TLC: Rf 0.65 (DCM/EtOAc 8:2). White solid. M.p. 133–135 °C. 1H NMR (acetone-d6): δ
7.99 (d, 1H, J5 ,6 5.4, H-6ꢀ), 7.58 (d, 2H, Jortho 7.3, H-2,6), 7.33 (t, 2H, Jortho 7.3, H-3,5), 7.26
(t, 1H, H-4), 6.51 (br s, 2H, NH2), 6.37 (d, 1H, H-5ꢀ). 13C NMR (acetone-d6): δ 170.0 (C-2ꢀ),
165.6 (C-4ꢀ), 157.4 (C-6ꢀ), 139.0 (C-1), 130.4, 130.0, and 128.8 (C-2–6), 104.0 (C-5ꢀ). Anal.
Calcd for C10H9N3S2 (235.33): C, 51.04; H, 3.85; N, 17.86. Found: C, 50.91; H, 3.84; N,
17.90.
ꢀ
ꢀ
2,2ꢀ-[1,4-Phenylenebis(methylenedithio)]bis-4-pyrimidinamine (5)
A solution of dimethyl 3,3ꢀ-[1,4-phenylenebis(methylenesulfinyl)]bispropanoate [8] (12)
(150 mg, 0.40 mmol) and thiocytosine (1) (212.3 mg, 1.67 mmol) in 3 mL of 1,4-dioxane
was maintained under stirring, at reflux temperature (101 °C) for 3 h and 40 min. The reac-
tion was monitored via TLC (DCM/EtOAc 7.5:2.5) and 1H NMR. The excess of thiocy-
tosine (1) was filtered. The solid residue was washed with acetone (3 × 5 mL) and the
solution was recovered. The solvent was removed and the crude was purified by col-
umn chromatography (acetone/hexane 4:6). Yield 88%. TLC: Rf 0.30 (acetone/hexane 8:2).
White solid. M.p. 85–90 °C. 1H NMR (acetone-d6): δ 8.01 (d, 2H, J5 ,6 5.9, 2 × H-
6ꢀ), 7.32 (s, 4H, benzeneH), 6.46 (br s, 4H, 2 × NH2), 6.36 (d, 2H, 2 × H-5ꢀ), 4.12
(s, 4H, 2 × CH2). 13C NMR (acetone-d6): δ 171.1 (2 × C-2ꢀ), 165.4 (2 × C-4ꢀ), 157.3
(2 × C-6ꢀ), 137.7 (C-1,4), 131.1 (C-2,3,5,6), 103.7 (2 × C-5ꢀ), 43.5 (2 × CH2). Anal. Calcd
for C16H16N6S4 (420.60): C, 45.69; H, 3.83; N, 19.98. Found: C, 45.56; H, 3.83; N,
19.95.
ꢀ
ꢀ

Journal of Sulfur Chemistry 323
2,2ꢀ,2ꢀꢀ-[1,3,5-Benzenetriyltris(methylenedithio)]tris-4-pyrimidinamine (6)
To a solution of trimethyl 3,3ꢀ,3ꢀꢀ-[1,3,5-benzenetriyltris(methylenesulfinyl)]trispropanoate [8]
(65 mg, 0.12 mmol) in 1,4-dioxane (3 mL) thiocytosine (1) was added (146 mg, 1.15 mmol). The
suspension was refluxed (101 °C) under stirring for 4 h and monitored by TLC (acetone/hexane
3:7). The excess of thiocytosine (1) was filtered. The solid residue was washed with acetone
(3 × 5 mL) and the solution was recovered. The solvent was removed and the crude was purified
by column chromatography (acetone/hexane 4:6).Yield 60%. TLC: Rf 0.32 (acetone/hexane 6:4).
1
White solid. M.p. 95–100 °C. H NMR (acetone-d6): δ 8.01 (d, 3H, J5 ,6 5.9, 3 × H-6ꢀ), 7.31
ꢀ
ꢀ
(s, 3H, benzeneH), 6.47 (br s, 6H, 3 × NH2), 6.34 (d, 3H, 3 × H-5ꢀ), 4.11 (s, 6H, 3 × CH2). 13
C
NMR (acetone-d6): δ 171.0 (3 × C-2ꢀ), 165.5 (3 × C-4ꢀ), 157.3 (3 × C-6ꢀ), 139.0 (C-1,3,5),
131.1 (C-2,4,6), 103.7 (3 × C-5ꢀ), 44.0 (3 × CH2). Anal. Calcd for C21H21N9S6 (591.84): C,
42.62; H, 3.58; N, 21.30. Found: C, 42.51; H, 3.57; N, 21.36.
2,2ꢀ,2ꢀꢀ-[(2,4,6-Trimethyl-1,3,5-benzenetriyl)tris(methylenedithio)]tris-4-pyrimidinamine (7)
To a solution of trimethyl 3,3ꢀ,3ꢀꢀ-[(2,4,6-trimethyl-1,3,5-benzenetriyl)tris(methylenesulfinyl)]
trispropanoate [8] (100 mg, 0.18 mmol) in 1,4-dioxane (3.6 mL) thiocytosine (1) was added
(203 mg, 1.60 mmol). The suspension was refluxed (101 °C) under stirring for 3 h and moni-
tored by TLC (EtOAc/MeOH 9.5:0.5). The excess of thiocytosine (1) was filtered. The solid
residue was washed with acetone (3 × 5 mL) and the solution was recovered. The solvent was
removed and the crude was purified by column chromatography (acetone/hexane 4:6). Yield
63%. TLC: Rf 0.42 (acetone/hexane 6:4). White solid. M.p. 208–212 °C. 1H NMR (acetone-d6):
δ 8.08 (d, 3H, J5 ,6 5.9, 3 × H-6ꢀ), 6.41 (br s, 6H, 3 × NH2), 6.39 (d, 3H, 3 × H-5ꢀ), 4.30 (s, 6H,
3 × CH2), 2.74 (s, 9H, 3 × CH3). 13C NMR (acetone-d6): δ 171.3 (3 × C-2ꢀ), 165.5 (3 × C-
4ꢀ), 157.4 (3 × C-6ꢀ), 139.5 (C-1,3,5), 132.7 (C-2,4,6), 103.7 (3 × C-5ꢀ), 40.8 (3 × CH2), 17.3
(3 × CH3). Anal. Calcd for C24H27N9S6 (633.92): C, 45.47; H, 4.29; N, 19.89. Found: C, 45.39;
H, 4.30; N, 19.91.
ꢀ
ꢀ
Antimicrobial activity test
Antimicrobial activity of compounds 2–8 was assessed against representative Gram-negative
(E. coli ATCC 25922, P. aeruginosa ATCC 27853), Gram-positive (B. cereus ATCC 11778,
S. aureus ATCC 29213 including three of our clinical isolates MRSA: CI26428, CI25967,
CI26348) and yeast (C. albicans ATCC 10231, C. neoformans ATCC MYA-565) strains. Bacte-
rial MIC of all compounds was determined in vitro using the broth dilution method in Mueller
Hinton medium as described in Clinical and Laboratory Standard Institute (CLSI) document
M07-A9.[19] Briefly, test compounds were dissolved in dimethyl sulfoxide and serial double
dilutions with Mueller Hinton broth were performed to obtain concentrations varying from 50
to 0.39 µg in a final volume of 1 mL. Each tested strain was previously grown in tryptic soy
agar for 24 h at 35 °C and bacterial suspensions were prepared to the turbidity of 0.5 McFar-
land (1.5 × 108 CFU/mL) with sterile saline solution. Suspensions were subsequently diluted to
provide a final inoculum density of 5 × 105 CFU/mL in the tube. All inoculated tubes, includ-
ing a control without any inhibitory substance, were incubated at 35 2 °C for 24 h. For yeast
strains antifungal activity was tested according to National Committee on Clinical Laboratory
Standards (NCCLS) reference document M27-A2.[20] The serial doubling dilutions of all com-
pounds were prepared in the RPMI-1640 medium supplemented with 2% of glucose and buffered
to pH 7.0 with 0.165 M 4-morpholinepropanesulfonic acid in concentrations ranging from 50 to
0.39 µg/mL. Compound-free and yeast-free controls were also included. Cultures (24 h-old) of

324 P. Bonaccorsi et al.
yeast strains in Sabouraud dextrose broth were used to prepare a 0.5 McFarland standard tur-
bidity (approximately 5 × 106 CFU/mL) and 5 µl was used to inoculate each tube containing 1
mL of RPMI-1640 medium and a specific compound. The tubes were incubated aerobically at
30 °C for 48–72 h and MICs were determined. Bacterial and fungal MICs were calculated by
two independent observers as the lowest compound concentration at which microbial growth
was inhibited. All experiments were performed in triplicate.
Cytotoxicity Assay. Fresh human blood (Hb) was collected from healthy adult donors. After
extraction, the whole blood was taken into anticoagulant ethylenediaminetetraacetic acid, were
incubated in a saline solution [composition in mM: 125 NaCl, 5 KCl, 1 MgSO4, 32 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 D-( + )-glucose, 1 CaCl2; pH 7.4 and
π 300 mOsm/kg] [21] Erythrocytes were resuspended in saline solution with compounds 4, 5,
and 8 at concentrations of 50 µg/mL.
Trypan blue assay
Cell viability by the trypan blue exclusion method was performed. The percentage of unstained
cells represented the percentage of viable cells in the suspension. This assay was performed in
different cell types and cytotoxicity based on alterations in plasma membrane permeability was
measured.
Cell viab. (%) = number of viable cells (unstained cells) × 100/total number of cells (stained
and unstained).
Hemolysis test
Hb samples were incubated with compounds and, after incubation at 37 °C overnight, the samples
were centrifuged and the supernatants were harvested. As a measure of hemolysis, Hb concen-
tration of the supernatants was determined photometrically (λ = 540 nm). The absorption of the
supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis. In the treat-
ment of blood with each of compounds 4, 5, and 8, hemolysis did not occur at 300 and 200
mOsm/kg, while it became clearly visible at 100 mOsm/kg to accomplish itself at 0 mOsm/kg,
as in the control conditions (Table 1). The maintenance of integrity of RBC membrane up to
150 mOsm/kg and the hemolysis occurrence at identical concentrations of the control conditions
suggest that none of the compounds under study showed toxicity effects in human erythrocytes
at the concentration tested.
Statistic analysis
The experiments were performed in triplicates and statistical analyses performed using Student’s
t-test, with a confidence level of 95% (p < .05) considered statistically significant.
Acknowledgements
Authors thank prof. Maria Chiara Aversa for useful discussion.
Disclosure statement
No potential conflict of interest was reported by the authors.

Journal of Sulfur Chemistry 325
Funding
Financial support by the Italian Ministry for the University and Research (MIUR) within PON01_01499 and PRIN
20109Z2XRJ_010 is gratefully acknowledged.
References
[1] Then RL. History and future of antimicrobial diaminopyrimidines. J. Chemother. 1993;5:361–368.
[2] Lee C-S, Ryu E-J, Oh SH, Paek K-S, Kim MY, Youn H. Synthesis and antibacterial activities of novel C(3)-
aminopyrimidinyl substituted cephalosporins including against respiratory tract pathogens. Bioorg Med Chem Lett.
2000;10:2123–2127. doi:10.1016/S0960-894X(00)00425-X
[3] Lee KS, Lim JH, Kang YK, Yoo KH, Kim DC, Shin KJ, Kim DJ. Synthesis and antibacterial activities of
new 1β-methylcarbapenems having aminopyrimidinylthioether moiety. Eur J Med Chem. 2006;41:1347–1351.
doi:10.1016/j.ejmech.2006.08.003
[4] Semenov VE, Mikhailov AS, Voloshina AD, Kulik NV, Nikitashina AD, Zobov VV, Kharlamov SV, Latypov SK,
Reznik VS. Antimicrobial activity of pyrimidinophanes with thiocytosine and uracil moieties. Eur J Med Chem.
2011;46:4715–4724. doi:10.1016/j.ejmech.2011.05.034
[5] Zhao Y, Tian Y, Cui Y, Liu W, Ma W, Jiang X. Small molecule-capped gold nanoparticles as potent antibacterial
agents that target gram-negative bacteria. J Am Chem Soc. 2010;132:12349–12356. doi:10.1021/ja1028843
[6] Palavecino EL. Clinical, epidemiologic, and laboratory aspects of methicillin-resistant Staphylococcus aureus
infections. Methods Mol Biol. 2014;1085:1–24. doi:10.1007/978-1-62703-664-1_1
[7] Aversa MC, Barattucci A, Bonaccorsi P, Marino-Merlo F, Mastino A, Sciortino MT. Synthesis and biological testing
of thioalkane- and thioarene-spaced bis-β-D-glucopyranosides. Bioorg Med Chem. 2009;17:1456–1463.
[8] Bonaccorsi P, Marino-Merlo F, Barattucci A, Battaglia G, Papaianni E, Papalia T, Aversa MC, Mastino A. Synthesis
and biological evaluation of a new class of glycoconjugated disulfides that exhibit potential anticancer properties.
Bioorg Med Chem. 2012;20:3186–3195.
[9] Barattucci A, Deni E, Bonaccorsi P, Ceraolo MG, Papalia T, Santoro A, Sciortino MT, Puntoriero F.
Oligo(phenylene ethynylene) Glucosides: modulation of cellular uptake capacity preserving light ON. J Org Chem.
2014;79:5113–5120.
[10] Papalia T, Siracusano G, Colao I, Barattucci A, Aversa MC, Serroni S, Zappalà G, Campagna S, Sciortino MT,
Puntoriero F, Bonaccorsi P. Cell internalization of BODIPY-based fluorescent dyes bearing carbohydrate residues.
Dyes Pigments. 2014;110:67–71.
[11] Aversa MC, Barattucci A, Bonaccorsi P. Efficient synthesis of unsymmetrical disulfides through sulfenic acids. Eur
J Org Chem. 2009;2009(36):6355–6359. doi:10.1002/ejoc.200900986
[12] Aversa MC, Barattucci A, Bonaccorsi P. From transient sulfenic acids to disulfide-functionalized tripodal structures.
Synlett. 2011;2011(2):254–258. doi:10.1055/s-0030-1259306
[13] Bonaccorsi P, Aversa MC, Barattucci A, Papalia T, Torre A, Trischitta F, Faggio C. Sulfenic acid - derived
glycoconjugated disulfides and sulfoxides: a biological evaluation on human red blood cells. J Sulfur Chem.
2013;34:684–691.
[14] Vacca A, Nativi C, Cacciarini M, Pergoli R, Roelens S. A new tripodal receptor for molecular recognition of
monosaccharides. A paradigm for assessing glycoside binding affinities and selectivities by 1H NMR spectroscopy.
J Am Chem Soc. 2004;126:16456–16465.
[15] Barattucci A, Di Gioia ML, Leggio A, Minuti L, Papalia T, Siciliano C, Temperini A, Bonaccorsi P. Stereoselective
synthesis of Dithia[3.3]cyclophane S,Sꢀ-dioxides with planar and central chirality. Eur J Org Chem. 2014;2099–
2104. doi:10.1002/ejoc.201301636
[16] Calvaresi EC, Hergenrother PJ. Glucose conjugation for the specific targeting and treatment of cancer. Chem Sci.
2013;4:2319–2333. doi:10.1039/c3sc22205e
[17] Hayes W, Osborn HMI, Osborne SD, Rastall RA, Romagnoli B. One-pot synthesis of multivalent arrays of mannose
mono- and disaccharides. Tetrahedron. 2003;59:7983–7996.
[18] Adams H, Anderson JC, Bell R, Jones DN, Peel MR, Tomkinson NCO. The synthesis and Diels-Alder reactions of
(E)- and (Z)-1-methoxy-3-(phenylsulfinyl)-1,3-butadienes. J Chem Soc Perkin Trans. 1998;1(23):3967–3974.
[19] Clinical and Laboratory Standards Institute. M07-A9. Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically; approved standard. 9th ed. Wayne, PA: CLSI; 2012.
[20] NCCLS - National Committee for Clinical Laboratory Standards. Reference Method For Broth Dilution Antifungal
Susceptibility Testing of Yeasts; Approved standard. 2nd ed. Wayne, PA: NCCLS document M27-A2; 2002.
[21] Mineo P, Faggio C, Micali N, Scamporrino E, Villari V. A star polymer based on a polyethylene glycol with
a porphyrinic core as a photosensitizing agent for application in photodynamic therapy: tests in vitro on human
erythrocytes. R Soc Chem Adv. 2014;4:19389–19395.