The antibacterial properties of sulfur containing flavonoids

Article abstract of DOI:10.1016/j.bmcl.2014.03.071

Some dithiocarbamic esters bearing a flavanone backbone, as well as their corresponding 1,3-dithiolium salts were tested against Staphylococcus aureus and Escherichia coli. The 1,3-dithiolium tricyclic flavonoids display good inhibitory properties against both Gram-positive and Gram-negative pathogens.

Full text of DOI:10.1016/j.bmcl.2014.03.071

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2315–2318
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The antibacterial properties of sulfur containing flavonoids
b,
Lucian G. Bahrin a, Mircea O. Apostu a, Lucian M. Birsa a, , Marius Stefan
⇑
⇑
a Department of Chemistry, Al.I. Cuza University of Iasi, 11 Carol I, 700506 Iasi, Romania
b Department of Biology, Al.I. Cuza University of Iasi, 11 Carol I, 700506 Iasi, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Some dithiocarbamic esters bearing a flavanone backbone, as well as their corresponding 1,3-dithiolium
salts were tested against Staphylococcus aureus and Escherichia coli. The 1,3-dithiolium tricyclic flavonoids
display good inhibitory properties against both Gram-positive and Gram-negative pathogens.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 7 February 2014
Revised 20 March 2014
Accepted 24 March 2014
Available online 2 April 2014
Keywords:
Flavonoids
Dithiolium salts
Dithiocarbamates
Antibacterial activity
Flavonoids represent a wide class of polyphenolic compounds
that play the role of secondary metabolites in the plant kingdom.
Over 9000 flavonoids have been identified,1 with more being dis-
covered or synthesized each year. The attention that they receive
is a direct consequence of the many biological activities that this
class of compounds displays. Flavonoids can potentially be used
as antimicrobial, antiviral, antifungal and antihelmintic agents.2–5
They are known anti-oxidants and can be used to regulate choles-
terol levels,6,7 or as cardioprotective agents.8,9 Some flavonoids dis-
play anti-cancer properties.10,11
Drug-resistant microbes represent a major cause of concern for
our modern day society,12 therefore it is imperative that new anti-
biotics be discovered which can be used against these pathogens.
The antimicrobial properties that some flavonoids display could
be exploited for this purpose. In principle, flavonoids can act
directly against the infectious microorganisms, they can be used
in combination with other antibiotics (synergistic relationship),
or can act against bacterial virulence factors, like cell-binding abil-
ity or toxins released by the pathogens. Many flavonoids, like quer-
cetin and naringenin,13 apigenin,14 or epigallocathechin gallate,15
to name but a few, are known to possess antibacterial activity.
More than that, epigallocathechin gallate was also shown to
enhance the activity of other antibiotics against drug-resistant
pathogens.16 Some flavonols were found to interfere with the path-
ogen’s binding ability to the host-cells,17 while genistein was
shown to neutralize toxins released by Vibrio vulnificus.18
This Letter presents the inhibitory activity of some previously
synthesized, as well as some new flavonoids, against Staphylococ-
cus aureus ATCC 25923 (Gram-positive) and Escherichia coli ATCC
25922 (Gram-negative). Compounds 3a–e display a flavanone
backbone with a dithiocarbamic moiety at the C(3) position.
Compounds 4a–c and 5a–e are tricyclic flavonoids containing a
1,3-dithiolium ring and are derived from flavanones 3a–e. The precur-
sors for the latter compounds, phenacyl carbodithioates 1a–d, have
been synthesized from the reaction of sodium N,N-diethyldithio-
carbamate with 2-bromo-1-(5-bromo-2-hydroxyphenyl)ethan-1-
one,19 2-bromo-1-(2-hydroxy-3,5-diiodophenyl)ethan-1-one,20,21
2-bromo-1-(2-hydroxy-3,5-dibromophenyl)ethan-1-one19 and
2-bromo-1-(2-hydroxyphenyl)ethan-1-one,19 respectively. Their
general structure, as well as the synthetic pathway, is presented
in Scheme 1.
Flavanones 3a and 3c, as well as their corresponding 1,3-dithio-
lium salts, 4a and 4c, have been reported by us in a previous Let-
ter.22 Compounds 3b, 3d, 3e, 4b have been obtained using a
similar protocol.22,23 Thus, flavanones 3b, 3d and 3e have been syn-
thesized by reacting the corresponding dithiocarbamic esters with
aminals 2a and 2b, in refluxing ethanol for 2 h, with a 54%, 62% and
55% yield, respectively. The formation of the chroman-4-one ring is
supported by the following series of spectral changes: the 1H NMR
spectrum shows the disappearance of the methylene doublet at
around 4.90 ppm and the appearance of two new doublets
between 5.80 ppm and 6.30 ppm—the H(2) and H(3) protons—as
well as the appearance of a new singlet at 3.80 ppm, corresponding
to the methoxy group hydrogen atoms, in the case of flavanone 3b.
The reaction leads to a mixture of syn and anti isomers, as the
H(2) and H(3) atoms are on the same side of the chroman-4-one
⇑
Corresponding authors. Tel.: +40 746798750 (L.M.B.).
E-mail addresses: lbirsa@uaic.ro (L.M. Birsa), stefanm@uaic.ro (M. Stefan).
http://dx.doi.org/10.1016/j.bmcl.2014.03.071
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2316
L. G. Bahrin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2315–2318
O
O
N
X
S
S
N
S
N
N
N
O
O
O
S
R2
S
R2
S
R2
ii, iii
R3
i
+
O
OH
R1
R1
R1
R3
R3
1
2
3
4, 5
4a, 5a: R1=H, R2=Br, R3=Cl
1a: R1=H, R2=Br
2a: R3=Cl
3a: R1=H, R2=Br, R3=Cl
1b: R1=I, R2=I
2b: R3=OCH3
3b: R1=H, R2=Br, R3=OCH3 4b, 5b: R1=H, R2=Br, R3=OCH3
1
2
1
2
3
1
2
3
1c
1d
3c:
3d
4c, 5c
: R =Br R =Br
R =I, R =I, R =Cl
: R =I, R =I, R =Cl
1
2
1
2
3
1
2
3
5d
: R =H, R =H
; R =Br R =Br, R =Cl
: R =Br R =Br, R =Cl
1
2
3
1
2
3
3e
; R =H, R =H, R =Cl
5e
; R =H, R =H, R =Cl
Scheme 1. Synthetic method for obtaining compounds 3a–e, 4a–c and 5a–e. Reaction conditions as follows: (i) EtOH, reflux 2 h; (ii) H2SO4/AcOH = 1/3 (v/v), 80 °C 10 min;
(iii) HClO4 70% for 4a–c, X = ClO4 or aqueous KBF4 for 5a–e, X = BF4.
ring, or on opposite sides of it (Fig. 1). The anti/syn ratio was deter-
mined from the 1H NMR spectra to be 70:30 for flavanones 3b and
3e and 46:54 for 3d. The structure of flavanone anti-3b was unam-
biguously proved by X-ray analysis (see Supplementary data).24
Because aminal 2b is obtained by reacting anisaldehyde with
two equivalents of morpholine,25 we decided to investigate the
possibility of a one-pot synthesis of flavanone 3b, by reacting dith-
iocarbamic ester 1a with one equivalent of anysaldehyde and two
equivalents of morpholine. The reaction mixture was refluxed in
ethanol for two hours and monitored using TLC. Upon workup, fla-
vanone 3b was isolated with a 40% yield. Encouraged by this result,
we decided to attempt a one-pot synthesis for flavanone 3c. Under
the same conditions, compound 3c was isolated with an 84% yield,
which is comparable to the previously reported value22 of 87%,
when aminal 2a was used. The reaction between dithiocarbamic
esters of type 1 and aminals of type 2 lead to the closure of a chro-
man-4-one ring and the elimination of the amine that is built-in
the molecule of the aminal. Because of the fact that all the amine
that is used for the formation of the aminal is later released into
the reaction medium, we decided to go one step further and try
a one-pot synthesis of flavanone 3c using a catalytic amount of
morpholine. Thus, by reducing the amount of amine from two
equivalents to 0.1 equiv, after two hours in refluxing ethanol, flava-
none 3c was isolated with an 80% yield. This modification of the
original procedure23 offers some interesting possibilities that will
require further investigations.
Tricyclic flavonoids 4a–c have been obtained via acid catalyzed
cyclization of flavanones 3a–c. The reaction is performed in a mix-
ture of sulfuric acid/acetic acid which acts at the same time as a
proton donor, as well as a dehydrating agent. After ten minutes
at 80 °C, perchloric acid is added, which leads to the desired prod-
uct. The cyclization step is accompanied by important spectral
changes. The 1H NMR spectrum shows the disappearance of the
two doublets between 5.80 ppm and 6.30 ppm and the appearance
of a new singlet at around 6.70–7.00 ppm belonging to the H(2)
proton of the former chroman-4-one ring. The 13C NMR spectrum
displays the disappearance of the carbonyl and thiocarbonyl sig-
nals and the appearance of a new signal at around 185 ppm, attrib-
uted to the positive C(2) atom of the 1,3-dithiolium ring. The IR
spectrum indicates the disappearance of the carbonyl signal at
1693 cmÀ1 and the appearance of a new one at 1081 cmÀ1 belong-
ing to the perchlorate anion.
The new flavonoids were tested against S. aureus and E. coli with
gentamicin as reference, using a disk diffusion assay. The samples
exhibiting antibacterial effect were further tested to determine
minimum inhibitory concentration (MIC) using a colorimetric
micro-dilution technique.
The growth inhibition zones measured by the disk diffusion
method are presented in Table 1. No antibacterial activity was
observed for flavanones 3a–e. In contrast, the tricyclic flavonoids
4a–c presented good antibacterial activity against both tested
microorganisms, depending on their concentration, with inhibition
zones ranging from 6.66 to 11.66 mm for E. coli and from 7.66 to
20.33 mm for S. aureus. The highest antimicrobial activity was
observed for 4a at a concentration of 1 mg/ml in the case of
S. aureus and the lowest (0.25 mg/ml) for 4b in the case of
E. coli. Compared with gentamycin, 4a showed an improvement
of antibacterial activity (Table 1). There was no inhibition of
growth observed when DMSO/H2O was used as control (see
Supplementary data).
The bacteriostatic concentrations of tricyclic flavonoids 4a–c
are provided in Table 2. All tested flavonoids were highly active
against S. aureus, the recorded MIC values being lower than
3.9
antibacterial activity against E. coli, generally less than 7.81
ml with the exception of 4b (125 g/ml). The lowest MIC was
recorded in the case of 4a for both Gram positive (0.48 g/ml)
and Gram negative (3.9 g/ml) tested bacteria.
l
g/ml. Also, the tricyclic flavonoids 4a–c displayed a potent
l
g/
l
l
l
Although antimicrobial properties of natural occurring or syn-
thetic flavonoids were previously reported,26–28 the smaller MIC
values of here reported flavonoids, as compared with the literature
data, make them very interesting compounds.29
Several perchlorate containing compounds have been previ-
ously reported to exhibit antimicrobial activity.30 Moreover, it is
well known that the perchlorate anion is the weakest oxidant
among the four chlorates.31 In order to clarify the impact of per-
chlorate anions on the antimicrobial activity of 1,3-dithiolium
derivatives 4a–c, we decided to investigate the behaviour of a ser-
ies of 1,3-dithiolium tetrafluoroborates, compounds 5a–e, against
the same bacteria. The synthesis of tetrafluoroborates 5a–e has
been accomplished by the addition, after the cyclization step, of
an aqueous solution of potassium tetrafluoroborate instead of per-
chloric acid. Using the same experimental procedures, tetrafluoro-
borates 5a–e have been found to exhibit even better antibacterial
properties than perchlorates 4a–c (Tables 1 and 2). Again, a higher
activity against Gram positive bacteria has been recorded. These
results bring support to the fact that the tricyclic flavonoid sub-
structure is responsible for the antibacterial activity. The slight
enhancement of antibacterial properties obtained by replacing
the perchlorate with a tetrafluoroborate anion is also supported
S
H
N
S
H
N
O
O
O
O
Br
S
Br
S
H
H
OCH3
OCH3
anti-3b
syn-3b
Figure 1. The two stereoisomers of flavanone 3b.

L. G. Bahrin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2315–2318
2317
Table 1
Antimicrobial activity of sulfur containing flavonoids against Staphylococcus aureus and Escherichia coli using disk diffusion assaya
Sample
Diameter of inhibition zone (mm)b
Bacterial strain
S. aureus
E. coli
Concentration (mg/ml)
0.25
0.5
1
0.25
—
0,5
—
1
c
3a
4a
3b
4b
3c
4c
3d
5d
3e
5e
—
—
—
—
15
1
17.33 1,15
—
9.66 0.57
—
20.33 0.57
—
13.66 0.57
—
16.66 0.57
—
21.33 0.57
—
8.33 1.15
—
6.66 0.57
—
10.66 1.15
—
7.33 0.57
—
11.66 0.57
—
7.66 0.57
—
9.33 0.57
—
10.66 0.57
—
—
—
7.66 0.57
—
8.33 0.57
14
1
1
1
7.66 0.57
8.33 0.57
—
—
—
—
—
—
—
—
—
—
17.66 1.15
—
7.33 0.57
18.66
—
14.33
16.33
1
Gentamicin (10
l
g/disc)
18.33 0.57
15.75 1.7
a
b
c
The diameter of inhibition zone includes the disc (5 mm).
Values are means of inhibition zones (mm) S.D. of three replicates.
No antimicrobial activity detected.
Table 2
The Gram positive tested bacterium was more sensitive to tricy-
Minimum inhibitory concentrations of tricyclic flavonoids 4a–c (
lg/ml, w/v) against
clic flavonoids 4 and 5 compared to the Gram negative one. Most
likely, this is the result of the different cell-wall structures of these
microbes. Gram-positive bacteria have a significantly simple cell
wall structure that is reasonably porous and thus foreign mole-
cules can easily traverse this outer layer. In contrast, Gram-nega-
tive bacteria have considerably complicated cell-wall structures
with a less porous outer layer.37 Moreover, it is well known that
the cell wall of Gram positive bacteria contains a considerable
amount of peptidoglycan as compared with that of Gram negative
bacteria.38 The susceptibility to nucleophilic attack of the C(2)
atom in the 1,3-dithiolium ring, correlated to the large number
of nucleophilic sites in peptidoglycans suggests that the higher
activity of the tricyclic flavonoids against S. aureus is related to a
Maxam–Gilbert type mechanism. Similar antimicrobial activity
has been observed when some of the above reported tricyclic flavo-
noids (5d and 5e) were used on Candida albicans ATCC 90028—an
eukaryotic microorganism (data not shown here). Being aware
about the difference between the prokaryotic and eukaryotic spe-
cies and taking into account that from a chemical point of view
the composition of the cell wall of C. albicans mainly consists of
glucans and chitin,39 polysaccharides with many nucleophilic sub-
stituents as in the case of peptidoglycan, these results bring sup-
plementary support to the proposed mechanism.
Different antibacterial activities were recorded for tricyclic
flavonoids 4 and 5. The highest antibacterial sensitivity was evi-
denced for 4–5a and the lowest one for 4b. The replacement of
the chlorine substituent at the para-position of the benzenic ring
in 4a with a methoxy group in 4b resulted in a notable decrease
of antibacterial activity against E. coli (32-fold). This might be the
result of the ability of the oxygen atom in the methoxy group to
act as an acceptor unit in hydrogen bonding; thus, the transport
of this flavonoid through the outer cell membrane of Gram nega-
tive bacteria might be considerably slower. An 8-fold decrease of
antibacterial effect against S. aureus was also observed.
Staphylococcus aureus and Escherichia coli
Sample
Bacterial strain
MIC (
g/ml)a
l
S. aureus
E. coli
4a
5a
4b
5b
4c
5c
5d
5e
M
0.48
0.48
3.9
1.95
1.95
0.97
1.95
1.95
250
3.9
3.9
125
125
7.81
15.62
7.81
62.5
250
a
MIC = minimum inhibitory concentration.
by literature data; the ion-dissociation or the formation of a tight
ionpair has been reported to affect the antimicrobial activities with
the following order chloride > tetrafluoroborate > perchlorate >
hexafluorophosphate.32
The ion-dissociation versus formation of a tight ionpair appears
to be of significant importance on how cationic tricyclic flavonoids
interact with bacteria. At the physiological pH, the outer cell walls
of all microbes become negatively charged. The major component
of bacterial cell wall is represented by negatively charged phospha-
tidylethanolamine (70%). Thus, the positively charged 1,3-dithioli-
um flavonoids target the oppositely charged biological structures
such as cell walls of microorganisms which leads to the leakage
of intracellular substances.33
As our results show, the very potent antibacterial activity of
flavonoids 4 and 5 compared with flavanones 3 is the consequence
of the appearance of the third fused cycle, the 1,3-dithiolium ring,
on the flavonoids 4 and 5 scaffold. 1,3-Dithiolium systems are well
known for the reactivity of the C(2)-position towards nucleo-
philes.34,35 Thus, the excellent antibacterial properties of tricyclic
flavonoids as compared with that of dithiocarbamic flavonoids 3,
can be the result of the interaction between nucleophilic moieties
of wall and/or membrane constituents (e.g., peptides, substituted
polysaccharides) or of purinic bases from bacterial DNA with the
electrophilic C(2) atom of the 1,3-dithiolium ring. The latter type
of interaction, the Maxam–Gilbert mechanism,36 has been pro-
posed to explain the antitumor activities of natural occurring
endiynes (e.g., Calicheamicin, Esperamicin, Dynemicin).
The substitution of the hydrogen atom at the C(8) position
(using chroman-4-one numbering) of 4–5a with an iodine atom
in 4–5c or a bromine atom in 5d lead to a decrease in antibacterial
sensitivity against both tested bacteria. A reasonable explanation
might be the increase in size of flavonoids 4–5c and 5d in compar-
ison with that of 4–5a, as the atomic radius of the bromine/iodine
atom is considerably bigger than that of the hydrogen atom; again,
the transport through cell membrane might be affected.

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L. G. Bahrin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2315–2318
10. Singh, R. P.; Gu, M.; Agarwal, R. Cancer Res. 2008, 68, 2043.
In conclusion, we have reported several flavonoid derivatives
11. Sung, B.; Pandey, M. K.; Aggarwal, B. B. Mol. Pharmacol. 2007, 71, 1703.
12. Gould, I. M. Int. J. Antimicrob. Agents 2008, 32, S2.
13. Rauha, J. P.; Remes, S.; Heinonen, M.; Hopia, A.; Kahkonen, M.; Kujala, T.;
Pihlaja, K.; Vuorela, H.; Vuorela, P. Int. J. Food Microbiol. 2000, 56, 3.
14. Sato, Y.; Suzaki, S.; Nishikawa, T.; Kihara, M.; Shibata, H.; Higuti, T. J.
Ethnopharmacol. 2000, 72, 483.
15. Ikigai, H.; Nakae, T.; Hara, Y.; Shimamura, T. Biochim. Biophys. Acta 1993, 1147,
132.
16. Zhao, W. H.; Hu, Z. Q.; Okubo, S.; Hara, Y.; Shimamura, T. Antimicrob. Agents
Chemother. 2001, 45, 1737.
which were evaluated for biological activity. A promising one-pot
synthetic procedure for the intermediate flavanones requires fur-
ther investigations. The presented tricyclic flavonoids have a good
potential for the design of new antimicrobial agents. Further stud-
ies are in progress in order to elucidate the antimicrobial activity
mechanisms, with emphasis on the effect on bacterial DNA.
Acknowledgments
17. Kang, S. S.; Kim, J. G.; Lee, T. H.; Oh, K. B. Biol. Pharm. Bull 2006, 29, 1751.
18. Oh, D. R.; Kim, J. R.; Kim, Y. R. Arch. Pharm. Res. 2010, 33, 787.
19. Buu-Hoi, Ng. Ph.; Lavit, D. J. Chem. Soc. 1955, 18.
L.M.B. is indebted to the Alexander von Humboldt Foundation
for a short stay in Braunschweig. Part of this work was supported
by a grant of the Romanian National Authority for Scientific
Research, CNDI–UEFISCDI, project number 51/2012.
20. Birsa, M. L. Synth. Commun. 2001, 31, 1271.
21. Birsa, M. L.; Asaftei, I. V. Monat. Chem. 2008, 139, 1433.
22. Bahrin, L. G.; Jones, P. G.; Hopf, H. Beilstein J. Org. Chem. 2012, 8, 1999.
23. Birsa, M. L. Synth. Commun. 2002, 32, 115.
24. CCDC-985190 contain the supplementary crystallographic data for flavanone
anti-3b. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
25. Sansone, M. F.; Koyanagi, T.; Przybyla, D. E.; Nagorski, R. W. Tetrahedron Lett.
2010, 51, 6031.
26. Alvarez, M. A.; Debattista, N. B.; Pappano, N. B. Folia Microbiol. 2008, 53, 23.
27. Mandalari, G.; Bennett, R. N.; Bisignano, G.; Trombetta, D.; Saija, A.; Faulds, C.
B.; Gasson, M. J.; Narbad, A. J. Appl. Microbiol. 2007, 103, 2056.
28. Takahashi, T.; Kokubo, R.; Sakaino, M. Lett. Appl. Microbiol. 2004, 39, 60.
29. Cushnie, T. P. T.; Lamb, A. J. Int. J. Antimicrob. Agents 2011, 38, 99.
30. Coates, J. D.; Achenbach, L. A. Nat. Rev. Microbiol. 2004, 2, 569.
31. Cotton, F. A.; Wilkinson, G. Advanced Inorganic Chemistry; Wiley-Interscience:
New York, 1988.
32. Kanazawa, A.; Ikeda, T.; Endo, T. J. Polym. Sci. A: Polym. Chem. 1993, 31, 467.
33. Lim, S. H.; Hudson, S. M. J. Macromol. Sci. Rev. 2003, 43, 223.
34. Prinzbach, H.; Futterer, E. Adv. Heterocycl. Chem. 1966, 7, 39.
35. Birsa, M. L. Synth. Commun. 2003, 33, 3071.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2014.03.071.
References and notes
1. Williams, C. A.; Grayer, R. J. Nat. Prod. Rep. 2004, 21, 539.
2. Wild, C.; Fasel, J. Am. J. Proct. 1969, 20, 60.
3. Jae, M. S.; Kwang, H. L.; Baik, L. S. Antivir. Res. 2005, 68, 66.
4. Sotres, C. M.; Albarrán, P. L.; Cruz-de-León, J.; Moreno, T. G.; Quiñones, J. G. R.;
Marrufo, G. V.; Mascarúa, J. T.; Bucio, R. H. Int. Biodeter. Biodegr. 2012, 69, 38.
5. Das, B.; Tandon, V.; Saha, N. Parasitology 2007, 134, 1457.
6. Chopra, M.; Fitzsimons, P. E.; Strain, J. J.; Thurnham, D. I.; Howard, A. N. Clin.
Chem. 2000, 46, 1162.
7. Kurowska, E. M.; Borradaile, N. M.; Spence, J. D.; Carrol, C. C. Nutr. Res. 2000, 20,
121.
8. Hertog, M. G. L.; Feskens, E. J. M.; Hollman, P. C. H.; Katan, M. B.; Kromhout, D.
Lancet 1993, 342, 1007.
36. Maxam, A. M.; Gilbert, W. Method Enzymol. 1980, 65, 499.
37. Ward, M.; Sanchez, M.; Elasri, M. O.; Lowe, A. B. J. Appl. Polym. Sci. 2006, 101,
1036.
38. Dunca, S.; Ailiesei, O.; Nimitan, E.; Stefan, M. Elements of Microbiology;
Junimea: Iasi, 2005, vol I.
39. Ruiz-Herrera, J.; Elorza, M. V.; Valentin, E.; Sentandreu, R. FEMS Yeast Res. 2006,
6, 14.
9. Hertog, M. G. L.; Kromhout, D.; Aravanis, C.; Blackburn, H.; Buzina, R.; Fidanza,
F.; Giampaoli, S.; Jansen, A.; Menotti, A.; Nedeljkovic, S. Arch. Intern. Med. 1995,
155, 381.