Syntheses, in vitro antibacterial and antifungal activities of a series of N-alkyl, 1,4-dithiines

Article abstract of DOI:10.1016/j.farmac.2005.06.015

A series of dithiines were synthesized by cyclization of 4-(alkylamino)-4-oxobutanoic acids under the action of SOCl₂. Their in vitro antibacterial and antifungal activities have been evaluated against reference strains and versus reference compounds. The so-called 'isoimides' 2a, 2b were totally inactive whereas some imides had low MICs for few bacteria and for few fungal microorganisms.

Full text of DOI:10.1016/j.farmac.2005.06.015

Il Farmaco 60 (2005) 944–947
http://france.elsevier.com/direct/FARMAC/
Original article
Syntheses, in vitro antibacterial and antifungal activities of a series
of N-alkyl, 1,4-dithiines
F. Zentz a,*, R. Labia a, D. Sirot b, O. Faure c, R. Grillot c, A. Valla a
a Chimie et biologie des substances naturelles, FRE 2125 CNRS, 6, rue de l’Université, 29000 Quimper, France
b Service de bactériologie–virologie, faculté de médecine, 28, place Henri-Dunant, B.P.38, 63001 Clermont-Ferrand, France
c Service de parasitologie–mycologie, CHU de Grenoble, B.P.217, 38043 Grenoble cedex 9, France
Received 14 July 2004; received in revised form 8 June 2005; accepted 23 June 2005
Available online 18 October 2005
Abstract
A series of dithiines were synthesized by cyclization of 4-(alkylamino)-4-oxobutanoic acids under the action of SOCl2. Their in vitro
antibacterial and antifungal activities have been evaluated against reference strains and versus reference compounds. The so-called ‘isoim-
ides’ 2a, 2b were totally inactive whereas some imides had low MICs for few bacteria and for few fungal microorganisms.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Dithiine; Imide; Isoimide; Antimicrobial agents
1. Introduction
solubility in the reaction medium. Otherwise, an isomeriza-
tion led to the green diimides 3, which were always poorly
soluble (Fig. 2).
In previous works [1–3], we have investigated the synthe-
ses and the biological properties of a series of maleimides
and succinimides. In order to obtain other succinimides
derivatives possessing antimicrobial and/or antifouling activi-
ties, we have studied the cyclization of 4-(alkylamino)-4-
oxobutanoic acids 1, easily obtained from aliphatic amines
and succininic anhydride (Fig. 1). Surprisingly a very few
papers are dealing with the synthesis and properties of dithi-
ines.
This methodology has been reported (without experimen-
tal details) by Michaïlidis et al. [4], and three of these 1,4-
dithiin-2,3,5,6-tetracarboxydiimides were used as synthons
for Diels–Alder cycloadditions, by Hayakawa et al. [5]. Only
one patent described a biological use of some 1,4-dithiin-
2,3,5,6-tetracarboxydiimides as anthelmintics [6]. These poor
data prompt us to synthesize a series of these compounds and
evaluate their biological properties. Hence, we report herein,
next to the synthesis, the in vitro antibacterial and antifungal
properties of a series of dithiines 2 and 3. One will observed
that dithiin diimide 3b was not obtained.
2. Chemistry
In these experimental conditions, the corresponding suc-
cinimides were not obtained and it could be shown that, when
the reaction was carried out in polar aprotic solvents (e.g.
THF, dioxane), the 4,8-dithiine-indacene-1,3,5,7-tetraones 3
(diimides 3) were produced via 3,7-bis-isoimino-4,8-dithia-
indacene-1,5-diones 2 (diisoimides 2). The diisomides 2 could
be considered as the kinetically controlled compounds and
may be isolated (as rose crystals), only when they had a low
* Corresponding author. Tel.: +33 2 98 90 85 30; fax: +33 2 98 90 85 40.
E-mail address: zentz@univ-brest.fr (F. Zentz).
Fig. 1
. Syntheses of succinamic acids. Substituents: a: R = benzyl; b:
R = cyclohexyl; c: R = isopropyl; d: R = propyl; e: R = butyl.
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.06.015

F. Zentz et al. / Il Farmaco 60 (2005) 944–947
945
Fig. 2
.
Syntheses and structures of dithiin diisoimides (2) and diimides (3).
3. Microbiological evaluation
E. faecalis with MICs ranging from 2 to 16 µg ml–1. Con-
cerning E. coli and P. aeruginosa MICs of 128 µg ml–1 are
considered as inactive. Nevertheless reference compounds
(ampicillin, kanamycin, erythromycin and ofloxacin) are
much more efficient with the exception of P. aeruginosa usu-
ally resistant to many antibiotics.
3.1. Antibacterial activities
The results of the screening tests of N-alkyl, 1,4-dithiines
isoimides 2a,b and N-alkyl, 1,4-dithiines 3a–e were reported
in Table 1 which showed the minimum inhibitory concentra-
tions (MICs) of dithiines against two Gram-positive bacteria
(S. aureus ATCC 25923, E. faecalis ATCC 29212) and two
Gram negative bacteria (E. coli ATCC 25922, P. aeruginosa
ATCC 27853). Dimethyl sulfoxide has no antibacterial effect
at a concentration up to 512 µg ml–1. The isoimides 2a and
2b are totally inactive versus the four strains. Conversely, the
imides 3a to 3e have some activities against S. aureus and
3.2. Antifungal activities
The antifungal activities of the compounds 2a,b and 3a–e
(see Table 2) were determined according to the guidelines of
the Clinical and Laboratory Standards Institute (formerly
NCCLS) for yeasts and for filamentous fungi [7,8]. Fungal
strains such as Candida albicans, Candida parapsilosis,
Aspergillus fumigatus were used in the aim to evaluate inter-
est of the investigational compounds against the species cur-
rently responsible for the majority of human mycoses. The
test strains included also some yeast species of which a char-
acteristic feature is the frequent resistance to major antifun-
gals: Candida krusei has an intrinsic resistance to flucona-
zole, while majority of Candida lusitaniae strains presents
high MIC to amphotericin B.
As observed for bacteria, the isoimides 2a and 2b are rela-
tively inactive, whereas the isomide 3d has some antifungal
activity especially against C. albicans (< 0.1 µg ml–1), and 3e
against C. parapsilosis and C. krusei (0.4 µg ml–1). The prod-
uct 3c shows a low MIC particularly against A. fumigatus
(0.1 µg ml–1), and also against C. albicans (0.8 µg ml–1). The
compound 3a shows a poorest spectrum of activity of N-alkyl,
1,4-dithiines molecules.
Table 1
In vitro antibacterial activities of N-alkyl, 1,4-dithiines isoimides 2 and
N-alkyl, 1,4-dithiines 3 compounds (MICs, µg ml–1
)
Compound
S. aureus
E. faecalis E. coli
P. aeruginosa
ATCC 25923
ATCC
ATCC
25922
256
ATCC 27853
29212
2a
2b
3a
3c
3d
3e
256
256
16
4
256
256
2
256
256
128
128
128
128
256
128
4
128
8
4
128
8
4
128
Reference compounds
Ampicillin
Kanamycin
0.5
2
1
4
> 16
> 16
> 4
2
32
2
2
Erythromycin 0.25
Ofloxacin 0.5
> 4
0.03
2
If we compare these results to the in vitro susceptibility of
these fungal species to the reference antifungals mainly to
a: R = benzyl; b: R = cyclohexyl; c: R = isopropyl; d: R = propyl; e: R = butyl.
Table 2
In vitro antifungal activities of N-alkyl, 1,4-dithiines isoimides 2 and N-alkyl, 1,4-dithiines 3 compounds (MICs 50, µg ml–1
)
Compound
C. parapsilosis
ATCC 22019
MIC50
> 51.2
C. krusei
ATCC 26258
MIC50
> 12.8
> 51.2
3.2
C. albicans
ATCC 90028
MIC50
> 51.2
> 51.2
3.2
C. lusitaniae
ATCC 410086
MIC50
> 51.2
> 51.2
3.2
A. niger
A. fumigatus
ATCC 980483435 wild strain a
MIC50
> 51.2
> 51.2
> 51.2
> 51.2
3.2
MIC50
> 25.6
> 51.2
> 51.2
0.1
2a
2b
3a
3c
3d
3e
> 51.2
6.4
> 51.2
51.2
0.8
12.8
3.2
1.6
< 0.1
6.4
1.6
0.4
0.4
1.6
6.4
> 51.2
0.8
Reference compounds (MIC range)
Amphotericin B
Fluconazole
Flucytosine
0.25–1
0.25–2
16–64
4–16
–
0.5–2
0.25–1
0.5–2
–
0.12–1
0.25–1
0.12–4
–
0.25–1
0.5–2
2–8
–
–
0.12–0.5
–
–
–
Itraconazole
0.5–12
0.12–1
a: R = benzyl; b: R = cyclohexyl; c: R = isopropyl; d: R = propyl; e: R = butyl.
a Fungal collection of the ‘Centre Hospitalier Universitaire de Grenoble’; Service de Parasitologie–Mycologie B.P.217, Grenoble cedex 9 France.

946
F. Zentz et al. / Il Farmaco 60 (2005) 944–947
amphotericin B, which remains the “gold standard” in mycol-
ogy because of its wide spectrum of activity, all the other
compounds can be considered as inactive.
However, we must note that the screening of antifungal
activity only on the basis of in vitro techniques can be uncer-
tain, as demonstrated these last years during the development
of the new class of echinocandins. Indeed, the fungal cell is
particularly complex, and according to the target of antifun-
gal classes and their site of activity, MIC results can show
wide variations between strains and species.
10 ml of dioxane was slowly added. The solution was warmed
at 80 °C for 30 min. and the succinamic acid 1 crystallize by
cooling. The white crystals were filtered off, dried and recrys-
tallized from dioxane.
1a: m.p. 144 °C (85%). IR (KBr): m CO 1691; 1642.
1b: m.p. 171 °C (90%). IR (KBr): m CO 1697; 1642.
1C: m.p. 108 °C (90%). IR (KBr): m CO 1731; 1641.
1d: m.p. 101 °C (85%). IR (KBr): m CO 1414; 1642.
1e: m.p. 96 °C (85%). IR (KBr): m CO 1695; 1650.
5.2. General procedure for the preparation
of compounds 2
4. Conclusions
One gram of the succinamic acid was dissolved into 10 ml
of ether and 13 ml of thionyl chloride was slowly added. The
solution was allowed to r.t. for 6 h and the diisoimide was
filtered off, washed with 2 × 5 ml of ether and recrystallized.
2a: Rose crystals, m.p.: 210 °C. IR: 1791, 1696 cm−1. 1H
NMR (CDCl3): 4.78 (s, 4H, CH2); 7.31–7.38 (m, 10H, Ar).
2b: Rose crystals, m.p.: 228 °C. IR: 1789, 1774, 1701 cm−1.
1H NMR (CDCl3): 1.23–2.13 (m, 20H, CH2); 3.75–3.82 (m,
2H, CH).
The reaction of thionyl chloride with succinamic acids
appeared to be a useful method for the synthesis of
S-containing heterocyclic compounds such as 1,4-dithiins,
which were usually obtained with yields close to 50%. In the
high majority of examples, the products crystallized sponta-
neously and the products did not require further purification.
Some dithiin diimides (compounds 3a, 3c, 3d and 3e) have
a moderate antibacterial activity against Gram-positive organ-
isms (S. aureus and E. faecalis), but E. coli and P. aerugi-
nosa were resistant.
The antifungal activity of the new dithiins compounds 3c,
3d and 3e against some yeasts and moulds of medical inter-
est could open a new way of investigation for future. To con-
firm the potential value of these new compounds, a large num-
ber of strains should be tested for the species which appear
sensitive to the products, especially C. albicans, C. krusei
and A. fumigatus. Indeed there is a need for research of new
antifungals to address infections due to an expanding num-
ber of opportunistic fungi.
5.3. General procedure for the preparation
of compounds 3
By the same method used for the preparation of the com-
pounds 2, using dioxane as solvent.
3a: Green needles, m.p.: 224 °C. IR: 1712, 1697 cm−1. 1H
NMR (CDCl3): 4.64 (s, 4H, CH2); 7.31–7.35 (m, 10H, Ar).
3c: Green needles, m.p.: 220 °C. IR: 1713, 1696 cm−1. 1H
NMR (DMSO d6): 1.35 (d, 12H, CH3, J = 6.9 Hz); 4.23–4.30
(m, 2H, CH).
3d: Green needles, m.p.: 211 °C. IR: 1714, 1693 cm−1. 1H
NMR (DMSO d6): 0.88 (t, 6H, CH3, J = 7.38 Hz); 1.48–1.61
(m, 4H, CH2); 3.45 (t, 4H, CH2, J = 7.09 Hz).
3e: Green needles, m.p.: 214 °C. IR: 1713, 1693 cm−1. 1H
NMR (DMSO d6): 0.91 (t, 6H, CH3, J = 7.35 Hz); 1.26–1.35
(m, 4H, CH2); 1.51–1.58 (m, 4H, CH2);.3.49 (t, 4H, CH2,
J = 7.05 Hz).
Conversely, dithin diisoimides (compounds 2a and 2b) are
totally inactive despites of their close structure similarities
with the dithin diimides. The present study adds new data in
the relationships of imides and their antimicrobial activities
[1–3].
5. Experimental part
6. Bacteriological assays
Melting points were taken on a Leitz 350 heated stage
microscope and are not corrected. H NMR spectra were
1
In vitro antimicrobial activities of the compounds were
determined by the twofold broth dilution method in Mueller
Hinton nutrient broth. The concentration of mother solutions
was 1024 µg ml–1 (50/50 water–methyl sulfoxide). Minimal
Inhibitory Concentration (MIC), was defined as the lowest
drug concentration resulting in complete inhibition of growth
after 18 h of incubation at 37 °C. Methyl sulfoxide has no
antibacterial activity at a concentration up to 20% in water.
The tested organisms were Staphylococcus aureus ATCC
25923, Enterococcus faecalis ATCC 29212, Escherichia coli
ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.
recorded on a BrukerAvance DPX 400 and a WP80 DS instru-
ments and were reported in ppm downfield from internal tet-
ramethylsilane. Chemical shifts were reported in ppm (d) rela-
tive to TMS. IR spectra were run on a Bruker IF
55 spectrometer. Elemental analyses were within 0.35 of
the theoretical values. Analytical thin layer chromatography
was performed on a plastic sheet (0.2 mm, silica gel 60 F254
,
Merck). Silica gel 60 (70–230 mesh, Merck) was used for
column chromatography.
5.1. General procedure for the preparation
of compounds 1
7. Antifungal assays
Fifteen millimols of succinic anhydride were dissolved into
10 ml of dioxane and 15 mmol of the correspondent amine in
Antifungal susceptibility testing was determined by the ref-
erence methods of the Clinical and Laboratory Standards Insti-

F. Zentz et al. / Il Farmaco 60 (2005) 944–947
947
tute (formerly NCCLS). Broth microdilution methodologies:
References
M27-A2 for yeasts and M38-A for filamentous fungi (7, 8).
Briefly the test isolates included two quality control (QC)
strains (C. parapsilosis ATCC 22019 and C. krusei ATCC
26258), three yeast and mould reference strains (C. albicans
ATCC 90028, C. lusitaniae ATCC 410086, Aspergillus niger
ATCC 9804.83435) and one A. fumigatus clinical isolate,
obtained from a proven invasive aspergillosis case at the Uni-
versity Hospital of Grenoble (France). Stock solutions of dithi-
ines were prepared in dimethyl sulfoxide (water–methyl sul-
foxide: 50/50), then they were diluted by the twofold broth
dilution method in RPMI 1640 medium buffered to pH
7.0 with MOPS, as recommended by the NCCLS. The final
solvent concentration was reduced to 1%, and at this concen-
tration it had no antifungal effect. Regarding susceptibility
testing, isolates were grown on Sabouraud medium at 35 °C
for a period of 24–48 h for yeasts and 7 days for moulds.
Final inoculum of yeast suspensions was 5 × 103 CFU ml–1
and suspensions of conidial spores of Aspergillus were
adjusted to obtain an inoculum of 1 × 104 CFU ml–1. The
final ranges of drug concentrations tested were 0.1–
51.2 µg ml–1 for all the dithiines compounds. Minimal inhibi-
tory concentrations (MICs) were determined after 24 h of
incubation of microdilution trays at 35 °C for Candida strains
and 48 h at 30 °C for Aspergillus strains. MICs were deter-
mined spectrophotometrically at 405 nm, and defined as the
lowest concentration of drug that produced 50% inhibition of
growth relative to the drug-free growth control.
[1] F. Zentz, R. Le Guillou, R. Labia, D. Sirot, B. Linard, A. Valla,
Syntheses, in vitro antibacterial and cytotoxic activities of series of
3-substituted succinimides, Farmaco 59 (2004) 879–886.
[2] F. Zentz, C. Hellio, A. Valla, D. De La Broise, G. Bremer, R. Labia,
Antifouling activities of N-substituted maleimides and succinimides:
antibacterial activities and inhibition of Mytilys edulis phenoloxidase,
Mar. Biotechnol. 4 (2002) 431–440.
[3] F. Zentz, A. Valla, R. Le Guillou, R. Labia, A.G. Mathot, D. Sirot,
Synthesis and antimicrobial activities of N-substituted imides, Far-
maco 57 (2002) 421–426.
[4] A. Michaïlidis, M. Giraud, D. Molho, in: ‘Organic Sulfur Chemistry’
Stirling C.J.M. Ed. Butterworths, London, 1975, pp. 466.
[5] K. Hayakawa, M. Mibu, E. Osawa, K. Kanematsu, Reagent design
and study of 1,4-dithinns as a promising class of reagents (synthons)
for cycloaddition. Diels–Alder with anthracene derivatives via
charge-transfert complexes, J. Am. Chem. Soc. 104 (1982) 7136–
7142.
[6] W. Draber, F. Korte, 1,4-Dithiin-2,3,5,6-tetracarboxydiimides, US
3364229 19680116.
[7] National committee for clinical laboratory standard, Reference
method for broth dilution antifungal susceptibility testing of yeasts,
second ed., Approved standard M27-A2, NCCLS, Wayne, PA, 2002.
Acknowledgements
[8] National committee for clinical laboratory standard, Reference
method for broth dilution antifungal susceptibility testing of filamen-
tous fungi. Approved Standard M38-A, NCCLS, Wayne, PA, 2002.
We acknowledged Dominique Rubio for technical assis-
tance.