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					FARMACIA, 2008, Vol.LVI, 5                                                                  491


           SCREENING OF THE ANTIMICROBIAL
           ACTIVITY OF SOME NEW
           DIBENZO[b,e]THIEPINE DERIVATIVES. NOTE 1
           CAMELIA ELENA STECOZA1*, CARMEN BALOTESCU CHIFIRIUC2,
           ANCA MICHAELA ISRAIL3
           2
             University of Medicine and Pharmacy “Carol Davila” Bucharest,
           Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 6 Traian
           Vuia, 020956, Bucharest, Romania
           1
             Microbiology-Immunology Department, Faculty of Biology, University
           of Bucharest, Ale. Portocalelor 1-3, sect. 5, Bucharest, Romania
           3
             National Institute for Research and Development in Microbiology and
           Immunology Cantacuzino
           *
             corresponding author: stecoza@rdslink.ro

            Abstract
            The emergence of the antimicrobials resistance and multiresistance of bacterial and
fungal infectious agents has urged the research for new antimicrobial substances and for new
strategies for the treatment of infectious diseases which still remain a top public health
problem in the entire world. The aim of this study was to evaluate the in vitro antimicrobial
activity of some newly synthesized compounds, O-acyloximino-dibenzo[b,e]thiepins (series
1) and O-acyloximino-dibenzo[b,e]thiepin-5,5-dioxides (series 2). The qualitative screening
of the susceptibility spectra of different microbial strains to these compounds was performed
by three adapted diffusion methods: paper filter disk impregnation with the tested substances
solutions, the disposal of tested solutions in agar wells and the spotting of tested solutions on
solid medium seeded with microbial inoculums. The in vitro antimicrobial testing was
performed by binary microdilution method, in 96 multi-well plates, in order to establish the
minimal inhibitory concentration (MIC), against Gram-positive: Staphylococcus aureus,
Bacillus sp., Listeria monocytogenes, Gram-negative Escherichia coli, Salmonella sp.,
Pseudomonas aeruginosa bacteria and Candida strains. The first series of compounds
exhibited different levels of antimicrobial activity, being specifically active on Pseudomonas
aeruginosa and Candida albicans, while the new sulfones (series 2) exhibited low MIC
values on Escherichia coli strains.
            Our studies demonstrated that among other biological activities of these
compounds, some of them also exhibit selective and effective antimicrobial properties that
could lead to the selection and use of these compounds as efficient antimicrobial agents,
especially for the treatment of multidrug resistant infections.

            Rezumat
            Apariţia fenomenelor de rezistenţă şi de multirezistenţă ale agenţilor infecţioşi la
medicamentele antimicrobiene a generat intensificarea eforturilor de cercetare pentru
găsirea de noi agenţi antimicrobieni şi dezvoltarea unor noi strategii pentru tratamentul
bolilor infecţioase, care rămân încă o problemă de sănătate publică în întreaga lume. Scopul
acestui studiu a fost evaluarea in vitro a activităţii antimicrobiene a unor substanţe chimice
nou-sintetizate, O-aciloximino-dibenzo[b,e]tiepine (seria 1) şi O-aciloximino-
492                                                            FARMACIA, 2008, Vol.LVI, 5

dibenzo[b,e]tiepin-5,5-dioxizi (seria 2). Evaluarea calitativă a sensibilităţii tulpinilor
microbiene faţă de aceşti compuşi a fost efectuată prin trei metode adaptate după metoda
difuzimetrică: discuri de hârtie de filtru impregnate cu soluţii ale substanţelor testate,
repartizarea soluţiilor testate în godeuri de agar şi repartizarea în spot a soluţiilor testate pe
medii solide însămânţate în pânză. Testarea cantitativă şi stabilirea concentraţiei minime
inhibitorii (CMI), a fost efectuată prin metoda microdiluţiilor binare în mediu lichid faţă de
următoarele genuri şi specii microbiene: Staphylococcus aureus, Bacillus sp., Listeria
monocytogenes, Escherichia coli, Salmonella sp., Pseudomonas aeruginosa şi Candida
albicans. Prima serie de compuşi a prezentat niveluri diferite de activitate antimicrobiană,
fiind în mod special activi pe Pseudomonas aeruginosa şi Candida albicans, în timp ce
sulfonele nou sintetizate (seria 2), au prezentat valori CMI scăzute faţă de tulpinile de
Escherichia coli. Studiile noastre au demonstrat că printre alte activităţi biologice ale
acestor compuşi, unii dintre ei prezintă o activitate antimicrobiană eficientă şi selectivă,
care ar putea conduce la selectarea şi utilizarea acestor compuşi ca agenţi antimicrobieni, în
special pentru tratamentul infecţiilor cu microorganisme rezistente la antibioticele existente.

                   dibenzo[b,e]thiepine
                   sulfones
                   antimicrobial activity

           INTRODUCTION
          Despite a century of often successful prevention and control efforts,
infectious diseases remain an important global problem in public health,
causing over 13 million deaths each year. Changes in society, technology
and the microorganisms themselves are contributing to the emergence of
new diseases, the re-emergence of diseases once controlled, and to the
development of antimicrobial resistance [1, 4].
          Since the first introduction of the antibacterial sulfonamides and
penicillin into clinical use, large numbers of antibiotics have been developed
and hence contributed to human health. But extensive use of antibiotics has
raised a serious public health problem due to multiantibiotic resistant
bacterial pathogens that inevitably develop resistance to every new drug
launched in the clinic. Consequently, there is a pressing need to develop
new antibiotics to keep pace with bacterial resistance [2, 3]. Although the
need for new antibacterial therapies and strategies is greater than it has been
in a quarter of a century and despite considerable effort, little progress has
been observed in the development of antimicrobial agents. Target-based
screening for new antibacterial agents has been particularly disappointing,
despite a plethora of potential targets, enhanced screening methodologies
and considerable investment [7, 8]. When a new class of antibiotics is
introduced, it is effective at first, but will eventually select for survival of
the small fraction of bacterial populations that have an intrinsic or acquired
FARMACIA, 2008, Vol.LVI, 5                                                                         493


resistance mechanism [3, 4]. In targeting the expression of resistance itself,
it appears that staying one step ahead of the beta-lactamases is an overly
optimistic goal; the best that can be expected is to avoid falling too far
behind [5, 6]. The purpose of this study was to evaluate the in vitro
antimicrobial activity of some newly synthesized compounds: O-
acyloximino-dibenzo[b,e]thiepins (series 1) and O-acyloximino-
dibenzo[b,e]thiepin-5,5-dioxides (series 2).

           MATERIAL AND METHODS
         a) Chemistry
         The synthesis of the new compounds, O-acyloximino-
dibenzo[b,e]thiepins 1 and O-acyloximino-dibenzo[b,e]thiepin-5,5-dioxides
(sulfones) 2 was performed in several stages. Thus by reaction of phtalide 3
with potassium salts of thiophenol 4a or p-thiocresol 4b we obtained 2-
(phenylthiomethyl)benzoic        acid        5a,      respectively     2-(4-
tolylthiomethyl)benzoic acid 5b. These acids were cyclized with
polyphosphoric acid (PPA) to the desired 6,11-dihydrodibenzo[b,e]thiepin-
11(6H)-one 6a and 2-methyl-6,11-dihydrodibenzo[b,e]thiepin-11(6H)-one
6b. The ketones 6 were converted to the oximes 7 by treatment with
hydroxylamine hydrochloride.
         The acylation of these oximes with various acid chlorides afforded
the new O-acyl-oximino-dibenzo[b,e]thiepins 1. The sulfones 2 were
obtained by the oxidation of O-acyloximino-dibenzo[b,e]thiepins 1 with
hydrogen peroxide in acetic acid, at boiling temperature.

                                                 CH2-S                                 CH2-S
                                (KOH)                                HCl
           O + HS           R
                                                - +                  -KCl
                                             COO K                  R                  COOH            R
  3        O       4                                                             5

                                   -
                                             S                          O
                   S          + Cl                           + R'   C                    S
PPA                        H3N-OH
                                                                        Cl
                           R (Py)                        R                                         R
       6       O                        7
                                            N-OH                                        N-O-C-R'
                                                                                  1
                                                                                             O
                            SO2
      + H2O2                                                   R= -H (a)
                                                               R= -CH3 (b)
                                        R                      R'= alkyl, aryl, etc.
                           N-O-C-R'
                       2
                                O
494                                              FARMACIA, 2008, Vol.LVI, 5


          b) Microbial strains
          The compounds were solubilised in DMF to a final concentration
of 1024 µg/mL. The antimicrobial activity of the new compounds was tested
against bacterial and fungal reference strains. The in vitro biological
activities were tested against a microbial inoculum of ~1.5x108 UFC/cm3,
corresponding to 0.5 McFarland density [9], represented by
Enterobacteriaceae (E. coli, Salmonella sp.,), Pseudomonadaceae
(Pseudomonas aeruginosa), Micrococcaceae (Staphylococcus aureus),
Bacillaceae (Bacillus sp., Listeria monocytogenes) and Candida strains.
          c) The qualitative screening of the susceptibility spectra of
different microbial strains to the tested compounds was performed by
adapted diffusion techniques: i) the study of the antimicrobial properties
by the adapted disk diffusion method -Petri dishes with Mueller Hinton
(for bacterial strains) /YPG (Yeast Peptone Glucose) (for yeasts) medium
were seeded with bacterial inoculum as for the classical antibiotic
susceptibility testing disk diffusion method (CLSI); 5 mm diameter paper
filter disks were placed on the seeded medium, at 30 mm distance.
Subsequently, the disks were impregnated with 5 L of compound solution.
The plates were left at room temperature for 20-30 minutes and then
incubated at 37oC for 24 hrs. The positive results were read as the
occurrence of a inhibition zone of microbial growth around the disk; ii) the
study of the antimicrobial properties by the agar well method-Petri
dishes with Mueller Hinton (for bacterial strains) /YPG (for yeasts) medium
were seeded with bacterial inoculum as for the previous method, but the 5
L compound solution were this time discarded in 5 mm diameter wells
cropped in the solid medium. The plates were left at room temperature for
20-30 minutes and then incubated at 37oC for 24 hrs. The positive results
were read as the occurrence of an inhibition zone of microbial growth
around the well; iii) the study of the antimicrobial properties by spotting
the tested solution on the culture medium seeded with the microbial
suspension, the antimicrobial activity being expressed as the absence of
microbial growth on the spot area [10, 11].
          d) The quantitative assay of minimal inhibitory concentration
(M.I.C., μg/mL) value was based on liquid medium serial microdilutions [9-
11]. Serial binary dilutions of the tested compounds (ranging between 1024
g/mL and 32 g/mL) were performed in a 200L volume of nutrient broth
and each well was seeded with 50 L of microbial inoculum (0.5 McFarland
density). The plates were incubated for 24 hours at 370C, and MICs were
read as the lowest concentration of compound which inhibited the microbial
growth.
FARMACIA, 2008, Vol.LVI, 5                                                                                   495


        RESULTS AND DISCUSSION
         The new compounds, O-acyloximino-dibenzo[b,e]thiepins 1 and
sulfones 2, are white crystalline substances, insoluble in water, but soluble
in organic solvents (acetone, chloroform, benzene, toluene, xylene).
         Structural and physical data for the new synthesized compounds
are given in Table I and Table II.
         The synthesis of the compounds 1 and 2, and their spectral data
 1
( H-NMR, 12C-NMR, IR) were published in previous papers [12- 19].

                                                                          Table I
    Structural and physical data for the new O-acyloximino-dibenzo[b,e]thiepins 1
                                                        5
                                               6
                                     7                  S            4

                             8                                               3


                                                   11
                                 9
                                                                         2
                                         10
                                                            12
                                                                 1               R
                                                   N-O-C-R'
                                     1
                                                      O
Comp                                           Formula/                              M.p.     Recryst.     Yield
          R            R’
 no.                                            mol. wt.                             (OC)     solvent       %
                                              C21H14NO2SF
  1.1    -H        -C6H4F(3)                                                     158-160     isopropanol   51.4
                                                 363.38
                                              C19H13NO2S2
  1.2    -H                                                                      156-158      methanol      52
                                                 351.42
                        S
                                     C18H17NO2S
  1.3    -H      -CH2-CH2-CH3                                                    128-130     isopropanol   51.2
                                        311.39
                                     C24H21NO5S                                  209.3-
  1.4    -H     -C6H2(OCH3)3(3,4,5)                                                            ethanol     55.3
                                        435.48                                   210.2
                                    C22H16NO2SI
 1.5.   -CH3         -C6H4I(2)                                                   143-144     isopropanol   70.4
                                        485.33
                                     C21H14NO2SI
  1.6    -H          -C6H4I(2)                                                   152-154     isopropanol    71
                                        471.30
                                    C21H14NO2SCl
  1.7    -H         -C6H4Cl(2)                                                   159-160     isopropanol   50.1
                                        379.85
                                    C21H14NO2SBr
  1.8    -H         -C6H4Br(3)                                                   170-173     isopropanol   67.2
                                        424.31
                                     C23H19NO3S
  1.9   -CH3      -C6H4OCH3(4)                                                   208-211     isopropanol   69.2
                                        389.45
                                    C22H16NO2SF
 1.10   -CH3         -C6H4F(3)                                                   148-152     isopropanol   50.6
                                        377.42
                                    C26H26N2O2S
 1.11    -H    -C6H4CH2N(C2H5)2(4)                                               136-140     isopropanol    62
                                        430.55
                                     C17H15NO2S
 1.12    -H          -CH2-CH3                                                        94-95   isopropanol   50.6
                                        297.36
496                                                                                      FARMACIA, 2008, Vol.LVI, 5

 Comp                                             Formula/                               M.p.      Recryst.     Yield
          R            R’
  no.                                              mol. wt.                              (OC)      solvent       %
                                                 C21H14N2O4S
 1.13     -H      -C6H4NO2(4)                                                           191-193   isopropanol    84
                                                    390.40
                                                 C23H19NO3S
  1.14   -CH3    -C6H4OCH3(3)                                                           190-192     ethanol      56
                                                    389.45
                                                 C23H19NO4S
  1.15    -H    -C6H3(OCH3)2(2,3)                                                       164-166   isopropanol    54
                                                    405.45
                                                 C23H19NO4S
 1.16     -H    -C6H3(OCH3)2(2,4)
                                                    405.45
                                                                                        172-174   isopropanol    58

                                                  C23H19NO4S
 1.17     -H    -C6H3(OCH3)2(2,6)
                                                    405.45
                                                                                        191-193     ethanol      49

                                                  C23H19NO4S
 1.18     -H    -C6H3(OCH3)2(3,4)
                                                    405.45
                                                                                        188-191     ethanol      57

                                                  C24H21NO4S
  1.19   -CH3   -C6H3(OCH3)2(2,3)                                                       147-149   isopropanol    55
                                                    419.48

                                                                                  Table II
                                       Structural and physical data for the new sulfones 2
                                                           5
                                                  6
                                        7                  SO2          4

                               8                                                3


                                                      11
                                   9
                                                                            2
                                            10
                                                               12
                                                                    1               R
                                                      N-O-C-R'
                                            2                  O
                                                 Formula/                               M.p.      Recryst.      Yield
 No.      R          R’
                                                 mol. wt.                               (OC)      solvent        %
                                        C21H14NO4SBr
  2.1    -CH3     -C6H4Br(4)
                                            456.31
                                                                                    232-235        ethanol       86
                                         C23H19NO5S
  2.2    -CH3   -C6H4OCH3(4)
                                           421.45
                                                                                    228-231        ethanol       91
                                         C23H19NO4S                                     231.1-
  2.3    -CH3    -C6H4CH3(4)
                                           405.45
                                                                                                   ethanol      79.5
                                                                                        233.3
                                         C22H17NO5S
  2.4     -H    -C6H4OCH3(4)                                                        235-238       acetic acid    88
                                           407.43
                                        C22H16NO4SF
  2.5    -CH3     -C6H4F(3)
                                           409.42
                                                                                    224-227        ethanol       69


         Antimicrobial activity assay
         Out of the three qualitative methods used for the screening of the
antimicrobial activity, the paper filter disk diffusion methods proved to be
the most efficient qualitative method, the results being very well correlated
with the results of the MIC quantitative assay.
FARMACIA, 2008, Vol.LVI, 5                                                               497


         In the case of the quantitative assay of the antimicrobial activity of
the tested compounds by the microdilution method in liquid medium, the
minimal inhibitory concentration was read by wells observation: in the first
wells containing high concentrations of compounds the culture growth was
not visible, the microbial cells being killed or inhibited by the tested
compound. At lower concentrations of the tested compounds, the microbial
culture becomes visible. The lowest concentration which inhibited the
visible microbial growth was considered the MIC (μg/mL) value for the
tested compound. In the next wells, including the standard culture growth
control wells, the medium become muddy as a result of the microbial
growth. In the sterility control wells series the medium had to remain clear.
From the last well without any visible microbial growth and from the first
one with a microbial growth, Gram stained smears were performed for the
results confirmation [10, 11].
         Concerning the qualitative assay of MIC values, the most active
compounds from the first series (Table III) proved to be the compounds no.
1.1 and 1.2, exhibiting antimicrobial activity against all tested species, with
a constant MIC value of 256 μg/mL, followed by the compounds 1.15, 1.16,
1.17 exhibiting good antimicrobial activity against the majority of the tested
species, excepting E. coli and S. aureus (MIC ranging from 64 to 256
μg/mL).
         The weakest antimicrobial activity was showed by the compounds
1.3 to 1.8, while the rest of the compounds exhibited variable levels and
spectra of antimicrobial activity.
         The most sensitive microorganisms to the tested compounds proved
to be P. aeruginosa and C. albicans, followed by Salmonella sp. and
Bacillus sp., while, curiously, the most resistant was the E. coli strain.

                                                                            Table III
                                The MIC values for the compounds of series 1 (μg/mL)
 Comp                                      Microbial strain
  no.    E. coli   Salmonella   Pseudomonas     Listeria        S.     Bacillus   Candida
                       sp.      aeruginosa monocytogenes      aureus     sp.        sp.
  1.1     256         256           256           256          256       256        256
  1.2     256         256          256            256          256       256       256
  1.3     512       >1024          256            1024         512     >1024       1024
  1.4     512       >1024         >1024          >1024        >1024    >1024      >1024
  1.5.   1024       >1024         >1024          >1024        >1024    >1024      >1024
  1.6    1024        1024          1024          >1024        1024       256      >1024
  1.7    1024        1024          1024          >1024        1024       256       512
  1.8    >1024      >1024         >1024          >1024        >1024      256      >1024
498                                                            FARMACIA, 2008, Vol.LVI, 5

 Comp                                       Microbial strain
  no.     E. coli   Salmonella   Pseudomonas     Listeria         S.     Bacillus   Candida
                        sp.      aeruginosa   monocytogenes     aureus     sp.        sp.
  1.9      256         256            32          >1024           32      >1024       64
  1.10     128         256           32           >1024         >1024    >1024       128
  1.11    1024         256           32           >1024          32      >1024       512
  1.12     512         512           32            1024          32      >1024       1024
  1.13    >1024      >1024           256          >1024         >1024    >1024       256
  1.14    >1024      >1024           256          >1024         >1024    >1024       256
  1.15    >1024        256           256           256          >1024      256       128
  1.16    >1024        256           256           256          >1024      128        64
  1.17    >1024        256           256           256          >1024      128        64
  1.18    >1024      >1024           256           128          >1024    >1024       128
  1.19    >1024      >1024         >1024          >1024         >1024    >1024       256
*in grey there are marked low MIC values demonstrating an efficient antimicrobial activity

        Concerning the antimicrobial activity of the sulfones series (Table
IV), the most active proved to be the compounds no. 2.3, 2.4 and 2.5 being
active at low MIC values (from 32 to 256 μg/mL) on the majority of the
tested strains, excepting the Gram-positive bacilli. In this case, E. coli
proved to be the most sensitive to all five tested compounds, while the most
resistant was in this case Listeria monocytogenes.

                                                                             Table IV
                                 The MIC values for the compounds of series 2 (μg/mL)
 Comp                                       Microbial strain
  no.     E. coli   Salmonella   Pseudomonas     Listeria         S.     Bacillus   Candida
                        sp.      aeruginosa   monocytogenes     aureus     sp.        sp.
  2.1      256       >1024          1024           1024         >1024      32        512
  2.2      256       >1024          1024            64           256       32        512
  2.3      128         256           32           >1024          32      >1024        32
  2.4      256         256           32           >1024          32      >1024        64
  2.5      128         256           32           >1024          32      >1024        32
*in grey there are marked low MIC values demonstrating an efficient antimicrobial activity


          CONCLUSIONS
        This paper presents the antimicrobial and antifungal activity of
some newly synthesized dibenzo[b,e]thiepine derivatives.
        Our studies demonstrated that among other biological activities of
these compounds (antidepressant, sedative, analgesic), some of them also
FARMACIA, 2008, Vol.LVI, 5                                                 499


exhibit selective and effective antimicrobial properties that could lead to the
selection and use of these compounds as efficient antimicrobial agents,
especially for the treatment of multidrug resistant infections.


        Acknowledgements
        The authors acknowledge support for this work from the Romanian
Ministry of Education, Research and Youth through project PN2 41-
055/2007.


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