Design, synthesis, characterization and antimicrobial evaluation of some heterocyclic condensed systems with bridgehead nitrogen from thiazolotriazole class

Article abstract of DOI:10.1248/cpb.c15-00379

In the present study, a series of new heterocyclic condensed systems with bridgehead nitrogen from the thiazolo[3,2-b][1,2,4]triazoles class was synthesized starting from some 4-(4-X-phenylsulfonyl)phenyl)-4H-1,2,4-triazole-3-thioles 1a-c (X=H, Cl, Br). The intermediates of S-alkylated 1,2,4-triazoles, 2-(5-(4-(4-X-phenylsulfonyl)phenyl)-2H-1,2,4-triazol-3-ylthio)-1-(4-fluorophenyl)ethanones 2a-c, were obtained by treatment of triazoles 1a-c with 2-bromo-4'-fluoroacetophenone. The 2-(4-(4-X-phenylsulfonyl)phenyl)-6-(4-fluorophenyl)thiazolo[3,2-b][1,2,4]triazoles 3a-c were obtained by cyclization of S-alkylated 1,2,4-triazoles 2a-c in sulfuric acid media, at 0°C. For the synthesis of 2-(4-(4-X-phenylsulfonyl)phenyl)-5-(4-fluorobenzylidene)-thiazolo[3,2-b][1,2,4]triazol-6(5H)-ones 4a-c, the triazoles 1a-c were treated with 4-fluorobenzaldehyde, chloroacetic acid and anhydrous sodium acetate, in the presence of acetic acid and acetic anhydride. The structures of the newly synthesized compounds have been confirmed by elemental analysis and spectral methods (IR, ¹H-NMR,¹³C-NMR, MS). The antimicrobial activity of all new compounds has been screened against some bacteria and yeasts.

Full text of DOI:10.1248/cpb.c15-00379

694
Chem. Pharm. Bull. 63, 694–700 (2015)
Vol. 63, No. 9
Regular Article
Design, Synthesis, Characterization and Antimicrobial Evaluation of
Some Heterocyclic Condensed Systems with Bridgehead Nitrogen from
Thiazolotriazole Class
,a
Stefania-Felicia Barbuceanu,* Constantin Draghici,b Florica Barbuceanu,c
Gabriela Bancescu,d and Gabriel Saramete
a Organic Chemistry Department, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy; 6
b
Traian Vuia, 020956, Bucharest, Romania: “C.D. Nenitescu” Institute of Organic Chemistry, Romanian Academy;
c
202B Splaiul Independentei, 060023, Bucharest, Romania: Institute for Diagnosis and Animal Health; 63 Dr.
d
Staicovici, 050557, Bucharest, Romania: Microbiology Department, Faculty of Dentistry, “Carol Davila” University
e
of Medicine and Pharmacy; 19 Calea Plevnei, 050051, Bucharest, Romania: and Pharmaceutical Technology and
Biopharmacy Department, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy; 6 Traian
Vuia, 020956, Bucharest, Romania.
Received April 29, 2015; accepted May 28, 2015
In the present study, a series of new heterocyclic condensed systems with bridgehead nitrogen from the
thiazolo[3,2-b][1,2,4]triazoles class was synthesized starting from some 4-(4-X-phenylsulfonyl)phenyl)-4H-
1,2,4-triazole-3-thioles 1a–c (X=H, Cl, Br). The intermediates of S-alkylated 1,2,4-triazoles, 2-(5-(4-(4-X-
phenylsulfonyl)phenyl)-2H-1,2,4-triazol-3-ylthio)-1-(4-fluorophenyl)ethanones 2a–c, were obtained by treat-
ment of triazoles 1a–c with 2-bromo-4ꢀ-fluoroacetophenone. The 2-(4-(4-X-phenylsulfonyl)phenyl)-6-(4-fluo-
rophenyl)thiazolo[3,2-b][1,2,4]triazoles 3a–c were obtained by cyclization of S-alkylated 1,2,4-triazoles 2a–c
in sulfuric acid media, at 0°C. For the synthesis of 2-(4-(4-X-phenylsulfonyl)phenyl)-5-(4-fluorobenzylidene)-
thiazolo[3,2-b][1,2,4]triazol-6(5H)-ones 4a–c, the triazoles 1a–c were treated with 4-fluorobenzaldehyde, chlo-
roacetic acid and anhydrous sodium acetate, in the presence of acetic acid and acetic anhydride. The struc-
tures of the newly synthesized compounds have been confirmed by elemental analysis and spectral methods
1
(IR, H-NMR, 13C-NMR, MS). The antimicrobial activity of all new compounds has been screened against
some bacteria and yeasts.
Key words thiazolo[3,2-b][1,2,4]triazole; 1,2,4-triazole-3-thiole; alkylation; antimicrobial activity
The chemistry of 1,2,4-triazoles and their heterocyclic con-
The importance of fluorine atom in medicinal chemistry
densed systems containing both nitrogen and sulfur atoms, has been demonstrated by a great number of fluorinated com-
has attracted increasing attention of researchers due to their pounds. Introduction of this atom into a molecule of organic
wide-ranging biological importance. From these fused het- compounds, may lead to significant changes in biological and
erocyclic compounds, thiazolotriazoles obtained by fusion of physical properties of these because of their higher metabolic
1,2,4-triazole and 1,3-thiazole ring together, have received stability, often increased lipophilicity and membrane perme-
considerable attention owing to their synthetic and biologi- ability.25)
cal properties. Both triazole and thiazole rings are known
Knowing that infectious microbial diseases are pressing
that possess various biological properties, being present in problems worldwide caused by the resistance of microorgan-
composition of numerous drugs, especially with antimicro- isms to existing antimicrobial agents, there is a vital need to
bial activity.1,2) Among of the antimicrobial drugs are distin- discover new antimicrobial agents.
guished Itraconazole, Fluconazole, Voriconazole (drugs with
Inspired by the aforesaid findings and keeping in mind our
1,2,4-triazole ring), Sulfathiazole and Abafungin (drugs with studies on different heterocycles with biological potential,26–32)
thiazole ring). The fusion of triazole and thiazole rings can we designed the synthesis of new heterocyclic compounds
lead to thiazolotriazoles with superior biological properties which contain in the same molecule the thiazolotriazole con-
due to mutual influence of the pharmacophores centers present densed system, diarylsulfone and 4-fluorophenyl fragments, in
in the molecule. Thus, literature data indicate that numerous order to obtain new compounds with potential antimicrobial
thiazolo[3,2-b][1,2,4]triazoles derivatives have been reported activity.
to possess antibacterial,3–9) antifungal,3,6–9) anti-inflamma-
Thus, in this paper we synthesized, characterized and
tory,3,10,11) analgesic,3,10) antiproliferative,12) anticonvulsant13) evaluated for their antimicrobial activity a series of new
properties. Also, thiazolo[3,2-b][1,2,4]triazol-ones have been heterocyclic condensed systems with bridgehead nitrogen
studied for their properties with respect to their biological from thiazolo[3,2-b][1,2,4]triazole and thiazolo[3,2-b][1,2,4]-
activities including anti-inflammatory,14–17) analgesic,15,17) anti- triazol-6(5H)-one class containing diarylsulfone and 4-fluoro-
cancer,18–20) antibacterial,21) antifungal21) and anticonvulsant.22) phenyl fragments.
Another interesting class are diarylsulfones which are im-
portant intermediates in organic synthesis because of their
chemical properties and biological activities being known es-
pecially as antibacterial agents.23,24)
Results and Discussion
Chemistry The synthesis pathway leading to the thiazolo-
triazoles is given in Chart 1.
*To whom correspondence should be addressed. e-mail: sbarbuceanu@gmail.com
© 2015 The Pharmaceutical Society of Japan

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
695
Chart 1.
Synthesis of the compounds 2a–c～4a–c has been achieved 2-bromo-4′-fluoroacetophenone was confirmed in the IR spec-
starting from the 5-(4-(4-X-phenylsulfonyl)phenyl)-4H-1,2,4- tra of compounds 2a–c by the presence of a new absorption
triazole-3-thioles 1a–c known in the literature.33) The 1,2,4-tri- band corresponding to the stretching vibration of the carbonyl
azoles intermediates were obtained by cyclization of the group, which occurs in the range 1680–1685cm−1. The ab-
corresponding acylthiosemicarbazides according to literature sence of νC=S and νSH absorption bands from 1,2,4-triazoles
data.33) Thus, the acylthiosemicarbazides were synthesized 1a–c33) in the IR spectra of derivatives 2a–c indicated that
starting from Friedel–Crafts reaction of benzene or haloben- alkylation took place at sulfur atom and not at nitrogen atom.
zene with p-tosyl chloride, followed by oxidation at the cor- In the 1H-NMR spectra of these S-alkylated compounds,
responding acids, esterification and hydrazinolysis. Finally, the presence of a new singlet signal at a chemical shift
the acylthiosemicarbazides were obtained by treatment of δ=4.93ppm, characteristic to protons from methylene group,
corresponding hydrazides with potassium thiocyanate in acid confirmed alkylation reaction. The singlet signal of NH het-
media.33)
erocyclic group appeared at 3.50ppm. In the 13C-NMR spectra
In order to obtain condensed heterocyclic compounds 3a–c, in characteristic is the carbon atom signal from carbonyl group
a first stage, new intermediates S-alkylates 1,2,4-triazole 2a–c which appeared at 191.94–192.24ppm and the carbon atom
were synthesized by the reaction of triazoles 1a–c with signal from methylene group at 39.60–39.70ppm.
2-bromo-4′-fluoroacetophenone. 2-(4-(4-X-Phenylsulfonyl)phenyl)-
The main proof that the cyclization of intermediates 2a–c
6-(4-fluorophenyl)thiazolo[3,2-b][1,2,4]triazoles 3a–c were took place is represented by the disappearance of absorption
synthesized from S-alkylated 1,2,4-triazoles 2a–c, by cycliza- bands and of signals characteristic to carbonyl and methylene
tion in sulfuric acid medium. 2-(4-(4-X-Phenylsulfonyl)- groups from the IR and NMR spectra of compounds 3a–c. In
1
phenyl)-5-(4-fluorobenzylidene)thiazolo[3,2-b][1,2,4]triazol- the H-NMR spectra of compounds 3a–c has appeared a new
6(5H)-one 4a–c were obtained by reaction of triazoles 1a–c singlet signal corresponding to the protons of methine group
with 4-fluorobenzaldehyde, chloroacetic acid and anhydrous from the thiazole ring which occurs at the chemical shift
sodium acetate, in the presence of acetic acid and acetic anhy- δ=7.96–7.97 ppm.3,8,11) In the 13C-NMR spectra appeared two
dride.
new characteristic signals. Thus, the methine carbon signal
The alkylation reaction of 1,2,4-triazoles 1a–c with appeared at 111.49–111.51ppm and the thiazole carbon signal,

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Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
adjacent to the methine carbon (which is linked by the 4-fluo- some reference bacterial and fungal strains belonging to the
rophenyl fragment), occurred at 130.61–130.62ppm. following species: Gram-positive strains (Staphylococcus au-
The condensation reaction of the triazoles 1a–c with reus ATCC 29213 and Bacillus cereus ATCC 13061), Gram-
4-fluorobenzaldehyde and chloroacetic acid is confirmed in negative strains (Escherichia coli ATCC 25922, Enterobacter
the IR spectra of the new compounds 4a–c by the presence cloacae ATCC 49141, Acinetobacter baumannii ATCC 19606,
of a new absorption band, of high intensity, in the range Pseudomonas aeruginosa ATCC 27853) and yeast strains
1733–1738 cm−1 characteristic to the stretching vibration of the (Candida albicans ATCC 90028, Candida parapsilosis ATCC
1
carbonyl group. The H-NMR spectra revealed a new singlet 22019, Candida glabrata ATCC 15126 and Candida tropicalis
signal corresponding to methine proton which resonated at 13803), using the broth microdilution method.
8.32–8.36ppm.15,16,18) On the other hand, the 13C-NMR spectra
The results of the antimicrobial activity screening of newly
confirmed the thiazolo[3,2-b][1,2,4]triazol-6-one structure by synthesized compounds are presented in the Table 1.
the presence of new signals characteristic to the carbon of
The aim of this study was to assess the antimicrobial activ-
the carbonyl group and to the carbon of the methine group ity and how this activity of new compounds synthesized is
that appeared at 167.05–167.29ppm and 142.82–143.74ppm, modified by:
respectively.
− the replacement of a hydrogen atom from 4 position of
Another proof for the obtaining of all new compounds is the diphenylsulfone fragment with a chlorine or bromine atom.
1
characteristic signals of protons in the H-NMR spectra and of
− creation of a new nucleus, thiazole, in condensed system,
carbon atoms in the 13C-NMR spectra from 4-fluorophenyl type of thiazolo[3,2-b][1,2,4]triazole, by cyclization of S-alkyl-
moiety. Also, the signals of the protons and carbon atoms of ated 1,2,4-triazoles 2a–c to compounds 3a–c.
the arylsulfonylphenyl moiety are found in the corresponding
regions (Experimental).
The mass spectra of the new thiazolotriazoles 3a–c and benzylidene fragment in compounds 4a–c.
− grafting on the condensed heterocyclic system of two
exocyclic double bonds, C=O and C=C through the 4-fluoro-
4a–c and of the S-alkylated 1,2,4-triazole intermediates 2a–c
The results obtained by the screening of the antibacterial
have confirmed the proposed structures through the presence activity against Gram-positive bacteria S. aureus and B. ce-
of the molecular ion corresponding to molecular weight. In reus indicated that all compounds tested had a better activity
case of compounds containing chlorine or bromine atom there against B. cereus bacterium. The presence of the chlorine or
was observed the molecular ion corresponding to the halo- bromine atom on the diphenylsulfone fragment in case of
gens isotopes (35Cl/37Cl and 79Br/81Br). The main fragments all tested compounds against the B. cereus strain produces
obtained through the fragmentation of the molecular ion are a significant improvement of the antibacterial activity (mini-
given in Experimental.
mum inhibitory concentration (MIC)=8µg/mL or MIC=16 µg/
The thiazolotriazoles can exist in two isomer forms: mL). The studies on the same B. cereus strain indicated that
thiazolo[3,2-b][1,2,4]triazole and thiazolo[2,3-c][1,2,4]triazole. the presence of the carbonyl group type of acyclic ketone in
The IR and NMR spectra of these isomers are similar and compounds 2a–c or type cyclic ketone in compounds 4a–c be-
can not be differentiated based on these data.11,34) Correct side of the chlorine or bromine atoms in para-position on the
assignment of the structure of the compounds 3a–c type of diphenylsulfone had as result the obtaining of the best values
thiazolo[3,2-b][1,2,4]triazoles was made by the mass spec- of MIC.
trometry. The absence of the fragment [M–28]+ corresponding
to the loss of nitrogen molecule (this type of cleavage took
place easier in case of [2,3-c] isomer) is a proof of the obtain-
ing of isomers type of [3,2-b].11,34)
Antimicrobial Activity Evaluation The antimicro-
bial activity of the investigated compounds was tested against
The antibacterial screening of compounds on Gram-negative
bacteria indicated that the best activity was obtained against
A. baumannii strain (MIC=16 µg/mL) in case of compounds
2b and 4b, both having the chlorine atom in the para-position
from diphenylsulfone fragment and the carbonyl group type of
ketone. From structural point of view, in these compounds 2b
Table 1. Antimicrobial Activities of Compounds 2a–c～4a–c as MIC Values (µg/mL)
Gram-positive bacteriaa)
Gram-negative bacteriab)
Yeastsc)
Compd
Sa
Bc
Ec
Ecl
Ab
Pa
Ca
Cp
Cg
Ct
2a
128
128
256
128
128
512
512
128
>512
2
32
8
128
128
128
128
128
256
128
64
64
128
128
128
128
256
128
64
32
16
64
64
32
64
64
16
32
—
—
128
128
128
128
128
>512
128
64
64
128
256
128
256
128
128
256
128
—
256
128
256
128
256
256
256
256
256
—
64
64
256
128
256
128
256
256
256
256
256
—
2b
2c
8
256
64
3a
64
16
16
16
8
3b
256
128
128
64
3c
4a
4b
4c
8
128
2
64
128
2
128
—
Control (AM)
Control (FL)
—
—
—
—
—
—
—
—
2
—
—
Note: a) Sa (Staphylococcus aureus ATCC 29213); Bc (Bacillus cereus ATCC 13061) b) Ec (Escherichia coli ATCC 25922); Ecl (Enterobacter cloacae ATCC 49141); Ab
(Acinetobacter baumannii ATCC 19606); Pa (Pseudomonas aeruginosa ATCC 27853) c) Ca (Candida albicans ATCC 90028); Cp (Candida parapsilosis ATCC 22019); Cg
(Candida glabrata ATCC 15126); Ct (Candida tropicalis ATCC 13803) Control: AM=amikacin; FL=fluconazole.

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Chem. Pharm. Bull.
697
and 4b, the π electrons of double bond permitted conjugation
with π electrons of p-fluorophenyl fragment or with exocyclic
double bond=CH-C6H4-F (p). Compounds 2a, 3b, 4c also had
a good activity against the same strain (MIC=32 µg/mL).
The results of the antifungal screening indicated insignifi-
cant behaviors between these classes of the tested compounds.
The presence of the chlorine atom on the diphenylsulfone
Fig. 1. Structure of Compounds 2a–c
fragment determined the best antifungal activity against C. formed by collision with argon at 1.5 mTorr. The elemental
glabrata fungus for compounds 2b and 4b (MIC=64µg/ analyses (% C, H, N) of the new compounds were performed
mL). Compound 2a had the same MIC value against the same with ECS-40-10-Costeh micro-dosimeter.
strain and against C. albicans.
General Procedure for the Synthesis of 2-(5-(4-(4-X-
The study of both the synthesis condition and structural and Phenylsulfonyl)phenyl)-2H-1,2,4-triazol-3-ylthio)-1-(4-fluo-
biological properties will be interesting both for ongoing and rophenyl)ethanone 2a–c Triazole 1 (5mmol) was dissolved
further researches on these heterocyclic classes.
in 160mL dimethylsulfoxide and to the solution obtained was
The promising results obtained for compounds 2b, c and added 2-bromo-4′-fluoroacetophenone (5mmol). The reaction
4b, c (MIC=8µg/mL) against B. cereus strain needs further mixture was stirred at room temperature for 10h, then it was
researches referring to the design of another structural models. poured onto crushed ice. The precipitate obtained was filtered
off, washed with water and finally with ethyl ether. The prod-
uct was recrystallized from ethanol (Fig. 1).
Conclusion
In summary, we synthesized and evaluated for their an-
1-(4-Fluorophenyl)-2-(5-(4-(phenylsulfonyl)phenyl)-2H-1,2,4-
timicrobial activity new heterocyclic condensed systems triazol-3-ylthio)ethanone 2a
with bridgehead nitrogen from thiazolo[3,2-b][1,2,4]triazole
mp=186–187°C; yield=89.8%; IR (KBr) cm−1: 3272m
and thiazolo[3,2-b][1,2,4]triazol-6(5H)-one class starting (NH), 3082 (CH), 2959, 2918 (CH2), 1680 (C=O), 1598 (C=N),
from some 4-(4-X-phenylsulfonyl)phenyl)-4H-1,2,4-triazole-3- 1507, 1449 (C=C), 1318, 1290, 1155 (SO2), 1107 (C-F);
thioles (X=H, Cl, Br) key intermediates. The target com- 1H-NMR (DMSO-d6) δ: 3.50 (1H, brs, NH); 4.93 (2H, s,
pounds from thiazolo[3,2-b][1,2,4]triazole class were syn- H-18); 7.40 (2H, t, J=8.8Hz, H-22, H-24); 7.63 (2H, brt,
thesized by cyclization of the S-alkylated 1,2,4-triazole J=7.6Hz, H-14, H-16); 7.70 (1H, tt, J=7.6Hz, J=1.6Hz, H-15);
intermediates (obtained from triazoles 1 with 2-bromo-4′- 7.98 (2H, dd, J=7.6Hz, J=1.6Hz, H-13, H-17); 8.06 (2H, d,
fluoroacetophenone) in sulfuric acid media. The thiazolo[3,2- J=8.5Hz, H-8, H-10); 8.11 (2H, d, J=8.5Hz, H-7, H-11); 8.13
b][1,2,4]triazol-ones were obtained by treatment of 1,2,4-tri- (2H, dd, J=8.8Hz, J=5.5Hz, H-21, H-25); 13C-NMR (DMSO-
azol-3-thioles with 4-fluorobenzaldehyde, chloroacetic acid d6) δ: 39.70 (C-18); 115.86 (d, J=22.1Hz, C-22, C-24); 126.84
and anhydrous sodium acetate, in presence of acetic acid and (C-8, C-10); 127.38 (C-7, C-11); 128.10 (C-6); 128.17 (C-13,
acetic anhydride. All the new compounds were confirmed by C-17); 129.82 (C-14, C-16); 131.42 (d, J=9.4Hz, C-21, C-25);
spectral techniques and elemental analysis. The results of the 132.60 (d, J=3.6Hz, C-20); 133.85 (C-15); 140.84 (C-12);
antimicrobial activity of the compounds synthesized indicated 141.50 (C-9); 153.57 (C-5); 156.92 (C-3); 162.25 (d, J=252.5Hz,
that the most active are thiazolo[3,2-b][1,2,4]triazol-6(5H)-ones C-23); 192.24 (C-19); ESI-MS, m/z (%): 454 [M+H]+; 436 (1)
4b, c and S-alkylated 1,2,4-triazoles 2b, c against B. cereus [M+H−H2O]+; 420 (2) [M+H−H2O−1/2O2]+; 392 (10)
(MIC=8µg/mL).
[M+H−HF−1/2O2−C2H2]+;
330
(28)
[C6H5SO2C6H4
TriazoleSCH2]+; 317 (35) [C6H5SO2C6H4TriazoleSH]+; 301 (21)
[C6H5SO2C6H4TriazoleSH−NH2]+; 244 (33) [C6H5SO2C6H4
Experimental
Chemistry All chemicals were of analytical reagent CNH]+; 217 (12) [C6H5SO2C6H4]+; 109 (100, BP) [C6H5S]+;
1
grade. The H-NMR and 13C-NMR spectra were recorded with Anal. Calcd for C22H16FN3O3S2: C, 58.26; H, 3.56; N, 9.25.
a Varian Gemini 300 BB instrument working at 300MHz for Found: C, 58.17; H, 3.64; N, 9.16.
1H and 75MHz for 13C, using dimethyl sulfoxide (DMSO)-
2-(5-(4-(4-Chlorophenylsulfonyl)phenyl)-2H-1,2,4-triazol-3-
d6 or CDCl3 with trifluoroacetic acid (TFA) as solvents and ylthio)-1-(4-fluorophenyl)ethanone 2b
tetramethylsilane (TMS) as internal standard. Chemical shifts
mp=177–179°C; yield=77.5%; IR (KBr) cm−1: 3274 (NH),
δ are reported in parts per million and coupling constant J 3076 (CH), 2966, 2913 (CH2), 1685 (C=O), 1598 (C=N), 1507,
values are in Hertz (Hz). The following abbreviations are used 1478 (C=C), 1320, 1286, 1160 (SO2), 1105 (C-F), 768 (C-Cl);
to explain the multiplicities: s (singlet), d (doublet), t (triplet), 1H-NMR (DMSO-d6) δ: 3.50 (1H, brs, NH); 4.93 (2H, s,
m (multiplet), dd (doublet of doublets), tt (triplet of triplets), H-18); 7.40 (2H, t, J=8.8Hz, H-22, H-24); 7.70 (2H, d,
br (broad). Melting points were taken with a Boetius appa- J=8.5Hz, H-14, H-16); 7.99 (2H, d, J=8.5Hz, H-13, H-17);
ratus and were uncorrected. The infrared absorption spectra 8.00–8.20 (6H, m, H-7, H-11, H-8, H-10, H-21, H-25);
(IR) were recorded on a Vertex 70 Bruker spectrometer using 13C-NMR (DMSO-d6) δ: 39.60 (C-18); 115.57 (d, J=21.8Hz,
potassium bromide pellets. Mass spectra were recorded on C-22, C-24); 126.68 (C-8, C-10); 127.56 (C-7, C-11); 128.09
a triple quadrupole mass spectrometer Varian 1200LC/MS/ (C-6); 129.30 (C-13, C-17); 131.09 (C-14, C-16); 131.17 (d,
MS with electrospray interface (ESI) coupled with a Varian J=9.8Hz, C-21, C-25); 131.96 (d, J=3.5Hz, C-20); 138.69
ProStar 240 SDM pump. The sample solution (2µg/mL in (C-15); 139.36 (C-12); 140.77 (C-9); 156.63 (C-3); 164.96 (d,
methanol–0.1% ammonia in water 10:1, v/v) was introduced, J=252.5Hz, C-23); 153.27 (C-5); 191.94 (C-19); ESI-MS, m/z
at a flow rate of 20µL/min, by direct infusion, in the ESI (%): 488 [M+H]+; 490 [M+H]+; 454 (21) [M+H−
interface. The instrument was operated in positive ions mode H2O−1/2O2]+; 456 (23) [M+H−H2O−1/2O2]+; 364 (54)
and the fragmentation of protonated molecular ion was per- [ClC6H5SO2C6H4TriazoleSCH2]+; 366 (57) [ClC6H5SO2C6H4

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TriazoleSCH2]+; 351 (18) [ClC6H4SO2C6H4TriazoleSH]+; 353 (C-16, C-18); 130.62 (C-6); 133.88 (C-17); 135.39 (C-8); 140.87
(21) [ClC6H4SO2C6H4TriazoleSH]+; 320 (60) [ClC6H4SO2C6H4 (C-14); 141.78 (C-11); 157.93 (C-2); 162.65 (d, J=246.8Hz,
Triazole+H]+; 322 (55) [ClC6H4SO2C6H4Triazole+H]+; 278 C-23); 163.95 (C-3a); ESI-MS, m/z (%): 436 [M+H]+; 295
(40) [ClC6H5SO2C6H4CNH]+; 280 (38) [ClC6H5SO2C6H4CNH]+; (100) [M+H−C6H5SO2]+; 152 (73) [FC6H4CHCS]+; Anal.
175 (63) [ClC6H5SO2]+; 177 (55) [ClC6H5SO2]+; 152 (100) Calcd for C22H14FN3O2S2: C, 60.67; H, 3.24; N, 9.65. Found: C,
[C7H4SO2]+; Anal. Calcd for C22H15ClFN3O3S2: C, 54.15; H, 60.56; H, 3.38; N, 9.73.
3.10; N, 8.61. Found: C, 54.04; H, 3.01; N, 8.74.
2-(5-(4-(4-Bromophenylsulfonyl)phenyl)-2H-1,2,4-triazol-3- thiazolo[3,2-b][1,2,4]triazole 3b
2-(4-(4-Chlorophenylsulfonyl)phenyl)-6-(4-fluorophenyl)-
ylthio)-1-(4-fluorophenyl)ethanone 2c
mp=239–240°C; yield=88.1%; IR (KBr) cm−1: 3087 (CH),
mp=186–188°C; yield=78.0%; IR (KBr) cm−1: 3268 (NH), 1604 (C=N), 1505, 1464, (C=C), 1325, 1278, 1159 (SO2), 1100
1
3085 (CH), 2974, 2917 (CH2), 1684 (C=O), 1598 (C=N), 1573, (C-F), 1159 (νSO2), 772 (C-Cl); H-NMR (DMSO-d6) δ: 7.43
1507, 1473 (C=C), 1321, 1288, 1160 (SO2), 1104 (C-F), 579 (2H, t, J=8.8Hz, H-22, H-24); 7.72 (2H, d, J=8.6Hz, H-16,
1
(C-Br); H-NMR (DMSO-d6) δ: 3.50 (1H, brs, NH); 4.93 (2H, H-18); 7.96 (1H, s, H-5); 8.02 (2H, d, J=8.6Hz, H-15, H-19);
s, H-18); 7.40 (2H, t, J=8.8Hz, H-22, H-24); 7.84 (2H, d, 8.11 (2H, d, J=8.5Hz, H-9, H-13); 8.29 (2H, dd, J=8.8Hz,
J=8.8Hz, H-14, H-16); 7.91 (2H, d, J=8.8Hz, H-13, H-17); J=5.2Hz, H-21, H-25); 8.32 (2H, d, J=8.5Hz, H-10, H-12);
8.06 (2H, d, J=8.8Hz, H-8, H-10); 8.12 (2H, d, J=8.8Hz, H-7, 13C-NMR (DMSO-d6) δ: 111.51 (C-5); 116.02 (d, J=21.8Hz,
H-11); 8.13 (2H, dd, J=8.8Hz, J=5.5Hz, H-21, H-25); C-22, C-24); 124.13 (d, J=3.2Hz, C-20); 127.36 (C-9, C-13);
13C-NMR (DMSO-d6) δ: 39.68 (C-18); 115.87 (d, J=22.0Hz, 128.26 (C-10, C-12); 128.74 (d, J=8.3Hz, C-21, C-25); 129.38
C-22, C-24); 126.91 (C-8, C-10); 128.10 (C-15); 128.27 (C-6); (C-15, C-19); 130.00 (C-16, C-18); 130.61 (C-6); 135.59 (C-8);
128.40 (C-7, C-11); 129.41 (C-13, C-17); 131.47 (d, J=9.5Hz, 138.99 (C-17); 139.69 (C-14); 141.27 (C-11); 157.93 (C-2);
C-21, C-25); 132.26 (d, J=3.6Hz, C-20); 132.94 (C-14, C-16); 162.63 (d, J=246.8Hz, C-23); 164.27 (C-3a); ESI-MS, m/z (%):
140.84 (C-12); 140.97 (C-9); 153.59 (C-5); 156.94 (C-3); 165.27 470 [M+H]+; 472 [M+H]+; 295 (49) [M+H−ClC6H4SO2]+;
(d, J=252.5Hz, C-23); 192.24 (C-19); ESI-MS, m/z (%): 532 175 (42) [ClC6H4SO2]+; 177 (38) [ClC6H5SO2]+; 152 (100, BP)
[M+H]+; 534 [M+H]+; 408 (100, BP) [BrC6H4SO2C6H4- [FC6H4CHCS]+; Anal. Calcd for C22H13ClFN3O2S2: C, 56.23;
TriazoleSCH2]+; 410 (100, BP) [BrC6H4SO2C6H4TriazoleSCH2]+; H, 2.79; N, 8.94. Found: C, 56.34; H, 2.88; N, 9.08.
395 (31) [BrC6H4SO2C6H4TriazoleSH]+; 397 (15) [BrC6H4SO2C6H4-
2-(4-(4-Bromophenylsulfonyl)phenyl)-6-(4-fluorophenyl)-
TriazoleSH]+; 322 (42) [BrC6H4SO2C6H4CNH]+; 324 (12) thiazolo[3,2-b][1,2,4]triazole 3c
[BrC6H4SO2C6H4CNH]+; 219 (22) [BrC6H4SO2]+; 221 (23)
mp=247–251°C; yield=93.0%; IR (KB) cm−1: 3084 (CH),
[BrC6H4SO2]+; Anal. Calcd for C22H15BrFN3O3S2: C, 49.63; H, 1603 (C=N), 1573, 1505, 1463, (C=C), 1325, 1277, 1160
1
2.84; N, 7.89. Found: C, 49.72; H, 2.91; N, 7.80.
(SO2), 1099 (C-F), 578 (C-Br); H-NMR (DMSO-d6) δ: 7.44
General Procedure for the Synthesis of 2-(4-(4-X-Phen- (2H, t, J=8.8Hz, H-22, H-24); 7.86 (2H, d, J=8.5Hz, H-16,
ylsulfonyl)phenyl)-6-(4-fluorophenyl)thiazolo[3,2-b][1,2,4]- H-18); 7.93 (2H, d, J=8.5Hz, H-15, H-19); 7.97 (1H, s, H-5);
triazole 3a–c To 100mL of concentrated sulfuric acid was 8.12 (2H, d, J=8.2Hz, H-9, H-13); 8.30 (2H, dd, J=8.8Hz,
added, on ice bath, S-alkylated 1,2,4-triazole 2 (2mmol). The J=5.5Hz, H-21, H-25); 8.33 (2H, d, J=8.2Hz, H-10, H-12);
mixture was stirred at 0°C for 3h and then, at room tempera- 13C-NMR (DMSO-d6) δ: 111.49 (C-5); 116.01 (d, J=21.7Hz,
ture, for 3h. The solution obtained was poured onto crushed C-22, C-24); 124.12 (d, J=3.2Hz, C-20); 127.37 (C-9, C-13);
ice, then precipitated obtained was filtered off, washed with 128.09 (C-17); 128.26 (C-10, C-12); 128.74 (d, J=8.3Hz, C-21,
water until pH ca. 7 and was recrystallized from ethanol (Fig. C-25); 129.40 (C-15, C-19); 130.61 (C-6); 132.94 (C-16, C-18);
2).
135.59 (C-8); 140.11 (C-14); 141.23 (C-11); 157.92 (C-2); 162.63
6-(4-Fluorophenyl)-2-(4-(phenylsulfonyl)phenyl)thiazolo- (d, J=246.8Hz, C-23); 163.87 (C-3a); ESI-MS, m/z (%): 514
[3,2-b][1,2,4]triazole 3a
[M+H]+; 516 [M+H]+; 295 (100) [M+H−BrC6H4SO2]+; 219
mp=259–261°C; yield=90.0%; IR (KBr) cm−1: 3080, 3062 (9) [BrC6H4SO2]+; 221 (22) [BrC6H4SO2]+; 155 (50) [BrC6H4]+;
(CH), 1602 (C=N), 1505, 1464 (C=C), 1322, 1277, 1158 (SO2), 157 (62) [BrC6H4]+; 152 (30) [FC6H4CHCS]+; Anal. Calcd for
1101 (C-F); H-NMR (DMSO-d6) δ: 7.44 (2H, t, J=8.8Hz, C22H13BrFN3O2S2: C, 51.37; H, 2.55; N, 8.17. Found: C, 51.22;
1
H-22, H-24); 7.65 (2H, br t, J=7.2Hz, H-16, H-18); 7.72 (1H, tt, H, 2.66; N, 8.33.
J=7.2Hz, J=1.7Hz, H-17); 7.97 (1H, s, H-5); 8.01 (2H, dd,
General Procedure for Synthesis of 2-(4-(4-X-Phenylsul-
J=7.3Hz, J=1.7Hz, H-15, H-19); 8.07 (2H, d, J=8.5Hz, H-9, fonyl)phenyl)-5-(4-fluorobenzylidene)thiazolo[3,2-b][1,2,4]-
H-13); 8.31 (2H, d, J=8.5Hz, H-10, H-12); 8.35 (2H, dd, triazol-6(5H)-one 4a–c A mixture of triazole 1 (3mmol),
J=8.8Hz, J=5.5Hz, H-21, H-25); 13C-NMR (DMSO-d6) δ: 4-fluorobenzaldehyde (3mmol), chloroacetic acid (4.5mmol),
111.49 (C-5); 116.04 (d, J=22.0Hz, C-22, C-24); 124.12 (d, anhydrous sodium acetate (0.4g) in 8mL glacial acetic acid
J=3.0Hz, C-20); 127.32 (C-9, C-13); 128.20 (C-10, C-12); and 6mL acetic anhydride was refluxed for 9h. The excess
128.80 (d, J=8.3Hz, C-21, C-25); 128.82 (C-15, C-19); 129.84 solvent evaporated under reduced pressure and a residue
was obtained. Petroleum ether was added to the residue and
Fig. 2. Structure of Compounds 3a–c
Fig. 3. Structure of Compounds 4a–c

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
699
the obtained solid was filtered and washed with water. The
Antimicrobial Assay The testing of antimicrobial activity
product was purified from CH2Cl2–petroleum ether (1:1, v/v) for the compounds synthesized was performed in vitro using
(Fig. 3). the broth microdilution method for detecting the MICs. For
5-(4-Fluorobenzylidene)-2-(4-(phenylsulfonyl)phenyl)- preparing the stock solutions, the compounds were dissolved
thiazolo[3,2-b][1,2,4]triazol-6(5H)-one 4a
in DMSO, at 2048µg/mL. The solvent has shown no antimi-
mp=269–272°C; yield=76.1%; IR (KBr) cm−1: 3098, 3059 crobial activity against the tested microbial strains. Series of
(CH), 1733 (C=O), 1612 (C=N), 1598, 1510 (C=C), 1311, binary dilutions of the tested compounds, from 1:2 to 1:1024,
1
1279, 1159 (SO2), 1105 (C-F); H-NMR (CDCl3+TFA) δ: 7.29 were performed in Mueller–Hinton broth, in 96-well plates
(2H, t, J=8.5Hz, H-23, H-25); 7.59 (2H, brt, J=7.5Hz, H-16, (Nunc, Denmark), in volume of 50µL broth per well.
H-18); 7.68 (1H, brt, J=7.5Hz, H-17); 7.72 (2H, dd, J=8.5Hz,
The protocol used for antimicrobial screening is accord-
J=5.2Hz, H-22, H-26); 8.00 (2H, brd, J=7.5Hz, H-15, H-19); ingly with other protocol presented by us previously.27) For
8.11 (2H, d, J=8.5Hz, H-9, H-13); 8.25 (2H, d, J=8.5Hz, H-10, testing the antibacterial activity, the initial inoculum of each
H-12); 8.36 (1H, s, H-20); 13C-NMR (CDCl3+TFA) δ: 117.65 bacterial strain was adjusted at 0.5 McFarland turbidity and
(d, J=22.3Hz, C-23, C-25); 121.46 (C-8); 127.96 (C-15, C-19); afterwards diluted to 1/100 in Mueller–Hinton broth. From
128.63 (C-9, C-13); 128.81 (C-10, C-12); 128.27 (d, J=3.4Hz, this diluted inoculum, 50µL were added in every well which
C-21); 130.04 (C-16, C-18); 134.06 (d, J=9.8Hz, C-22, C-26); contained the tested compounds and in the wells with the
134.73 (C-17); 139.44 (C-14); 143.46 (C-11); 143.74 (C-20); positive growth control, the last ones containing already 50µL
143.84 (C-5); 156.87 (C-2); 165.58 (d, J=257.3Hz, C-24); of compound-free broth. The wells containing the negative
166.92 (C-3a); 167.29 (C-6); ESI-MS, m/z (%): 464 [M+H]+; growth control (the sterility control) were filled in only with
436 (25) [M+H−CO]+; 323 (25) [M+H−FC6H4CHSH]+; 244 compound-free Mueller–Hinton broth (100µL). Then, the 96-
(38) [C6H5SO2C6H4CN+H]+; 152 (100) [FC6H4CHCS]+; Anal. well plates were sealed with sterile adhesive sheet, covered
Calcd for C23H14FN3O3S2: C, 59.60; H, 3.04; N, 9.07. Found: C, by a proper lid and incubated at 37°C for 24h. The MICs
59.94; H, 2.86; N, 8.80.
of the tested compounds were read after 20h by naked-eye
2-(4-(4-Chlorophenylsulfonyl)phenyl)-5-(4-fluorobenzylidene)- examination of the aspect of the growth controls and of the
thiazolo[3,2-b][1,2,4]triazol-6(5H)-one 4b
wells containing the compounds. The MIC was considered the
mp=266–269°C; yield=77.8%; IR (KBr) cm−1: 3084, 3073 lowest concentration of the tested compounds which was able
(CH), 1736 (C=O), 1614 (C=N), 1597, 1507, 1496 (C=C), to inhibit the bacterial growth, which was the concentration of
1315, 1280, 1159 (SO2), 1106 (C-F), 771 (C-Cl); 1H-NMR the compound in the last well (in each dilution series) showing
(CDCl3+TFA) δ: 7.29 (2H, t, J=8.2Hz, H-23, H-25); 7.54 (2H, no turbidity.
d, J=8.5Hz, H-16, H-18); 7.70 (2H, dd, J=8.2Hz, J=5.2Hz,
For the antifungal testing, the initial inoculum of each yeast
H-22, H-26); 7.93 (2H, d, J=8.5Hz, H-15, H-19); 8.09 (2H, d, strain was adjusted at 0.5 McFarland turbidity. Afterwards, it
J=8.5Hz, H-9, H-13); 8.26 (2H, d, J=8.5Hz, H-10, H-12); 8.32 was diluted in Sabouraud broth in order to obtain an inoculum
(1H, s, H-20); 13C-NMR (CDCl3+TFA) δ: 117.56 (d, J= with a microbial density of 1×105 colony forming units (CFU)/
22.3Hz, C-23, C-25); 121.81 (C-8); 128.27 (d, J=3.3Hz, C-21); mL of which 50µL were added in every well containing the
128.54 (C-9, C-13); 128.72 (C-10, C-12); 129.40 (C-15, C-19); tested compounds and in the wells with the positive growth
138.55 (C-14); 132.93 (C-16, C-18); 133.82 (d, J=9.7Hz, C-22, control, the last ones containing already 50µL of compound-
C-26); 140.82 (C-17); 141.26 (C-5); 142.82 (C-20); 143.30 free broth. The wells containing the negative growth control
(C-11); 156.52 (C-2); 165.32 (d, J=261.8Hz, C-24); 166.90 (the sterility control) were filled in only with compound-free
(C-3a); 167.05 (C-6); ESI-MS, m/z (%): 498 [M+H]+; 500 Sabouraud broth (100µL). Then, the 96-well plates were
[M+H]+; 470 (38) [M+H−CO]+; 472 (24) [M+H−CO]+; 152 sealed with sterile adhesive sheet, covered by a proper lid and
(100) [FC6H4CHCS]+; Anal. Calcd for C23H13ClFN3O3S2: C, incubated at 37°C for 24h. The MICs of the tested compounds
55.48; H, 2.63; N, 8.44. Found: C, 55.16; H, 2.77; N, 8.24.
were read after 24h by naked-eye examination of the aspect
2-(4-(4-Bromophenylsulfonyl)phenyl)-5-(4-fluorobenzylidene)- of the growth controls and of the wells containing the com-
thiazolo[3,2-b][1,2,4]triazol-6(5H)-one 4c
pounds. The MIC was considered the lowest concentration
mp=272–274°C; yield=78.3%; IR (KBr) cm−1: 3083 (CH), of the tested compounds which was able to inhibit the yeast
1738 (C=O), 1614 (C=N), 1598, 1507, 1497 (C=C), 1315, 1280, growth, which was the concentration of the compound in the
1
1160 (SO2), 1103 (C-F), 584 (C-Br); H-NMR (CDCl3+TFA) δ: last well (in each dilution series) showing no turbidity.
7.30 (2H, t, J=8.4Hz, H-23, H-25); 7.71 (2H, dd, J=8.4Hz,
In addition, for all 10 microbial strains, an inoculum control
J=5.3Hz, H-22, H-26); 7.73 (2H, d, J=8.6Hz, H-16, H-18); was performed by removing 10µL from the positive growth
7.86 (2H, d, J=8.6Hz, H-15, H-19); 8.10 (2H, d, J=8.4Hz, control wells (just after they had been inoculated), which were
H-9, H-13); 8.26 (2H, d, J=8.4Hz, H-10, H-12); 8.36 (1H, s, further diluted into 10mL of Mueller–Hinton broth when con-
H-20); 13C-NMR (CDCl3+TFA) δ: 117.65 (d, J=22.6Hz, C-23, troling the bacterial inoculum and of Sabouraud broth when
C-25); 128.21 (C-17); 121.46 (C-8); 128.31 (d, J=3.3Hz, C-21); controling the yeast inoculum. After vortexing, 100µL of this
128.63 (C-9, C-13); 128.87 (C-10, C-12); 129.42 (C-15, C-19); dilution were spread onto a Columbia blood agar plate or onto
133.96 (C-16, C-18); 134.02 (d, J=9.2Hz, C-22, C-26); 138.70 a Sabouraud agar plate, respectively, that were afterwards
(C-14); 143.16 (C-11); 143.63 (C-20); 143.73 (C-5); 156.82 (C-2); incubated for 24h, followed by counting the total number of
165.54 (d, J=257.3Hz, C-24); 166.29 (C-3a); 167.25 (C-6); ESI- CFU/mL.
MS, m/z (%): 542 [M+H]+; 544 [M+H]+; 514 (13) [M+H−
The following reference strains: E. coli ATCC 25922, P.
CO]+; 516 (9) [M+H−CO]+; 152 (100) [FC6H4CHCS]+; Anal. aeruginosa ATCC 27853 and S. aureus ATCC 25923 were
Calcd for C23H13BrFN3O3S2: C, 50.93; H, 2.42; N, 7.75. Found: tested against amikacin (for quality control) and C. parapsi-
C, 51.32; H, 2.63; N, 7.43.
losis ATCC 22019 was tested against fluconazole (as quality

700
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
paran B., Bioorg. Med. Chem., 23, 2518–2528 (2015).
control) by the same microdilution broth method. The MIC
value of amikacin was 2µg/mL for the 3 tested bacterial
strains and the MIC value of fluconazole was also 2µg/mL,
for the yeast reference strain. The investigation of the antimi-
crobial activity of the compounds was done in duplicate.
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Acknowledgments This paper is supported by the
Sectoral Operational Programme Human Resources De-
velopment, financed from the European Social Fund and
by the Romanian Government under the contract number
POSDRU/89/1.5/S/64109.
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Conflict of Interest The authors declare no conflict of
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