Synthesis, characterization and evaluation of antimicrobial and antituberculosis activities of new N-(substituted-thioureido)aminobicyclo dicarboximide and 3,4-disubstituted 1,2,4-triazolino-5-thione

Article abstract of DOI:10.1002/jccs.201300362

The novel N-(substituted-thioureido)aminobicyclo dicarboximide and 3,4-disubstituted 1,2,4-triazolino-5-thione were synthesized and their antimicrobial and antituberculosis activities were examined. Selected dicarboxylic acid anhydrides were used to obtai

Full text of DOI:10.1002/jccs.201300362

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis, Characterization and Evaluation of Antimicrobial and Antituberculosis
Activities of New N-(Substituted-thioureido)aminobicyclo Dicarboximide and
3,4-Disubstituted 1,2,4-Triazolino-5-thione
Anna Pachuta-Stec,a* Urszula Kosikowska,b Liliana Mazur,c Marta Swatko-Ossor,d
Monika Pitucha,a Anna Malmb and Graóyna Ginalskad
aDepartment of Organic Chemistry, Faculty of Pharmacy, Medical University, Chodïki 4a, 20-093 Lublin, Poland
bDepartment of Pharmaceutical Microbiology with Laboratory for Microbiological Diagnostics, Faculty of Pharmacy,
Medical University, Chodïki 1, 20-093 Lublin, Poland
cDepartment of General and Coordination Chemistry, Maria Curie-Sklodowska University, M. Curie-Sklodowska 3,
20-031 Lublin, Poland
dDepartment of Biochemistry and Biotechnology, Faculty of Pharmacy, Medical University, Chodïki 1, 20-093 Lublin,
Poland
(Received: Jul. 16, 2013; Accepted: Oct. 21, 2013; Published Online: Dec. 19. 2013; DOI: 10.1002/jccs.201300362)
The novel N-(substituted-thioureido)aminobicyclo dicarboximide and 3,4-disubstituted 1,2,4-triazolino-
5-thione were synthesized and their antimicrobial and antituberculosis activities were examined. Selected
dicarboxylic acid anhydrides were used to obtain derivatives of thiosemicarbazide and dicarboximide.
Dicarboximides were also cyclized in the presence of an alkaline solution giving 1,2,4-triazolino-5-
1
thione. The chemical structure of all compounds was confirmed by IR, H NMR, 13C NMR, the X-ray
crystallography (6, 11) and elemental analysis. Antimicrobial and antituberculosis activities of the deriva-
tives were measured.
Keywords: Dicarboximide; Triazole; Synthesis; Antimicrobial activity; Antituberculosis activity.
INTRODUCTION
Bacterial infection is an important medicinal problem
On the other hand, substituted 1,2,4-triazole and its open-
chain counterpart thiosemicarbazide are an important class
of heterocycles that also show antimicrobial activity.17-23
In the light of the above observations the purpose of
the present work was to design, synthesize and investigate
the in vitro antibacterial and antitubercular activities of
some novel thiosemicarbazides and of 1,2,4-triazoles.
all over the world. Among them are deadly epidemics and
common infections e.g. pneumonia, ear infections, diar-
rhea, and urinary tract infections. Even today tuberculosis
is one of the most dangerous infections. Infectious diseases
have been the cause of death of children and the elderly in
all countries. Such infections are very dangerous especially
to people with a weakened immune system. Recently, there
are new alarming cases of bacterial diseases caused by
drug-resistant microbial pathogens that are resistant to an-
tibiotics currently in use, including vancomycin.1-5 Such
cases must be treated with drugs which are less effective
and can cause serious side effects. Therefore one possible
solution of this problem is to search and synthesize novel,
highly effective and less toxic antimicrobial agents.6-11
It is well known that molecules including 1,2,4-tri-
azole have a wide range of therapeutic properties. Several
compounds, like Flutrox, Nefazodone, Trazodone, Letra-
zole, Vorozole, Voriconazole, Posaconazole and etc., are
used as drugs in modern medicine. Moreover 1,2,4-triazole
and its derivatives possess antifungal,12 antitubercular,13
anticancer,14 anticonvulsant,15 and anti-HIV16 properties.
RESULTS AND DISCUSSION
In this paper we reported the synthesis of N-(substi-
tuted-thioureido)aminobicyclo dicarboximide and 3,4-di-
substituted 1,2,4-triazolino-5-thione.
The synthetic strategies adopted to obtain the target
compounds are depicted in Scheme I and Scheme II.
As the starting material in the present study selected
dicarboxylic acid anhydrides were used. These compounds
reacted with 4-substituted-3-thiosemicarbazide24,25 in boil-
ing dry chloroform, giving linear derivatives of thiosemi-
carbazide (1, 2) in Scheme I and N-(substituted-thioureido)
aminobicyclo dicarboximide (5-7, 13) in Schemes I and II.
The obtained compounds were cyclized in alkaline media
to a new group of 3,4-disubstituted 1,2,4-triazolino-5-
thione (8-12, 14). In addition, the cyclization reactions of
* Corresponding author. E-mail: anna.pachuta@umlub.pl
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Pachuta-Stec et al.
fore.26,27 The spectral characterization of the compound we
synthesized were consistent with the published data.
Single-crystal X-ray analysis confirmed the struc-
tures and composition of 6 and 11. The molecular structure
of both compounds, along with the atom numbering scheme
and selected geometric parameters are given in Figs. 1 and
2. Compounds 6 and 11 crystallize in the centrosymmetric
P21/c and P-1 space groups, respectively with one mole-
cule in the asymmetric unit. As each of the molecules con-
tains four stereogenic centers (C3, C4, C6, C7), both en-
antiomeric forms (a racemic mixture) are present in the
studied crystals.
Reagents and conditions: (a) CHCl3, reflux,
1 h; (b) CH3COOH, H2SO4, room tempera-
ture, 1 h; (c) 2% NaOH, reflux, 2 h
Scheme I
The central thiosemicarbazide moiety in 6 is almost
planar with cis configuration of S1 with respect to the hy-
drazide N2 atom. All bond lengths are within normal
ranges for this unit.28,29 The dicarboximidonorbornene unit
Reagents and conditions: (a) CHCl3, re-
flux, 1 h; (c) 2% NaOH, reflux, 2 h
Scheme II
Fig. 1. Molecular structure of 6 with the atom-number-
ing scheme. Displacement ellipsoids are drawn
at the 30% probability level. Selected bond dis-
tances (Å): C1-N3 1.326(4), C1-N1 1.354(4),
C1-S1 1.658(3), Nl-N2 1.373(3), N2-C2
1.385(4), N2-C10 1.384(4), C2-O2 1.196(3),
C2-C3 1.488(4), C3-C7 1.535(4), C7-C10
1.493(4), C10-O1 1.203(3).
linear derivatives of thiosemicarbazide (1, 2) in a mixture
of concentrated sulfuric acid and glacial acetic acid were
carried out. It was found that N-(substituted-thioureido)
aminobicyclo dicarboximide (3, 4) was also obtained in the
last reaction Scheme I. Interestingly, in the case of exo-
7-oxa-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic anhy-
dride, the nucleophilic ring-opening occurred14 to give
3-(4-phenyl-5-thione-1,2,4-triazol-3-yl) propenoic acid
(12) (Scheme I).
Unfortunately, in the case of exo-7-oxa-bicyclo-
[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride, endo-bicy-
clo[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride and bicy-
clo[2.2.2]oct-7-ene-2,3,5,6-tetracarboxylic anhydride, the
attempts to obtain the linear thiosemicarbazides failed and
N-(substituted-thioureido)aminobicyclo dicarboximides
were formed directly.
Fig. 2. Molecular structure of 11 with the atom-num-
bering scheme. Displacement ellipsoids are
drawn at the 30% probability level. Selected
bond distances (Å): C1-N3 1.364(3), C1-N1
1.324(3), C1-S1 1.674(3), N1-N2 1.366(3),
N2-C2 1.298(3), N3-C2 1.378(3), C10-O2
1.223(3), C10-O 11.296(3).
N-(Phenyl-thioureido)aminobicyclo[2.2.1]hept-5-
ene dicarboximide (5) was obtained another way be-
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Dicarboximide and 1,2,4-Triazoline-5-thione
in 6 has its typical boat conformation (endo configura-
tion)30 with the dihedral angle of 120.4(1)° between the
dicarboximido fragment and the best-plane of norbornene
C3/C4/C6/C7 atoms. In the norbornene skeleton the flap-
ping angles of the five-membered envelope rings C3/C4/
C5/C6/C7 and C4/C5/C6/C8/C9 are 60.6(3)° and 51.6(3)°,
respectively. The planar dicarboximido moiety (r.m.s. de-
viation = 0.0096 Å) enclose a dihedral angle of 40.6(3)°
with the terminal phenyl ring plane. At the same time, the
above mentioned units form the dihedral angles of 84.6(3)°
and 68.3(3)° with the central thiosemicarbazide function,
respectively.
[N1…S1 = 3.419(2) Å, <N1H…S1 = 153°, (ii): x, 1/2-y,
1/2+z] and weak C-H…O/S and p…p contacts.
In crystal 11 the adjacent “head-to-head” arranged
molecules form centrosymmetric dimers through strong
O1-H1…O2(iii) [O1…O2 = 2.613(2) Å, <O1H…O2 =
168°, (iii): 1-x, 2-y, 1-z] hydrogen bonds. The dimers are
further connected by centrosymmetric N1-H1n···S1(iv) in-
teractions [N1…S1 = 3.255(2) Å, <N1H…S1 = 172°, (iv):
-x, 1-y, -z] into the “zig-zag” molecular chains, propagated
along [111] crystallographic direction. The interactions be-
tween adjacent chains are proceed mainly through weak
C-H…O/S contacts.
The cyclic derivative 11 exists in its thione tautomeric
form in the solid state, as supported by the C1-S1 distance
of 1.674(3) Å, which is much shorter than a typical single
C-S bond, as well as from the presence of hydrogen atom
on the triazole N1 atom. The bond lengths within the planar
triazole ring are not regular. The shortest C2-N2 distance
(1.298(3) Å) is indicative of significant double-bond char-
acter whereas all remaining bonds (Fig. 2) are of intermedi-
ate character. The conformation of the norbornene skeleton
in 11 is almost the same as in 6, with the flapping angles of
the five-membered rings of 61.4(4)° and 51.7(4)°. The two
norbornene substituents, namely carboxylic and triazole
functions, occupy axial positions with the dihedral angle
between their best-planes being of 51.0(2)°. The phenyl
ring is twisted from the plane of central heterocycle by
59.2(3)°.
The chemical character of obtained derivatives and
their substituents may determine the antimicrobial activity
against the broad spectrum of microorganisms - either
against Gram-positive pathogenic bacteria and opportunis-
tic bacteria or against opportunistic fungi belonging to
Candida spp.
The inhibitory effect in vitro of thiosemicarbazide,
N-(substituted-thioureido)aminobicyclo dicarboximide
and 3,4-disubstituted 1,2,4-triazolino-5-thione derivatives
against the growth of Gram-positive and Gram-negative
bacteria or Candida albicans belonging to yeasts was pre-
liminary evaluated using the agar dilution technique with
1000 mg/mL concentration of the compounds and next, for
potentially active compounds, by broth microdilution tech-
nique. Among all tested compounds, six compounds showed
potential antimicrobial activity. Inhibitory effect for four of
the tested derivatives (2, 4, 6 and 7) on the growth of
Gram-positive bacteria showed MIC = 125 - >1000 mg/mL
(Table 1). The most sensitive Gram-positive species were
M. luteus ATCC 10240 (MIC = 125 – 500 mg/mL) or S.
epidermidis ATCC 12228 and S. aureus ATCC 25923
(MIC = 250 – 500 mg/mL). Bacillus spp. strains (MIC =
125 - >1000 mg/mL) and methicillin-resistant S. aureus
The most characteristic feature of crystal 6 is the pres-
ence of extended molecular chains formed via strong hy-
drogen bonds between one of the NH groups and the car-
bonyl O1 atom of the next translation-related molecule
[N3-H3n···O1(i); D…A = 2.988(4) Å, <DH…A = 143°, (i):
x+1, y, z]. The additional stabilization of the 3D structure is
provided by strong N1-H1n···S1(ii) hydrogen bonds
Table 1. The influence of compounds 2, 4, 6 and 7 on the growth of Gram-positive and Gram-negative
bacteria on the basis of MIC (mg/mL) values in the broth microdilution method (OD600
)
Species Sa 25923 Sa 14001 Se 12228 Bs 6638 Bc 10876 Ml 10240 Ec 25922 Pm 12453 Pa 9027
2
4
6
7
500
500
250
250
1000
>1000
500
500
500
250
500
500
1000
500
>1000
>1000
1000
250
250
500
125
250
>1000
>1000
500
>1000
>1000
>1000
62.5
>1000
>1000
>1000
1000
1000
125
>1000
Abbreviations: Sa25923 - Staphylococcus aureus ATCC 25923, Sa14001 - Staphylococcus aureus
MICROBANK 14.001, Se12228 - Staphylococcus epidermidis ATCC 12228, Bs6638 - Bacillus subtilis
ATCC 6638, Bc10876 - Bacillus cereus ATCC 10876, Ml10240 - Micrococcus luteus ATCC 10240,
Ec25922 - Escherichia coli ATCC 25922, Pm12453 - Proteus mirabilis ATCC 12453, Pa9027 -
Pseudomonas aeruginosa ATCC 9027
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MICROBANK 14.001 (MIC = 500 - >1000 mg/mL) were
less sensitive to the tested derivatives. Furthermore, two in-
vestigated compounds showed inhibitory effect against the
growth of Gram-negative bacteria against E. coli ATCC
25922 (MIC = 500 mg/mL) for derivative 6, while deriva-
tive 7 was active against P. mirabilis ATCC 12453 (MIC =
62.5 mg/mL) and P. aeruginosa ATCC 9027 (MIC = 1000
mg/mL).
karyotic (bacteria) and eukaryotic (yeasts) cells.
Antituberculosis activity of the synthesized com-
pounds was screened using the broth dilution method for
determination of the Minimum Inhibitory Concentration
(MIC) to inhibit the growth of microorganisms. The MIC
was defined as the lowest concentration which inhibited
the growth of microorganisms judged by lack of turbidity
in the tube.31
In addition, compounds (2-7) were active against
strains of yeasts. These compounds had incubation-time
dependent activity against both reference strains and clini-
cal isolates of C. albicans. MIC of compounds 2, 4, 6 and 7
after 24 h incubation ranged from 15.63 to 1000 mg/mL,
while after 48 h incubation - from 62.5 to ³ 1000 mg/mL.
Derivatives 3 and 5 had showed inhibitory activity against
the growth of C. albicans only after 24 h incubation with
MIC = 62.5 - 1000 mg/mL (Table 2). In conclusion, only
linear derivative of thiosemicarbazide (2) and N-(substi-
tuted-thioureido)aminobicyclo dicarboximide (4, 6, 7)
containing phenyl group showed moderate activity against
both bacteria and fungi, while N-(substituted-thioureido)-
aminobicyclo dicarboximide (3, 5) containing ethyl substi-
tuent showed activity only against C. albicans. This may
suggest a presence of more than one mechanism of antimi-
crobial action of these derivatives – both against pro-
Based on the preliminary results, only two com-
pounds (7 and 12) among the tested agents showed moder-
ate activity against the tested mycobacteria. The MIC val-
ues of these compounds ranged from 256 to 512 mg/mL, re-
spectively These results are based on preliminary tests; fur-
ther biological studies in these groups of compounds are in
progress.
EXPERIMENTAL
Melting points (°C) were determined using Fischer-Johns
block and presented without any corrections. Elemental analyses
were performed on a Perkin-Elmer 2400 CHN Analyzer and the
data were within 0.4% of the theoretical values. IR spectra were
recorded in KBr on a Perkin-Elmer 1725X FTIR spectrometer.
NMR spectra (1H and 13C) were recorded on a Bruker Avance 300
MHz spectrometer in solution noted and with TMS as an internal
standard. Chemicals were purchased from Sigma-Aldrich or Lan-
Table 2. The influence of compounds 2-7 on the growth of Candida albicans on the basis of MIC
(mg/mL) values in the broth microdilution method (OD600
)
Incubation
time
Species
2
3
4
5
6
7
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
MIC24
MIC48
125
125
62.5
125
15.63 31.25
Candida albicans ATCC 2901
Candida albicans 1
Candida albicans 2
Candida albicans 3
Candida albicans 4
Candida albicans 5
Candida albicans 6
Candida albicans 7
Candida albicans 8
Candida albicans 9
1000 >1000 1000 >1000
500 1000 250 >1000
>1000 >1000 >1000 >1000
250 500 500 >1000
>1000 >1000 >1000 >1000 >1000 1000
15.63
500
125
250
500
500
62.5
125
500
250
125
>1000
500
15.63
500
125
62.5
>1000
>1000
250
250
125
500
500
500
250
250
125
500
250
125
500
250
500
125
>1000
250
1000
250
500
250
500
>1000 >1000 1000 >1000
500 500 1000 >1000
1000 >1000 >1000 >1000
500
500
250
>1000 >1000 >1000
500 500 1000
500
>1000
1000
>1000 >1000 >1000 >1000
125
500
500
62.5
250
62.5
>1000 >1000 >1000 1000
>1000 250 >1000 250
1000
250
250
>1000 >1000 >1000 >1000 1000
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Dicarboximide and 1,2,4-Triazoline-5-thione
caster Synthesis and used without further purification. The mass
spectra were obtained with an AMD-604 mass spectrometer a
70-eV electrons beam. The homogeneity of the obtained com-
pound was checked by TLC on aluminum oxide 60 F254 plates
(Merck) in a CHCl3/C2H5OH (10:1 and 10:2) solvent system with
UV or iodine visualization.
2.30-2.39 (m, 4H, 2CH2), 3.40 (s, 2H, 2CH), 5.89 (s, 2H,
CH=CH), 7.07-7.22 (m, 1H, p-Ph), 7.30-7.58 (m, 4H, o-Ph+m-
Ph), 10.05 (s, 2H, 2NH) ppm. 13C NMR (DMSO-d6) d: 22.80
(2CH2), 36.89 (2CH-CO), 124.78 (p-PhC), 127.37 (o-PhC),
128.48 (m-PhC+CH=CH), 138.51 (i-PhC), 176.79 (2CO), 180.93
(CS) ppm. MS (EI) m/z (%): 301 (11) [M+], 135 (100). Synthesis
of N-(substituted-thioureido)aminobicyclo dicarboximide
(5-7, 13): 10 mmol of selected dicarboxylic acid anhydride and
10 mmol or 20 mmol (for bicyclo[2.2.2]oct-7-ene-2,3,5,6-tetra-
carboxylic dianhydride) of appropriate 4-substituted-3-thio-
semicarbazide in dry chloroform were refluxed for 1 h. After
cooling the precipitate was filtered off and crystallized from etha-
nol or ethanol-water. N-(Ethyl-thioureido)aminobicyclo-
[2.2.1]-hept-5-ene dicarboximide (5): Yield: 73%, mp 196-198
oC. 1H NMR (DMSO-d6) d: 1.09 (t, 3H , J = 7.5 Hz, CH3), 1.62 (s,
2H, CH2), 3.32-3.41 (m, 2H, CH2CH3), 3.44 (s, 2H, 2CH), 3.46 (s,
2H, 2CH), 6.08 (s, 2H, CH=CH), 8.21 (s, 1H, NH), 9.73 (s, 1H,
NH) ppm. 13C NMR (DMSO-d6) d: 14.56 (CH3), 39.17 (CH2CH3),
44.17 (2CH), 44.8 (CHCH2CH), 51.53 (CHCH2CH), 133.14
(CH=CH), 173.31 (2CO), 179.31 (CS) ppm. N-(Phenyl-thio-
ureido)aminobicyclo[2.2.1]hept-5-ene dicarboximide (6):
Yield: 79%, mp 184-186 °C. 1H NMR (DMSO-d6) d: 1.57 (s, 2H,
CH2), 3.44 (s, 2H, 2CH), 3.46 (s, 2H, 2CH), 6.08 (s, 2H, CH=CH),
8.21 (s, 1H, NH), 9.73 (s, 1H, NH) ppm. 13C NMR (DMSO-d6) d:
14.56 (CH3), 39.17 (CH2CH3), 44.17 (2CH), 44.8 (CHCH2CH),
51.53 (CHCH2CH), 133.14 (CH=CH), 173.31 (2CO), 179.31
(CS) ppm. N-(Phenyl-thioureido)amino-7-oxabicyclo[2.2.1]-
hept-5-ene dicarboximide (7): Yield: 64%, mp 219-221 oC. 1H
NMR (DMSO-d6) d: 3.00 (s, 2H, 2CH), 5.21 (s, 2H, 2CH), 6.57 (s,
2H, CH=CH), 7.17-7.23 (m, 2H, m-Ph), 7.34-7.36 (m, 3H,
o-Ph+p-Ph), 9.35 (bs, 1H, NH), 10.33 (s, 1H, NH) ppm. 13C NMR
(DMSO-d6) d: 45.33 (2CH), 80.34 (CHOCH), 125.67 (p-PhC),
128.40 (o-PhC), 136.34 (m-PhC+CH=CH), 138.49 (i-PhC),
173.65 (2CO), 180.80 (CS) ppm. Bis N-(phenyl-thioureido)-
aminobicyclo[2.2.2]oct-7-ene-2,3,5,6-tetracarboximide (13):
Yield: 83%, mp 220-222 oC. 1H NMR (DMSO-d6) d: 3.33 (s, 4H,
4CH), 3.47 (s, 2H, 2CH) 6.38 (s, 2H, CH=CH), 7.08-7.36 (m, 4H,
m-Ph), 7.44-7.62 (m, 6H, o-Ph+p-Ph), 9.92 (bs, 2H, 2NH), 10.18
(s, 2H, 2NH) ppm. 13C NMR (DMSO-d6) d: 33.23 (2CH), 40.33
(4CH-CO), 125.62 (2 p-PhC), 128.45 (2 o-PhC), 130.90 (2
m-PhC+CH=CH), 138.43 (2 i-PhC), 174.18 (4CO), 180.69 (2CS)
ppm. Synthesis of 3,4-disubstituted 1,2,4-triazolino-5-thione.
Method A (8-12, 14): A solution of N-(substituted-thioureido)-
aminobicyclo dicarboximide 3-7, 13 (2 mmol) in 2% sodium hy-
droxide (5 mL) was refluxed for 2 h. After cooling, the reaction
mixture was filtered off and isolated solution was acidified with 3
M HCl. The precipitate was filtered off and crystallized from eth-
Synthesis of N4-substituted-N1-(2-carboxy-1,2,3,6-tetra-
hydrobenzoyl)thiosemicarbazide (1-2): 1.52 g (10 mmol) of
1,2,3,6-tetrahydrophtalic anhydride and 10 mmol of appropriate
4-substituted-3-thiosemicarbazide in dry chloroform were re-
fluxed for 1 h. After cooling the precipitate was filtered off and
crystallized from ethanol-water. N4-Ethyl-N1-(2-carboxy-
1,2,3,6-tetrahydrobenzoyl)thiosemicarbazide (1): Yield: 65%,
mp 158-159 oC. 1H NMR (DMSO-d6) d: 1.05 (t, 3H , J = 7.5 Hz,
CH3), 2.26 (s, 2H, CH2), 2.49 (s, 2H, CH2), 2.74 (s, 1H, CH), 3.02
(s, 1H, CH), 3.33-3.46 (m, 2H, CH2), 5.62 (s, 2H, CH=CH), 7.38
(s, 1H, NH), 9.15 (s, 1H, NH), 9.71 (s, 1H, NH), 12.48 (s, 1H,
COOH) ppm. 13C NMR (DMSO-d6) d: 17.70 (CH3), 25.38 (CH2),
26.70 (CH2), 38.22 (CH-CONH), 38.93 (CH2), 39.86 (CH-
COOH), 123.33 (CH=CH), 123.91 (CH=CH), 171.09 (CONH),
174.09 (COOH), 179.91 (CS) ppm. N4-Phenyl-N1-(2-carboxy-
1,2,3,6-tetrahydrobenzoyl)thiosemicarbazide (2): Yield: 86%,
mp 143-145 oC. 1H NMR (DMSO-d6) d: 2.31 (s, 2H, CH2), 2.49
(s, 2H, CH2), 2.84 (s, 1H, CH), 3.09 (s, 1H, CH), 5.65 (s, 2H,
CH=CH), 7.12-7.17 (m, 1H, p-Ph), 7.29-7.35 (m, 2H, m-Ph),
7.53-7.56 (m, 2H, o-Ph), 9.06 (s, 1H, NH), 9.64 (s, 1H, NH), 9.98
(s, 1H, NH), 12.53 (s, 1H, COOH) ppm. 13C NMR (DMSO-d6) d:
22.83 (CH2), 26.24 (CH2), 37.82 (CH-CONH), 39.77 (CH-
COOH), 124.58 (p-PhC), 124.86 (o-Ph2C), 125.31 (CH=CH),
128.11 (m-Ph2C), 138.92 (i-PhC), 172.67 (CONH), 175.80
(COOH), 180.50 (CS) ppm. MS (EI) m/z (%): 135 (100). Synthe-
sis of N-(substituted-thioureido)aminobicyclo(1,2,3,6-tetra-
hydrophthaloyl)dicarboximide (3-4): The thiosemicarbazide 1,
2 (2 mmol) was dissolved in a mixture of glacial acetic acid (4
mL) and concentrated sulfuric acid (2 mL). The solution was left
at room temperature for 1h, then poured into crushed ice and kept
for 2 h at room temperature. The precipitated solid was filtered off
and crystallized from ethanol. N-(Ethyl-thioureido)aminobi-
cyclo(1,2,3,6-tetrahydrophthaloyl)dicarboximide (3): Yield:
62%, mp 164-166 oC. 1H NMR (DMSO-d6) d: 1.11 (t, 3H , J = 7.5
Hz, CH3), 2.29-2.50 (m, 4H, 2CH2), 2.58 (s, 1H, CH), 3.41 (s, 1H,
CH), 3.46-3.51 (m, 2H, CH2), 5.93 (s, 2H, CH=CH), 8.28 (s, 1H,
NH), 9.70 (s, 1H, NH) ppm. 13C NMR (DMSO-d6) d: 14.29 (CH3),
23.12 (2CH2), 37.20 (CH2CH3), 38.90 (CH), 39.90 (CH), 127.68
(CH=CH), 177.32 (2CO), 181.38 (CS) ppm. N-(Phenyl-thio-
ureido)aminobicyclo(1,2,3,6-tetrahydrophthaloyl)dicarbox-
imide (4): Yield: 82%, mp 164-166 oC. 1H NMR (DMSO-d6) d:
J. Chin. Chem. Soc. 2014, 61, 369-376
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Pachuta-Stec et al.
anol. Method B (8, 9): A solution of N4-substituted-N1-(2-car-
boxy-1,2,3,6-tetrahydrobenzoyl) thiosemicarbazide 1, 2 (2
mmol) in 2% sodium hydroxide (5 mL) was refluxed for 2 h. After
cooling, the reaction mixture was filtered off and isolated solution
was acidified with 3 M HCl. The precipitate was filtered off and
crystallized from ethanol. 1-(4-Ethyl-1,2,4-triazol-5-thione-
3-yl)-cyclohex-4-ene-2-carboxylic acid (8): Yield: 58%, mp
204-206 oC. 1H NMR (DMSO-d6) d: 1.30 (t, 3H , J = 7.5 Hz, CH3),
3.02 (s, 1H, CH), 3.39 (s, 4H, 2CH2), 3.62 (s, 1H, CH), 4.07 (q,
2H, J = 7.5 Hz, CH2CH3), 3.44 (s, 2H, 2CH), 5.60 (d, 1H, J = 10.0
Hz, CH=CH), 5.79 (d, 1H, J = 10.0 Hz, CH=CH), 12.36 (bs, 1H,
NH), 13.49 (s, 1H, COOH) ppm. 13C NMR (DMSO-d6) d: 13.81
(CH3), 25.42 (CH2), 28.12 (CH2), 29.85 (CH-triazole) 38.52
(CH2CH3), 39.95 (CH-COOH) 123.46 (CH=CH), 126.54
(CH=CH), 153.64 (Ctriazole), 166.16 (COOH), 174.55 (CS) ppm.
1-(4-Phenyl-1,2,4-triazol-5-thione-3-yl)-cyclohex-4-ene-2-
carboxylic acid (9): Yield: 78%, mp 271-273 oC. 1H NMR
(DMSO-d6) d: 2.16-2.18 (m, 4H, 2CH2), 2.68-2.74 (m, 1H, CH),
3.19 (q, 1H, J = 5.2 Hz, CH), 5.52 (d, 1H, J = 10.1 Hz, CH=CH),
5.70 (d, 1H, J = 9.8 Hz, CH=CH), 7.37-7.39 (m, 2H, m-Ph),
7.52-7.63 (m, 3H, o-Ph+p-Ph), 12.31 (bs, 1H, NH), 13.65 (s, 1H,
COOH) ppm. 13C NMR (DMSO-d6) d: 25.13 (CH2), 26.61 (CH2),
29.97 (CH-triazole), 38.93 (CH-COOH) 123.22 (CH=CH),
125.91 (CH=CH), 128.47 (o-PhC), 129.60 (m-PhC+p-PhC),
133.95 (i-PhC), 153.38 (Ctriazole), 167.37 (COOH), 174.05 (CS)
ppm. MS (EI) m/z (%): 301 (100) [M+], 256 (50). 2-(4-Ethyl-
1,2,4-triazol-5-thione-3-yl)-bicyclo[2.2.1]hept-5-ene-3-car-
boxylic acid (10): Yield: 64%, mp 224-226 oC. 1H NMR
(DMSO-d6) d: 1.29 (t, 3H , J = 7.5 Hz, CH3), 1.43 (d, 1H, J = 7.5
Hz, CH2), 1.57 (d, 1H, J = 7.5 Hz, CH2), 3.22 (s, 2H, 2CH), 3.63
(q, 2H, J = 5.0 Hz, CH2CH3), 3.92-4.04 (m, 2H, 2CH), 6.19 (dd,
1H, J = 6.25, 3.75 Hz, CH=CH), 6.33 (dd, 1H, J = 6.25, 3.75 Hz,
CH=CH), 11.95 (bs, 1H, NH), 13.30 (s, 1H, COOH) ppm. 13C
NMR (DMSO-d6) d: 13.77 (CH3), 38.69 (CH2CH3), 38.91
(CH-triazole+CH-COOH), 46.45 (CH-CH2-CH), 47.21 (CH-
CH2-CH), 48.78 (CH2), 135.15 (CH=CH), 136.42 (CH=CH),
153.30 (Ctriazole), 163.88 (COOH), 171.23 (CS) ppm. 2-(4-
Phenyl-1,2,4-triazol-5-thione-3-yl)-bicyclo[2.2.1]hept-5-
COOH), 128.67 (o-Ph2C), 129.28 (m-Ph2C+p-PhC), 133.95
(i-PhC), 134.57 (CH=CH), 135.99 (CH=CH), 152.88 (Ctriazole),
166.94 (COOH), 172.59 (CS) ppm. 3-(4-Phenyl-1,2,4-triazol-
5-thione-3-yl)propenoic acid (12): Yield: 58%, mp 283-285 °C.
1H NMR (DMSO-d6) d: 6.34 (d, 1H, J = 8.9 Hz, CH=CH), 6.39 (d,
1H, J = 12.8 Hz, CH=CH), 7.36-7.53 (m, 2H, m-Ph), 7.56-7.66
(m, 3H, o-Ph+p-Ph), 12.99 (bs, 1H, NH), 14.29 (bs, 1H, COOH)
ppm. 13C NMR (DMSO-d6) d: 122.80 (CH=CH), 126.46 (p-PhC),
128.96 (CH=CH), 130.21 (o-Ph2C), 133.75 (m-Ph2C), 147.70
(i-PhC), 166.48 (Ctriazole), 169.30 (COOH), 172.23 (CS) ppm.
2,6-[Bis-(4-phenyl-1,2,4-triazol-5-thione-3-yl)]bicyclo[2.2.2]-
oct-7-ene-3,5-dicarboxylic acid (14): Yield: 57%, mp 287-289
1
oC. H NMR (DMSO-d6) d: 2.71-3.24 (m, 6H, 6CH), 6.21-6.30
(m, 1H, CH=CH), 6.36-6.41 (m, 1H, CH=CH), 7.28-7.30 (m, 4H,
m-Ph), 7.45-7.59 (m, 6H, o-Ph+p-Ph), 12.08 (bs, 2H, 2NH), 13.50
(s, 1H, COOH), 13.54 (s, 1H, COOH) ppm. 13C NMR (DMSO-d6)
d: 34.59 (2CH), 37.75 (2CH-triazole), 47.29 (2CH-COOH),
128.76 (2p-PhC), 129.52 (2o-PhC), 131.47 (2m-PhC+CH=CH),
133.76 (2i-PhC), 152.90 (2Ctriazole), 166.90 (2COOH), 174.34
(2CS) ppm. X-ray Crystallography: Single-crystal diffraction
data were measured at room temperature in the w/2q mode on an
Oxford Diffraction Xcalibur diffractometer with the graph-
ite-monochromated Mo-Ka radiation (l = 0.71073 Å). The pro-
grams CrysAlis CCD and CrysAlis Red32 were used for data col-
lection, cell refinement and data reduction. The structures were
solved by direct methods using SHELXS-9733 and refined by the
full-matrix least-squares on F2 using SHELXL-97.33 All non-hy-
drogen atoms were refined with anisotropic displacement param-
eters. The H-atoms bonded to N1 (6 and 11) and N3 atoms (6)
were found from the difference-Fourier maps and refined
isotropically. All remaining H-atoms were positioned geometri-
cally and allowed to ride on their parent atoms, with Uiso(H) = 1.2
Ueq(C, O).
Crystal data for 6: C16Hl5N3O2S, monoclinic, space group
P21/c, a = 6.561(2) Å, b = 22.816(4) Å, c = 9.771(5) Å, b =
95.49(4)°, V = l456.0(9) Å3; Z = 4; dcalc = 1.430 g cm-3; m = 0.233
mm-1; GooF = 0.997; data/restraints/parameters 3339/0/207 [Rint
= 0.034]; final R indices (I > 2s(I)): R1 = 0.0534, wR2 = 0.1022; R
indices (all data): R1 = 0.1554, wR2 = 0.1387; largest diff. peak and
hole: 0.26 and -0.28 e Å-3.
o
1
ene-3-carboxylic acid (11): Yield: 70%, mp 233-234 C. H
NMR (DMSO-d6) d: 1.27 (d, 1H, J = 8.8 Hz, CH2), 1.30 (d, 1H, J
= 8.8 Hz, CH2), 2.89 (dd, 1H, J = 9.6, 3.6 Hz, CH-CH2-CH), 3.03
(s, 1H, CH), 3.15 (s, 1H, CH), 3.29 (dd, 1H, J = 9.9, 3.0 Hz,
CH-CH2-CH), 6.09 (dd, 1H, J = 5.5, 2.9 Hz, CH=CH), 6.30 (dd,
1H, J = 5.5, 2.9 Hz, CH=CH), 7.34-7.49 (m, 2H, m-Ph), 7.52-7.59
(m, 3H, o-Ph+p-Ph), 11.94 (s, 1H, NH), 13.49 (s, 1H, COOH)
ppm. 13C NMR (DMSO-d6) d: 38.93 (CH-triazole), 46.12 (CH-
CH2-CH), 46.53 (CH-CH2-CH), 48.16 (CH2), 48.34 (CH-
Crystal data for 11: C16Hl5N3O2S, triclinic, space group
P-1, a = 7.311(5) Å, b = 10.446(6) Å, c = 11.389(6) Å, a =
111.24(4)°, b = 101.89(5)°, g = 94.05(5)°, V = 783.2(8) Å3; Z = 2;
dcalc = 1.329 g cm-3; m = 0.217 mm-1; GooF = 1.025; data/re-
straints/parameters 3588/0/203 [Rint = 0.037]; final R indices (I >
2s(I)): R1 = 0.0537, wR2 = 0.1092; R indices (all data): R1=
0.1315, wR2 = 0.1328; largest diff. peak and hole: 0.22 and -0.25 e
374
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J. Chin. Chem. Soc. 2014, 61, 369-376

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Dicarboximide and 1,2,4-Triazoline-5-thione
Å-3. Supplementary Materials: Crystallographic data for the
structures reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publi-
cations Nos. CCDC-929645 (6) and CCDC-929646 (11). Copies
of available materials can be obtained free of charge on applica-
tion to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
(+44) 1223-336-033;e-mail:data_request@ccdc.cam.ac.uk or
www:http//www.ccdc.cam.ac.uk/data_request/cif).
on the growth of the tested microorganisms MIC was estimated
by using the broth microdilution technique, according to Euro-
pean Committee on Antimicrobial Susceptibility Testing
(EUCAST) for bacteria36 or according to the Clinical and Labora-
tory Standards Institute (CLSI) standards M27-A337 and
EUCAST, Subcommittee on Antifungal Susceptibility Testing38
for yeasts, with some modifications. Microdilution technique was
developed using sterile 96-well microplates, which were inocu-
lated with a microorganism suspension of optical density of 0.5
McFarland standard. 2 µL of the suspension was put into 198 µL
of Mueller Hinton broth (for bacteria) or Mueller Hinton broth
buffered at pH 5.6 and supplemented with 2% glucose (for fungi)
containing two-fold dilution of the tested compound at final con-
centration from 7.82 to 1000 mg/mL. The medium without the
tested compound was used as negative control.
Antimicrobial Screening
The antimicrobial activity of the tested compounds was
screened using the panel of seven reference bacterial strains from
the American Type Culture Collection (ATCC) and one reference
methicillin-resistant Staphylococcus aureus (MRSA) Microbank
14.001 strain from the collection of National Medicines Institute
in Warsaw. Four Gram-positive strains of bacteria (Staphylococ-
cus aureus ATCC 25923, Staphylococcus epidermidis ATCC
12228, Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC
10240), and three Gram-negative strains of bacteria (Escherichia
coli ATCC 25922, Proteus mirabilis ATCC 12453, Pseudomonas
aeruginosa ATCC 9027) were included. Moreover, the yeasts be-
longing to Candida spp. - reference strain C. albicans ATCC 2091
and nine clinical isolates of C. albicans from the Department of
Pharmaceutical Microbiology of Medical University of Lublin
collection were used.
Reading of MIC (minimal inhibitory concentration) was
done after incubation (at 35 oC for 24 h for bacteria or at 35 oC for
24 or 48 h for yeasts); the optical density (OD600) measurements
were determined spectrophotometrically for microbial cultures in
broth medium. The MIC values, defined as the lowest concentra-
tion of a compound that inhibits a visible growth of the tested mi-
croorganisms,38 were determined after 24 h (for bacteria) or 24
and 48 h (for yeasts) by comparison with the growth in control
(compound free) medium. The blank control wells with two-fold
dilution of each of the tested compounds added to broth medium
(total volume - 200 µL) without the microbial suspension were in-
cubated under the same conditions. Antimycobacterial Screen-
ing: New compounds we screened for their antimycobacterial ac-
tivity in vitro against two strains of mycobacteria: Mycobacte-
rium smegmatis and Mycobacterium H37Ra, too.
Microbial suspensions were prepared in sterile 0.85% NaCl
with an optical density of 0.5 McFarland standard - 150 ´ 106
CFU/mL (CFU - colony forming units). All stock solutions of the
tested compounds were prepared in dimethyl sulfoxide (DMSO).
Mueller-Hinton agar (for bacteria) or Mueller-Hinton agar sup-
plemented with 2% glucose and buffered at pH 5.6 (for yeasts)
were used for examination of antimicrobial activity. The
Mueller-Hinton medium plus 2% glucose used instead of RPMI
1640 is readily available, possesses good batch-to-batch repro-
ducibility and supports the growth of most pathogenic yeasts.34
In the first step the in vitro antimicrobial potency all of the
tested compounds were screened by the agar dilution method on
the basis of the microbial growth inhibition on the medium to
which the tested compounds were added at a concentration of
1000 mg/mL, as described elsewhere.35 The medium with DMSO
at the final concentration and without the tested compounds
served as a negative control; no microbial growth inhibition was
observed. The plates were poured on the day of testing. 10 mL of
each microbial suspension was put onto prepared solid media.
The plates were pre-incubated at room temperature for 1-1.5 h,
Inocula for susceptibility testing were prepared from the
suspension of microorganisms stored on Löwenstein medium,
equivalent to a McFarland No. 1 standard. Turbidity of the sus-
pensions was measured using nefelometer BD Phoenix Spec.
Becton, Dickinson and Company USA. Then 12 µL of the cul-
tures were added to each vial containing the tested drug and were
incubated for 5 days at 37 ºC. The control was prepared in the
same manner as a vial containing drug and stored at 4 ºC. The an-
tibiotic Streptomycin was used as a standard.
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© 2014 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Chin. Chem. Soc. 2014, 61, 369-376