Synthesis, characterization, and tuberculostatic activity of novel 2-(4-nitrobenzoyl)hydrazinecarbodithioic acid derivatives

Article abstract of DOI:10.1007/s00706-011-0708-y

A series of novel S-esters of 2-(4-nitrobenzoyl) hydrazinecarbodithioic acid and S,S'-diesters of (4-nitrobenzoyl)carbonohydrazonodithioic acid were synthesized by reaction of 4-nitrobenzohydrazide and N-methyl- 4-nitrobenzohydrazide with carbon disulfide and alkyl halides in the presence of triethylamine. Novel 5-(4-nitrophenyl)- 1,3,4-oxadiazoles were also obtained. The structures were confirmed by IR, NMR, and mass spectroscopy, and by elemental analysis. All the compounds obtained were screened in vitro for their tuberculostatic activity. Promising preliminary results were obtained for some of the compounds. The crystal structure of the most active compound was determined. The Author(s) 2012.

Full text of DOI:10.1007/s00706-011-0708-y

Monatsh Chem (2012) 143:607–617
DOI 10.1007/s00706-011-0708-y
ORIGINAL PAPER
Synthesis, characterization, and tuberculostatic activity of novel
2-(4-nitrobenzoyl)hydrazinecarbodithioic acid derivatives
•
Katarzyna Gobis Henryk Foks Ewa Augustynowicz-Kopec
•
•
•
•
•
Agnieszka Napiorkowska Malgorzata Szczesio Andrzej Olczak
Marek L. Glowka
Received: 3 October 2011 / Accepted: 18 December 2011 / Published online: 24 January 2012
Ó The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract A series of novel S-esters of 2-(4-nitro-
benzoyl)hydrazinecarbodithioic acid and S,S0-diesters of
(4-nitrobenzoyl)carbonohydrazonodithioic acid were syn-
thesized by reaction of 4-nitrobenzohydrazide and N-methyl-
4-nitrobenzohydrazide with carbon disulfide and alkyl
halides in the presence of triethylamine. Novel 5-(4-nitro-
phenyl)-1,3,4-oxadiazoles were also obtained. The structures
were confirmed by IR, NMR, and mass spectroscopy, and by
elemental analysis. All the compounds obtained were
screened in vitro for their tuberculostatic activity. Promising
preliminary results were obtained for some of the com-
pounds. The crystal structure of the most active compound
was determined.
Introduction
Microorganisms of the Mycobacterium genus cause serious
infectious diseases. Tuberculosis is one of these [1]. In
recent years an increase in the incidence of that disease has
been observed, and not only in developing countries [2].
Tuberculosis remains a global problem [3]. Failure of the
therapy because of inadequately maintained treatment,
abuse of antimicrobial drugs, and discontinuation of the
treatment when symptoms disappear, favour the spread of
the disease [4]. Improperly conducted therapy results in the
appearance of resistant disease varieties, multidrug-resis-
tant tuberculosis (MDR-TB), and extremely drug-resistant
tuberculosis (XDR) [5]. The resistance of Mycobacterium
strains is the cause of the increased risk of tuberculosis
active form development and mortality in immunocom-
promised individuals, for example patients during
immunosuppressive therapy or HIV-positive patients [6].
For many years isonicotinic acid hydrazide (isoniazid)
has been one of the most commonly used anti-tuberculosis
chemotherapeutics [7]. Unfortunately, mycobacteria rap-
idly develop resistance to this drug. Nevertheless, the
isoniazid molecule is a leading structure for discovery of
new chemotherapeutic agents [8]. In the course of this
research many new compounds have been synthesized, for
example hydrazones [9] and arylhydrazones [10]. Some of
these have promising anti-tuberculosis activity [11].
Keywords Drug research ꢀ
5-(4-Nitrophenyl)-1,3,4-oxadiazole- ꢀ
Tuberculostatic activity ꢀ Crystal structure ꢀ
Structure–activity relationship
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-011-0708-y) contains supplementary
material, which is available to authorized users.
K. Gobis (&) ꢀ H. Foks
Department of Organic Chemistry,
Medical University of Gdansk, 107 Gen. Hallera Str.,
80-416 Gdansk, Poland
Our previous studies on the synthesis of new compounds
with antituberculous activity helped formulate the
hypothesis of the relationship between the tuberculostatic
activity of compounds in the isoniazid analog group and
the coplanar structure of their molecules. Among others
S-methyl and S,S0-dimethyl amino(pyridin-2-ylmethyl-
ene)carbonohydrazonodithioates have been synthesized.
These compounds had high tuberculostatic activity in vitro,
e-mail: kgobis@gumed.edu.pl
E. Augustynowicz-Kopec ꢀ A. Napiorkowska
Department of Microbiology, Institute of Tuberculosis
and Pulmonary Diseases, Warsaw, Poland
M. Szczesio ꢀ A. Olczak ꢀ M. L. Glowka
Institute of General and Ecological Chemistry,
Technical University of Lodz, Lodz, Poland
123

608
K. Gobis et al.
Results and discussion
O
NH2
N
SR
SR
N
SR
N
H
N
H
Chemistry
N
N
S
The synthetic route of the target compounds is outlined in
Schemes 1 and 2. First, symmetrical S,S0-diesters of 4-nitro-
benzoylocarbonohydrazonodithioic acid and N-methyl-4-
nitrobenzoylcarbonohydrazonodithioic acid (1–9) were
synthesized. Modification within the thioxyl group gave
methyl, isopropyl, n-butyl, n-decyl, and benzyl S,S0-diesters.
Substituents were chosen to differ substantially in size. Esters
of 1,3-dithiolane and 1,3-dithiane with cyclic structures were
also obtained (11–14, Scheme 1).
Fig. 1 Intramolecular hydrogen bonds formed in molecules of S,S0-
diesters of picolinoylcarbonohydrazonodithioic acid and amino(pyr-
idine-2-yl)methylenecarbonohydrazonodithioic acid
O
O
N
SCH3
SCH3
N
SCH3
SCH3
N
H
N
H
O2N
NO2
4-Nitrobenzoyl chloride and methyl 4-nitrobenzoate
were the starting compounds for synthesis of all the
derivatives obtained. Methyl 4-nitrobenzoate in dioxane
under the action of hydrazine hydrate formed 4-nitro-
benzohydrazide [15]. N-Methyl-4-nitrobenzohydrazide was
obtained by reaction of 4-nitrobenzoyl chloride with a
twofold excess of N-methylhydrazine in anhydrous
dichloromethane [16]. Linear S,S0-diesters 1–9 and cyclic
diesters 11–14 were then synthesized from 4-nitro-
benzohydrazide and N-methyl-4-nitrobenzohydrazide. The
reactions were conducted in methanol with a twofold
excess of triethylamine (TEA). The products obtained by
addition of carbon disulfide were alkylated with a twofold
excess of the corresponding halides (methyl iodide, iso-
propyl iodide, n-butyl iodide, benzyl chloride, n-decyl
iodide) or an equimolar amount of dihalides (1,2-dibro-
moethane, 1,3-dibromopropane). The reactions proceeded
rapidly at room temperature and generally afforded the
desired products in good yields (50–96%).
A
B
Fig. 2 Structures of dimethyl 2-nitrobenzoylcarbonohydrazonodithio-
ate (a) and dimethyl 4-nitrobenzoylcarbonohydrazonodithioate (b)
with MIC (minimum concentrations inhibiting the growth
of mycobacteria) of 3.13 lg/cm3 [12, 13]. Crystallographic
studies performed on these derivatives indicated their
molecules have a planar structure. The adoption of such a
structure is a result of intramolecular hydrogen bond for-
mation (Fig. 1). This condition is also met by the S-esters
and S,S0-diesters of benzoylhydrazinecarbodithioic acids
derived from benzohydrazides, especially those that have
the appropriate substituents at C-2 position of the benzene
ring. For some of the compounds in this group high
tuberculostatic activity in vitro has been detected [14].
Unexpectedly, we found no correlation between the almost
planar structure of a derivative with an NO2 group in C-2
position and its activity towards Mycobacterium tubercu-
losis. The structure of the most active compound with an
NO2 group in C-4 position has no ability to form a
hydrogen bond and adopt a planar structure (Fig. 2). That
compound was the most active against H37Rv standard
strain and sensitive 192 strain (MICs 3.1 lg/cm3).
Its activity against resistant 210 strain was lower (MIC
25 lg/cm3).
In this work 2-(4-nitrobenzoyl)hydrazinecarbodithioates
and (4-nitrobenzoyl)carbonohydrazonodithioates have been
the synthesized for further verification of the hypothesis that
planarity of hydrazinecarbodithioic acid derivatives corre-
lates with their high tuberculostatic activity. We also
expanded the synthesized group by preparing S,S0-diesters of
N-methyl-4-nitrobenzoylcarbonohydrazonodithioic acid. In
their structure, spatial hindrance by a methyl group attached
to the nitrogen atom of the carbonohydrazonodithioic moiety
prevents intramolecular hydrogen bond formation and, in
accordance to our hypothesis, these compounds should have
rather low tuberculostatic activity.
The interaction of 4-nitrobenzohydrazide with CS2
and isopropyl iodide in the presence of TEA did not lead
to formation of diisopropyl 4-nitrobenzoylcarbonohy-
drazonodithioate, as is apparent from the absence of C=O
and NH vibrations in the IR spectrum. In addition, in the
1H NMR spectrum a signal suggesting the presence of the
NH group was not observed and results from integration
were indicative of half the number of alkyl protons. By
means of elemental analysis and mass spectrometry, the
chemical formula C11H11N3O3S was suggested and MW
265 was determined; these correspond to cyclic 2-(iso-
propylthio)-5-(4-nitrophenyl)-1,3,4-oxadiazole (10). Such
cyclization of methyl hydrazinecarbodithioates to
2-(methylthio)-1,3,4-oxadidiazoles in alkaline solution is
known and has been already described [17, 18].
S-Esters 15–17 were also obtained from 4-nitro-
benzohydrazide (Scheme 2). Reactions were carried out in
a methanol solution of TEA using the corresponding
iodides. For methyl ester 15 slightly less methyl iodide was
used to prevent formation of the S,S0-diester, because of
easy methylation [19]. In the synthesis of S-isopropyl ester
123

Synthesis, characterization, and tuberculostatic
609
Scheme 1
O
O
O
NH2
NH2NH2
CH3NHNH2
O2N
OCH3
N
R1
Cl
O2N
O2N
R1: H, Me
2 Et3N / CS2
2 R2X, X = I or Cl
2 Et3N / CS2
(CH2)nBr2
O
SR2
SR2
S
O
N
for R1 = H
2 Et3N / CS2
N
(CH2)n
N
R1
N
S
2 i-Pr
R1
O2N
O2N
1 9
-
11 14
-
3
7
9
2
4
5
6
8
1
11 12 13 14
R1
R2 Me
H
H
Me Me Me
Me
Me
Bn
R1
n
H
H
H
0
H
1
Me Me
0
1
n-Bu n-C10H21 Bn
i-Pr n-Bun-C10H21
Me
N N
S-i-Pr
O
O2N
10
Et3N / R2X,
X = I or Cl
O
Scheme 2
O
SMe
SR2
S
NHNH
SR1
NHN
15
for
O2N
19 20
O2N
15-17
19-22
Et3N / CS2
R1I or 1/2 Me2SO4
21
22
17
15
R1 Me n-Bu
R2 i-Pr n-Bu
n-C10H21
Bn
O
NHNH2
O2N
Et3N / CS2
i-PrI
Et3N / CS2
O
N
NH
N
NH
S
S
S
NHNH
S
O
O
i-Pr
O2N
O2N
O2N
16a
16
n-BuI
N
N
S-n-Bu
O
O2N
18
16 a side product 16a was also formed, as is apparent from
the absence of C=O vibrations and the presence of NH
5-(4-nitrophenyl)-1,3,4-oxadiazole-2(3H)-thione described
elsewhere by Chau [20]. That conclusion was confirmed by
the measured melting point of 204 °C. We have already
reported cyclization of methyl hydrazinecarbodithioates to
1,3,4-oxadiazole-2(3H)-thiones [21]. 5-(4-Nitrophenyl)-1,3,4-
oxadiazole-2(3H)-thione was unsuccessfully alkylated with
isopropyl iodide in a methanol solution of TEA, affording
1,3,4-oxadiazole 10. After 2 days of stirring, both at room
1
vibrations in the IR spectrum of that product. In the H
NMR spectrum no signals of the isopropyl group were
observed, and the singlet of the NH group was detected at
13.92 ppm. By means of elemental analysis and mass spec-
trometry, the chemical formula C8H5N3O3S was suggested
and MW 223 was determined; these correspond to cyclic
123

610
K. Gobis et al.
temperature and under reflux, no 2-(isopropylthio)-5-(4-
nitrophenyl)-1,3,4-oxadiazole was found in the reaction
mixture. Alkylation in the presence of KOH, as strong base,
in absolute ethanol was also attempted. The desired product
was obtained in a yield of 25% only. The moderate efficiency
of 5-phenyl and 5-pyridyl-1,3,4-oxadiazole-2(3H)thione
alkylation (30–40%) with isopropyl iodide has been descri-
bed by Muhi-eldeen and co-workers [22]. Other authors have
conducted that type of alkylation in dioxane containing
KOH, KI, Bu4NBr, and the corresponding halide [23, 24].
They also recommended addition of a catalytic amount of
crown ether (18-crown-6 or 15-crown-5). It is, therefore,
concluded that isopropyl 2-(4-nitrobenzoyl)hydrazinecar-
bodithioate (16) at room temperature readily cyclizes to
2-(isopropylthio)-5-(4-nitrophenyl)-1,3,4-oxadiazole (10),
whereas 5-(4-nitrophenyl)-1,3,4-oxadiazol-2(3H)-thione
undergoes alkylation with a secondary halide, i.e. isopropyl
iodide, with unsatisfactory yield. Easy cyclization of S-iso-
propyl ester 16 prevents alkylation of the second sulfur atom
in the synthesis of a diisopropyl S,S0-ester. This method
seems more useful for obtaining 2-(isopropylthio)-5-(4-
nitrophenyl)-1,3,4-oxadiazole (10).
oxadiazoles 10 and 18 were examined in vitro for their
tuberculostatic activity against M. tuberculosis H37Rv strain
and two ‘‘wild’’ strains isolated from tuberculosis patients:
one (spec. 210) resistant to p-aminosalicylic acid (PAS),
isonicotinic acid hydrazide (INH), etambutol (ETB), and
rifampicine (RMP), and the another (spec. 192) fully sensi-
tive to the administered tuberculostatics (Table 1).
The results from measurement of tuberculostatic activity
indicated that some of the title compounds had moderate
activity against tested strains in vitro and were less active
than isoniazid (INH) but of comparable activity to pyra-
zinamide (PZA), used as reference drugs. The MIC values
ranged from 25 to 100 lg/cm3 for most of the compounds
tested, from 0.5 to 1.1 lg/cm3 for INH, and from 25 to
40 lg/cm3 for PZA. There were no differences in sensi-
tivity to the tested compounds between sensitive 192 and
resistant 210 strain. Dimethyl 4-nitrobenzoylcarbonohy-
drazonodithioate (1) had the highest tuberculostatic activity
in vitro among all the S-esters and symmetrical S,S0-
Table 1 In-vitro tuberculostatic activity of synthesized compounds
1–3, 5–7, and 10–22
Incontrastwith the 1,3,4-oxadiazolederivative10 itsbutyl
analogue 18 was obtained easily in methanol by addition of
carbon disulfide to 4-nitrobenzohydrazide in the presence of
TEA. The reaction was conducted at room temperature for
24 h. The resulting 1,3,4-oxadiazole-2(3H)-thione was then
alkylated with n-butyl iodide without isolation.
No.
MIC/lg cm-3
H37Rv
Spec. 192
Spec. 210
1
25
12.5
50
50
50
50
50
50
100
25
50
50
50
50
50
50
50
25
25
50
50
50
1.1
40
2
50
3
50
50
Methyl S-ester 15 when treated with the respective
halides in a methanol solution of TEA gave unsymmetrical
S,S0-diesters 19–22 with different substituents at both sulfur
atoms. Introduction of different substituents on sulfur
atoms would provide a greater variety of compound
structures and enable determination of the effect of sub-
stituent size on the tuberculostatic activity of these
derivatives.
5
50
50
6
50
50
7
100
12.5
50
100
6.2
25
10
11
12
13
14
15
16
17
18
19
20
21
22
INH
PZA
50
50
100
50
50
50
The newly synthesized compounds were characterized
by IR, NMR, and mass spectroscopy, and elemental anal-
ysis. Results from spectral analysis were in accordance
50
25
50
25
1
with the assigned structures. In the H NMR spectra of
50
25
compounds 2, 4, 5, 11, and 13 the alkyl groups of the
thioalkyl moieties were detected as separate signals. For
example, for compound 5 two singlets of two methyl
groups were detected at chemical shifts 2.32 and 2.49 ppm.
This indicates the magnetic inequivalence of both groups.
We have already observed a similar phenomenon for other
dimethyl benzoylcarbonohydrazonodithioates [14].
6.2
25
6.2
12.5
6.2
12.5
12.5
0.5
25
25
25
25
0.5
25
Minimum inhibitory concentrations for the bacterial strains were
determined by the twofold serial dilution method for microdilution
plates, and for the mycobacterial strains by the twofold classical test-
tube method of successive dilution
Tuberculostatic activity
The newly synthesized 4-nitrobenzoylcarbonohydrazono-
dithioates 2–9, 11–14, and 19–22, 2-(4-nitrobenzoyl)hy-
drazinecarbodithioates 15–17, and 5-(4-nitrophenyl)-1,3,4-
INH, isoniazid; PZA, pyrazinamide
M. tuberculosis H37Rv, spec. 192, spec. 210
123

Synthesis, characterization, and tuberculostatic
611
Fig. 3 The molecular structure
of 18, showing the atom-
numbering scheme.
Displacement ellipsoids are
drawn at the 50% probability
level
diesters obtained. The MIC values for compound 1 were 25
(H37Rv), 12.5 (spec. 192), and 50 lg/cm3 (spec. 210).
Compound 19, i.e. isopropyl methyl ester, was the most
active in the group of unsymmetrical S,S0-diesters. The MIC
values determined for that compound were 12.5 and 25 lg/
cm3. Given that all S,S0-symmetric diesters had rather weak
tuberculostatic activity, the size of substituents at sulfur
atoms seemed to be important for the activity—at least one
should be small in volume. These results enabled inclusion
of the obtained S-esters and S,S0-diesters of 4-nitrobenzoyl-
carbonohydrazonodithioic acid among the compounds with
moderate tuberculostatic activity comparable with that of
clinically used PZA (MICs 25–40 lg/cm3).
Table 2 Crystal data and details of structure determination for
compound 18
Formula
C12H13N3 O3S
279.32
Formula weight
Crystal system
Space group
Monoclinic
P21/c
˚
a/A
14.6575(4)
10.0627(2)
8.8996(2)
92.665(1)
1311.22(5)
4
˚
b/A
˚
c/A
b/°
3
˚
V/A
Z
Calculated density/g cm-3
1.415
5-(4-Nitrophenyl)-1,3,4-oxadiazoles 10 and 18 had the
highest activity among the group of compounds tested. The
MIC values determined for these compounds were
6.2–12.5 lg/cm3 against sensitive strain 192 and the stan-
dard H37Rv strain, and 25 lg/cm3 against resistant 210
strain. These results indicated that 1,3,4-oxadiazoles have
antimycobacterial activity in vitro, as reported in the lit-
erature [25, 26]. Therefore, further studies of this group of
compounds seem appropriate.
F(000)
584
˚
Radiation/A
1.54178
No of reflections: measured, independent
Rint
13846, 2398
0.029
Observed reflections [I [ 2.0 r(I)]
No of parameters refined
R, wR2, S
2237
173
0.0467, 0.1406, 1.09
-0.20, 0.34
-3
˚
Min. and max. residual density/e A
Molecular structure of compound 18 in the crystal
Unexpectedly, two cyclic products were obtained. 2-(Iso-
propylthio)-5-(4-nitrophenyl)-1,3,4-oxadiazole was obtained
instead of the diisopropyl ester and 5-(4-nitrophenyl)-1,3,4-
oxadaizole-2(3H)-thione was synthesized in addition to the
S-isopropyl ester. Because of the easy cyclization, syn-
thesis of 2-(butylthio)-5(4-nitrophenyl)-1,3,4-oxadiazole
was conducted. The structures of the new compounds were
confirmed by IR, NMR, and mass spectroscopy, and by
elemental analysis. Their tuberculostatic activity was
evaluated against M. tuberculosis H37Rv standard strain,
sensitive strain 192, and 210 strain resistant to commonly
administrated drugs (PAS, INH, ETB, RMP). MIC values
were determined by use of the twofold serial dilution
technique. The results showed that most of the synthesized
derivatives had moderate tuberculostatic activity in vitro.
X-ray diffraction was performed for the most active 1,3,4-
oxadiazole 18. Its molecular structure is shown in Fig. 3.
Crystal data, details of data collections, reduction, and
refinement are shown in Table 2.
˚
Partial stacking (with interplanar distance \3.5 A) is
observed for the planar fragments of the molecules,
resulting in a semi-layered architecture of the crystal with
butyl groups fitting between the layers (Fig. 4). The only
other packing forces present in the structure are weak
intermolecular H11B–C11ꢀꢀꢀN3 contacts.
In summary, a series of novel S-esters and S,S0-diesters
of 4-nitrobenzoylcarbonohydrazonodithioic acid and 5-
(4-nitrophenyl)-1,3,4-oxadiazoles with different alkyl
moieties on sulfur atoms were synthesized successfully.
123

612
K. Gobis et al.
General procedure for synthesis of symmetrical
S,S0-diesters 1–9
4-Nitrobenzohydrazide or N-methyl-4-nitrobenzohydrazide
(2.5 mmol) was suspended in 5 cm3 methanol and
0.87 cm3 TEA (6.25 mmol) and 0.23 cm3 CS2
(3.75 mmol) were added. The mixture was stirred at room
temperature for 1 h. Then 6.25 mmol of the respective
halide was added and the mixture was stirred for another
2 h. Then 20 g ice was added, and the precipitate of the
dimethyl ester (1, 5) was isolated by filtration, dried, and
recrystallized from methanol. All other products were
extracted from the methanol–water solution with chloro-
form (3 9 10 cm3), the extract was dried with MgSO4, and
the solvent was evaporated under vacuum. Crude products
6–9 were treated with dry diethyl ether, isolated by filtra-
tion, dried, and recrystallized from methanol. Products 2–4
were purified by column chromatography.
Fig. 4 The molecular packing of 18 in the crystal, viewed perpen-
dicular to the b axis
The most active compounds were 1,3,4-oxadiazoles 10 and
18. The MIC values determined for those compounds
ranged from 6.2 to 25 lg/cm3. X-ray diffraction was per-
formed for 1,3,4-oxadiazole 18. These results enabled
inclusion of the 5-(4-nitrophenyl)-1,3,4-oxadiazoles
obtained among compounds with promising activity
against Mtb.
4-Nitrobenzoic acid 2-[bis(butylthio)methylene]hydrazide
(2, C16H23N3O3S2)
This product was purified by column chromatography with
chloroform–cyclohexane (2:1) as eluent. The compound
was obtained as an orange solid in 75% yield (0.69 g).
M.p.: 58–60 °C; IR (KBr):vꢀ = 3,253 (m N–H), 2,958,
2,928, 2,873 (m C–H), 1,648 (m C=O), 1,545, 1,527 (m NO2),
1,484 (m C=C), 1,341, 1,329 (d C–H), 850 (m C–N), 707 (c
C–C) cm-1; 1H NMR (200 MHz, CDCl3): d = 0.82 (t, 3H,
CH3, J = 7 Hz), 0.94 (t, 3H, CH3, J = 7 Hz), 1.26–1.38
(m, 2H, CH2), 1.42–1.49 (m, 4H, 2 CH2), 1.62–1.66 (m,
2H, CH2), 2.90 (t, 2H, SCH2, J = 7.3 Hz), 3.03 (t, 2H,
SCH2, J = 7.3 Hz), 8.10 (d, 2H, Ph, J = 8.8 Hz), 8.17 (d,
2H, Ph, J = 8.8 Hz), 9.12 (s, 1H, NH) ppm; 13C NMR
(50 MHz, CDCl3): d = 14.03 (CH3), 22.41 (CH2), 31.21
(SCH2), 32.73 (CH2), 123.94 (Ph), 128.93 (Ph), 142.75
(Ph-CO), 149.34 (Ph-NO2), 157.25 (C=N), 168.75 (C=O)
ppm; MS (70 eV): m/z = 369.
Experimental
Melting points were determined with a Boethius apparatus
(Franz Ku¨stner, Dresden, Germany). IR spectra of KBr pel-
lets or paraffin oil suspensions were acquired on a Satellite
FT-IR spectrophotometer (Mattson Instruments, Madison,
WI, USA). 1H and 13C NMR spectra were acquired in CDCl3
and DMSO-d6 on Varian Gemini (200 MHz) and Varian
Unity Plus (500 MHz) instruments (Varian, Palo Alto, CA,
USA). Results from elemental analysis (% C, H, N) were in
agreement with calculated values within the 0.3% range for
all the compounds. Mass spectra for compounds 2, 10, 12, 16,
16a, 18, and 19 were acquired on a Biosystem Qstar XL MS/
MS spectrometer with electrospray ionization (70 eV).
Gravity column chromatography for compounds 2–4, 7, 8,
and 17 was performed on Fluka silica gel 60 of particle size
0.063–0.2 mm and 70–230 mesh ASTM (Sigma–Aldrich
Chemie, Steinheim, Germany). Synthesis of 4-nitro-
benzohydrazide and N-methyl-4-nitrobenzohydrazide is
described elsewhere [15, 16]; reaction yields and compound
characteristics were found to be identical with those reported.
4-Nitrobenzoic acid 2-[bis(methylthio)methylene]hydrazide
(1) was synthesized by the method reported recently [14];
reaction details and compound characteristics corresponded
with literature data.
4-Nitrobenzoic acid 2-[bis(decylthio)methylene]hydrazide
(3, C28H47N3O3S2)
This product was purified by column chromatography with
chloroform–cyclohexane (2:1) as eluent. The compound
was obtained as a pale yellow oil in 73% yield (0.98 g).
IR:vꢀ = 3,280 (m N–H), 2,925, 2,853 (m C–H), 1,673 (m
C=O), 1,602 (m C=C), 1,527 (m NO2), 1,467 (m C=C), 1,341
(d C–H), 1,268 (m C–N), 865, 849 (m C–N), 715 (c C–C)
1
cm-1; H NMR (200 MHz, CDCl3): d = 0.88 (t, 6H, 2
CH3, J = 6 Hz), 1.26–1.44 (m, 28H, 14 CH2), 1.56–1.76
(m, 4H, 2 CH2), 2.75–3.21 (m, 4H, 2 SCH2), 7.98 (d, 2H,
Ph, J = 8.7 Hz), 8.22–8.35 (m, 2H, Ph), 9.68 (brs, 1H,
NH) ppm; 13C NMR (50 MHz, CDCl3): d = 14.27 (CH3),
21.98 (CH2), 26.53 (SCH2), 27.89 (CH2), 28.05 (CH2),
28.99 (CH2), 23.31 (CH2), 30.98 (CH2), 31.67 (CH2),
123

Synthesis, characterization, and tuberculostatic
613
1
122.97 (Ph), 127.56 (Ph), 137.34 (Ph-CO), 149.78 (Ph-
NO2), 162.28 (C=N), 169.60 (C=O) ppm.
863 (m C–N), 719 (c C–C) cm-1; H NMR (200 MHz,
CDCl3): d = 0.77–0.98 (m, 6H, 2 CH3), 1.21–1.73 (m, 8H,
4 CH2), 2.86–3.00 (m, 4H, 2 SCH2), 3.29 (s, 3H, NCH3),
7.68 (d, 2H, Ph, J = 9 Hz), 8.19 (d, 2H, Ph, J = 8.6 Hz)
ppm; 13C NMR (50 MHz, CDCl3): d = 13.90 (CH3), 13.96
(CH3), 22.27 (CH2), 31.63 (CH2), 31.91 (SCH2), 35.14
(NCH3), 123.32 (Ph), 129.10 (Ph), 142.61 (Ph-CO), 148.81
(Ph-NO2), 163.96 (C=N), 176.11 (C=O) ppm.
4-Nitrobenzoic acid 2-[bis(phenylmethylthio)-
methylene]hydrazide (4, C21H17N3O3S2)
This product was purified by column chromatography with
chloroform as eluent. The compound was obtained as a red
solid in 50% yield (0.53 g). M.p.: 159–160 °C; IR
(KBr):vꢀ = 3,289 (m N–H), 2,924, 2,853 (m C–H), 1,689 (m
C=O), 1,552 (m NO2), 1,522, 1,497, 1,478 (m C=C), 1,337
4-Nitrobenzoic acid 2-[bis(decylthio)methylene]-1-
methylhydrazide (8, C29H49N3O3S2)
1
(d C–H), 849 (m C–N), 711, 698 (c C–C) cm-1; H NMR
(200 MHz, CDCl3): d = 4.08 (s, 2H, SCH2), 4.38 (s, 2H,
SCH2), 7.03–7.37 (m, 8H, Ph), 7.52–7.67 (m, 4H, Ph), 8.24
(d, 2H, Ph, J = 8.3 Hz), 9.45 (s, 1H, NH) ppm; 13C NMR
(50 MHz, DMSO-d6): d = 36.36 (SCH2), 37.29 (SCH2),
122.98 (Ph), 127.34 (Ph), 127.41 (Ph), 128.67 (Ph), 128.93
(Ph), 129.27 (Ph), 129.36 (Ph), 129.63 (Ph), 137.66 (Ph-
CO), 146.05 (Ph-NO2), 163.85 (C=N), 174.03 (C=O) ppm.
This product was purified by column chromatography with
chloroform as eluent. The compound was obtained as a
yellow oil in 96% yield (1.32 g). IR:vꢀ = 2,924, 2,853 (m C–
H), 1,648 (m C=O), 1,601 (m C=C), 1,525 (m NO2), 1,491,
1,464 (m C=C), 1,347 (d C–H), 1,220 (m C–C), 1,071, 1,043,
994 (d C–H), 862, 850 (m C–N), 719 (c C–C) cm-1; 1H NMR
(200 MHz, CDCl3): d = 0.85–0.92 (m, 6H, 2 CH3), 1.12–
1.27 (m, 32H, 16 CH2), 2.83–2.90 (m, 4H, 2 SCH2), 3.29 (s,
3H, NCH3), 7.68 (d, 2H, Ph, J = 8.9 Hz), 8.18 (d, 2H, Ph,
J = 8.7 Hz) ppm; 13C NMR (50 MHz, CDCl3): d = 14.38
(CH3), 22.85 (CH2), 27.58 (SCH2), 28.49 (CH2), 29.31
(CH2), 29.86 (CH2), 30.05 (CH2), 31.97 (CH2), 32.11 (CH2),
39.86 (N-CH2), 122.79 (Ph), 128.52 (Ph), 142.61 (Ph-CO),
149. 76 (Ph-NO2), 162.31 (C=N), 175.89 (C=O) ppm.
4-Nitrobenzoic acid 2-[bis(methylthio)methylene]-1-
methylhydrazide (5, C11H13N3O3S2)
This compound was obtained as yellow needles in 62%
yield (0.46 g). M.p.: 98–100 °C; IR (KBr):vꢀ = 3,068,
3,046, 3,007 (m C–H), 1,642 (m C=O), 1,598 (m C=C),
1,518 (m NO2), 1,415 (m C=C), 1,348 (d C–H), 1,023, 950
1
(d C–H), 870, 852 (m C–N), 718 (c C–C) cm-1; H NMR
(200 MHz, CDCl3): d = 2.32 (s, 3H, SCH3), 2.49 (s, 3H,
SCH3), 3.30 (s, 3H, NCH3), 7.71 (d, 2H, Ph, J = 8.9 Hz),
8.18 (d, 2H, Ph, J = 8.7 Hz) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 13.95 (SCH3), 15.18 (SCH3), 35.20
(NCH3), 123.31 (Ph), 129.01 (Ph), 141.81 (Ph-CO),
148.26 (Ph-NO2), 166.79 (C=N), 176. 85 (C=O) ppm.
4-Nitrobenzoic acid 2-[bis(phenylmethylthio)methylene]-1-
methylhydrazide (9, C23H21N3O3S2)
This compound was obtained as colorless crystals in 65%
yield (0.73 g). M.p.: 78–79 °C; IR (KBr):vꢀ = 3,029, 2,983,
2,936 (m C–H), 1,634 (m C=O), 1,601 (m C=C), 1,520 (m
NO2), 1,481 (m C=C), 1,354, 1,340 (d C–H), 994 (m C–N),
711 (c C–C) cm-1; 1H NMR (500 MHz, CDCl3): d = 3.30
(s, 3H, NCH3), 4.25 (s, 4H, 2 SCH2), 7.28–7.38 (m, 10H, 2
Ph), 7.59 (d, 2H, Ph, J = 7.3 Hz), 8.04–8.08 (m, 2H, Ph)
ppm; 13C NMR (50 MHz, CDCl3): d = 36.00 (NCH3),
36.59 (SCH2), 122.85 (Ph), 127.70 (Ph), 128.15 (Ph),
128.59 (Ph), 128.78 (Ph), 128.88 (Ph), 129.00 (Ph), 134.82
(Ph-CH2), 141.63 (Ph-CO), 149.51 (Ph-NO2), 161.83
(C=N), 173.21 (C=O) ppm.
4-Nitrobenzoic acid 2-[bis[(1-methylethyl)thio]methylene]-
1-methylhydrazide (6, C15H21N3O3S2)
This compound was obtained as a pale yellow solid in 27%
yield (0.24 g). M.p.: 89–90 °C; IR (KBr):vꢀ = 2,961, 2,927,
2,865 (m C–H), 1,639 (m C=O), 1,600 (m C=C), 1,521 (NO2),
1,497 (m C=C), 1,352 (d C–H), 989, 866 (m C–N), 723 (c C–
C) cm-1; 1H NMR (200 MHz, CDCl3): d = 1.14 (d, 6H, 2
CH3, J = 6 Hz), 1.38 (d, 6H, 2 CH3, J = 6.7 Hz), 3.27 (s,
3H, NCH3), 3.65–3.72 (m, 2H, 2 SCH), 7.68 (d, 2H, Ph,
J = 8.8 Hz), 8.18 (d, 2H, Ph, J = 8.4 Hz) ppm; 13C NMR
(50 MHz, CDCl3): d = 21.34 (CH3), 23.94 (SCH), 40.95
(NCH3), 122.81 (Ph), 124.09 (Ph), 128.35 (Ph), 130.71
(Ph), 139.23 (Ph-CO), 148.27 (Ph-NO2), 159.93 (C=N),
172.71 (C=O) ppm.
2-[(1-Methylethyl)thio]-5-(4-nitrophenyl)-1,3,4-oxadiazole
(10, C11H11N3O3S)
Method A: the compound was synthesized from 0.45 g
4-nitrobenzohydrazide (2.5 mmol), 0.87 cm3 TEA (6.25
mmol), 0.23 cm3 CS2 (3.75 mmol), and 0.62 cm3 isopropyl
iodide (6.25 mmol) by the method described for symmet-
rical diesters 1–9. The reaction mixture was stirred for
5 days. Ice (20 g) was then added, the solution was
extracted with chloroform (3 9 10 cm3), the extract was
dried with MgSO4, and the solvent was evaporated under
vacuum. The crude product was treated with dry diethyl
ether, isolated by filtration, dried, and recrystallized from
methanol to give yellow flakes in 63% yield (0.42 g).
4-Nitrobenzoic acid 2-[bis(butylthio)methylene]-1-
methylhydrazide (7, C17H25N3O3S2)
This product was purified by column chromatography with
chloroform as eluent. The compound was obtained as a
light yellow oil in 61% yield (0.58 g). IR:vꢀ = 2,959, 2,931,
2,872 (m C–H), 1,643 (m C=O), 1,600 (m C=C), 1,523 (m
NO2), 1,348 (d C–H), 1,221 (m C–C), 1,045, 992 (d C–H),
123

614
K. Gobis et al.
Method B: 0.456 g 5-(4-nitrophenyl)-1,3,4-oxadiazole-
4-Nitrobenzoic acid 2-(1,3-dithian-2-ylidene)hydrazide
(12, C11H11N3O3S2)
2(3H)-thione (2 mmol) was suspended in 35 cm3 anhy-
drous ethanol and 0.12 g KOH (2 mmol) was added and
the solution was heated under reflux for 30 min. Then
0.25 cm3 isopropyl iodide (2.5 mmol) was added dropwise
and the mixture was heated under reflux for another 5 h.
The reaction mixture was cooled, filtered, and the filtrate
was poured on 100 g ice. The precipitate was isolated by
filtration, dried, and recrystallized to give yellow flakes in
25% yield (0.132 g). M.p.: 91–93 °C; IR:vꢀ = 2,974, 2,924,
2,870 (m C–H), 1,606 (m C=C), 1521 (m NO2), 1,465 (m
C=C), 1,347, 1,070 (d C–H), 863, 850 (m C–N), 726, 705 (c
This compound was obtained as a yellow solid in 39%
yield (0.29 g). M.p.: 140–142 °C; IR (KBr):vꢀ = 3,372 (m
N–H), 2,976 (m C–H), 1,660 (m C=O), 1,599 (m C=C), 1,520
(m NO2), 1,343 (d C–H), 1,281 (m C–C), 1,108, 1,052 (d C–
1
H), 849 (m C–N), 715 (c C–C) cm-1; H NMR (200 MHz,
CDCl3): d = 2.21–2.34 (m, 2H, CH2), 3.10–3.27 (m, 4H, 2
SCH2), 8.00 (d, 2H, Ph, J = 8.7 Hz), 8.30 (d, 2H, Ph,
J = 8.1 Hz), 8.92 (s, 1H, NH) ppm; 13C NMR (50 MHz,
CDCl3): d = 36.11 (CH2), 39.36 (SCH2), 124.41 (Ph),
128.93 (Ph), 139.04 (Ph-CO), 152.11 (Ph-NO2), 161. 25
(C=N), 169.15 (C=O) ppm; MS (70 eV): m/z = 297.
1
C–C) cm-1; H NMR (200 MHz, CDCl3): d = 1.55 (d,
6H, 2 CH3, J = 6.7 Hz), 3.95–4.09 (m, 2H, 2 SCH), 8.19
(d, 2H, Ph, J = 8.9 Hz), 8.35 (d, 2H, Ph, J = 8.9 Hz) ppm;
13C NMR (50 MHz, CDCl3): d = 23.78 (CH3), 39.81
(SCH), 124.85 (Ph), 127.98 (Ph), 129.64 (Ph-1,3,4-oxadi-
azole), 149.90 (Ph-NO2), 164.21 (1,3,4-oxadiazole-Ph),
166.29 (1,3,4-oxadiazole-S) ppm; MS (70 eV): m/z = 265.
4-Nitrobenzoic acid 2-(1,3-dithiolan-2-ylidene)-1-
methylhydrazide (13, C11H11N3O3S2)
This compound was obtained as colorless crystals in 93%
yield (0.69 g). M.p.: 158–159 °C; IR (KBr):vꢀ = 2,974,
2,936 (m C–H), 1,645 (m C=O), 1,601 (m C=C), 1,538, 1,513
(m NO2), 1,353 (d C–H), 985 (m C–N), 862 (c C–C) cm-1
;
1H NMR (500 MHz, CDCl3): d = 3.37 (s, 3H, NCH3),
3.51 (t, 2H, SCH2, J = 5.9 Hz), 3.58 (t, 2H, SCH2,
J = 5.4 Hz), 7.73 (d, 2H, Ph, J = 8.8 Hz), 8.22 (d, 2H,
Ph, J = 8.8 Hz) ppm; 13C NMR (125 MHz, CDCl3):
d = 34.73 (NCH3), 36.54 (SCH2), 123.44 (Ph), 129.12
(Ph), 141.70 (Ph-CO), 148.37 (Ph-NO2), 166. 55 (C=N),
175.39 (C=O) ppm.
General procedure for synthesis of cyclic diesters
11–14
4-Nitrobenzohydrazide or N-methyl-4-nitrobenzohydrazide
(2.5 mmol) was suspended in 5 cm3 methanol and
0.87 cm3 TEA (6.25 mmol) and 0.23 cm3 CS2
(3.75 mmol) were added. The mixture was stirred for 1 h at
room temperature. Then 3.75 mmol 1,2-dibromoethane or
1,3-dibromopropane was added and the mixture was stirred
for another 2 h. Ice (20 g) was then added. The precipitates
of products 11 and 12 were isolated by filtration, dried, and
recrystallized from dioxane–water (1:1). The solutions of
products 13 and 14 were extracted with chloroform
(3 9 10 cm3), the extracts were dried with MgSO4, and the
solvent was evaporated under vacuum. The crude products
were treated with dry diethyl ether, isolated by filtration,
dried, and recrystallized from methanol.
4-Nitrobenzoic acid 2-(1,3-dithian-2-ylidene)-1-
methylhydrazide (14, C12H13N3O3S2)
This compound was obtained as pale yellow crystals in
75% yield (0.58 g). M.p.: 87–89 °C; IR (KBr):vꢀ = 2,927
(m C–H), 1,634 (m C=O), 1,597 (m C=C), 1,518 (m NO2),
1,351 (d C–H), 853 (m C–N), 713 (c C–C) cm-1; 1H NMR
(500 MHz, CDCl3): d = 2.20–2.22 (m, 2H, CH2), 3.07 (s,
3H, NCH3), 3.24–3.39 (m, 4H, SCH2), 7.75 (d, 2H, Ph,
J = 8.8 Hz), 8.23 (d, 2H, Ph, J = 8.3 Hz) ppm; 13C NMR
(125 MHz, CDCl3): d = 33.98 (CH2), 36.07 (SCH2), 38.99
(NCH3), 122.95 (Ph), 128.43 (Ph), 142.67 (Ph-CO), 149.78
(Ph-NO2), 159.93 (C=N), 167.83 (C=O) ppm.
4-Nitrobenzoic acid 2-(1,3-dithiolan-2-ylidene)hydrazide
(11, C10H9N3O3S2)
4-Nitrobenzoic acid 2-[(methylthio)thioxomethyl]hydrazide
(15, C9H9N3O3S2)
This compound was obtained as pale yellow crystals in
84% yield (0.59 g). M.p.: 199–200 °C; IR (KBr):vꢀ = 3,228
(m N–H), 2,980, 2,936 (m C–H), 1,646 (m C=O), 1,599 (m
C=C), 1,564 (m C=C), 1,511 (m NO2), 1,343 (d C–H), 1,285
4-Nitrobenzohydrazide (0.45 g, 2.5 mmol) was suspended in
10 cm3 methanol and 0.348 cm3 TEA (2.5 mmol) and
0.151 cm3 CS2 (2.5 mmol) were added. The mixture was
stirred for 1 h at room temperature and 0.12 cm3 methyl
iodide (2 mmol) was added. The mixture was stirred for
another 15 min and 20 g ice was added. A small amount of
precipitate was removed by filtration and the filtrate was
acidified with 0.183 cm3 acetic acid (2.5 mmol). The
precipitate of the monoester was isolated by filtration, dried,
and recrystallized from methanol to give a white solid in 46%
(m C–C), 1,040 (d C–H), 849 (m C–N), 721 (c C–C) cm-1
;
1H NMR (200 MHz, CDCl3): d = 3.50–3.56 (m, 2H,
SCH2), 3.67–3.73 (m, 2H, SCH2), 8.00 (d, 2H, Ph,
J = 8.8 Hz), 8.31 (d, 2H, Ph, J = 8.1 Hz), 9.15 (s, 1H,
NH) ppm; 13C NMR (50 MHz, DMSO-d6): d = 35.91
(SCH2), 123.81 (Ph), 129.37 (Ph), 139.35 (Ph-CO), 149.37
(Ph-NO2), 161.33 (C=N), 173.05 (C=O) ppm.
123

Synthesis, characterization, and tuberculostatic
615
yield(0.31 g). M.p.: 186–188 °C;IR(KBr):vꢀ = 3,285, 3,152
(m N–H), 2,842 (m C–H), 1,650 (m C=O), 1,598 (m C=C), 1,525
(m NO2), 1,345 (d C–H), 1,265 (m C–N), 985 (d C–H), 865 (m
acidified with 0.183 cm3 acetic acid (2.5 mmol) and
extracted with chloroform (3 9 10 cm3). The chloroform
fractions were combined, dried with MgSO4, and the
solvent was evaporated under vacuum. Components of the
mixture were separated and purified by column chroma-
tography with chloroform as eluent. The product was
obtained as a pale yellow solid in 29% yield (0.23 g). M.p.:
110–112 °C; IR (KBr):vꢀ = 3,151 (m N–H), 2,960, 2,930 (m
C–H), 1,665 (m C=O), 1,600 (m C=C), 1,524 (m NO2), 1,439
1
C–N), 728 (c C–C) cm-1; H NMR (200 MHz, CDCl3):
d = 2.73 (s, 3H, SCH3), 8.05 (d, 2H, Ph, J = 8.8 Hz), 8.37
(d, 2H, Ph, J = 8.8 Hz), 10.05 (s, 1H, NH), 10.55 (s, 1H, NH)
ppm; 13C NMR (50 MHz, CDCl3): d = 19.21 (SCH3),
122.92 (Ph), 127.99 (Ph), 139.88 (Ph-CO), 148.51 (Ph-
NO2), 165.78 (C=O), 202.64 (C=S) ppm.
(m C=C), 1,340 (d C–H), 1,292 (m C–N), 708 (c C–C) cm-1
;
Synthesis of 4-nitrobenzoic acid 2-[[(1-
methylethyl)thio]thioxomethyl]hydrazide (16) and
5-(4-nitrophenyl)-1,3,4-oxadiazole-2(3H)-thione (16a)
1H NMR (200 MHz, DMSO-d6): d = 0.84–0.94 (m, 3H,
CH3), 1.30–1.44 (m, 2H, CH2), 1.55–1.66 (m, 2H, CH2),
3.13–3.29 (m, 2H, SCH2), 8.10 (d, 2H, Ph, J = 8.7 Hz),
8.35–8.41 (m, 2H, Ph), 11.27 (s, 1H, NH), 11.61 (s, 1H,
NH) ppm; 13C NMR (50 MHz, DMSO-d6): d = 13.79
(CH3), 21.76 (CH2), 30.66 (CH2), 33.56 (SCH2), 124.23
(Ph), 129.33 (Ph), 137.58 (Ph-CO), 149.96 (Ph-NO2),
164.10 (C=O), 203.45 (C=S) ppm.
4-Nitrobenzohydrazide (0.45 g, 2.5 mmol) was suspended
in 10 cm3 methanol and 0.383 cm3 TEA (2.75 mmol) and
0.227 cm3 CS2 (3.75 mmol) were added. The mixture was
stirred for 1 h and 0.250 cm3 isopropyl iodide (2.5 mmol)
was added. The mixture was stirred for another 48 h and
20 g ice was added. The precipitate of monoester 16 was
isolated by filtration, dried, and recrystallized from meth-
anol–water (1:1). The filtrate was acidified with 0.183 cm3
acetic acid (2.5 mmol). The precipitate of 1,3,4-oxadiaz-
ole-2-thione 16a was isolated by filtration, dried, and
recrystallized from methanol.
2-(Butylthio)-5-(4-nitrophenyl)-1,3,4-oxadiazole
(18, C12H13N3O3S)
4-Nitrobenzohydrazide (0.91 g, 5 mmol) was suspended in
30 cm3 methanol, 0.836 cm3 TEA (6 mmol) and 0.453 cm3
CS2 (7.5 mmol) were added, and the mixture was stirred
overnight at room temperature. The precipitate of an
unidentified side product was removed by filtration and
0.569 cm3 butyl iodide (5 mmol) was added to the filtrate.
The mixture was stirred for 3 h at room temperature, and the
precipitate was isolated by filtration, dried, and recrystallized
from methanol to give bright needles in 82% yield (1.15 g).
M.p.: 99–100 °C; IR (KBr):vꢀ = 2,960, 2,943 (m C–H), 1,605,
1,560 (m C=C), 1,516 (m NO2), 1,455 (m C=C), 1,344, 1,185
4-Nitrobenzoic acid 2-[[(1-methylethyl)thio]-
thioxomethyl]hydrazide (16, C11H23N3O3S2)
This compound was obtained as a pale beige solid in 19%
yield (0.14 g). M.p.: 172–174 °C; IR (KBr):vꢀ = 3,182 (m N–
H), 2,968, 2,922 (m C–H), 1,672 (m C=O), 1,601 (m C=C),
1,524 (m NO2), 1,324 (d C–H), 1,283 (m C–N), 1,035 (d C–H),
864 (m C–N), 718 (c C–C) cm-1
;
1H NMR (200 MHz,
(dd C–H), 853 (m C–N), 706 (c C–C) cm-1 1H NMR
;
CDCl3): d = 1.46 (d, 6H, 2 CH3, J = 6.9 Hz), 4.03–4.17 (m,
1H, SCH), 8.05 (d, 2H, Ph, J = 8.8 Hz), 8.37 (d, 2H, Ph,
(200 MHz, CDCl3): d = 0.98 (t, 3H, CH3, J = 7.3 Hz),
1.46–1.61 (m, 2H, CH2), 1.78–1.94 (m, 2H, CH2), 3.35(t, 2H,
SCH2, J = 7.3 Hz), 8.20 (d, 2H, Ph, J = 9 Hz), 8.37 (d, 2H,
Ph, J = 9 Hz) ppm; 13C NMR (50 MHz, CDCl3): d = 13.62
(CH3), 21.33 (CH2), 31.24 (CH2), 32.10 (SCH2), 124.85 (Ph),
127.97(Ph), 128.87(Ph-1,3,4-oxadiazole), 149.32(Ph-NO2),
163.96 (1,3,4-oxadiazole-Ph), 165.65 (1,3,4-oxadiazole-S)
ppm; MS (70 eV): m/z = 279.
J = 8.8 Hz), 10.21 (s, 1H, NH), 10.67 (s, 1H, NH) ppm; 13
C
NMR (50 MHz, DMSO-d6): d = 22.45 (CH3), 39.28 (SCH),
123.81 (Ph), 129.08 (Ph), 137.28 (Ph-CO), 149.72 (Ph-NO2),
163.74 (C=O), 202.31 (C=S) ppm; MS (70 eV): m/z = 299.
5(4-Nitrophenyl)-1,3,4-oxadiazole-2(3H)-thione (16a)
This compound was obtained as pale yellow needles in
55% yield (0.31 g). M.p.: 203–205 °C; the 1H NMR
spectrum corresponded to the literature spectrum [20].
General procedure for the synthesis of unsymmetrical
S,S0-diesters 19–22
4-Nitrobenzoic acid 2-[(butylthio)thioxomethyl]hydrazide
(17, C12H15N3O3S2)
Methyl 2-(4-nitrobenzoyl)hydrazinecarbodithioate (15,
0.35 g, 1.3 mmol) was dissolved in 5 cm3 methanol and
0.272 cm3 TEA (1.95 mmol) and 1.95 mmol of the respec-
tive halide were added. The mixture was stirred for 1 h at
room temperature. In the reaction with isopropyl iodide
(product 19) the reaction time was prolonged to 24 h. The
mixturewasthencooledinanicebath, andtheprecipitatewas
isolated by filtration, dried, and recrystallized from methanol.
4-Nitrobenzohydrazide (0.45 g, 2.5 mmol) was suspended
in 10 cm3 methanol and 0.383 cm3 TEA (2.75 mmol) and
0.227 cm3 CS2 (3.75 mmol) were added. The mixture was
stirred for 1 h at room temperature and 0.285 cm3 butyl
iodide (2.5 mmol) was added. The mixture was stirred for
45 min and the solvent was evaporated under vacuum.
Then 20 g ice was added to the residue. The solution was
123

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K. Gobis et al.
4-Nitrobenzoic acid 2-[[(1-methylethyl)thio]-
(m C=C), 1,535 (m NO2), 1,342 (d C–H), 1,288 (m C–N), 1,071
(methylthio)methylene]hydrazide (19, C12H15N3O3S2)
This compound was obtained as a light yellow solid in 32%
yield (0.13 g). M.p.: 107–108 °C; IR (KBr):vꢀ = 3,163 (m
N–H), 2,961, 2,923 (m C–H), 1,667, 1,643 (m C=O), 1,602
(m C=C), 1,524 (m NO2), 1,481 (m C=C), 1,342 (d C–H),
1,277 (m C–N), 1,055 (d C–H), 867, 849 (m C–N), 721 (c C–
C) cm-1; 1H NMR (200 MHz, CDCl3): d = 1.42 (d, 6H, 2
CH3, J = 6.6 Hz), 2.62 (s, 3H, SCH3), 3.66–3.75 (m, 1H,
SCH), 8.01 (d, 2H, Ph, J = 8.7 Hz), 8.28–8.37 (m, 2H,
Ph), 9.50 (s, 1H, NH) ppm; 13C NMR (50 MHz, CDCl3):
d = 15.34 (SCH3), 23.94 (CH3), 30.96 (CH), 124.09 (Ph),
128.36 (Ph), 142.31 (Ph-CO), 146.28 (Ph-NO2), 151.21
(C=N), 165.73 (C=O) ppm; MS (70 eV): m/z = 313.
(d C–H), 865 (m C–N), 719 (c C–C) cm-1 1H NMR
;
(200 MHz, CDCl3): d = 2.62 (s, 3H, SCH3), 4.19 (s, 2H,
SCH2), 7.30–7.44 (m, 5H, Ph), 7.69–7.83 (m, 2H, Ph), 8.25
(d, 2H, Ph, J = 8.7 Hz), 9.25 (s, 1H, NH) ppm; 13C NMR
(50 MHz, CDCl3): d = 15.29 (SCH3), 37.81 (SCH2), 124.85
(Ph), 128.01 (Ph), 128.18 (Ph), 128.61 (Ph), 128.64 (Ph),
128.78 (Ph), 128.83 (Ph), 135.02 (Ph-CH2), 135.79 (Ph-CO),
148.47 (Ph-NO2), 153.43 (C=N), 165.97 (C=O) ppm.
Tuberculostatic assay
Investigations were performed by a classical test-tube
method of a successive dilution in Youmans’ modification of
Proskauer and Beck’s liquid medium containing 10% bovine
serum [27, 28]. Bacterial suspensions were prepared from
14-day-old cultures of slowly growing strains and from
48-hour-oldculturesofsaprophytic strains [29, 30]. Solutions
of the compounds in ethylene glycol were tested. Stock
solutions contained 10 mg of the compounds in 1 cm3.
Dilutions (in a geometric progression) were prepared in
Youmans’ medium. Medium containing no investigated
substances or containing isoniazid (INH) or pyrazinamide
(PZA) as reference drugs were used for comparison. Dime-
thyl 4-nitrobenzoylcarbonohydrazonodithioate (1) described
earlier [14] was also included in the tests. Incubation was
performed at 37 °C. The MIC values were determined as the
minimum concentration inhibiting the growth of the tested
tuberculous strains in relation to the probe with no tested
compound.
4-Nitrobenzoic acid 2-[(butylthio)(methylthio)-
methylene]hydrazide (20, C13H17N3O3S2)
This compound was obtained as pale yellow crystals in
78% yield (0.33 g). M.p.: 60–62 °C; IR (KBr):vꢀ = 3,170
(m N–H), 2,959, 2,928 (m C–H), 1,672, 1,647 (m C=O),
1,522 (m NO2), 1,456 (m C=C), 1,344 (d C–H), 1,291, 864 (m
1
C–N), 721 (c C–C) cm-1; H NMR (200 MHz, CDCl3):
d = 0.95 (t, 3H, CH3, J = 7.3 Hz), 1.35–1.57 (m, 2H,
CH2), 1.62–1.89 (m, 2H, CH2), 2.60 (s, 1H, SCH2), 3.06 (t,
2H, SCH2, J = 6.8 Hz), 8.00 (d, 2H, Ph, J = 8.6 Hz), 8.19
(d, 2H, Ph, J = 8.5 Hz), 9.70 (s, 1H, NH) ppm; 13C NMR
(50 MHz, CDCl3): d = 14.18 (CH3), 15.21 (SCH3), 21.82
(CH2), 28.13 (SCH2), 32.57 (CH2), 124.58 (Ph), 128.14
(Ph), 142.91 (Ph-CO), 146.57 (Ph-NO2), 151.23 (C=N),
166.17 (C=O) ppm.
4-Nitrobenzoic acid 2-[(decylthio)(methylthio)-
methylene]hydrazide (21, C19H29N3O3S2)
X-ray crystallography
Single crystals of compound 18 suitable for X-ray dif-
fraction were obtained from ethanol by slow evaporation
of the solvent at room temperature. X-ray data were
collected on a Bruker SMART diffractometer with
APEX II area detector. Data reduction was performed by
means of the Bruker software SAINT (Version 6.45) and
SADABS (Version 2.10) [31]. The structure was solved
by direct methods using SHELXTL [32] and refined by
full-matrix least squares [33]. Final coordinates and
details of measurement, solution, and refinement have
been deposited with the Cambridge Crystallographic
Data Centre (no. 845158). All details of the crystal data
may be obtained free of charge via http://www.ccdc.
cam.ac.uk/conts/retrieving.html or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cam-
bridge CB2 1EZ, UK; fax: ?44 1223 336033; or
deposit@ccdc.cam.ac.uk.
This compound was obtained as a yellow solid in 77%
yield (0.41 g). M.p.: 43–44 °C; IR (KBr):vꢀ = 3,181 (m N–
H), 2,921, 2,853 (m C–H), 1,672, 1,647 (m C=O), 1,523 (m
NO2), 1,464 (m C=C), 1,344 (d C–H), 863 (m C–N), 720 (c
C–C) cm-1; 1H NMR (200 MHz, CDCl3): d = 0.87 (t, 3H,
CH3, J = 5.9 Hz), 1.26–1.46 (m, 12H, 6 CH2), 1.62–1.73
(m, 2H, CH2), 1.78–1.89 (m, 2H, CH2), 2.62 (s, 3H, SCH3),
3.06 (t, 2H, SCH2, J = 7 Hz), 8.00 (d, 2H, Ph,
J = 8.8 Hz), 8.19 (d, 2H, Ph, J = 8.9 Hz), 9.37 (s, 1H,
NH) ppm; 13C NMR (50 MHz, DMSO-d6): d = 14.20
(CH3), 15.17 (SCH3), 22.36 (CH2), 28.20 (SCH2), 28.66
(CH2), 28.71 (CH2), 28.96 (CH2), 29.19 (CH2), 29.80
(CH2), 31.56 (CH2), 124.87 (Ph), 127.98 (Ph), 139.50 (Ph-
CO), 149.35 (Ph-NO2), 155.17 (C=N), 164.28 (C=O) ppm.
4-Nitrobenzoic acid 2-[(methylthio)-
(phenylmethylthio)methylene]hydrazide
(22, C16H15N3O3S2)
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and source are credited.
This compound was obtained as yellow flakes in 77% yield
(0.36 g). M.p.: 124–125 °C; IR (KBr):vꢀ = 3,175 (m N–H),
3,029, 2,999, 2,931 (m C–H), 1,677, 1,651 (m C=O), 1,600
123

Synthesis, characterization, and tuberculostatic
617
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