Synthesis of novel isothiazolopyridines and their in vitro evaluation against Mycobacterium and Propionibacterium acnes

Article abstract of DOI:10.1016/j.bmc.2013.06.027

In this paper we describe synthesis, structures and some physicochemical properties of 20 isothiazolopyridines 8-13 substituted differently into an isothiazole ring as well as their in vitro antibacterial assays against Mycobacterium tuberculosis H37Rv, Mycobacterium fortuitum PCM 672 and Propionibacterium acnes PCM 2400. Compound 13a was found to be the most active derivative against M. tuberculosis H37Rv, demonstrating 100% growth inhibition of microorganisms in the primary screen (minimum inhibitory concentration [MIC] 6.25 μg/mL). Nineteen of the prepared compounds were evaluated against M. fortuitum PCM 672 and P. acnes PCM 2400 and only compounds 9 and 12d exhibited excellent activity against individual strains of microorganisms with MIC ₉₀ <1 μg/mL. The inhibitory action of the remaining isothiazolopyridines towards the tested strains of the microorganism was low, absent, or a non-linear correlation prohibited accurate determination of MIC values. Unexpectedly, seven of the remaining isothiazolopyridines tested against M. fortuitum and P. acnes stimulated growth of the microorganisms in the range 10-50% or even more (10b) under experimental conditions.

Full text of DOI:10.1016/j.bmc.2013.06.027

Accepted Manuscript
Synthesis of novel isothiazolopyridines and their in vitro evaluation against
Mycobacterium and Propionibacterium acnes
Wiesław Malinka, Piotr Świątek, Małgorzata Śliwińska, Bogumiła Szponar,
Andrzej Gamian, Zbigniew Karczmarzyk, Andrzej Fruziński
PII:
S0968-0896(13)00558-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.06.027
BMC 10920
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 January 2013
7 June 2013
11 June 2013
Please cite this article as: Malinka, W., Świątek, P., Śliwińska, M., Szponar, B., Gamian, A., Karczmarzyk, Z.,
Fruziński, A., Synthesis of novel isothiazolopyridines and their in vitro evaluation against Mycobacterium and
Propionibacterium acnes, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2013.06.027
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Synthesis of novel isothiazolopyridines and their in vitro evaluation against
Mycobacterium and Propionibacterium acnes
Wiesław Malinka a, f, Piotr Świątek a, *, Małgorzata Śliwińska a, Bogumiła Szponar b, Andrzej
Gamian b, c, Zbigniew Karczmarzyk d, Andrzej Fruziński e
a Department of Chemistry of Drugs, Wrocław Medical University, Borowska 211,
50-556 Wrocław, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla
12, 53-114 Wrocław, Poland
c Department of Medical Biochemistry, Wrocław Medical University, Chałubińskiego 10,
50-368 Wrocław, Poland
d Department of Chemistry, University of Podlasie, 3 Maja 54, 08-110 Siedlce, Poland
e Institute of General and Ecological Chemistry, Technical University, Żeromskiego115,
90-924 Łódź, Poland
f College of Physiotherapy, Kościuszki 4, 50-038 Wrocław, Poland
In this paper we describe synthesis, structures and some physicochemical properties of
20 isothiazolopyridines 8-13 substituted differently into an isothiazole ring as well as their in
vitro antibacterial assays against Mycobacterium tuberculosis H37Rv, Mycobacterium
fortuitum PCM 672 and Propionibacterium acnes PCM 2400. Compound 13a was found to
be the most active derivative against M. tuberculosis H37Rv, demonstrating 100% growth
inhibition of microorganisms in the primary screen (minimum inhibitory concentration [MIC]
6.25 μg/mL). Nineteen of the prepared compounds were evaluated against Mycobacterium
fortuitum PCM 672 and Propionibacterium acnes PCM 2400 and only compound 9 and 12d
exhibited excellent activity against individual strains of microorganisms with MIC90 < 1
μg/mL. The inhibitory action of the remaining isothiazolopyridines towards the tested strains
of the microorganism was low, absent, or a non-linear correlation prohibited accurate
determination of MIC values. Unexpectedly, seven of the remaining isothiazolopyridines
tested against M. fortuitum and P. acnes stimulated growth of the microorganisms in the range
10-50% or even more (10b) under experimental conditions.

1. Introduction
The treatment of mycobacterial infection has become an important problem due to
emergence of multiple-drug-resistant microorganisms towards conventional therapeutic
agents. Therefore there is a need to search for new antimycobacterial drugs with potent
bioactivity and low toxicity. The new antimycobacterial agents are developed by modification
of existing antimycobacterial drugs or by development of new classes of compounds. Among
numerous new classes of compounds synthesized for this purpose, derivatives of bicyclic
structures (benzoxazole, benzothiazole, benzisothiazole, quinolone, quinoxaline) have
exhibited promising results.1-9
One of our research targets is synthesis of isothiazolo[5,4-b]pyridine derivatives for
pharmaceutical purposes. We reported that several 4,6-dimethylisothazolo[5,4-b]pyridines,
depending on substitution on the isothiazole ring, have biological activities including
significant analgesic10 or anorectic11 effects. Other authors have also suggested
antiaggregatory12 and antiacne action13 of derivatives of isothiazolo[5,4-b]pyridine.
Additionally, derivatives of this system attracted our considerable interest as 7-azaanalogues
of benzisothiazoles, which are cited for their promising activity against standard strains of M.
tuberculosis as well as against mycobacteria isolated from blood of infected patients (MIC 16-
8 μg/mL).3
However, the mechanism of antimycobacterial action of benzisothiazoles remain
unknown and there is poor knowledge about the structure–activity relationship which would
be useful for rational projection of new bioactive compounds of this series.
Taking into account the interesting antimycobacterial activity of benzisothiazoles, a few of
our 4,6-dimethylisothiazolo[5,4-b]pyridines possessing 4-aryl(benzyl)piperazinyl(piperidinyl)
substructure linked to the N2 or 3-O- atom of the isothiazolopyridine through different linkage
(Fig. 1; 1 and 2, respectively) and synthesized in connection with another project were
additionally tested against Mycobacterium tuberculosis H37Rv.
Fig. 1 The general formula of isothiazolopyridines 1 and 2
By comparing the data concerning antimycobacterial potency against M. tuberculosis
H37Rv within series 1 and 2 it was evident that insertion of the side chain in the N2 position
(series 1) but not in the 3-O position (series 2), generates interesting activity against this strain

of mycobacterium.14, 15 The most active compounds 1a-d (Table 4) exhibited 100% inhibition
of the mycobacteria after application at 6.25 μg/mL under preliminary testing.
The aim of this study was synthesis and evaluation under preliminary in vitro
antibacterial screening of several classes of new isothiazolo[5,4-b]pyridines designed
specially as potential antimycobacterial agents.
The new series of antimycobacterial isothiazolopyridines include derivatives with 4-
nitrophenylpiperazine grouping in the structure of the side chain (8, 9; Scheme 1), because
such a substituent is characteristic for quinoxaline derivatives with significant
antimycobacterial activity (MIC<1 μg/mL).6
Pagani et al. revealed that carbamic esters of N2-(2-hydroxyethyl)benzoisothiazole
were interesting compounds for their promising in vitro antimycobacterial results (MIC 16-32
μg/mL).4 In this context we prepared series of isomeric isothiazolopyridines of carbamic ester
type 10 and 11 (Scheme 2).
As reported in the literature, N-alkylbenzylamines are compounds with specific action
against mycobacteria.16 On the other hand, our N2-hydroxymethylisothiazolopyridine 3
(Fig. 2) showed 65% inhibition of Mycobacterium tuberculosis H37Rv.15 A similar effect was
exhibited by 2-hydroxymethylbenzoisothiazole 4 (Fig. 2).5
Fig. 2. Antimycobacterial activity of 2-hydroxymethyl derivatives of isothiazolopyridine 3
and benzisothiazole 4
Based
on
these
findings
and
combining
benzylamines
and
hydroxymethylisothiazolopyridine 3 we prepared a series of hybrid compounds of specific
Mannich base of type 12 as potential antimycobacterial agents (Scheme 3).
Additionally, to develop the structure-antimycobacterial relationship, we included in
our investigation isomeric isothiazolopyridines 13a and 13b devoid of a base nitrogen atom
within the structure of the side chain (Scheme 4).
Compounds 8-13 were screened against Mycobacterium.
In 1984 and 1985, several patented N-(cyclo)alkyl, hydroxyalkyl, (un)substituted benzyl
and carbamoylisothiazolo[5,4-b]pyridin-3-one compounds were claimed as useful agents
against Propionibacterium acnes,13 which hydrolyzes sebum triglycerides to form the fatty
acids responsible for inflammation in acne. Therefore all of our new isothiazolopyridines
(except 11c) were also tested against Propionibacterium acnes in order to complete their in
vitro microbiological profile.

2. Chemistry
Different synthetic routes were used in preparation of the final isothiazolopyridines
8-13 (Schemes 1-4) with satisfactory yield.
In general, the new compounds were prepared starting from 4,6-
dimethylisothiazolo[5,4-b]pyridin-3(2H)-one 7 (Scheme 4) or its hydroxy(chloro)alkyl
derivatives 3, 5, 6 (Scheme 1, 2, 3). Intermediates 3, 5-7 were described recently.11, 17-19
Preparation of the 4-nitrophenylpiperazinylalkyl derivatives of isothiazolopyridine 8
and 9 involves alkylation of 4-nitrophenylpiperazine with corresponding intermediates 3 or 5,
respectively (Scheme 1).
Scheme 1. Synthetic route of compounds 8, 9
The isomeric carbamates 10a-d and 11a-d were prepared after treatment of N2- or 3-
O-(2-hydroxyethyl)derivatives of isothiazolopyridine 6a and 6b, respectively, with
appropriate alkyl and phenyl isocyanates (Scheme 2).
Scheme 2. Synthetic route of compounds 10a-d and 11 a-d
Alkylation of the commercially available benzylamines (un)substituted with different
substituents in the o- , m- or p-positions (X=CH) or 2-aminomethylpyridine (X=N) with
excess of 2-hydroxymethylisothiazolopyridine 3 led to formation of the specific Mannich
bases of type 12 (Scheme 3).
Scheme 3. Synthetic route of compounds 12a-h
Finally, alkylation of isothiazolopyridine 7 with 2-(2-phenoxy)ethoxyethyl chloride
afforded the isomeric compounds 13a and 13b (Scheme 4).
Scheme 4. Synthetic route of compounds 13a and 13b
Analytical purification of all new products 8-13 (20 compounds) was achieved by
crystallization from the appropriate solvent. Final compounds were characterized by a sharp

melting point, correct elemental (C, H, N) analyses, proton or carbon nuclear magnetic
resonance (1H NMR, 13C NMR) spectra, and in the case of 12d by X-ray data.
The structures of the isomeric isothiazolopyridines 13a and 13b (Scheme 4) were
1
assigned on the basis of H NMR. In the N2-substituted isomer 13a the signal of the
methylene protons adjacent to the N2-nitrogen of the isothiazolopyridine was recorded ~ 3.8
ppm. The 3-O-substitution produced downfield shift of these protons (~ 4.7 ppm). The above
spectral data agree with those previously reported by us for related isomeric N2- and 3-O-
hydroxyethyl substituted intermediates 6a and 6b (Scheme 2), respectively.
1
The H NMR spectra of compounds 12, independent of the type and position of
R-substitution on the phenyl ring of the benzyl substructure (Scheme 3), showed a 2H singlet
~ 4.0 and 4H singlet ~ 5.0 ppm corresponding to the 2x NCH2N methylene group and 1x
arylmethylene group ArCH2N. It may suggest a specific configuration of these compounds.
Therefore the structure of the Mannich bases 12 were additionally determined by the X-ray
diffraction method taking into account 12d as a model compound (section 2.1.).
Most of the compounds 8-13 were highly lipophilic substances and were characterized
by logPcalc. 2.5-6.5 (Table 3). LogPcalc. was calculated using the ChemPlus program from
Hypercube, Inc., IBM PC version, implemented in the HyperChem program package.
The new synthesized compounds presented above, except for 8 and 9, were tested in
vitro for their activity against strains of Mycobacterium tuberculosis (18 compounds), and
additionally all compounds 8-13, except for 11c, were evaluated against M. fortuitum and
Propionibacterium acnes.
2.1. Crystal structure of isothiazolopyridine 12d
The structure of compound 12d was unambiguously confirmed by X-ray analysis
(Fig. 3).
Fig. 3. A view of the X-ray molecular structure of 12d with the atomic labeling scheme
(probability 50%)
The geometry (bond lengths, angles and planarity) of the isothiazolopyridine rings in
12d is very similar to that found in 4,6-dimethylisothiazolo[5,4-b]pyridine-3(2H)-one20 and
other related structures containing this ring.21 The N atom of the tertiary amino group has
pyramidal configuration with the sum of angles around it of 337.2° characteristic for sp3

hybridization. The methylene bridges linking the isothiazolopyridine and phenyl rings with
the central amino group have the gauche-gauche-trans, gauche-trans-gauche and trans-
gauche-trans conformation in relation to the isothiazolopyridine rings A and B and phenyl
ring C, respectively (Table 1).
Table 1. Selected torsion angles (°)
The dihedral angles between the mean planes of isothiazolopyridine and phenyl rings are: A/B
= 55.74(4)°, A/C = 32.19(8)° and B/C = 87.72(9)°. The methoxy group, coplanar with the
phenyl ring, is in trans conformation with respect to the methylene chain with the torsion
angle C31–C32–O37–C38 of -178.1(3)°. The conformation of the molecule as a whole is
stabilized by the C–H…X (X = O, N) intramolecular hydrogen bonds listed in Table 2.
Table 2. Hydrogen-bond geometry (Å,°) for 12d
The packing of molecules in the crystal structure of 12d (Fig. 3) is governed by combination
of a weak C12 –H12C…O3A hydrogen bond linking the inversion-related molecules into
dimers and a C11A–H11D…O3A hydrogen bond linking the dimers into molecular chains
parallel to the X axis direction (Table 2).
Fig. 4. The molecular packing in crystal of 12d. Dashed lines indicate intermolecular
hydrogen bonds [symmetry codes: (i) = 1 + x, y, z; (ii) = 1 – x, 2–y, –z)].
Significant π–π interactions observed in the packing form the pairs of parallel isothiazole
S1A…C8A rings and pairs of the parallel pyridine N7B…C6B rings belonging to inversion-
related molecules with the centroid-to-centroid separations of 3.7348(15) and 3.9392(18) Å,
respectively.
3. Microbiology and discussion
Structures of the compounds evaluated under the microbiological study are shown in
Table 3.
Initially all compounds, except 8 and 9, were screened against Mycobacterium
tuberculosis H37Rv strain at a single concentration of 6.25 g/mL. Rifampicin, an antibiotic

known for its potent activity against many strains of M. tuberculosis, was used as a reference
drug. The microbiological data were provided by the GWL Hansen’s Disease Center
(Colorado State University) within the Tuberculosis Acquisition and Coordinating Facility
(TAACF: NIH, NIAID Contract AI45264) screening program for the discovery of novel
drugs for treatment of tuberculosis.
All the new compounds, except 11c, were additionally tested for their in vitro action
against Mycobacterium fortuitum PCM 672 as well for Propionibacterium acnes PCM 2400
obtained from the Polish Collection of Microorganisms (PCM). Isoniazid or erythromycin,
respectively, were used as reference drugs.
The results of antibacterial studies of isothiazolopyridines 8-13 with the data for the
control drugs are presented in Table 3.
Table 3. In vitro antibacterial activity against Mycobacterium tuberculosis, Mycobacterium
fortuitum, Propionibacterium acnes of isothiazolopyridines 8-13
To develop the preliminary S-A relationship, microbiologically tested compounds
8-13 were divided into four series (Table 3):
Series I) N2-substituted isothiazolopyridines 8, 9, 10d, 13a bearing in the structure of the side
chain a terminal phenyl ring separated from the N2-methyleneisothiazolopyridine fragment
with a different spacer (X).
Series II) N2-substituted carbamates 10, except for 10d, which was included in series I.
Series III) 3-O-substituted carbamates 11. In this series 3-O- isomer 13b was also included.
Series IV) Specific Mannich bases of type 12.
The data from Table 3 showed that carbamates 10a-c (series II), 10d (series I) and 11
(series III), which represent a group of compounds related to the antimycobacterial
benzisothiazoles,4 in contrast to their precursors, were completely inactive against
Mycobacterium tuberculosis H37Rv.
Similarly, lack of activity against this strain of mycobacterium was shown by highly
lipophilic (logPcalc. ~5.5 - 6.6) Mannich bases 12 (Table 3, series IV), which combine
corresponding benzylamines and two N2-methyleneisothiazolopyridine moieties in a single
molecule.
Significant action against Mycobacterium tuberculosis H37Rv (100% inhibition) was
exhibited only by isothiazolopyridine 13a (Table 3), devoid of a nitrogen atom within the
structure of the side chain. At the same time, its 3-O-substituted isomer 13b (Table 3, series

III) was completely inactive under preliminary screening. The above results correspond with
our recent observation within the series of isothiazolopyridines shown in Fig. 1. For example,
isothiazolopyridine 1a (Table 4) exhibited 100% inhibition of mycobacteria under preliminary
screening whereas its O-isomer (Fig. 1, 2 (R=m-Cl, n=2)) was a practically inactive
compound.
Because the new isothiazolopyridines 10-12 and 13b exhibited <20% inhibition in the
preliminary screen against M. tuberculosis H37Rv at the assayed concentration of 6.25 g/mL,
these compounds were not further evaluated.
For compound 13a causing >90% inhibition in the primary screen at concentration of
6.25 g/mL a confirmatory advanced screening was performed against M. tuberculosis H37Rv
in order to determine an actual MIC. In this investigation we also included recently
synthesized and evaluated isothiazolopyridines 1a-d (Fig. 1, Table 4), which under
preliminary investigation exhibited significant action against M. tuberculosis H37Rv (100%
inhibition, MIC 6.25 g/mL).14, 15 Among these, compound 1b resulted in the most inhibiting
effect, with an MIC value of 1.56 g/mL. For the remaining compounds MIC values were 6.25
g/mL. So, the above results showed that none of the investigated compounds exhibited higher
activity than that of rifampicin and the most interesting compound was ~12-fold less active
than the reference drug, which showed an inhibition activity of 98% at a concentration of
0.125 g/mL.
On the other hand, the significant inhibitory activity of 13a, whose N2-substituent
structure represents a marked departure from those present in piperazine (piperidine)
derivatives 1a-d (Table 4), suggests that the nature of the central linkage X within series I
(Table 3) does not play a relevant role in antimycobacterial activity.
The balance of the therapeutic versus toxicological effects of bioactive agents is an
important parameter when verifying their applicability as drugs. Therefore the most active
isothiazolopyridines 1a-d and 13a were also tested in VERO cells for determination of
cytotoxicity (IC50) and the selectivity indexes (SI), defined as IC50/MIC. All compounds
demonstrate a degree of cytotoxicity ranging from 1.6 to 7 g/mL and a low level of
selectivity index SI=0.25-2.69 (Table 4). The value of SI<10 indicates significant
cytotoxicity, and these compounds were not considered to be evaluated further against
Mycobacterium tuberculosis.

Table 4. Antimycobacterial activity against M. tuberculosis H37Rv and cytotoxic activity of
isothiazolopyridines 1a-d and 13a
To determine whether the activity of isothiazolopyridines 1a-d was exclusive to
Mycobacterium tuberculosis H37Rv, compounds 1a and 1b were also tested against
Mycobacterium avium, a naturally drug-resistant opportunistic pathogen, using clarithromycin
as a standard. The tested compounds proved inactive after application at a concentration of
12.5 g/mL, whereas clarithromycin exhibited inhibition of 98% of microorganisms at a
concentration of 2 g/mL.
Some authors suggest that activity against the rapidly growing and less hazardous
microorganism Mycobacterium fortuitum may be used as a measure of anti-M. tuberculosis
action.3 To verify this hypothesis, our new isothiazolopyridines 8-13 (except for 11c) were
also evaluated against this strain of microorganisms in vitro at concentrations of 0.97-250
g/mL. The results of anti-Mycobacterium fortuitum activity studies are presented in Table 3.
Initially the MIC50 values were determined. Isoniazid was used as a standard drug (MIC50
=
0.322 g/mL). The data from Table 3 showed that only Mannich base 12d significantly
reduced growth of microorganisms and this effect was observed for concentration below 1
g/mL, similar as for the reference drug. It should be noted that isothiazolopyridine 12d was
also active against M. fortuitum at MIC90 < 1 g/mL, and this compound will be the subject of
further investigation to confirm the preliminary results.
The fact that very similar analogues 12 of isothiazolopyridine 12d did not inhibit
mycobacteria suggests that a specific interaction may exist between compound 12d and some
components specific for the Mycobacterium fortuitum strain.
The action of the remaining isothiazolopyridines against Mycobacterium fortuitum
was generally poor, with MIC50 values of 125 g/mL for carbamates 10b and 11b and 197
g/mL for nitrophenylpiperazine derivative 8. In this context it should be noted that
isothiazolopyridines 9, 10d and 12f were not active against Mycobacterium fortuitum at the
MIC50 level; however, these compounds inhibited growth of 20-50% of microorganisms at all
concentrations used. Thus, these isothiazolopyridines exhibited predominantly a bacteriostatic
effect.
Taking into account the fact that the new isothiazolopyridines tested were inactive in
general against M. fortuitum as well as against M. tuberculosis H37Rv, it is difficult to

conclude whether the in vitro anti-M. tuberculosis H37Rv activity of our compounds can be
related to their anti-M. fortuitum action.
The results of an initial in vitro microbiological evaluation of nineteen of our
isothiazolopyridines against Propionibacterium acnes revealed that four of them (9, 10a, 11a,
12d) were efficient antibacterial agents at the MIC50 level (Table 3). However, only 9 showed
strong activity and produced > 90% inhibition of microorganisms (MIC90 > 1 g/mL).
Compound 9 also demonstrated higher activity at the concentration range of 1-0.25 g/mL
when compared to the reference drug (erythromycin).22
Studies are currently ongoing to explain the mode of unique action of 9 against
Propionibacterium acnes. It should be noted that isothiazolopyridine 12h inhibited 21-32% of
microorganisms at all concentrations used. Thus, the compound has a bacteriostatic effect
against Propionibacterium acnes.
The remaining isothiazolopyridines tested were inactive against Mycobacterium
fortuitum and Propionibacterium acnes (0, Table 3), non-linear correlations prohibited
accurate determination of MIC values (nc, Table 3) or unexpectedly 7 of them (carbamates
10c, 11a, b and Mannich bases 12a, e, f, g) stimulated bacterial growth (st, Table 3). The
compounds which stimulated growth of microorganisms were classified as high stimulating
(stimulation >50%) and low stimulating (stimulation 10-50%) agents. However, it is unclear
whether the low increase in bacterial replication ~10% is a consequence of enhancement of
bacterial growth by the preparations or a lack of activity of the compounds to allow the
natural growth of the microorganisms.
A low growth stimulation effect (10-50%) of the Mycobacterium fortuitum strain was
shown by carbamates 10c and 11a. Compound 11a stimulated at all concentrations used
(0.97-250 g/mL) whereas 10c exhibited the best stimulation effect within the range of 15.6-
31.25 g/mL (Table 3).
The best stimulant of Propionibacterium acnes was carbamate 11b, which exhibited
the effect of stimulation >50% observed at a concentration of 3.9-15.6 g/mL. Mannich bases
12a, e, f, g have been found to stimulate growth of this strain of bacteria in the range of 10-
50% at different concentrations (Table 3).
In this context of bacterial growth stimulation by compounds 11 and 12, it should be
noted that carbamates, like Mannich bases, are unstable substances and it cannot be ruled out
that nitrogen-containing products of degradation of the side chain may be utilized by
microorganisms to increase growth stimulation.

4. Conclusion
To investigate the influence of the introduction of various substituents in the
isothiazolopyridine nucleus on antibacterial activity, twenty compounds 8-13 were
synthesized and in vitro microbiologically evaluated, including activity against
Mycobacterium tuberculosis H37Rv, Mycobacterium fortuitum PMC 672 and
Propionibacterium acnes PCM 2400.
We found that the most active against Mycobacterium tuberculosis H37Rv, compound
13a and isothiazolopyridines 1 (Table 4) recently investigated in this test, were characterized
by MIC values within the range 1.56-6.25 g/mL (Table 3). However, this activity was about
10-50 times lower than that observed for the reference drug rifampicin (0.125 g/mL).
Additionally, these compounds were characterized by a low value of the selectivity index (SI
0.25-2.69) and therefore they could not be considered for further evaluation. The investigation
also proved rather low anti-Mycobacterium fortuitum activity of the tested compounds 8-13,
because only derivative 12d produced 90% reduction of this microorganism at concentration
<1 g/mL. Only compound 9 exhibited significant activity against Propionibacterium acnes
(MIC90 <1 g/mL). The activity of 9 was better than that of erythromycin within the
concentration range of 1-0.25 g/mL. Unexpectedly, 7 of the 19 isothiazolopyridines tested
against M. fortuitum and P. acnes stimulated growth of microorganisms in the range of 10-
50% or even more (11b).
The promising isothiazolopyridines 9 and 12d will be subjected to advanced
biochemical investigation.
5. Experimental
5.1. Chemistry
5.1.1. Chemical experimental section
Melting points were determined with a Mel-Temp II apparatus (Laboratory Devices, USA)
1
and were uncorrected. The H NMR and 13C NMR spectra were recorded on a Bruker 300
MHz spectrometer in CDCl3 using tetramethylsilane (TMS) as an internal reference (chemical
shift in δ ppm). The IR (KBr) spectra were recorded on a Specord-75 IR Spectrometer.

Elemental C, H, N analyses were run on a Carlo Erba NA-1500 analyzer. The results were
within ±0.4% of the values calculated for the corresponding formulae. Chromatographic
separations were performed on a silica gel [Kieselgel 60 (70-230mesh), Merck] column(CC).
Progress of the reaction was monitored by TLC on silica gel plates with fluorescent indicator
(Fluka) and visualized by UV light at 254 nm.
5.1.1.1. 2-[4-nitrophenyl(piperazinyl)methyl]-4,6-dimethylisothiazolo[5,4-b]pyridin-3(2H)-
one 8
To a stirred mixture of 2.4 mmol of 2-hydroxymethyl-4,6-dimethylisothiazolo 5,4-b pyridin-
3(2H)-one 3 in 20 mL of ethanol 2.4 mmol of 4-nitrophenylpiperazine was added. Next the
mixture was stirred for 2 h at room temperature and then refluxed for 5 h. After cooling, the
precipitated, crude product was filtered off and crystallized from methanol.
M.p.: 201-203°C, yield: 70%
IR: 1650 (CO)
1H NMR: 2.60 (s, 3H, CH3), 2.76 (s, 3H, CH3), 2.81-2.97 (m, 4H, CH2-piperazine), 3.10-3.25 (m,
4H, CH2-piperazine), 4.70 (s, 2H, N-CH2-N), 6.76 (s, 1H, Hβ-pyridine), 6.90-6.99 (m, 2H, ArH),
8.05-8.20 (m, 2H, ArH)
5.1.1.2. 2-{2-[4-nitrophenyl(piperazinyl)]ethyl}-4,6-dimethylisothiazolo[5,4-b]pyridin-3(2H)-
one 9
To a stirred mixture of 1.2 mmol of 2-(2-chloro)ethyl-4,6-dimethylisothiazolo 5,4-b pyridin-
3(2H)-one 5 in 30 mL of ethanol 2.4 mmol of 4-nitrophenylpiperazine was added. Next the
mixture was refluxed for 20 h. After cooling, the solvent was distilled off. The product was
isolated from the resulting residue by column chromatography (ethyl acetate, Rf =0.61).
M.p.: 164-166°C, yield: 37%
IR: 1650 (CO)
1H NMR: 2.62 (s, 3H, CH3), 2.70-2.85 (m, 9H, CH3 and N(CH2)3), 3.40-3.55 (m, 4H, CH2-
piperazine), 4.00-4.25 (m, 2H, Nisothiazole-CH2), 6.76 (s, 1H, Hβ-pyridine), 6.85-7.01 (m, 2H, ArH),
8.10-8.25 (m, 2H, ArH)
13C NMR: 164.9, 163.7, 162.8, 154.7, 149.8, 138.8, 125.9, 122.5, 114.8, 112.9, 56.5, 52.4,
47.0, 40.2, 24.5, 17.5

5.1.1.3. General procedure for preparation of the isomeric carbamates 10a-d and 11a-d
To a stirred mixture of a catalytic amount of 1,4-diazabicyclo[2.2.2]octane (DABCO) and
3.75 mmol of appropriate alkyl or phenyl isocyanate in 15 mL of dry xylene the solution of
2.5 mmol of 2-N- or 3-O-(2-hydroxyethyl) derivatives of isothiazolopyridine 6a and 6b in 10
mL of dry xylene was added. The reaction mixture was refluxed for 5 h. Next the solvent was
distilled off and the resulting residue was chromatographed (except for 11b). The obtained,
crude product was crystallized from the appropriate solvent.
2-[2-(N-ethylcarbamoiloxy)ethyl]-4,6-dimethylisotiazolo 5,4-b pyridin-3(2H)-one 10a
M.p.: 101-103°C (n-hexane), yield: 54%
CC (ethyl acetate, Rf =0.56)
IR: 1660 (CO), 1720 (CO), 3340 (NH)
1H NMR: 1.12 (m, 3H, CH3CH2), 2.59 (s, 3H, CH3), 2.73 (s, 3H, CH3), 3.16-3.25 (m, 2H,
CH3CH2), 4.09 (t, J=4.8 Hz, 2H, N2-CH2), 4.34 (t, J=4.8 Hz, 2H, CH2O), 4.86 (brs, 1H, NH),
6.93 (s, 1H, Hβ-pyridine
)
2-[2-(N-n-buthylcarbamoiloxy)ethyl]-4,6-dimethylisothiazolo 5,4-b pyridin-3(2H)-one 10b
M.p.: 79-81°C (cyclohexane), yield: 61.7%
CC (ethyl acetate/chloroform 1:1, Rf =0.54)
IR: 1670 (CO), 1735 (CO), 3420 (NH)
1H NMR: 0.89 (t, J=7.2 Hz, 3H, CH3CH2CH2CH2), 1.25-1.37 (m, 2H, CH3CH2CH2CH2),
1.40-1.50 (m, 2H, CH3CH2CH2CH2), 2.57 (s, 3H, CH3), 2.71 (s, 3H, CH3), 3.14-3.16 (m, 2H,
CH3CH2CH2CH2), 4.07 (t, J=5.1 Hz, 2H, N2-CH2), 4.32 (t, J=5.1 Hz, 2H, CH2O), 4.89 (brs,
1H, NH), 6.91 (s, 1H, Hβ-pyridine
)
2-[2-(N-tert-buthylcarbamoiloxy)ethyl]-4,6-dimehtylisothiazolo 5,4-b pyridin-3(2H)-one 10c
M.p.: 106-107°C (cyclohexane), yield: 18%
CC (ethyl acetate/chloroform 1:1, Rf =0.51)
IR: 1670 (CO), 1730 (CO), 3310 (NH)
1H NMR: 1.31 (s, 9H, C(CH3)3), 2.60 (s, 3H, CH3), 2.75 (s, 3H, CH3), 4.07 (t, J=5.2 Hz, 2H,
N2-CH2), 4.32 (t, J=5.1 Hz, 2H, CH2O), 4.72 (brs, 1H, NH), 6.92 (s, 1H, Hβ-pyridine
)
2-[2-(N-phenylcarbamoiloxy)ethyl]-4,6-dimethylizothiazolo 5,4-b \pyridin-3(2H)-one 10d
M.p.: 135-136°C (cyclohexane), yield: 61.3%
CC (ethyl acetate/chloroform 1:1, Rf=0.60)
IR: 1670 (CO), 1710 (CO), 3310 (NH)

1H NMR: 2.59 (s, 3H, CH3), 2.73 (s, 3H, CH3), 4.15 (t, J=5.4 Hz, 2H, N2-CH2), 4.45 (t, J=5.4
Hz, 2H, CH2O), 4.76 (brs, 1H, NH), 6.93 (s, 1H, Hβ-pyridine), 7.03-7.08 (m, 2H, ArH), 7.29-
7.39 (m, 3H, ArH)
13C NMR: 164.5, 162.4, 152.8, 137.5, 129.1, 123.7, 122.7, 119.8, 118.9, 63.2, 42.7, 24.2, 17.5
3-[2-(N-ethylcarbamoiloxy)ethoxy]-4,6-dimethylisothiazolo 5,4-b pyridine 11a
M.p.: 125-127°C (cyclohexane), yield: 33.8%
CC (ethyl acetate/chloroform 1:1, Rf=0.63)
IR: 1690 (CO), 3330 (NH)
1H NMR: 1.12 (m, 3H, CH3CH2), 2.62 (s, 3H, CH3), 2.66 (s, 3H, CH3), 3.05-3.43 (m, 2H,
CH3CH2), 4.53 (t, J=4.8 Hz, 2H, CH2O), 4.68 (t, J=4.8 Hz, 2H, OCH2), 4.94 (brs, 1H, NH),
6.93 (s, 1H, Hβ-pyridine
)
3-[2-(N-n-buhtylcarbamoiloxy)ethoxy]-4,6-dimethylisothiazolo 5,4-b pyridine 11b
M.p.: 119-121°C (cyclohexane), yield: 49.4%
IR: 1690 (CO), 3320 (NH)
1H NMR: 0.91 (t, J=7.2 Hz, 3H, CH3CH2CH2CH2), 1.01-1.12 (m, 2H, CH3CH2CH2CH2),
1.24-1.48 (m, 2H, CH3CH2CH2CH2), 2.63 (s, 3H, CH3), 2.64 (s, 3H, CH3), 3.09-3.17 (m, 2H,
CH3CH2CH2CH2), 4.46 (t, J=5.2 Hz, 2H, CH2O), 4.66 (t, J=5.2 Hz, 2H, OCH2), 4.92 (brs,
1H, NH), 6.92 (s, 1H, Hβ-pyridine
)
3-[2-(N-tert-buthylcarbamoiloxy)ethoxy]-4,6-dimethylisothiazolo 5,4-b pyridine 11c
M.p.: 109-111°C (n-heptane), yield: 30.5%
CC (ethyl acetate/chloroform 1:1, Rf=0.55)
IR: 1730 (CO), 3260 (NH)
1H NMR: 1.31 (s, 9H, C(CH3)3), 2.62 (s, 3H, CH3), 2.65 (s, 3H, CH3), 4.42-4.52 (m, 2H,
CH2O), 4.64-4.72 (m, 3H, OCH2 and NH), 6.92 (s, 1H, Hβ-pyridine
)
3-[2-(N-phenylcarbamoiloxy)ethoxy]-4,6-dimethylisothiazolo 5,4-b pyridine 11d
M.p.: 144-146°C (cyclohexane), yield: 32.8%
CC (ethyl acetate/chloroform 1:1, Rf=0.65)
IR: 1740 (CO), 3230 (NH)
1H NMR: 2.61 (s, 6H, 2CH3), 4.64-4.67 (m, 4H, OCH2CH2O), 4.99 (brs, 1H, NH), 6.93 (s,
1H, Hβ-pyridine), 7.05-7.37 (m, 5H, ArH)
13C NMR: 160.3, 152.2, 136.8, 128.1, 122.8, 121.0, 118.0, 65.7, 62.1, 24.1, 18.4

5.1.1.4. General procedure for preparation of benzylamine derivatives of isothiazolo 5,4-
b pyridine 12
To a stirred mixture of 10 mmol 2-hydroxymethyl-4,6-dimethylisothiazolo 5,4-b pyridin-
3(2H)-one 3 in 20 mL of ethanol 5 mmol of an appropriate benzylamine was added. Next the
mixture was refluxed for 2 h. After cooling, the precipitated, crude product (12a-c, 12e, 12g)
was filtered off and crystallized from the appropriate solvent. In the case of compounds 12d
and 12h-f the ethanol solution was evaporated and the oil residue was cleaned by
crystallization with charcoal from appropriate solvent.
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-benzylamine
12a
M.p.: 192-194°C (ethanol), yield: 36.7%
IR: 1670 (CO)
1H NMR: 2.59 (s, 6H, 2CH3), 2.72 (s, 6H, 2CH3), 3.97 (s, 2H, NCH2), 4.94 (s, 4H, 2N2-CH2),
6.92 (s, 2H, 2Hβ-pyridine), 7.22-7.52 (m, 5H, ArH)
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-2-
chlorobenzylamine 12b
M.p.: 190-192°C (ethanol), yield: 45.6%
IR: 1670 (CO)
1H NMR: 2.59 (s, 6H, 2CH3), 2.72 (s, 6H, 2CH3), 4.14 (s, 2H, NCH2), 5.00 (s, 4H, 2N2-CH2),
6.90 (s, 2H, 2Hβ-pyridine), 7.15-7.36 (m, 4H, ArH)
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-4-
chlorobenzylamine 12c
M.p.: 162-164°C (ethanol), yield: 38%
IR: 1670 (CO)
1H NMR: 2.60 (s, 6H, 2CH3), 2.71 (s, 6H, 2CH3), 3.94 (s, 2H, NCH2), 4.93 (s, 4H, 2N2-CH2),
6.93 (s, 2H, 2Hβ-pyridine), 7.14-7.30 (m, 4H, ArH)
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-2-
methoxybenzylamine 12d
M.p.: 178-180°C (cyclohexane), yield: 28.7%
IR: 1660 (CO)
1H NMR: 2.57 (s, 6H, 2CH3), 2.70 (s, 6H, 2CH3), 3.80 (s, 3H, OCH3), 4.06 (s, 2H, NCH2),
4.97 (s, 4H, 2N2-CH2), 6.89 (s, 2H, 2Hβ-pyridine), 7.21-7.51 (m, 4H, ArH)

13C NMR: 165.2, 163.4, 162.8, 157.8, 150.0, 130.7, 128.9, 125.2, 122.4, 120.5, 115.3, 110.4,
61.0, 55.2, 48.7, 24.5, 17.5
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-4-
methoxybenzylamine 12e
M.p.: 152-154°C (ethanol), yield: 46%
IR: 1670 (CO)
1H NMR: 2.59 (s, 6H, 2CH3), 2.72 (s, 6H, 2CH3), 3.78 (s, 3H, OCH3), 3.94 (s, 2H, NCH2),
4.93 (s, 4H, 2N2-CH2), 6.88 (s, 2H, 2Hβ-pyridine), 7.22-7.49 (4H, ArH)
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-2-(3,4-
dimethoxyphenylo)methylamine 12f
M.p.: 157-159°C (acetone), yield: 30%
IR: 1670 (CO)
1H NMR: 2.59 (s, 6H, 2CH3), 2.72 (s, 6H, 2CH3), 2.81-3.10 (m, 4H, NCH2CH2), 3.79 (s, 3H,
OCH3), 3.81 (s, 3H, OCH3), 4.97 (s, 4H, 2N2-CH2), 6.70-6.74 (m, 3H, 3ArH), 6.94 (s, 2H,
2Hβ-pyridine
)
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-3,4-
methylenodioxybenzylamine 12g
M.p.: 179-181°C (ethanol), yield: 48.5%
IR: 1650 (CO
1H NMR: 2.59 (s, 6H, 2CH3), 2.71 (s, 6H, 2CH3), 3.84 (s, 2H, NCH2), 4.93 (s, 4H, 2N2-CH2),
5.91 (s, 2H, OCH2O) 6.66-7.31 (m, 5H, 2Hβ-pyridine and 3ArH)
N,N-bis(4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo 5,4-b]pyridin-2-ylmethyl)-N-2-
pyridylmethylamine 12h
M.p.: 181-183°C (cyclohexane/toluene 1:2), yield: 26.4%
IR: 1660 (CO)
1H NMR: 2.59 (s, 6H, 2CH3), 2.72 (s, 6H, 2CH3), 4.14 (s, 2H, NCH2), 5.05 (s, 4H, 2N2-CH2),
7.03 (s, 2H, 2Hβ-pyridine), 7.16-7.89 (m, 3H, 2Hβ+Hγ-pyridine), 8.67-8.77 (m, 1H, Hα-pyridine
)
5.1.1.5. 2-[2-(2-Phenoxyethoxy)ethyl]-4,6-dimethylisothiazolo[5,4-b]pyridin-3(2H)-one 13a
and 3-[2-(2-Phenoxyethyl)ethoxy]-4,6-dimethylisothiazolo[5,4-b]pyridine 13b
To a solution of sodium ethoxide prepared from 0.1 mol of sodium and 100 mL of anhydrous
ethanol 0.1 mol of phenol and 0.2 mol of bis(2-chloroethyl)ether were added. The reaction
mixture was refluxed 12 h and then filtered. The solvent was removed and the oily residue

was distilled under reduced pressure. The 136°C/5 mmHg fraction was collected to give 6 g
of oily 2-(2-phenoxy)ethoxyethyl chloride. 0.02 mol of 2-(2-phenoxy)ethoxyethyl chloride
were added to the stirred mixture of 0.01 mol of 4,6-dimethylisothiazolo[5,4-b]pyridin-3(2H)-
one 7 and 0.01 mol of sodium hydride (~60% suspension in mineral oil) in 50 mL of
anhydrous dimethylformamide (DMF). The reaction mixture was heated at 100°C for 10 h
and the solvent was distilled off. The isomers 13a and 13b were isolated by column
chromatography (toluene/ethyl acetate – 10:1). Evaporation fraction of Rf = 0.53 afforded
1.15 g of O-isomer 13b m.p.: 67-69°C (n-hexane). Evaporation fraction of Rf = 0.28 gave
0.44g of N-isomer 13a m.p.: 59-61°C.
1
13a H NMR: 2.58 (s, 3H, CH3), 2.72 (s, 3H, CH3), 3.79-3.9 (4H, 2xCH2), 4.02-4.19 (m, 4H,
2xCH2), 6.83-6.99 (m, 4H, 3ArH and H-pyridine), 7.16-7.35 (m, 2H, 2ArH)
1
13b H NMR: 2.62 s (3H, CH3), 2.64 s (3H, CH3), 3.92-4.15 m (6H, OCH2CH2OCH2), 4.63-
4.69 m (2H, 3-OCH2), 6.84-6.93 m (4H, 3ArH and H-pyridine), 7.18-7.35 (m, 2H, 2ArH)
5.2 Crystallography
5.2.1. X-ray structure determinations of 12d
X-ray data of 12d were collected on the Bruker SMART APEX II CCD
diffractometer; crystal sizes 0.21x0.13x0.04 mm, MoKα (λ = 0.71073 Å) radiation, ω scans,
absorption correction: multi-scan SADABS,23 Tmin/Tmax = 0.9509/0.9904. The structure was
solved by direct methods using SIR9224 and refined by full-matrix least-squares with
SHELXL97.25 The H atoms were positioned geometrically and treated as riding on their
parent C atoms with C-H distances of 0.93 Å (aromatic), 0.97 Å (CH2) and 0.96 Å (CH3). All
H atoms were refined with isotropic displacement parameters taken as 1.5 times those of the
respective parent atoms. All calculations were performed using WINGX version 1.64.05
package.26 CCDC-859050 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or
from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2
1EZ, UK; fax: +44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk].
5.2.1.1. Crystal data of 12d: C26H27N5O3S2, M = 521.65, monoclinic, space group P21/c, a =
8.2284(3), b = 16.8045(6), c = 20.1143(6) Å, β = 110.743(1)o, V = 2601.00(15) Å3, Z = 4, dcalc
= 1.332 Mg m-3, F(000) = 1096, μ(Mo Kα) = 0.242 mm-1, T=293K, 21949 measured

reflections (θ range 1.62–19.58o), 2282 unique reflections (Rint = 0.026), final R = 0.029, wR =
0.078, S = 1.068 for 2013 reflections with I>2σ(I).
5.3. Pharmacology
5.3.1. In vitro evaluation of antimycobacterial activity against M. tuberculosis H37Rv
Primary screening was conducted at 6.25 g/mL against M. tuberculosis H37Rv in
BACTEC 12B medium using the BACTEC 460 radiometric system.27 Compounds causing
<90% inhibition in the primary screen (MIC > 6.25 g/mL) were not considered for further
evaluation. Compounds demonstrating at least 90% inhibition in the primary screen were re-
tested by serial dilution beginning at the concentration of 6.25 g/mL to determine the actual
minimum inhibitory concentration (MIC). The MIC is defined as the lowest concentration
inhibiting 99% of the inoculum.
Also, compounds were screened by serial dilution to assess toxicity to a VERO cell line
(IC50), beginning at 10 x MIC if sample solubility in culture media was permitted. The
selectivity index (SI) is defined as the ratio of the measured IC50 in VERO cells to the MIC
described above.
5.3.2. In vitro evaluation of antibacterial activity against Mycobacterium fortuitum (PCM
672), Staphylococcus aureus (PCM 2602) and Propionibacterium acnes (PCM 2400)
5.3.2.1. Bacterial strains and growing conditions
The strains of Mycobacterium fortuitum (PCM 672) and Staphylococcus aureus (PCM 2602)
and Propionibacterium acnes (PCM 2400) (obtained from the Polish Collection of
Microorganisms (PCM) of the Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences) were used throughout the study. Bacteria were cultivated on liquid 79
culture medium (for M. fortuitum), Luria-Bertani (LB) medium (for S. aureus) at 37°C for 24
h under aerobic conditions. Then bacterial cells were diluted with the same media,
respectively, to obtain suspension of about 2 x 105 cfu/mL of each strain.
5.3.2.2. Antibacterial susceptibility test
The antibacterial activities of synthesized compounds were determined against bacterial
strains by the microplate Alamar Blue assay according to Ahmed et al.28 Stock solutions of

the compounds were prepared in dimethyl sulfoxide (DMSO) 1 mg/mL and were diluted with
appropriate media in the range 0.03-1000 μg/mL on a cell culture microtitration plate. To the
wells containing 100 μL of drug compound, aliquots of 100 μL of the diluted suspension of
the strain were added. The control wells consisting of either bacteria only or medium only and
those containing different drug concentrations (100 μL) were inoculated with 100 μL of the
diluted bacterial cells. Plates were incubated at 37°C for 48 h and after that 20 μL of Alamar
Blue (10x diluted) and 12.5 μL of 20% Tween 80 solutions were added to the wells and
incubation was continued at 37°C for 2 h. Fluorescence was measured using Victor apparatus
(Wallac, Perkin Elmer). The experiment was repeated two or three times. The minimal
inhibitory concentration (MIC) was defined as the lowest drug concentration which prevented
a color change from blue to pink, inhibiting the bacterial growth by ≥ 90%. The means and
standard error values were determined using the program Statistica.
References
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1985, 28, 1661.

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2000, 55, 416.
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2012
[23] Bruker 2005. SADABS (Version 2004/1). Bruker AXS Inc., Madison, Wisconsin, USA.
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26, 343.
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CH3
N
CH3
N
O
O
( )
n
N
N
N
X
N
H3C
S
N
H3C
S
Y
R
( )
2
1
R
0,1
X= (CH2)0-3, CH(OH)CH2, C=O
Y= N, CH
n = 2,3
1, 2 R = H, Cl, CF3, OCH3
Fig. 1 The general formula of isothiazolopyridines 1 and 2

CH3
N
O
O
N
OH
N
OH
H3C
S
S
3
4
M. tuberculosis H37Rv (MIC 12.5 µg/mL)
65% inhibition 100% inhibition
Fig. 2. Antimycobacterial activity of 2-hydroxymethyl derivatives of isothiazolopyridine 3
and benzisothiazole 4

CH3
N
O
+
HN
N
NO2
N
OH
H3C
S
3
C2H5OH, reflux
CH3
N
O
N
N
n
N
NO2
H3C
S
8 n=1
9 n=2
C2H5OH, reflux
CH3
O
+
HN
N
NO2
N
H3C
N
S
Cl
5
Scheme 1. Synthetic route of compounds 8, 9

CH3
N
CH3
N
O
O
O
O
O
O=C=NR
O=C=NR
N
N
R
N
N
H3C
S
O
NH
O
H3C
S
S
OH
10a-d
CH3
6a
CH3
R
OH
O
NH
H3C
N
H3C
N
S
11a-d
6b
10, 11a (R=C2H5)
10, 11b (R=C4H9)
10, 11c (R=C(CH3)3)
10, 11d (R=C6H5)
Scheme 2. Synthetic route of compounds 10a-d and 11 a-d

CH3
O
2
H3C
+
N
OH
R
H2N
X
N
S
3
C2H5OH, reflux
R
X
CH3
O
S
N
CH3
N
N
N
H3C
N
S
12a-h
O
CH3
X=CH: 12a (R=H), 12b (o-Cl), 12c (R=p-Cl), 12d (R=o-OCH3),
12e (p-OCH3), 12f (R=m-OCH3, p-OCH3), 12g (R=3,4-OCH O-)
2
X=N: 12h (R=H)
Scheme 3. Synthetic route of compounds 12a-h

CH3
N
O
Cl
O
O
+
NH
H3C
S
7
o
NaH, DMF, 100 C
CH3
N
O
O
O
CH3
N
O
H3C
S
13b
N
O
H3C
N
S
O
13a
Scheme 4. Synthetic route of compounds 13a and 13b

Fig. 3. A view of the X-ray molecular structure of 12d with the atomic labeling scheme
(probability 50%)

Table 1. Selected torsion angles (°)
___________________________________________________________________________
Torsion angle
Torsion angle
___________________________________________________________________________
S1A–N2A–C12A–N21
N2A–C12A–N21–C12B
N2A–C12A–N21–C22
C32–C31–C22–N21
72.1(3)
72.7(3)
S1B–N2B–C12B–N21
N2B–C12B–N21–C12A
N2B–C12B–N21–C22
C31–C32–O37–C38
52.7(3)
-153.6(2)
78.9(3)
-159.4(2)
-164.0(2)
81.5(3)
-178.1(3)
C31–C22–N21–C12A
C31–C22–N21–C12B
-149.8(2)
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Table 2. Hydrogen-bond geometry (Å,°) for 12d
________________________________________________________________________
D – H …A
D – H
H…A
D…A
D – H … A
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C12A-H12C…O3A
C10B-H10F…O3B
C12B-H12F…O3B
C36-H361…N21
C11A-H11D…O3A(i)
C12A-H12C…O3A(ii)
0.97
0.96
0.97
0.93
0.96
0.97
2.46
2.33
2.51
2.50
2.57
2.44
2.850(3)
3.085(6)
2.879(4)
2.843(4)
3.512(4)
3.258(4)
104
135
102
102
166
142
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Symmetry codes: (i) = 1 + x, y, z; (ii) = 1 – x, 2–y, –z