Synthesis and biological activity of novel 3-heteroaryl-2H-pyrido[4,3-e][1, 2,4]thiadiazine and 3-heteroaryl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxides

Article abstract of DOI:10.1007/s00706-013-0988-5

A series of novel 1,2,4-thiadiazine 1,1-dioxides were synthesized by condensation of 2-chlorobenzenesulfonamide and 4-chloropyridine-3-sulfonamide with heterocyclic methyl carbimidates obtained from heterocyclic carbonitriles and used at the time of their creation. Substituted amidines were isolated as the intermediates in the reaction with 2-chlorobenzenesulfonamide. Those intermediates were successfully cyclized to corresponding 1,2,4-thiadiazine 1,1-dioxides in pyridine with the addition of DBU. The newly synthesized compounds were evaluated for their tuberculostatic and anticancer activities. Eight compounds were able to inhibit the growth of some renal and non-small cell lung cancer cell lines.

Full text of DOI:10.1007/s00706-013-0988-5

Monatsh Chem (2013) 144:1197–1203
DOI 10.1007/s00706-013-0988-5
ORIGINAL PAPER
Synthesis and biological activity of novel 3-heteroaryl-2H-
pyrido[4,3-e][1,2,4]thiadiazine and 3-heteroaryl-2H-
benzo[e][1,2,4]thiadiazine 1,1-dioxides
•
Katarzyna Gobis Henryk Foks Jarosłw Sławinski
•
•
´
Ewa Augustynowicz-Kopec Agnieszka Napiorkowska
•
´
´
Received: 23 November 2012 / Accepted: 31 March 2013 / Published online: 14 May 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract A series of novel 1,2,4-thiadiazine 1,1-dioxides
were synthesized by condensation of 2-chlorobenzene-
sulfonamide and 4-chloropyridine-3-sulfonamide with het-
erocyclic methyl carbimidates obtained from heterocyclic
carbonitriles and used at the time of their creation. Substi-
tuted amidines were isolated as the intermediates in the
reaction with 2-chlorobenzenesulfonamide. Those interme-
diates were successfully cyclized to corresponding 1,2,4-
thiadiazine 1,1-dioxides in pyridine with the addition of
DBU. The newly synthesized compounds were evaluated for
their tuberculostatic and anticancer activities. Eight com-
pounds were able to inhibit the growth of some renal and
non-small cell lung cancer cell lines.
polymerase, and aldose reductase [5–7]. Benzothiadiazine
1,1-dioxides also constitute an important class of cyclic
sulfonamides with broad-spectrum activity against bacteria,
fungi, and Mycobacterium tuberculosis [8–10]. In an effort
to develop new and effective chemotherapeutic agents for
the treatment of tuberculosis, several series of heterocyclic
compounds based on a sulfonamidine scaffold have recently
been synthesized by our laboratory [11–13]. Here, we
disclose the synthesis of novel carbimidate-derived 1,2,4-
benzothiadiazine 1,1-dioxides and 1,2,4-pyridothiadiazine
1,1-dioxides with different heterocyclic rings at the 3-position.
The synthesized compounds were screened for their anti-
tubercular and anticancer activities in vitro.
Keywords Sulfonamidine Á Heterocycles Á Synthesis Á
Anticancer activity Á Structure–activity relationship
Results and discussion
The aim of the study was to investigate the reactivity of
heterocyclic methyl carbimidates towards sulfonamides that
possess a chlorine atom as a substituent at the ortho position
to the sulfonamide group. The use of such sulfonamides
facilitates the cyclization of sulfonated amidines, formed in
the first stage of the reaction, to 1,2,4-thiadiazine 1,1-diox-
ides. The literature describes methods for the synthesis of
1,2,4-thiadiazine 1,1-dioxides. The most common method is
the reaction of 2-aminobenzenesulfonamides with carbox-
ylic acids, their halides, or anhydrides [14, 15]. Synthesis via
the reaction of 2-aminosulfonamides with aldehydes is
another method that has been used [16]. Other authors have
reported the reaction of 2-halobenzenesulfonyl chlorides
with amidines and aminopyridines in the presence of
potassium carbonate [17]. The synthetic method in which
substituted amidines react with TosNSO (N-sulfinyl-p-
toluenesulfonamide) in acetic acid and hydrogen peroxide
has also been described [18].
Introduction
1,2,4-Benzothiadiazine 1,1-dioxides are well known for
their cardiovascular and hypertensive effects [1, 2]. They
also act as ATP-sensitive potassium channel openers, like
their pyridyl analogs the 1,2,4-pyridothiadiazine 1,1-diox-
ides [3, 4]. Insulin release is inhibited as a result of that
activity. Compounds of this group are the inhibitors of
some enzymes, such as xanthine oxidase, HCV NS5B
´
K. Gobis (&) Á H. Foks Á J. Sławinski
Department of Organic Chemistry, Medical University
´
of Gdansk, Gdansk, Poland
e-mail: kgobis@gumed.edu.pl
´
´
´
E. Augustynowicz-Kopec Á A. Napiorkowska
Department of Microbiology, Institute of Tuberculosis
and Pulmonary Diseases, Warsaw, Poland
123

1198
K. Gobis et al.
Scheme 1
NH
OMe
i
R CN
R
1
2
3
4
5
No
R
MeO
N
N
N
for R=Ph
N
O
O
O
O
N
N
N
N
S
S
N
H2N
iii
H2N
i or vi
NH2
Cl
Cl
O
O
N
O
O
O
O
H
S
N
H
S
S
ii
N
N
R
N
R
R
N
Cl
1-5
6-10, 18
11-17
No 6, 11 7, 14 8, 15
9, 16
10, 17
12
13
18
MeO
N
N
N
N
R
N
N
N
N
N
N
.
The method presented in this paper involved the use of
heterocyclic methyl carbimidates as they are synthesized
from the corresponding carbonitriles (Scheme 1). The car-
bimidates were reacted with 2-chlorobenzenesulfonamide
and 4-chloropyridine-3-sulfonamide in methanol. We have
previously described the diazabicyclo products of this
reaction when it is carried out with a catalytic amount of
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene). This gave a
linear amidine [13]. When DBU was equimolar to the sul-
fonamide, the reaction with 2-chlorbenzenesulfonamide led
to linear structures 1–5. However, when 4-chloropyridine-3-
sulfonamide was used, the corresponding 3-heteroaryl-
substituted pyrido[4,3-e][1,2,4]thiadiazine 1,1-dioxides
11–17 were the reaction products. Reducing the electron
density on the carbon atoms of the pyridine ring at positions a
and c increases their vulnerability to nucleophilic attack.
A halogen at the c position of the pyridine ring is readily
exchanged for a nucleophilic NH group. c-Halopyridines are
even more reactive than a-isomers [19]. Therefore, products
that were cyclized to 1,2,4-thiadiazine 1,1-dioxides were
easily obtained.
H
N
N
N
N
X
X
N
N
S
S
H
O
O
O
O
6A X=CH
11A X=N
6B X=CH
11B X=N
Fig. 1 Structures of possible tautomers of compound 6: 2H (6A) and
4H (6B)
alone. The 3-phenylpyrido[1,2,4]thiadiazine derivative 18
was obtained from ethyl benzimidate hydrochloride and
4-chloropyridine-3-sulfonamide in methanol with excess
DBU.
1
The H NMR signals for the aromatic protons and NH-
group protons were observed at 12–13 ppm. To elucidate
the possible tautomeric forms of the representative com-
pounds 6 (Fig. 1) and 11, we estimated the total energies of
the isolated molecules shown in Table 1.
Calculations were performed using ab initio Hartree–
Fock and DFT methods in the gas phase. From the data
presented in Table 1, one can infer that the 2H tautomers of
compounds 6 and 11 are more energetically favorable than
the 4H tautomers by 42.94–93.19 kJ/mol according to
ab initio RHF as well as the density functional B3LYP
method with the 6-31G* basis set [20]. Moreover, the
possible optimized structures for compound 6 indicated
conditions favoring hydrogen-bond formation between the
hydrogen at nitrogen atom N-2 and the nitrogen atom of the
pyridine substituent at carbon C-3. In this way, a stable
Using an equimolar amount of DBU in the case of
methyl 6-chloropyrazinecarbimidate led to the creation of a
mixture of linear and thiadiazine (16) structures that were
quite easy to separate. However, the chlorine atom was
replaced with a methoxy group.
Cyclization of amidines substituted with a 2-chloro-
benzenesulfonamide moiety (1–5) to 3-heteroaryl-2H-
benzo[e][1,2,4]thiadiazine 1,1-dioxides 6–10 was carried
out by refluxing the substrates in pyridine in the presence
of equimolar DBU. Cyclization did not occur in pyridine
123

Synthesis and biological activity of 1,2,4-thiadiazine 1,1-dioxides
1199
Table 1 Calculated energies (E) and relative energies (DE) of tau-
tomers 6A–6B and 11A–11B
Table 2 Tuberculostatic activities of the newly synthesized com-
pounds 11 and 15
Tautomer
Method
E/hartrees
DE/kJ mol-1
Compound
MIC/lg cm-3
H37Rv
Spec. 192
Spec. 210
6A (2H)
6B (4H)
6A (2H)
6B (4H)
11A (2H)
11B (4H)
11A (2H)
11B (4H)
RHF/6-31G*
-1170.176389
-1170.158428
-1175.525612
-1175.490116
-1186.167699
-1186.150241
-1191.559916
-1191.543559
47.15
0
RHF/6-31G*
11
50
50
50
0.5
25
25
1.1
B3LYP/6-31G*
B3LYP/6-31G*
RHF/6-31G*
93.18
0
15
100
0.5
INH
45.83
0
Minimum inhibitory concentrations for bacterial strains were deter-
mined by a twofold serial dilution method for microdilution plates
and by a classical test-tube method of twofold successive dilution for
mycobacterial strains. M. tuberculosis H37Rv, Spec. 192, Spec. 210
RHF/6-31G*
B3LYP/6-31G*
B3LYP/6-31G*
42.94
0
Energy values were calculated using ab initio RHF and B3LYP with
the 6-31G* basis set
antitumor activities [13]. It was interesting to see how the
closure of the open structure to form the 1,2,4-thiadiazine
1,1-dioxide system affects this activity, especially consid-
ering that we have already reported the high affinity of the
1,2,4-thiadiazine 1,1-dioxide derivatives for isozyme CA
IX (cancer-associated), an isoform of zinc enzyme carbonic
anhydrase (CA, EC 4.2.1.1) [21], and the significant anti-
tumor activities of these derivatives [22].
five-membered cyclic structure can form, which addition-
ally stabilizes that tautomer (Figs. 1, 2).
Biological activity
Two of the 1,2,4-thiadiazine 1,1-dioxides obtained (11, 15)
were evaluated for their in vitro tuberculostatic activity
against the Mycobacterium tuberculosis H37Rv strain and
two ‘‘wild’’ strains isolated from tuberculosis patients: one
(Spec. 210) resistant to p-aminosalicylic acid (PAS),
isonicotinic acid hydrazide (INH), ethambutol (ETB), and
rifampicin (RFP), and another (Spec. 192) that was fully
sensitive to the tuberculostatics administered (Table 2).
Isoniazid (INH) was used as a reference drug.
Compounds were tested in the framework of the
Developmental Therapeutics Program (DTP) at the
National Cancer Institute (Bethesda, MD, USA) on a panel
of 60 human tumor cell lines derived from nine different
cancer types: leukemia, lung, colon, CNS, melanoma,
ovarian, renal, prostate, and breast. Among the compounds
tested (6–18) in the preliminary NCI-60 one-dose screening
test, eight of them (62 %) exhibited distinct growth inhi-
bition (DGI) properties (Table 3). Six compounds (6, 9, 10,
12, 13, 16) were active towards renal cancer cell lines:
A498 (6, 10), TK-10 (9, 16), and UO-31 (10, 13). These
compounds inhibited the growth of those cell lines, with
DGI ranging from 19.2 to 24.2 %. Derivative 10 exhibited
activity against two renal cancer cell lines A498 and UO-
31. Moreover, it was potent towards the colon cancer HT29
cell line (DGI 20.1 %). Derivatives 15 and 17 were active
towards non-small cell lung cancer cell lines. Compound
15 was potent towards the HOP-92 cell line (DGI 25.9 %)
and compound 17 was potent towards EKVX (DGI
19.2 %).
The tested compounds showed weak tuberculostatic
activity, much lower than the reference INH (MIC 0.5–
1.0 lg/cm3). The MIC values obtained when the com-
pounds were tested against three strains ranged from 25 to
100 lg/cm3 for both compounds. Interestingly, both com-
pounds were more active against the resistant 210 strain
than the sensitive 192 one.
All of the newly synthesized compounds were also
tested for antitumor activity. We previously described the
synthesis of open sulfonamidine derivatives that are ana-
logs of the 1,2,4-thiadiazine 1,1-dioxides presented here.
We established that these compounds have notable
Fig. 2 The optimized structures
of the possible tautomers of
compound 6 (calculated via the
B3LYP/6-31G* method): 2H
(left) and 4H (right)
123

1200
K. Gobis et al.
Table 3 One-dose screening
data on the in vitro tumor
growth inhibitory activities of
compounds 6–18 at a dose of
10 lM
Compound Mean growth values (MG_MIDa)/% Panel
Cell line Growth
inhibition DGI/%
6
103.8
102.1
102.9
Renal cancer
Renal cancer
Colon cancer
Renal cancer
A498
19.2
9
TK-10 19.7
10
HT29
A498
20.1
21.7
UO-31 51.1
UO-31 21.7
UO-31 24.5
Data obtained from the NCI-60
DTP human tumor cell line
screening
12
13
15
16
17
104.3
102.3
101.8
101.4
95.1
Renal cancer
Renal cancer
a
Non-small cell lung cancer HOP-92 22.0
Renal cancer TK-10 24.2
MIG_MID mean graph
midpoint (i.e., the arithmetical
mean growth for all tested cell
lines)
Non-small cell lung cancer EKVX 19.2
Conclusion
The synthesis of sulfonylcarboximidamides 1–5 was
described previously [13].
A series of novel 1,2,4-thiadiazine 1,1-dioxides with different
six-membered nitrogen heterocyclic systems at the C-3 posi-
tion were successfully synthesized by the reaction of
heterocyclic methyl carbimidates with 2-chlorobenzene-
sulfonamide and 4-chloropyridine-3-sulfonamide. Substi-
tuted amidines were isolated as the intermediates in the
reaction with 2-chlorobenzenesulfonamide. Those interme-
diates were successfully cyclized to the corresponding 1,2,
4-thiadiazine 1,1-dioxides in pyridine with the addition of
DBU. The syntheses of these new compounds were confirmed
by analyzing their IR and NMR spectra as well as elemental
analysis. The tuberculostatic and anticancer activities of the
synthesized compounds were evaluated. The results showed
that the synthesized 1,2,4-thiadiazine 1,1-dioxides exhibited
rather poor tuberculostatic activities in vitro. Eight com-
pounds (6, 9, 12, 13, 15–17) were able to inhibit the growth of
some cancer cell lines derived mainly from renal cancer and
non-small cell lung cancer.
General method for the synthesis of 3-heteroaryl-2H-
benzo[e][1,2,4]thiadiazine 1-1-dioxides 6–10
The respective sulfonamide derivative 1–5 (5 mmol) was
refluxed with 1.8 cm3 DBU (12 mmol) in 3 cm3 of pyri-
dine for 2 h. The mixture was cooled down and 30 g of ice
were added. The clear solution was acidified with glacial
acetic acid. The precipitate was filtered off and purified by
crystallization from a suitable solvent with activated
carbon.
3-(Pyridin-2-yl)-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (6, C12H9N3O2S)
This compound was recrystallized from dioxane, affording
0.791 g (61 %) of 6. M.p.: 295–297 °C; IR (KBr):
ꢀv = 3,268 (m N–H), 3,066 (m C–H), 1,615 (m C=N),
1,595, 1,567 (m C=C), 1,526 (d N–H), 1,301, 1,173 (m SO2),
1
826, 761 (c C–H), 679, 555 (c N–H), 499 cm-1; H NMR
(200 MHz, DMSO-d6): d = 7.52 (t, 1H, J = 7.3 Hz, Ph),
7.72–7.80 (m, 2H, 1H Ph and 1H pyridine), 7.86–8.00 (m,
2H, 1H Ph, 1H pyridine), 8.10 (t, 1H, J = 7.7 Hz, Ph), 8.32
(d, 1H, J = 7.3 Hz, pyridine), 8.85 (d, 1H, J = 4.4 Hz,
pyridine), 12.62 (br s, 1H, NH ? D2O exchangeable) ppm;
13C NMR (50 MHz, DMSO-d6): d = 119.70, 122.01,
123.34, 123.54, 127.02, 127.93, 133.35, 135.79, 138.71,
148.24, 149.40, 152.23 ppm.
Experimental
All materials and solvents were of analytical reagent grade.
Thin-layer chromatography was performed on Merck
(Darmstadt, Germany) silica gel 60F254 plates and visual-
ized with UV. Elemental analyses for C, H, N were
performed on a Carlo Erba 1108 instrument (Thermo Sci-
entific, Waltham, MA, USA) and the results for all of the
obtained compounds were in agreement with calculated
values to within 0.3 %. NMR spectra in DMSO-d6 were
recorded on Varian (Palo Alto, CA, USA) Unity Plus
(500 MHz) and Gemini (200 MHz) instruments. IR spectra
were determined as KBr pellets of the solids on a Satellite
FT-IR spectrophotometer (Mattson Instruments, Madison,
WI, USA). Melting points were determined with a Boethius
apparatus (Franz Ku¨stner Nachf. K.G., Dresden, Germany).
3-(Pyrimidin-2-yl)-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (7, C11H8N4O2S)
This compound was recrystallized from a DMSO-dioxane
mixture (1:1), affording 0.703 g (54 %) of 7. M.p.:
307–310 °C; IR (KBr): ꢀv = 3,277 (m N–H), 1,616 (m
C=N), 1,597, 1,568 (m C=C), 1,525 (d N–H), 1,410
(m C=C), 1,302, 1,159 (m SO2), 818, 766 (c C–H), 675,
1
555 (c N–H), 500 cm-1; H NMR (500 MHz, DMSO-d6):
123

Synthesis and biological activity of 1,2,4-thiadiazine 1,1-dioxides
1201
d = 7.54 (t, 1H, J = 7.3 Hz, Ph), 7.76 (t, 1H, J = 7.3 Hz,
Ph), 7.85 (t, 1H, J = 4.4 Hz, pyridine), 7.90–7.92 (m, 2H,
Ph), 9.14 (d, 2H, J = 4.9 Hz, pyridine), 12.79 (br s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 119.32, 121.71, 123.69, 124.14, 127.42,
133.54, 135.33, 150.68, 156.74, 158.55 ppm.
General method for the synthesis of 3-heteroaryl-2H-
pyrido[4,3-e][1,2,4]thiadiazine 1,1-dioxides 11–18
The respective heteroarylcarbonitrile (5 mmol) was
refluxed with 0.6 cm3 DBU (4 mmol) in 10 cm3 of meth-
anol for 0.5 h. Then 0.77 g 4-chloropyridine-3-sulfonamide
(4 mmol) were added and the mixture was refluxed for
another 3 h. Methanol was removed under vacuum and
30 cm3 of water were added to the residue. The clear
solution was acidified with glacial acetic acid. The precip-
itate was filtered off and recrystallized from a suitable
solvent.
3-(Pyrazin-2-yl)-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (8, C11H8N4O2S)
This compound was recrystallized from a dioxane–ethanol
mixture (1:1), affording 0.755 g (58 %) of 8. M.p.:
275–278 °C; IR (KBr): ꢀv = 3,255 (m N–H), 1,598, 1,570
(m C=C), 1,526 (d N–H), 1,481 (m C=C), 1,304, 1,165
(m SO2), 1,017 (d C–H), 824, 773 (c C–H), 596, 556 (c N–
H), 499 cm-1; 1H NMR (200 MHz, DMSO-d6): d = 7.49–
7.57 (m, 1H, Ph), 7.70–7.80 (m, 1H, Ph), 7.87–7.93 (m, 2H,
Ph), 8.90-9.01 (m, 2H, pyrazine), 9.43-9.44 (m, 1H,
pyrazine), 12.67 (br s, 1H, NH ? D2O exchangeable)
ppm; 13C NMR (50 MHz, DMSO-d6): d = 119.57, 122.06,
123.63, 127.35, 133.53, 135.60, 143.98, 144.43, 148.52,
151.10 ppm.
3-(Pyridin-2-yl)-2H-pyrido[4,3-e][1,2,4]thiadiazine
1,1-dioxide (11, C11H10N4O2S)
This compound was recrystallized from a dioxane–water
mixture, affording 0.708 g (68 %) of 11. M.p.:
331–333 °C; IR (KBr): ꢀv = 3,227 (m N–H), 2,933 (m C–
H), 1,615 (m C=N), 1,584, 1,497 (m C=C), 1,305, 1,166 (m
SO2), 820, 742 (m C–H, c C–H), 605, 550 (c N–H),
507 cm-1 1H NMR (200 MHz, DMSO-d6): d = 7.77–
;
7.89 (m, 2H, pyridine), 8.11–8.19 (m, 1H, pyridine),
8.31–8.35 (m, 1H, pyridine), 8.77 (d, 1H, J = 5.9 Hz,
pyridine), 8.86–8.88 (m, 1H, pyridine), 9.06 (s, 1H,
pyridine), 12.93 (br s, 1H, NH ? D2O exchangeable)
ppm; 13C NMR (50 MHz, DMSO-d6): d = 113.04, 118.10,
123.67, 128.51, 138.92, 141.75, 146.06, 147.32, 149.50,
152.93 ppm.
3-(6-Methoxypyrazin-2-yl)-2H-benzo[e][1,2,4]-
thiadiazine 1,1-dioxide (9, C12H10N4O3S)
This compound was recrystallized from a dioxane–ethanol
mixture (1:1), affording 0.755 g (52 %) of 9. M.p.:
292–295 °C; IR (KBr): ꢀv = 3,298 (m N–H), 1,601, 1,576,
1,548 (m C=C), 1,522 (d N–H), 1,392 (m C=C), 1,303, 1,170
(m SO2), 1,010 (d C–H), 831, 765 (c C–H), 672 (c N–H),
3-(Pyridin-3-yl)-2H-pyrido[4,3-e][1,2,4]thiadiazine
1,1-dioxide (12, C11H8N4O2S)
502 cm-1 1H NMR (500 MHz, DMSO-d6): d = 7.54–
;
7.58 (m, 1H, Ph), 7.75–7.95 (m, 3H, Ph), 8.66 (s, 1H,
pyrazine), 8.96 (s, 1H, pyrazine), 12.20 (br s, 1H, NH ?
D2O exchangeable) ppm; 13C NMR (50 MHz, DMSO-d6):
d = 54.90, 119.61, 122.12, 123.67, 127.76, 133.48, 135.49,
136.01, 139.48, 140.53, 151.30, 159.25 ppm.
This compound was recrystallized from ethanol, affording
0.260 g (25 %) of 12. M.p.: 300–303 °C; IR (KBr):
ꢀv = 3,351 (m N–H), 2,923, 2,808 (m C–H), 1,617 (m
C=N), 1,579 (m C=C), 1,508 (d N–H), 1,482 m C=C), 1,351,
1,299, 1,173 (m SO2), 1,100 (d C–H), 827, 808, 715 (c
1
C–H), 603, 551 (c N–H), 511 cm-1; H NMR (200 MHz,
3-(Quinolin-2-yl)-2H-benzo[e][1,2,4]thiadiazine
DMSO-d6): d = 7.50 (d, 1H, J = 5.7 Hz, pyridine),
7.64–7.70 (m, 1H, pyridine), 8.36–8.42 (m, 1H, pyridine),
8.76 (d, 1H, J = 5.8 Hz, pyridine), 8.86 (d, 1H,
J = 4.8 Hz, pyridine), 9.07 (s, 1H, pyridine), 9.19 (s, 1H,
pyridine), 12.50 (br s, 1H, NH ? D2O exchangeable) ppm;
13C NMR (50 MHz, DMSO-d6): d = 112.95, 118.08,
124.07, 128.18, 136.60, 142.81, 145.87, 149.36, 152.40,
153.64, 154.69 ppm.
1,1-dioxide (10, C16H11N3O2S)
This compound was recrystallized from dioxane–ethanol
mixture (1:1), affording 0.619 g (40 %) of 10. M.p.:
323–324 °C; IR (KBr): ꢀv = 3,441, 3,357, 3,242 (m N–H),
2,957, 2,849 (m C–H), 1,644, 1,596, 1,527 (m C=C), 1,276,
1,136 (m SO2), 1,084 (d C–H), 828 (c C–H), 556 (c N–H)
1
cm-1; H NMR (500 MHz, DMSO-d6): d = 7.56 (t, 1H,
J = 7.8 Hz, Ph), 7.78–7.83 (m, 2H, Ph), 7.93–7.99 (m, 2H,
quinoline), 8.03 (d, 1H, J = 8.3 Hz, quinoline), 8.18 (d,
1H, J = 7.8 Hz, pyridine), 8.33–8.39 (m, 2H, quinoline),
8.70 (d, 1H, J = 8.3 Hz, quinoline), 12.54 (br s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 119.27, 119.51, 121.86, 123.69, 127.22,
128.58, 129.22, 129.43, 129.55, 131.33, 133.56, 135.30,
138.85, 146.50, 148.19, 152.00 ppm
3-(Pyridin-4-yl)-2H-pyrido[4,3-e][1,2,4]thiadiazine
1,1-dioxide (13, C11H8N4O2S)
This compound was recrystallized from DMSO, affording
0.437 g (42 %) of 13. M.p.:[340 °C; IR (KBr): ꢀv = 3,057
(m C–H), 1,629 (m C=N), 1,489, 1,426 (m C=C), 1,406,
1,287, 1,158 (m SO2), 843, 688 (c C–H), 601, 550 (c N–H)
1
cm-1; H NMR (500 MHz, DMSO-d6): d = 7.54 (d, 1H,
123

1202
K. Gobis et al.
J = 5.9 Hz, pyridine), 7.99 (d, 2H, J = 4.9 Hz, pyridine),
8.77 (d, 1H, J = 5.4 Hz, pyridine), 8.89 (d, 2H,
J = 5.4 Hz, pyridine), 9.10 (s, 1H, pyridine), 12.70 (br s,
1H, NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 113.34, 118.16, 122.26, 139.76, 143.25,
145.74, 150.72, 152.06, 154.78 ppm.
3-(Quinolin-2-yl)-2H-pyrido[4,3-e][1,2,4]thiadiazine
1,1-dioxide (17, C15H10N4O2S)
This compound was recrystallized from DMSO, affording
0.720 g (76 %) of 17. M.p.: 347–349 °C; IR (KBr):
ꢀv = 3,233 (m N–H), 1,618 (m C=N), 1,584, 1,489 (m
C=C), 1,321, 1,301, 1,161 (m SO2), 770 (c C–H), 591 (c N–
1
H), 515 cm-1; H NMR (200 MHz, DMSO-d6): d =7.78–
3-(Pyrimidin-2-yl)-2H-pyrido[4,3-e][1,2,4]thiadiazine
1,1-dioxide (14, C10H7N5O2S)
7.82 (m, 1H, pyridine), 7.86–8.02 (m, 2H, quinoline),
8.17–8.20 (m, 1H, quinoline), 8.32–8.39 (m, 2H, 1H
pyridine and 1H quinoline), 8.71 (d, 1H, J = 8.4 Hz,
quinoline), 8.81 (d, 1H, J = 5.8 Hz, quinoline), 9.10 (s,
1H, pyridine), 12.81 (br s, 1H, NH ? D2O exchangeable)
ppm; 13C NMR (50 MHz, DMSO-d6): d = 113.24, 118.08,
119.36, 128.63, 129.49, 129.59, 131.46, 136.48, 138.97,
141.91, 146.04, 146.47, 147.66, 152.76, 152.83 ppm.
This compound was recrystallized from a DMSO–water
mixture (1:1), affording 0.397 g (58 %) of 14. M.p.:
326–329 °C; IR (KBr): ꢀv = 3,195, 3,160 (m N–H), 1,623 (m
C=N), 1,589, 1,560, 1,503 (m C=C), 1,297, 1,159 (m SO2),
1,102 (d C–H), 816 (c C–H), 551 (c N–H), 510 cm-1; H
1
NMR (200 MHz, DMSO-d6): d = 7.84 (d, 1H,
J = 5.9 Hz, pyridine), 7.88 (t, 1H, J = 4.9 Hz, pyrimi-
dine), 8.80 (d, 1H, J = 5.9 Hz, pyridine), 9.09 (s, 1H,
pyridine), 9.16 (d, 2H, J = 4.9 Hz, pyrimidine), 13.06 (br
s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz, DMSO-d6): d = 112.97, 118.17, 141.77,
143.25, 144.00, 144.66, 146.10, 148.93, 151.84,
153.02 ppm.
3-Phenyl-2H-pyrido[4,3-e][1,2,4]thiadiazine
1,1-dioxide (18, C12H9N3O2S)
A mixture of 0.75 g ethyl benzimidate hydrochloride
(4 mmol), 0.58 g 4-chloropyridine-3-sulfonamide (3 mmol),
and 1.5 cm3 DBU (10 mmol) in 10 cm3 of dioxane was
refluxed for 2.5 h. Then solvent was removed under vacuum
and 30 cm3 of cold water were added to the residue. The
mixture was acidified with 6 M HCl. The precipitate was
filtered off and recrystallized from methanol, affording
0.506 g (65 %) of 18. M.p.: 312–315 °C; IR (KBr):
ꢀv = 3,274 (m N–H), 3,089 (m C–H), 1,609 (m C=N), 1,490 (m
C=C), 1,323, 1,290, 1,158 (m SO2), 1,096 (d C–H), 821, 700 (c
3-(Pyrazin-2-yl)-2H-pyrido[4,3-e][1,2,4]thiadiazine
1,1-dioxide (15, C10H7N5O2S)
This compound was recrystallized from ethanol affording
0.543 g (38 %) 15. M.p.: 284–287 °C; IR (KBr):
ꢀv = 3,204 (m N–H), 2,923 (m C–H), 1,631 (m C=N),
1,588, 1,499 (m C=C), 1,310, 1,161 (m SO2), 1,021 (d C–H),
1
C–H), 602, 543 (c N–H), 511 cm-1; H NMR (200 MHz,
812 (c C–H), 604, 551 (c N–H), 511 cm-1 1H NMR
;
DMSO-d6): d = 7.53–7.77 (m, 4H, 3H Ph and 1H pyridine),
8.03–8.07 (m, 2H, Ph), 8.77 (d, 1H, J = 5.7 Hz, pyridine),
9.05 (s, 1H, pyridine), 8.73 (s, 1H, pyrazine), 12.45 (br s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 112.66, 117.99, 128.72, 129.21, 131.66,
133.55, 142.37, 145.98, 152.67, 155.84 ppm.
(500 MHz, DMSO-d6): d = 7.82 (d, 1H, J = 5.7 Hz,
pyridine), 8.80 (d, 1H, J = 5.7 Hz, pyridine), 8.91-8.93
(m, 1H, pyridine), 9.02 (d, 1H, J = 2.4 Hz, pyrazine), 9.08
(s, 1H, pyrazine), 9.44 (s, 1H, pyrazine), 13.04 (br s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 112.93, 117.86, 124.43, 141.80, 146.20,
151.29, 152.95, 156.19, 158.62 ppm.
Tuberculostatic activity
Investigations were performed by a classical test-tube
method of successive dilution in Youmans’ modification of
Proskauer and Beck’s liquid medium containing 10 %
bovine serum [23, 24]. Bacterial suspensions were prepared
from 14-day-old cultures of slow-growing strains and from
48-hour-old cultures of saprophytic strains [25, 26]. Solu-
tions of the compounds in ethylene glycol were tested.
Stock solutions contained 10 mg of the compounds in
1 cm3. Dilutions (geometric progression) were prepared in
Youmans’ medium. A sample of the medium containing
isoniazid (INH) as a reference drug but none of the
investigated substances was used for comparison. Incuba-
tion was performed at a temperature of 37 °C. The MIC
values were determined as the minimum concentration that
3-(6-Methoxypyrazin-2-yl)-2H-pyrido[4,3-e][1,2,4]-
thiadiazine 1,1-dioxide (16, C11H9N5O3S)
This compound was recrystallized from DMSO affording
0.886 g (52 %) 16. M.p.: 285–286 °C (decomp.); IR
(KBr): ꢀv = 3,283 (m N–H), 1,615 (m C = N), 1,590,
1,494 (m C = C), 1,409, 1,392, 1,311, 1,169 (m SO2),
1,005 (d C–H), 894, 837, 710 (c C–H), 603, 560 (c N–H),
1
506 cm-1; H NMR (200 MHz, DMSO-d6): d = 4.17 (s,
3H, OCH3), 7.78 (d, 1H, J = 5.8 Hz, pyridine), 8.67 (s,
1H, pyrazine), 8.80 (d, 1H, J = 5.8 Hz, pyridine), 8.97 (s,
1H, pyridine), 9.09 (s, 1H, pyrazine), 12.28 (br s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 55.43, 113.46, 118.68, 136.71, 140.25,
140.39, 146.53, 149.50, 152.41, 153.38, 159.70 ppm.
123

Synthesis and biological activity of 1,2,4-thiadiazine 1,1-dioxides
1203
8. Kamal A, Ahmed SK, Reddy KS, Khan MNA, Shetti RVCRNC,
Siddardha B, Murthy USN, Khan IA, Kumar N, Sarma S, Ram
AB (2007) Bioorg Med Chem Lett 17:5419
inhibited the growth of the tested tuberculosis strains in
relation to the probe with no tested compound.
9. Kamal A, Shetti RVCRNC, Azeeza S, Ahmed SK, Swapna P,
Reddy AM, Khan JA, Sharma S, Abdullah ST (2010) Eur J Med
Chem 45:4545
Anticancer activity
10. Kamal A, Shetti RVCRNC, Azeeza S, Swapna P, Khan MNA,
Khan IA, Sharma S, Abdullah ST (2011) Eur J Med Chem 46:893
Compounds were tested at one concentration (10 lM). A
mean graph midpoint (MG_MID) was calculated to give
the average activity parameter over all cell lines. Cell lines
that were insensitive in the screen were included in the
calculate the MG_MID. Selectivity of a compound with
respect to one or more cell lines of the screen was char-
acterized by a high deviation of the particular cell line
parameter from the MG-MID value. Details of the system
and the information encoded by the activity pattern over all
cell lines have been published [27–29].
´
11. Gobis K, Foks H, Wisniewska K, Da˛browska-Szponar M, Au-
gustynowicz-Kopec E, Napiorkowska A, Sikorski A (2012)
´
´
Monatsh Chem 143:1161
´
12. Gobis K, Foks H, Wisniewska K, Da˛browska-Szponar M, Au-
gustynowicz-Kopec E, Napiorkowska A (2012) Arch Pharm
´
´
345:911
13. Gobis K, Foks H, Sławinski J, Sikorski A, Trzybinski D, Au-
´
´
´
´
gustynowicz-Kopec E, Napiorkowska A, Bojanowski K (2013)
Monatsh Chem. doi:10.1007/s00706-012-0888-0
14. Tait A, Luppi A, Franchini S, Preziosi E, Parent C, Buccioni M,
Marucci G, Leonardi A, Poggesi E, Brasili L (2005) Bioorg Med
Chem Lett 15:1185
Acknowledgments The authors are very grateful to Dr. Joel Morris,
Chief of Drug Synthesis and Chemistry Branch, National Cancer
Institute (Bethesda, MD), for the in vitro screening.
15. Kamal A, Reddy KS, Ahmed SK, Khan MNA, Sinha RK, Yadav
IS, Arora SK (2006) Bioorg Med Chem 14:650
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heterocyclic chemistry III, vol 7. Elsevier, Amsterdam, p 41
20. Wavefunction Inc. (2008) Spartan’08, Irvine http://www.
wavefun.com
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 the source are credited.
´
21. Brzozowski Z, Sławinski J, Gdaniec M, Innocenti A, Supuran CT
(2011) Eur J Med Chem 46:4403
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