Synthesis, structure, and biological activity of novel heterocyclic sulfonyl-carboximidamides

Article abstract of DOI:10.1007/s00706-012-0888-0

A series of novel heterocyclic sulfonyl-carboximidamides were synthesized in satisfactory yields via condensation of heterocyclic methyl carbimidates with 2-chlorobenzenesulfonamide and 4-chloropyridine-3-sulfonamide. New structures were confirmed by IR and NMR spectra as well as elemental analyses. X-ray crystallography of two derivatives was performed. The single-crystal structures confirmed the presence of a primary amine group in the amidine moiety. All the compounds were screened for their tuberculostatic, antibacterial, and anticancer activities. Preliminary results indicated that target compounds exhibited weak tuberculostatic and antibacterial activities. Seven compounds inhibited the growth of some cancer cell lines, whereas one of the 2-quinoline derivatives displayed favorable activity against all tested cancer cells with GI ₅₀ values of 0.92-13 μM.

Full text of DOI:10.1007/s00706-012-0888-0

Monatsh Chem (2013) 144:647–658
DOI 10.1007/s00706-012-0888-0
ORIGINAL PAPER
Synthesis, structure, and biological activity of novel heterocyclic
sulfonyl-carboximidamides
•
Katarzyna Gobis Henryk Foks Jarosław Sławinski Artur Sikorski
•
•
•
´
•
•
Damian Trzybinski Ewa Augustynowicz-Kopec Agnieszka Napiorkowska
•
´
Krzysztof Bojanowski
´
´
Received: 23 May 2012 / Accepted: 22 November 2012 / Published online: 25 January 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract A series of novel heterocyclic sulfonyl-car-
boximidamides were synthesized in satisfactory yields via
condensation of heterocyclic methyl carbimidates with
2-chlorobenzenesulfonamide and 4-chloropyridine-3-sul-
fonamide. New structures were confirmed by IR and NMR
spectra as well as elemental analyses. X-ray crystallogra-
phy of two derivatives was performed. The single-crystal
structures confirmed the presence of a primary amine group
in the amidine moiety. All the compounds were screened
for their tuberculostatic, antibacterial, and anticancer
activities. Preliminary results indicated that target com-
pounds exhibited weak tuberculostatic and antibacterial
activities. Seven compounds inhibited the growth of some
cancer cell lines, whereas one of the 2-quinoline deriva-
tives displayed favorable activity against all tested cancer
cells with GI50 values of 0.92–13 lM.
Introduction
Sulfonamides are compounds with diverse pharmacologi-
cal activity. They are known the most for their antibacterial
[1] and antihypoglycemic [2] activities. Some of the sul-
fonamides act as antimycobacterial [3] and antifungal [4]
agents. Intensive studies on the antitumor activity of sul-
fonamides were also carried out [5]. One of the most potent
compounds is chloroquinoxaline sulfonamide (CQS) pres-
ently in the second phase of clinical trials [6, 7]. This
compound has a chlorine atom in its structure linked to the
quinoxaline ring (C-5) substituted at the C-2 position with
a sulfanilamide moiety. The antitumor activity of this
sulfonamide is associated with the inhibition of topoiso-
merase II [8]. Other sulfonamides have also been described
as apoptosis promoters [9, 10] including sulfonyl-carbox-
imidamides [11].
Keywords Sulfonamidine ꢀ Heterocycles ꢀ
Crystal structure ꢀ Antimicrobial activity ꢀ
Antitumor activity ꢀ Structure–activity relationship
The amidine functional group is an important structural
element of compounds with established pharmacological
activity. Amidine derivatives have antidegenerative [12],
antitumor [13], and antiplatelet action [14]. They also act
as serine protease inhibitors [15] and nitric oxide synthase
(NOS) inhibitors [16]. Compounds with anti-HIV [17],
antibacterial, and antifungal activities [18] also were found
among them. Moreover, the amidine group may be a per-
fect linker unit that could connect two pharmacophores,
e.g., the sulfanilamide moiety and the pyridine or pyrazine
system.
´
K. Gobis (&) ꢀ H. Foks ꢀ J. Sławinski
Department of Organic Chemistry, Medical University
´
of Gdansk, Gdansk, Poland
e-mail: kgobis@gumed.edu.pl
´
´
A. Sikorski ꢀ D. Trzybinski
´
Department of Physical Chemistry, University of Gdansk,
´
Gdansk, Poland
These findings prompted us to extend our search for
biologically active compounds among nitrogen heterocy-
clic derivatives. We undertook the synthesis of structures
that were condensates of heterocyclic methyl carbimidates
with 2-chlorobenzenesulfonamide and 4-chloropyridine-
3-sulfonamide. The synthesized compounds were evalu-
ated for their biological activity in vitro: tuberculostatic,
´
´
E. Augustynowicz-Kopec ꢀ A. Napiorkowska
Department of Microbiology, Institute of Tuberculosis
and Pulmonary Diseases, Warsaw, Poland
K. Bojanowski
Sunny BioDiscovery, Santa Paula, CA, USA
123

648
K. Gobis et al.
antibacterial, cytotoxic, and anticancer. We have also
determined their crystal structure.
amino-imine structure adopted by the obtained derivatives
(Fig. 1, structure B), as suggested by Northey and co-
workers [20]. They could also be the result of the magnetic
inequivalence of NH protons in the amine moiety upon
formation of a hydrogen bond in the case of heterocyclic
compounds in which the amidine group is in the a position
to the nitrogen atom of the heterocyclic ring (structure A),
as shown in the previous article [21].
Results and discussion
The subject of this work was to study the reactions of
heterocyclic carbimidates with sulfonamides that have a
chlorine atom as a substituent in the ortho position to the
sulfonamide group. Carbimidates are compounds of great
reactivity. Among others they react with amines giving
amidines as the products. A few reactions of alkyl- and
phenylcarbimidates with sulfonamides have been also
described. As a result, sulfonamidines are formed [19].
One method presented in this article is to use 2-, 3-, and
4-pyridine-, 2-pyrimidine-, 2-pyrazine-, 6-chloro-2-pyra-
zine-, 6-methoxypyrazine-, and 2-quinolinecarbimidate. An
important element of the chosen synthetic route is that there
is no need for isolation of carbimidates and they are used
in situ after generation from the corresponding carbonitriles
in methanol in the presence of DBU (1,8-diazabicy-
cloundec-7-ene). Isolated carbimidates are easy to obtain in
pure form and can be also used for 2-pyrazine, 6-chloro-
pyrazine, and 6-methoxypyrazine derivatives. The
carbimidates mentioned above underwent reaction with
2-chlorobenzenesulfonamide and 4-chloropyridine-3-sul-
fonamide. The reactions were carried out in a methanol
solution of DBU. That led to the formation of amidine
structures with the 2-chlorobenzenesulfonyl or 4-chloro-
pyridine-3-sulfonyl substituent (1–12, 15, 16; Scheme 1).
The amidine structures 9, 10, 13, and 14 were prepared
from pure isolated carbimidates by refluxing equimolar
amounts of the reagents in diglyme (bis(2-methoxyethyl)
ether).
Such a structure could also be stabilized by hydrogen
bonding between the second proton of the amine group and
the oxygen atom of the sulfonyl moiety. This issue was
resolved by X-ray studies performed on derivatives 3 and
4. The obtained results revealed the tautomeric structure A
(Fig. 2a, b). Thus, formation of hydrogen bonds is the
reason for the magnetic inequivalence of the protons of the
amino group and separate signals in the 1H NMR spectra of
the synthesized compounds.
Crystal structure of compounds 3 and 4
In the molecules of the title compounds (Fig. 2a, b) the
bond lengths and angles characterizing the geometry of the
pyridine skeleton, amino and sulfonyl groups, and benzene
ring are typical for this group of compounds [21], although
earlier reports from other authors suggested the existence
of imine tautomeric structures. Comparison of molecules of
3 and 4 shows that the structures of both compounds are
very similar (Table 1).
However, wecan observe differences in the dihedralangles
and molecular interactions in the crystal packing. With
respective average deviations from planarity of 0.006(1) and
˚
0.007(3) A, the nearly planar pyridine ring and aromatic
fragment (2-chlorobenzene in compound 3 and 2-chloro-3-
pyridine in compound 4) are oriented at an angle of 60.6(2)
and 69.1(3)° to each other, for I and II, respectively. In the
packing of both compounds, the amino group participates in
the intramolecular N–HꢀꢀꢀO and intermolecular N–HꢀꢀꢀO
interactions (Fig. 2c, d). This group is also engaged in
N–HꢀꢀꢀN intermolecular interactions, where the acceptor of
the H atom is the endocyclic N atom from the pyridine
(compound 3) or chloropyridine (compound 4) rings. The
neighboring molecules in 3 and 4 are also linked through the
C–HꢀꢀꢀCl hydrogen bond. Additionally, weak C–HꢀꢀꢀO
hydrogen bonds are observed in the crystal packing of 4.
In the 1H NMR spectra of the target amidines the signals
for all the protons of aromatic systems were observed and
two signals for the NH groups were shifted from each other
at about 1 ppm. These separated signals could be due to the
Biological activity
Four of the obtained sulfonyl-carboximidamides (2, 7, 8,
10) were evaluated for their in vitro tuberculostatic activity
against the Mycobacterium tuberculosis H37Rv strain and
two ‘‘wild’’ strains isolated from tuberculosis patients: one
(sp. 210) resistant to p-aminosalicylic acid (PAS), isoni-
cotinic acid hydrazide (INH), ethambutol (ETB), and
Scheme 1
123

Heterocyclic sulfonyl-carboximidamides
649
Fig. 1 Possible tautomeric
structures of sulfonyl-
carboximidamides
A
B
Fig. 2 Structure of 3 (a) and 4 (b) showing 25 % probability
displacements for ellipsoid. H atoms shown as small spheres of
arbitrary radius. The arrangement of the molecules in the crystal
structure of 3 (c) and 4 (d) viewed along b axis. Dashed lines
N–HꢀꢀꢀO, N–HꢀꢀꢀN, C–HꢀꢀꢀCl, and C–HꢀꢀꢀO interactions
rifampicin (RFP), and the another (sp. 192) fully sensitive
to the administrated tuberculostatics. Isoniazid (INH) was
used as a reference drug. The tested compounds showed
rather weak tuberculostatic activity, weaker than the ref-
erence INH (MIC 0.5–1.0 lg/cm3). For all the compounds
the determined MIC values were 25–50 lg/cm3 against the
three tested strains (Table 2).
tested on P. acnes. Only three of the tested compounds (1,
2, 15) exhibited moderate antibacterial activity with MIC
values of 12.5–50 lg/cm3. For all other compounds the
MIC values were above 100 lg/cm3. Compound 15 was
the most active (MIC 12.5 lg/cm3).
The most antibacterially potent sulfonylcarboximida-
mides 1, 2, and 15 were then tested for their effects on the
proliferation of neonatal human dermal fibroblasts (ATCC
PCS-201-010). MAP (magnesium ascorbyl phosphate) and
bFGF (basic fibroblast growth factor) were used as the
positive control (Fig. 3). Compound 2 had no cytotoxic
activity. Irrespective to the compound concentration the cell
growth remained at the level corresponding to the water-
treated control. Compound 1 had a weak inhibitory activity.
For low compound concentrations (6.25–12.5 lg/cm3) no
The compounds were also tested for their antibacterial
activity against P. acnes (ATCC 11827) and Brevibacte-
rium linens (ATCC 9174). All of the synthesized sulfonyl-
carboximidamides (1–13) exhibited activity with MICs
greater than 256 lg/cm3, which meant that those values did
not fit standard test concentrations. All synthesized com-
pounds were tested on B. linens but no test compound had a
MIC of less than 100 lg/cm3. The compounds were then
123

650
K. Gobis et al.
Table 1 Crystal data and
structure refinement for
compounds 3 and 4
Compound 3
Compound 4
Empirical formula
Formula weight
Temperature/K
C12H10ClN3O2S
295.74
C11H9ClN4O2S
296.73
295(2)
295(2)
˚
Wavelength/A
1.54184
1.54184
Crystal system
Space group
Orthorhombic
Pbca
Monoclinic
P21/n
Unit cell dimensions
˚
a/A
7.7435(2)
10.9172(2)
29.3315(6)
5.5337(5)
20.8163(9)
10.6688(6)
104.298(8)
1,190.88(14)
4
˚
b/A
˚
c/A
b/°
3
˚
V/A
2,479.61(9)
Z
8
D
calcd/mg m-3
1.584
1.655
Absorption coefficient/mm-1
4.330
4.536
F(000)
Crystal size/mm3
1,216
608
0.40 9 0.12 9 0.10
3.01–67.18
0.34 9 0.18 9 0.13
4.25–67.35
H range for data collection/deg
Limiting indices
-7 B h B 8, -13 B k B 13,
-34 B l B 34
7,923/2,179 [Rint = 0.0581]
98.2
-5 B h B 6, -24 B k B 24,
-12 B l B 12
Reflections collected/unique
Completeness 2H = 50.00°/%
Refinement method
5,600/20,929 [Rint = 0.0961]
97.6
Full-matrix least-squares on F2
Data/restraints/parameters
Goodness-of-fit on F2
2,179/0/172
2,092/0/178
1.058
1.079
Final R indices [I [ 2r(I)]
R1 = 0.0523
wR2 = 0.1316
R1 = 0.0555
wR2 = 0.1352
0.313 and -0.494
R1 = 0.0645
wR2 = 0.1567
R1 = 0.1037
wR2 = 0.1811
0.352 and -0.580
R indices (all data)
-3
˚
Largest diff. peak and hole/e A
cytotoxic effect was evident. In the range 25–100 lg/cm3 a
linear relation of the cytotoxic effect was observed with an
88 % growth inhibitory activity at a concentration of
100 lg/cm3. Compound 15 was strongly cytotoxic, even at
the lowest concentration tested (6.25 lg/cm3—70 %
growth inhibitory activity).
that cell line with DGI of from 19.4 to 21.1 %. Derivatives
4, 10, and 12 exhibited activity against two cell lines.
Compound 4 was potent towards the melanoma MALME-
3M cell line (DGI 20.0 %) and the renal cancer A498 cell
line (DGI 24.1 %). Compound 10 exhibited activity against
CNS cancer SNB-75 cell line (DGI 19.3 %) and compound
11 against melanoma MALME-3M cell line (DGI 22.6 %).
Derivative 12 was active towards two cell lines, non-small
cell lung cancer HOP-92 (DGI 25.9 %) and NCI-H522,
against which the compound exhibited cytotoxic activity
(DGI 142.9 %). Derivative 15 was the most potent com-
pound. That compound exhibited activity towards eleven
cell lines: leukemia HL-60 (DGI 19.7 %) and K-562 (DGI
35.9 %), non-small cell lung cancer NCI-H460 (DGI
19.6 %), colon cancer HT29 (DGI 60.4 %), KM12 (DGI
23.0 %) and SW-620 (DGI 24.7 %), melanoma MDA-MB-
435 (DGI 80.4 %), ovarian cancer NCI/ADR RES (DGI
42.9 %), renal cancer TK-10 (DGI 21.7 %), breast cancer
MCF7 (DGI 15.7 %) and MDA-MB-468 (DGI 42.2 %).
In view of the cytotoxic activity of compound 15, it was
of interest to determine whether those compounds had an
antitumor potential. Compounds 2–16 were tested in the
framework of the Development Therapeutic 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 2–16 tested in the preliminary NCI-60 one-
dose screen test seven of them (44 %) exhibited distinct
growth inhibition (DGI) properties (Table 3). Three com-
pounds (2, 7, 11) were active towards one renal cancer
UO-31 cell line. These compounds inhibited the growth of
123

Heterocyclic sulfonyl-carboximidamides
651
Table 2 Antibacterial activity
No.
MIC/lg/cm3
of the synthesized compounds
against P. acnes and
M. tuberculosis
P. acnesa
M. tuberculosis
H37Rv
sp. 192
sp. 210
1
2
3
4
5
25
–
–
–
50
50
–
50
–
50
–
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
12.5
–
–
–
–
–
–
6
–
–
–
Minimum inhibitory
concentrations for P. acnes were
assessed optically as the lowest
concentration of a test material
which caused no bacteria
growth. MICs for M.
tuberculosis strains were
determined by twofold serial
dilution method for
microdilution plates and for
mycobacterial strains by
twofold classical test-tube
method of successive dilution
7
50
50
–
50
50
–
50
50
–
8
9
10
11
12
13
14
15
16
INH
50
–
25
–
50
–
–
–
–
–
–
–
–
–
–
–
–
–
[100
–
–
–
–
INH isoniazid, – not tested
a
P. acnes ATCC 11827
0.5
0.5
1.1
Compound 15 was selected for further studies in five
concentrations in the range of 10-4 to 10-8 M. The activity
of the compound was expressed by three dose–response
parameters: GI50—the molar concentration that inhibits
50 % net cell growth, TGI—the molar concentration leading
to total growth inhibition, LC50—molar concentration lead-
ing to 50 % net cell death (Table 4). Derivative 15 was the
most potent towards the leukemia SR cell line (GI50
0.92 lM), the non-small cell lung cancer NCI-H522 (GI50
2.28 lM), the CNS cancer SNB-75 cell line (GI50 2.52 lM),
the melanoma MDA-MB-435 cell line (GI50 1.13 lM), the
ovarian cancer NCI/ADR-RES cell line (GI50 2.32 lM), and
the breast cancer MDA-MB-468 cell line (GI50 2.01 lM). In
general, compound 15 exhibited the highest activity against
leukemia cell lines. The GI50 mean value for that cell line
panel was 3.52 lM.
tautomeric structure. The antimicrobial activities of the
synthesized compounds were evaluated against P. acnes and
B. linens as well as M. tuberculosis. The results showed that
thesynthesizedsulfonamidederivativesexhibited ratherpoor
antimicrobial activities in vitro. Seven compounds (2, 4, 7,
10–12, 15) were able to inhibit the growth of some cancer cell
lines, whereas the 2-quinoline derivative 15 showed the
highest activity with GI50 values of from 0.92 to 13.00 lM.
Experimental
All materials and solvents were of analytical reagent grade.
Thin-layer chromatography was performed on Merck silica
gel 60F254 plates and visualized with UV. The results of
elemental analyses (% C, H, N) for all obtained compounds
were in agreement with calculated values within 0.3 %.
1H NMR spectra in DMSO-d6 were recorded on Varian
Unity Plus (500 MHz) and Varian Gemini (200 MHz)
instruments (Varian, Palo Alto, CA). IR spectra were
determined as KBr pellets of the solids on a Satellite FT-IR
spectrophotometer (Mattson Instruments, Madison, WI).
Electrospray MS analyses for compounds 1, 6, 9, 14, and
16 were performed on an HCT Ultra Bruker Daltonics
spectrometer operating in positive- and negative-ion modes
(sheath gas N2, temperature 300 °C, flow 7 dm3/min,
pressure 10 psi (689.48 hPa); capillary voltage in posi-
tive ion mode ?4 kV, in negative ion mode -4 kV).
Conclusion
A series of novel heterocyclic sulfonyl-carboximidamides
with different nitrogen heterocyclic systems were synthe-
sized successfully via condensation of heterocyclic
methyl carbimidates with 2-chlorobenzenesulfonamide and
4-chloropyridine-3-sulfonamide. Structures of all these
new compounds were confirmed by IR and NMR spectra as
well as elemental analyses. X-ray crystallography of com-
pounds 3 and 4 demonstrated the presence of the amine
123

652
K. Gobis et al.
220
210
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
Fig. 3 Total cell/protein count for compounds 1, 2, and 15
Table 3 One-dose screening
No.
Growth percent
mean values
Panel
Cell line
Growth
inhibition
DGI/%
data of in vitro tumor growth
inhibition for compounds 2, 4,
7, 10–12, and 15 at a dose of
10 lM
(MG_MIDa)/%
2
4
98.2
Renal cancer
UO-31
20.4
20.0
24.1
19.4
19.3
22.6
25.9
142.9b
19.7
35.9
19.6
60.4b
23.0
24.7
80.4b
42.9
21.7
15.7
42.2
104.8
Melanoma
MALME-3 M
A498
Renal cancer
7
103.0
101.9
102.1
97.8
Renal cancer
UO-31
10
11
12
CNS cancer
SNB-75
Renal cancer
UO-31
Non-small cell lung cancer
HOP-92
NCI-H522
HL-60
15
53.4
Leukemia
K-562
Non-small cell lung cancer
Colon cancer
NCI-H460
HT29
KM12
Data obtained from the NCI-60
DTP human tumor cell line
screening
SW-620
MDA-MB-435
NCI/ADR-RES
TK-10
Melanoma
a
Ovarian cancer
Renal cancer
Breast cancer
MIG_MID mean graph
midpoint, i.e., arithmetical mean
value of growth percent for all
tested cell lines
MCF7
MDA-MB-468
b
Cytotoxic effect (lethality)
Compound samples were prepared in acetonitrile (1, 9, 14,
16) or methanol (6). Melting points were determined with a
Boethius apparatus (Franz Ku¨stner Nachf. KG, Dresden,
Germany). Methyl pyrazine-2-carbimidate and methyl
6-methoxypyrazine-2-carbimidate required for syntheses of
compounds 9, 10 and 13, 14 were obtained according to the
method described earlier by Foks and co-workers [22, 23].
Reaction yield and compound characteristics were found to
be identical with those described (m.p. 115–116 and
100–101 °C, respectively).
123

Heterocyclic sulfonyl-carboximidamides
653
Table 4 Inhibition of in vitro human cancer cell lines growth by
compound 15
Table 4 continued
Panel/cell line
GIa50/lM
TGIb/lM
Panel/cell line
GIa50/lM
TGIb/lM
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
2.32
3.93
50.50
c
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
c
3.65
2.29
3.11
5.91
5.27
0.92
c
c
8.41
c
4.94
8.08
7.19
5.21
4.02
7.67
6.02
7.26
A498
c
c
MOLT-4
ACHN
13.10
c
RPMI-8226
SR
CAKI-1
21.40
RXF 393
SN 12C
35.90
c
Non-small cell lung cancer
A549/ATCC
EKVX
c
c
c
5.37
8.75
5.37
9.34
10.80
5.44
5.19
4.02
2.28
TK-10
7.08
UO-31
48.50
HOP-62
Prostate cancer
PC-3
c
c
HOP-92
90.80
c
9.59
4.96
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
DU-145
c
c
c
Breast cancer
MCF-7
c
c
c
3.28
13.00
8.98
5.08
7.04
2.01
MDA-MB-231/ATCC
HS 578T
BT-549
7.62
56.00
c
3.68
9.63
4.66
4.23
3.40
3.29
3.45
16.00
T-47D
59.40
c
MDA-MB-468
6.19
Data obtained from the NCI-60 DTP human tumor cell line screening
c
Omitted LC50 values (molar concentration leading to 50 % net cell
death) were[100 lM except SK-MEL-5 cell line in melanoma panel
(36.2 lM)
HT29
14.90
c
KM12
c
a
SW-620
GI50 the molar concentration that inhibits 50 % net cell growth
b
CNS cancer
SF-268
TGI the molar concentration leading to total growth inhibition
c
c
6.79
3.83
4.19
5.61
2.52
4.48
Determined TGI values were [100 lM
SF-295
86.30
c
SF-539
General method A for the synthesis of sulfonyl-
carboximidamides 1–12, 15, 16
c
SNB-19
SNB-75
30.60
c
U251
The respective carbonitrile (5 mmol) was dissolved in
10 cm3 of methanol and 0.1 cm3 (0.7 mmol) of DBU was
added. The mixture was refluxed for 0.5 h required for
methyl carbimidate formation. Then 2-chlorobenzene-
sulfonamide or 4-chloropyridine-3-sulfonamide (4 mmol)
was added. The mixture was refluxed for another 3 h. The
solvent was evaporated under vacuum and 30 g of ice was
added. The precipitate was filtered and recrystallized from
a suitable solvent.
Melanoma
LOX IMVI
MALME-3M
M-14
c
c
c
4.05
5.42
3.61
1.13
3.40
5.45
3.31
11.70
6.07
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
3.78
c
12.00
50.50
c
c
Alternative method B for the synthesis of sulfonyl-
carboximidamides 9, 10, 13, 14
c
9.42
9.27
9.15
7.71
5.74
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
3.78
Methylpyrazine-2-carbimidateormethyl6-methoxypyrazine-
2-carbimidate (3 mmol) and the respective sulfonamide
(3 mmol) were refluxed in 5 cm3 of diglyme for 15 min. Then
the mixture was cooled and 30 g of ice was added. The
9.53
c
12.00
123

654
K. Gobis et al.
3,330 (m N–H), 1,620 (m C=N), 1,554, 1,514 (m C=C), 1,283,
precipitate was filtered and recrystallized from a suitable sol-
vent with addition of activated carbon.
1
1,146 (m SO2), 836, 795 (c C–H), 592 (c N–H) cm-1; H
NMR (200 MHz): d = 7.49–7.56 (m, 1H, pyridine), 7.78
(d, J = 5.4 Hz, 1H, 4-chloropyridine), 8.17–8.19 (m, 1H,
pyridine), 8.22 (br s, 1H, NH ? D2O exchangeable), 8.74–
8.77 (m, 2H, 1H pyridine and 1H 4-chloropyridine), 9.00 (d,
J = 2.4 Hz, 1H, pyridine), 9.20 (s, 1H, 4-chloropyridine),
9.48 (br s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz): d = 124.08, 126.78, 129.60, 135.75 (2C),
141.93, 148.58, 149.38, 152.60, 154.39, 162.03 ppm.
N0-[(2-Chlorophenyl)sulfonyl]-2-pyridinecarboximidamide
(1, C12H10ClN3O2S)
The crude product was recrystallized from methanol
affording 1.1 g (93 %) 1. M.p.: 140–142 °C; IR (KBr):
ꢀ
m = 3,423, 3,211 (m N–H), 1,613 (m C=N), 1,583 (m C=C),
1,537 (m N–H), 1,292, 1,284, 1,157 (m SO2), 1,041 (d C–H),
828, 759 (c C–H), 587 (c N–H) cm-1; 1H NMR (200 MHz):
d = 7.51–7.70 (m, 4H, 2H Ph and 2H pyridine), 7.95–8.18
(m, 3H, 2H Ph and 1H pyridine), 8.40 (br s, 1H, NH ? D2O
exchangeable), 8.70 (d, J = 4.7 Hz, 1H, pyridine), 9.18
(br s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz): d = 123.26, 127.75, 127.92, 129.60, 131.56,
131.60, 134.13, 138.46, 139.63, 148.69, 149.28, 159.76 ppm;
MS (-): m/z = 611 (100 %, [2M?Na-3H]-), 294 (32 %,
[M-2H]-), 258 (28 %, [M-2H–Cl]-); MS (?): m/z = 613
(25 %, [2M?Na-H]?), 318 (100 %, [M?Na-H]?), 298
(71 %, [M?2H]?).
N0-[(2-Chlorophenyl)sulfonyl]-4-pyridinecarboximidamide
(5, C12H10ClN3O2S)
This compound was recrystallized from methanol/water
(1:1) affording 0.65 g (55 %) 5. M.p.: 182–185 °C; IR
ꢀ
(KBr): m = 3,387 (m N–H), 3,175 (m C–H), 1,658 (m C=N),
1,581 (m C=C), 1,526 (d N–H), 1,289, 1,145 (m SO2), 850,
1
749 (c C–H), 591 (c N–H) cm-1; H NMR (500 MHz):
d = 7.56 (t, J = 7.8 Hz, 1H, Ph), 7.65 (m, 2H, Ph), 7.75
(d, J = 5.4 Hz, 1H, pyridine), 8.14 (d, J = 7.3 Hz, 1H,
Ph), 8.56 (br s, 1H, NH ? D2O exchangeable), 8.74 (d,
J = 5.9 Hz, 1H, pyridine), 9.42 (br s, 1H, NH ? D2O
exchangeable) ppm; 13C NMR (50 MHz): d = 121.92
(2C), 127.70, 129.56, 131.53, 131.99, 134.11, 139.61,
141.12, 150.59 (2C), 161.79 ppm.
N0-[(4-Chloropyridin-3-yl)sulfonyl]-2-pyridine-
carboximidamide (2, C11H9ClN4O2S)
This compound was recrystallized from ethanol affording
ꢀ
0.82 g (69 %) 2. M.p.: 150–152 °C; IR (KBr): m = 3,430,
3,390, 3,323 (m N–H), 1,629 (m C=N), 1,587 (m C=C),
N0-[(4-Chloropyridin-3-yl)sulfonyl]-4-pyridine-
carboximidamide (6, C11H9ClN4O2S)
This compound was recrystallized from dioxane affording
1,559 (m N–H), 1,297, 1,275, 1,151 (m SO2), 1,115, 833 (d
1
C–H), 592 (c N–H) cm-1; H NMR (200 MHz): d = 7.67
(t, J = 5.9 Hz, 1H, pyridine), 7.96–8.13 (m, 2H, 1H
pyridine and 1H 4-chloropyridine), 8.57 (br s, 1H,
NH ? D2O exchangeable), 8.70–8.77 (m, 2H, 1H pyridine
and 1H 4-chloropyridine), 9.22 (s, 1H, 4-chloropyridine),
9.32 (br s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz): d = 123.41, 126.75, 128.04, 135.75, 138.50,
141.92, 148.50 (2C), 149.38, 154.45, 160.03 ppm.
ꢀ
0.71 g (60 %) 6. M.p.: 215–218 °C; IR (KBr): m = 3,385,
3,330 (m N–H), 3,088, 2,964 (m C–H), 1,660 (m C=N), 1,570
(m C=C), 1,533 (d N–H), 1,280, 1,116 (m SO2), 854, 769 (c
C–H), 594 (c N–H) cm-1; 1H NMR (200 MHz): d = 7.76–
7.80 (m, 3H, 2H pyridine and 1H 4-chloropyridine), 8.72–
8.77 (m, 4H, 2H pyridine and 1H 4-chloropyridine and 1H
NH ? D2O exchangeable), 9.19 (s, 1H, 4-chloropyridine),
9.55 (br s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz): d = 122.52 (2C), 126.53, 135.64, 141.84,
141.95, 149.34 (2C), 149. 67, 154.48, 161.86 ppm; MS
(-): m/z = 297 (26 %, [M]-), 295 (66 %, [M-2H]-), 259
(100 %, [M-2H–Cl]-); MS (?): m/z = 297 (70 %, [M]?),
153 (100 %, [M-H–Cl–C5H3N–NH2–O]?).
(Z)-N0-[(2-Chlorophenyl)sulfonyl]-3-pyridine-
carboximidamide (3, C12H10ClN4O2S2)
This compound was recrystallized from dioxane affording
ꢀ
0.51 g (43 %) 3. M.p.: 188–190 °C; IR (KBr): m = 3,341
(m N–H), 3,156 (m C–H), 1,644 (m C=N), 1,591 (m C=C),
1,546 (d N–H), 1,295, 1,281, 1,162, 1,146 (m SO2), 1,042 (d
1
C–H), 839, 761 (c C–H), 591 (c N–H) cm-1; H NMR
(200 MHz): d = 7.49–7.65 (m, 4H, 2H Ph and 2H
pyridine), 8.12–8.21 (m, 2H, Ph), 8.50 (br s, 1H,
NH ? D2O exchangeable), 8.74 (d, J = 4.4 Hz, 2H,
pyridine), 9.00 (s, 1H, pyridine), 9.39 (br s, 1H, NH ? D2O
exchangeable) ppm; 13C NMR (50 MHz): d = 123.76,
127.65 (2C), 129.59, 129.71, 131.49, 131.97, 133.99,
136.04, 139.81, 149.03, 153.09, 161.94 ppm.
N0-[(2-Chlorophenyl)sulfonyl]pyrimidine-2-
carboximidamide (7, C11H9ClN4O2S)
This compound was recrystallized from methanol affording
ꢀ
0.99 g (83 %) 7. M.p.: 191–192 °C; IR (KBr): m = 3,396,
3,309 (m N–H), 1,623 (m C=N), 1,554, 1,391 (m C=C),
1,279, 1,151 (m SO2), 838, 693 (c C–H), 589 (c N–H)
1
cm-1; H NMR (200 MHz): d = 7.51–7.65 (m, 3H, Ph),
(Z)-N0-[(4-Chloropyridin-3-yl)sulfonyl]-3-pyridine-
carboximidamide (4, C11H9ClN4O2S)
This compound was recrystallized from dioxane affording
7.70 (t, J = 5.0 Hz, 1H, pyrimidine), 8.13 (d, J = 7.2 Hz,
2H, Ph), 8.54 (br s, 1H, NH ? D2O exchangable), 9.00
(d, J = 5.0 Hz, 2H, pyrimidine), 9.15 (br s, 1H, NH ?
D2O exchangable) ppm; 13C NMR (50 MHz): d = 123.73,
ꢀ
0.36 g (30 %) 4. M.p.: 192–194 °C; IR (KBr): m = 3,439,
123

Heterocyclic sulfonyl-carboximidamides
655
127.73, 129.49, 131.66, 131.89, 134.08, 139.76, 158.20
(2C), 158.89, 159.83 ppm.
6-Chloro-N0-(2-chlorophenylsulfonyl)pyrazine-2-
carboximidamide (11, C11H8Cl2N5O2S)
This compound was recrystallized from dioxane/methanol
N0-[(4-Chloropyridin-3-yl)sulfonyl]pyrimidine-2-
carboximidamide (8, C10H8ClN5O2S)
This compound was recrystallized from dioxane affording
(1:1) affording 1.1 g (85 %) 11. M.p.: 195–198 °C; IR
ꢀ
(KBr): m = 3,394, 3,288, 3,236 (m N–H), 1,642 (m C=N),
1,554 (m C=C), 1,522 (d N–H), 1,362, 1,297, 1,152, 11,07 (m
SO2), 892, 790, 757 (c C–H), 567 (c N–H) cm-1; 1H NMR
(500 MHz): d = 7.56–7.59 (m, 1H, Ph), 7.64–7.69 (m, 2H,
Ph), 8.18 (d, J = 7.8 Hz, 1H, Ph), 8.56 (br s, 1H, NH ? D2O
exchangeable), 9.07 (s, 1H, pyrazine), 9.13 (s, 1H, pyrazine),
9.34 (br s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz): d = 127.77, 129.69, 132.04, 134.30, 139.27,
142.45, 144.56, 147.52, 148.42, 157.80, 158.70 ppm.
ꢀ
0.62 g (52 %) 8. M.p.: 162–163 °C; IR (KBr): m = 3,288
(m N–H), 1,623 (m C=N), 1,560, 1,397 (m C=C), 1,291,
1,219, 1,147 (m SO2), 840 (c C–H), 600 (c N–H) cm-1; 1H
NMR (200 MHz): d = 7.69–7.77 (m, 2H, 1H pyrimidine
and 1H 4-chloropyridine), 8.75 (d, J = 4.3 Hz, 1H,
4-chloropyridine), 8.85 (br s, 1H, NH ? D2O exchanga-
ble), 8.97 (d, J = 4.5 Hz, 2H, pyrimidine), 9.14 (s, 1H,
4-chloropyridine), 9.39 (br s, 1H, NH ? D2O exchangeable)
ppm; 13C NMR (50 MHz): d = 123.73, 126.67, 136.07,
141.94, 149.25, 154.26, 158.37 (2C), 160.53, 161.25 ppm.
6-Chloro-N0-[(4-chloropyridin-3-yl)sulfonyl]pyrazine-2-
carboximidamide (12, C10H7Cl2N5O2S)
This compound was recrystallized from methanol/water
N0-[(2-Chlorophenyl)sulfonyl]pyrazine-2-
carboximidamide (9, C11H9ClN4O2S)
(1:1) affording 0.64 g (48 %) 12. M.p.: 160–162 °C; IR
(KBr): m = 3,372 (m N–H), 3,092 (m C–H), 1,656 (m N=C),
ꢀ
This compound was recrystallized from ethanol affording
0.55 g (46 %) 9 for method A and 0.68 g (76 %) for
1,563, 1,547 (m C=C), 1,518 (d N–H), 1,368, 1,302, 1,150
(m SO2), 796 (c C–H), 601 (c N–H) cm-1 1H NMR
;
ꢀ
method B. M.p.: 173–174 °C; IR (KBr): m = 3,438, 3,356,
(500 MHz): d = 7.80 (d, J = 5.4 Hz, 1H, 4-chloropyri-
dine), 8.76 (br s, 1H, NH ? D2O exchangeable), 8.78 (d,
J = 5.4 Hz, 1H, 4-chloropyridine), 9.07 (s, 1H, pyrazine),
9.16 (s, 1H, 4-chloropyridine), 9.22 (s, 1H, pyrazine), 9.48
(br s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz): d = 126.80, 135.48, 142.03, 142.61, 144.45,
147.49, 148.49, 149.42, 154.55, 158.13 ppm.
3,329, 3,254 (m N–H), 3,092 (m C–H), 1,615 (m C=N),
1,337, 1,278, 1,182, 1,153 (m SO2), 1,041 (d C–H), 592 (c
N–H) cm-1; H NMR (500 MHz): d = 7.57–7.59 (m, 1H,
1
Ph), 7.66–7.67 (m, 2H, Ph), 8.18 (d, J = 7.3 Hz, 1H, Ph),
8.53 (br s, 1H, NH ? D2O exchangeable), 8.20 (t,
J = 2.4 Hz, 1H, pyrazine), 8.81 (d, J = 2.4 Hz, 1H,
pyrazine), 9.20 (d, J = 1.4 Hz, 1H, pyrazine), 9.34 (br s,
1H, NH ? D2O exchangable) ppm; 13C NMR (50 MHz):
d = 127.76, 129.69, 131.58, 132.03, 134.22, 139.42,
144.03, 144.28 (2C), 148.58, 158.79 ppm; MS (-):
m/z = 613 (92 %, [2M?Na-3H]-), 297 (43 %, [M]-),
295 (100 %, [M-2H]-), 259 (79 %, [M-2H–Cl]-); MS
(?): m/z = 615 (29 %, [2M?Na-H]?), 319 (100 %,
[M?Na-H]?), 153 (71 %, [M-Cl–C6H4–NH2–O]?).
N0-(2-Chlorophenylsulfonyl)-6-methoxypyrazine-2-
carboximidamide (13, C12H11ClN4O3S)
This compound was recrystallized from methanol affording
ꢀ
0.75 g (76 %) 13. M.p.: 227–230 °C; IR (KBr): m = 3,401,
3,248 (m N–H), 1,643 (m C=N), 1,541 (m C=C), 1,381,
1,320, 1,281, 1,146, 1,106 (m SO2), 804 (c C–H), 584, 561
(c N–H) cm-1 1H NMR (200 MHz): d = 4.03 (s, 3H,
;
OCH3), 7.51–7.65 (m, 3H, Ph), 8.15–8.19 (m, 1H, Ph), 8.55
(s, 1H, pyrazine), 8.73 (s, 1H, pyrazine), 8.80 (br s, 1H,
NH ? D2O exchangeable), 9.00 (br s, 1H, NH ? D2O
exchangeable) ppm; 13C NMR (50 MHz): d = 54.59,
127.73, 129.61, 131.55, 132.03, 134.16, 135.74, 139.81
(2C), 140.87, 158.70, 159.08 ppm.
N0-[(4-Chloropyridin-3-yl)sulfonyl]pyrazine-2-
carboximidamide (10, C10H8ClN5O2S)
This compound was recrystallized from ethanol affording
0.79 g (66 %) 10 for method A and 0.38 g (42 %) for
ꢀ
method B. M.p.: 187–188 °C; IR (KBr): m = 3,380, 3,239
N0-[(4-Chloropyridin-3-yl)sulfonyl]-6-methoxypyrazine-2-
carboximidamide (14, C11H10ClN5O3S)
This compound was recrystallized from DMSO affording
(m N–H), 1,637 (m C=N), 1,560 (m C=C), 1,547 (d N–H),
1,285, 1,153, 1,120 (m SO2), 853, 793 (c C–H), 600
(c N–H) cm-1
;
1H NMR (200 MHz): d = 7.80
(d, J = 4.8 Hz, 1H, 4-chloropyridine), 8.57 (br s, 1H,
NH ? D2O exchangeable), 8.77–8.80 (m, 2H, 1H pyrazine
and 1H 4-chloropyridine), 8.92 (d, J = 3.2 Hz, 1H,
4-chloropyridine), 9.21–9.23 (m, 2H, pyrazine), 9.43 (br s,
1H, NH ? D2O exchangable) ppm; 13C NMR (50 MHz):
d = 126.79, 135.57, 141.98, 144.03, 144.43 (2C), 148.68,
149.44, 154.53, 159.11 ppm.
0.32 g (33 %) 14. M.p.: 265–270 °C (decomp.); IR (KBr):
m = 3,337, 3,302 (m N–H), 3,087 (m C–H), 1,642 (m C=N),
ꢀ
1,547, 1,450 (m C=C), 1,383, 1,280, 1,131 (m SO2), 1,008
(d C–H), 822, 777 (c C–H), 581 (c N–H) cm-1 1H
;
NMR (200 MHz): d = 4.04 (s, 3H, OCH3), 7.08 (d,
J = 5.1 Hz, 1H, 4-chloropyridine), 7.55 (s, 1H, pyrazine),
8.75–8.77 (m, 3H, 2H 4-chloropyridine and 1H NH ? D2O
123

656
K. Gobis et al.
exchangeable), 9.22 (s, 1H, pyrazine), 9.27 (br s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz):
d = 54.64, 126.82, 135.66, 135.88, 140.01 (2C), 140.68,
141.96, 149.33, 154.49, 159.04 ppm; MS (-): m/z = 326
(15 %, [M-2H]-), 290 (100 %, [M-2H–Cl]-); MS (?):
m/z = 341 (8 %, [M?2Na-H–NH2–O]?), 153 (100 %,
[M?H-Cl–C5H3N–NH2–O]?).
obtained by least-squares fit to the optimized setting angles
of the collected reflections by means of CrysAlis CCD
[24]. Data were reduced by using CrysAlis RED [24]
software by applying multi-scan absorption corrections
(empirical absorption correction using spherical harmonics,
implemented in the SCALE3 ABSPACK scaling algo-
rithm). The structural resolution procedure was made using
the SHELXS-97 package solving the structures by direct
methods and carrying out refinements by full-matrix least-
squares on F2 using the SHELXL-97 program [25].
N0-(2-Chlorophenylsulfonyl)quinoline-2-
carboximidamide (15, C16H12ClN3O2S)
This compound was recrystallized from ethanol affording
ꢀ
All H atoms bound with aromatic C atoms were placed
geometrically and refined using a riding model with
˚
C–H = 0.93 A and Uiso(H) = 1.2 Ueq(C). All H atoms
bound with N atoms were placed geometrically and refined
0.97 g (70 %) 15. M.p.: 161–162 °C; IR (KBr): m = 3,446,
3,331 (m N–H), 1,640 (m C=N), 1,616 (m C=C), 1,527 (d N–H),
1,277, 1,180, 1,106 (m SO2), 1,030 (d C–H), 801, 770, 628
(c C–H), 575 (c N–H) cm-1 1H NMR (200 MHz):
;
˚
using a riding model with N–H = 0.86 A and Uiso(H) =
d = 7.53–7.77 (m, 4H, 2H Ph and 2H quinoline), 7.88 (t,
J = 7.0 Hz, 1H, quinoline), 8.06–8.23 (m, 4H, 2H Ph and
2H quinoline), 8.52 (d, J = 8.2 Hz, 1H, quinoline), 8.60
(br s, 1H, NH ? D2O exchangeable), 9.30 (br s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz):
d = 119.26, 127.79, 128.34, 129.02, 129.39, 129.58,
129.64, 131.12, 131.64, 132.03, 134.20, 138.52, 139.58,
146.33, 148.98, 159.57 ppm.
1.2 Ueq(N). All interactions demonstrated were found
by the PLATON program [26]. The following programs
were used to prepare molecular graphics: ORTEPII [27],
PLUTO-78 [28], and Mercury [29].
Full crystallographic details for compounds 3 and 4,
excluding structures features, have been deposited with the
Cambridge Crystallographic Data Centre (deposition no.
CCDC 869834 & 869835). These data may be obtained, on
request, from the Director, CCDC, 12 Union Road, Cam-
bridge, CB2 1EZ, UK (Tel.: ?44-1223-336408; Fax: ?44-
1223-336033; E-mail: deposit@ccdc.cam.ac.uk or http://
www.ccdc.cam.ac.uk).
N0-[(4-Chloropyridin-3-yl)sulfonyl]quinoline-2-
carboximidamide (16, C15H11ClN4O2S)
This compound was recrystallized from dioxane affording
1.1 g (80 %) 16. M.p.: 347–350 °C (decomp.); IR (KBr):
ꢀ
m = 3,380, 3,182 (m N–H), 1,637 (m C=N), 1,560 (m C=C),
1,536 (d N–H), 1,300, 1,148, 1,121 (m SO2), 813, 771, 627
Tuberculostatic activity
1
(c C–H), 605, 592 (c N–H) cm-1; H NMR (200 MHz):
d = 7.72–7.81 (m, 2H, 1H quinoline and 1H 4-chloropyr-
idine), 7.91 (t, J = 7.0 Hz, 1H, quinoline), 8.09–8.19 (m,
3H, quinoline), 8.57 (d, J = 8.8 Hz, 1H, quinoline), 8.69
(br s, 1H, NH ? D2O exchangeable), 8.77 (d, J = 5.4 Hz,
1H, 4-chloropyridine), 9.25 (s, 1H, 4-chloropyridine), 9.42
(br s, 1H, NH ? D2O exchangeable) ppm; 13C NMR
(50 MHz): d = 119.36, 126.80, 128.38, 129.12, 129.44,
129.61, 131.19, 135.71, 138.61, 141.98, 146.35, 148.82,
149.40, 154.54, 159.90 ppm; MS (-): m/z = 713 (7 %,
[2M?Na-3H]-), 613 (41 %, [2M?Na-2Cl-2NH2]-),
283 (61 %, [M?Na-3H-Cl-NH2-2O]-), 255 (100 %,
[M?Na-2H–Cl–C5H3N]-); MS (?): m/z = 715 (25 %,
[2M?Na-H]?), 353 (100 %, [M?Na-H–NH2]?), 153
(25 %, [M-H–NH2–Cl–C5H3N–SO2]?).
The newly synthesized compounds were examined in vitro
for their tuberculostatic activity against M. tuberculosis
H37Rv strain and two ‘‘wild’’ strains isolated from tuber-
culosis patients: one (sp. 210) resistant to p-aminosalicylic
acid (PAS), isonicotinic acid hydrazide (INH), etambu-
tol (ETB), and rifampicine (RFP), and the another (sp.
192) fully sensitive to the administered tuberculostat-
ics (Table 2). Investigations were performed by a classical
test-tube method of successive dilution in Youmans’
modification of the Proskauer and Beck liquid medium
containing 10 % of bovine serum [30, 31]. Bacterial sus-
pensions were prepared from 14-day-old cultures of slowly
growing strains and from 48-h-old cultures of saprophytic
strains [32, 33]. Solutions of compounds in ethylene glycol
were tested. Stock solutions contained 10 mg of com-
pounds in 1 cm3. Dilutions (in geometric progression) were
prepared in Youmans’ medium. Media containing no
investigated substances and containing INH as reference
drug were used for comparison. The incubation was per-
formed at a temperature of 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.
X-ray crystallography
Good quality single-crystal specimens were selected for the
X-ray diffraction experiments at T = 295(2) K. They were
mounted with epoxy glue at the tip of glass capillaries.
Diffraction data were collected on an Oxford Diffraction
Gemini R ULTRA Ruby CCD diffractometer with CuKa
˚
radiation (k = 1.54184 A). The lattice parameters were
123

Heterocyclic sulfonyl-carboximidamides
657
Antibacterial activity
preliminary screening, was then tested at five different
concentrations. Details of the system and the information
which is encoded by the activity pattern over all cell lines
have been published [36–38]. The antitumor activity of a
test compound is given by the parameters for each cell line:
GI50, i.e., the molar concentration of the compound that
inhibits 50 % net cell growth, TGI, i.e., the molar con-
centration of compound leading to total growth inhibition,
and LC50, i.e., the molar concentration of the compound
leading to 50 % net cell death. Furthermore, a mean graph
midpoint (MG_MID) is calculated for each of the men-
tioned parameters, giving an averaged activity parameter
over all cell lines. For the calculation of the MG_MID,
insensitive cell lines of the screen are included with the
highest concentration tested. The selectivity of a compound
with respect to one or more cell lines of the screen is
characterized by the high deviation of the particular cell
line parameter compared to the MG_MID value.
Compounds 1–15 were dissolved immediately before use
in 100 % DMSO at 5 mg/cm3 and further to 1 mg/cm3 in
10 % DMSO. Compounds were tested at serial dilutions in
bacterial broth starting at 100 lg/cm3 (final concentration).
P. acnes (ATCC 11827, lot 419697) was grown in thio-
glycollate nutrient broth (Hardy Diagnostics K29) for 72 h
at 33 °C, then inoculated at the density equivalent to
0.5 McFarland standard and incubated with the test mate-
rials for another 72 h in an anaerobic environment.
B. linens (ATCC 9174, lot 419862) culture was started
from an agar plate, grown in nutrient broth (Hardy Diag-
nostics K243) for 24 h at 30 °C, then inoculated at the
density equivalent to 1 McFarland standard and incubated
with the test materials for another 24 h. At the end of the
incubation the MIC was assessed optically as the lowest
concentration of a test material which caused no bacteria
growth [34]. This optical assessment was further confirmed
by measuring the absorbance at 655 nm with the BioRad
3550-UV microplate reader. The MIC was defined as at
least 50 % inhibition of the increase of OD.
Acknowledgments The authors are very grateful to Dr. Joel Morris,
Chief of Drug Synthesis & Chemistry Branch, National Cancer
Institute (Bethesda, MD) for the in vitro screening.
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.
Cytotoxic activity
Passage for normal neonatal human dermal fibroblasts
(ATCC PCS-201-010, lot 58243223) were grown in
DMEM with 5 % calf serum (Hyclone). For the experi-
ment, cells were plated in DMEM/5 % serum at
2,000 cells/well in 96 well plates (plate 598) and were
exposed to test materials for 96 h. MAP and bFGF were
used as the positive controls and water, 1, 0.5, and 0.25 %
DMSO were used as the negative controls. Plate growth
was stopped and cells were stained with a sulforhodamine
B dye [35]. The dye was then dissolved and a colorimetric
signal proportional to total cell/protein count was quanti-
fied with the BioRad microplate spectrophotometer
3550-UV at 570 nm with background subtraction at
660 nm and analyzed with Microplate Manager v.2 soft-
ware for Macintosh (BioRad). Error bars represent standard
errors of the mean (SEM). P values representing statistical
significance were calculated using the t test.
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