Synthesis and antimycobacterial evaluation of N-substituted 3-aminopyrazine-2,5-dicarbonitriles

Article abstract of DOI:10.1016/j.bmcl.2011.12.129

A series of 14 new compounds related to pyrazinamide were synthesized, characterized with analytical data and screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii and two types of Mycobacterium avium. The series comprised of N-substituted 3-aminopyrazine- 2,5-dicarbonitriles derived from 3-chloropyrazine-2,5- dicarbonitrile by nucleophilic substitution of chlorine by various non-aromatic amines (alkylamines, cycloalkylamines and heterocyclic amines). Noteworthy antimycobacterial activity against M. tuberculosis was found among the alkylamino derivatives, for example, 3-(heptylamino)pyrazine-2,5-dicarbonitrile inhibited M. tuberculosis at MIC = 51 lmol/L. 3-(Hexylamino)pyrazine-2,5- dicarbonitrile inhibited M. kansasii at MIC = 218 lmol/L. Basic structure-activity relationships are discussed. A comparison between calculated and experimentally determined lipophilicity parameters within the series is included.

Full text of DOI:10.1016/j.bmcl.2011.12.129

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1598–1601
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimycobacterial evaluation of N-substituted
3-aminopyrazine-2,5-dicarbonitriles
Jan Zitko a, , Josef Jampílek , Lukáš Dobrovolny , Michaela Svobodová , Jirí Kuneš , Martin Dolezal
⇑
b
a
c
a
a
´
ˇ
ˇ
a Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, Hradec Králové 500 05, Czech Republic
b Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 612 42 Brno, Czech Republic
c Department of Microbiology, Regional Hospital, Kyjevská 44, Pardubice 532 03, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 14 new compounds related to pyrazinamide were synthesized, characterized with analytical
data and screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacte-
rium kansasii and two types of Mycobacterium avium. The series comprised of N-substituted 3-aminopyr-
azine-2,5-dicarbonitriles derived from 3-chloropyrazine-2,5-dicarbonitrile by nucleophilic substitution
of chlorine by various non-aromatic amines (alkylamines, cycloalkylamines and heterocyclic amines).
Noteworthy antimycobacterial activity against M. tuberculosis was found among the alkylamino
derivatives, for example, 3-(heptylamino)pyrazine-2,5-dicarbonitrile inhibited M. tuberculosis at
Received 8 December 2011
Revised 26 December 2011
Accepted 27 December 2011
Available online 3 January 2012
Keywords:
Alkyl chain homology
Antimycobacterial activity
Lipophilicity
Pyrazinamide derivatives
Structure–activity relationships
MIC = 51 lmol/L. 3-(Hexylamino)pyrazine-2,5-dicarbonitrile inhibited M. kansasii at MIC = 218 lmol/L.
Basic structure–activity relationships are discussed. A comparison between calculated and experimen-
tally determined lipophilicity parameters within the series is included.
Ó 2012 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) is chronic infectious disease and some esti-
mates suggest that one third of global population might be infected
with latent form of TB.1 Pyrazinamide (PZA) belongs to first-line
antituberculotics and is an important part of all basic therapeutic
regimes of TB. PZA possesses sterilizing activity against Mycobacte-
rium tuberculosis and it is well known that PZA is even more active
against dormant bacilli with low metabolic activity.2 The killing of
these persisters is a crucial factor in shortening the therapy course
and avoiding relapses. PZA as a prodrug is converted by mycobac-
terial pyrazinamidase/nicotinamidase (pncA) into pyrazinoic acid
(POA). The mechanism of action of PZA/POA was suggested by
Zhang et al.3 In acidic environment and mainly due to ineffective
efflux mechanisms for POA in M. tuberculosis,4 POA accumulates
inside the mycobacterial cell and leads to impairment of cytoplas-
mic enzymes. The disruption of proton gradient on cytoplasmic
membrane affects the membrane transport systems as well. How-
ever, the debate whether there is a specific cellular target for PZA
or POA continues and new theories have been raised recently.5,6
Therefore PZA derivatives as potential antituberculotics remain in
the focus of scientists.7–11
6-amino-5-cyanopyrazine-2-carboxamides derived from the chloro
precursor by nucleophilic substitution with various alkyl, cycloalkyl
and heterocyclic amines.14 The antimycobacterial activity was
strongly dependent on lipophilicity and culminated in the deriva-
tive with heptylamino substitution. Therefore it was decided to in-
crease the lipophilicity by the exchange of the carboxamide group
for carbonitrile to obtain a series of N-substituted 3-aminopyr-
azine-2,5-dicarbonitriles presented in this Letter, that is N-alkyl/
cycloalkyl analogues of compounds described by Palek et al.13
The starting compound 0 was prepared according to Refs.13,15
Briefly, PZA was treated with peroxoacetic acid to yield the mixture
of pyrazinecarboxamide-N-oxides. This unseparated mixture was
chlorinated with POCl3 to yield three position isomers of chloro-
pyrazine-2-carbonitrile. The main product 6-chloropyrazine-2-car-
bonitrile was separated by flash chromatography and underwent
homolytic amidation to produce 3-chloro-5-cyanopyrazine-2-car-
boxamide, which was dehydrated by POCl3 to 3-chloropyrazine-
2,5-dicarbonitrile (0). Fourteen new compounds were prepared
from dinitrile 0 by means of nucleophilic substitution (Scheme 1,
a) of chlorine by various non-aromatic amines.16 When 2.5 excess
of primary amine was used to neutralize the originating HCl, an
amidine by-product was formed by nucleophilic addition of amine
to one of the carbonitrile groups (Scheme 1, b). The structure was
confirmed by NMR and MS techniques. This type of reaction con-
cerning pyrazine carbonitrile group was already described before
in literature.14,17,18 In this series, primary amines were the most
willing to form the by-product and if enough time and enough
amine were applied, the amidine by-product dominated the
3-Chloropyrazine-2,5-dicarbonitrile (0), the starting compound
for the discussed series, was synthesized previously and also
showed excellent antimycobacterial activity.12 Based on this fact,
a series of 3-arylaminopyrazine-2,5-dicarbonitriles was published
recently.13 Another paper reported a series of 14 N-substituted
⇑
Corresponding author. Tel.: +420 495067272; fax: +420 495067167.
E-mail addresses: jan.zitko@faf.cuni.cz, jan_zitko@seznam.cz (J. Zitko).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.129

J. Zitko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1598–1601
1599
analytical data for the three most active compounds 6, 7 and 8 can
be found as Ref.,19 complete data are enclosed in the Supplementary
data.
As discussed above, the antimycobacterial activity of different
trisubstituted pyrazines was strongly dependent on lipophilic-
ity,13,14 therefore attention was paid to lipophilicity determination.
Most frequently drugs cross biological membranes through the pas-
sive transport, which strongly depends on their lipophilicity, there-
fore it has been studied and applied as an important drug property
for decades.20 Different lipophilicity descriptors such as logkw, logP,
logD, etc. are used for SAR description and prediction. However, the
algorithms used in their calculation do not take into account config-
uration specificity and sometimes even intramolecular interactions
of these N-substituted pyrazine derivatives. Lipophilicity parame-
ters (logP/ClogP) of all the compounds were calculated using two
commercially available programs (ChemOffice Ultra 10.0 and ACD/
LogP ver. 1.0)21 and also measured by means of the RP-HPLC deter-
mination of capacity factors k expressed as logk. The procedure22
was performed under isocratic conditions with methanol as an or-
ganic modifier in the mobile phase using an end-capped non-polar
C18 stationary RP column. The results are shown in Table 1 and illus-
trated in Figure 1.
Scheme 1. Synthesis of compounds 1–14 and formation of amidine by-product.
reaction mixture. The by-products had almost identical Rf values
with corresponding intended products (silica, hexane/ethyl-ace-
tate). The modification of mobile phase by addition of 1% (v/v) of
acetic acid increased the d Rf sufficiently (>0.2). The by-products
were beyond the interest of this study and were not further evalu-
ated. To minimize the production of this amidine by-product the
excess of amine was substituted with indifferent organic base (tri-
ethylamine) and prolonged dropwise addition of the amine to the
reaction mixture was introduced.
The results obtained with compounds 4–11 show that the
experimentally-determined lipophilicity (logk) of the discussed
compounds are generally in accordance with the calculated values
as shown in Figure 1A, nevertheless it is important to notice that
The prepared compounds were characterized with 13C NMR, 1H
NMR and IR spectroscopy as well as elementary analysis. The
Table 1
Antimycobacterial activity23 expressed as MIC [
lg/mL] and comparison of calculated lipophilicity parameters (log P/Clog P) with determined log k values. Numbers in brackets
represent the MICs converted to molar concentrations [
lmol/L].
Structure
Lipo/hydrophobicity
Antimycobacterial activity (
l
g/mL) (
lmol/L)
a
b
c
No.
R1
R2
logk
logP/ClogP (ChemOffice)
logP (ACD/LogP)
M. tbc H37Rv
M. kansasii
M. avium
M. avium
0
1
2
3
4
5
6
7
—
CH3
C2H5
C3H7
C4H9
C5H11
C6H13
C7H15
—
H
H
H
H
H
H
H
H
H
H
H
C2H5
0.5792
0.5859
0.6146
0.6497
0.7088
0.7901
0.9133
1.0243
1.1953
0.7273
0.7896
0.8789
0.6685
0.5946
0.5830
0.0514
1.17/À0.539
0.49/À0.283
0.82/0.246
1.31/0.775
1.73/1.304
2.15/1.833
2.56/2.362
2.98/2.891
3.40/3.420
1.62/1.189
2.03/1.748
2.45/2.307
1.95/0.820
1.03/À1.018
0.87/À0.583
À1.31/À0.676
0.72 0.40
À0.07 0.42
0.46 0.42
0.99 0.42
1.52 0.42
2.05 0.42
2.58 0.42
3.12 0.42
3.65 0.42
1.42 0.42
1.99 0.42
2.55 0.42
2.62 0.42
1.03 0.54
0.59 0.52
À0.37 0.35
25 (152)
100 (628)
100 (577)
200 (1068)
100 (497)
50 (232)
25 (109)
12.5 (51)
25 (97)
>200
>200
>200
100 (497)
>200
>200
100 (608)
>200
>200
200 (1068)
100 (497)
100 (465)
100 (436)
>200
>200
>200
>200
>200
100 (608)
>200
>200
200 (1068)
100 (497)
100 (465)
100 (436)
>200
>200
>200
>200
>200
100 (628)
200 (1155)
200 (1068)
100 (497)
100 (465)
50 (218)
>200
100 (389)
>200
>200
>200
100 (497)
>200
200 (869)
>200
8
9
C8H17
Cyclopentyl
Cyclohexyl
Cycloheptyl
C2H5
–(CH2)2NCH3(CH2)2–
–(CH2)2O(CH2)2–
10
11
12
13
14
PZA
>200
>200
>200
>200
>200
>200
>200
>200
200 (869)
12.5–25 (102–203)
–
–
a
b
c
CNCTC My 235/80
CNCTC My 80/72
CNCTC My 152/73)
A
B 1.3
3.8
2.8
1.8
k
1.1
0.9
0.7
0.8
-0.2
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Compounds
log P [ChemOffice]
Clog P [ChemOffice]
log P [ACD/LogP]
0.5
-1.2
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Compounds
Figure 1. Relationships between calculated logP/ClogP and ACD/LogP parameters and experimentally found logk values.

1600
J. Zitko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1598–1601
A
B
7.5
7.2
6.9
6.6
6.3
6.0
5.7
6.9
6
7
8
6.6
0
6
8
4
12
5
6.3
5
1
12
2
4
14
1
6.0
14
3
2
3
5.7
0.5
0.6
0.7
0.8
0.9
log k
1.0
1.1
1.2
1.3
0.5
0.6
0.7
0.8
0.9
log k
1.0
1.1
1.2
1.3
Figure 2. Dependences of antimycobacterial activity expressed as log(1/MIC) on lipophilicity parameter logk for M. tuberculosis (A) and M. kansasii (B). Marks: rhombs–
alkylamino substituted derivatives, squares–dialkylamino substituted derivatives, triangles–amino moiety as a part of heterocyclic system.
the real linear dependence of logk on the length of the alkyl chain
is only in the range n-butyl (4)-n-octyl (8). Pyrazines substituted
by short alkyl 1–3 did not possess any linear relation, see Figure
1B. Great differences between experimental and calculated lipo-
philicity values can be also observed for compounds 12–14, that
is, derivatives with the trisubstituted amino moiety. All these facts
indicate probable intramolecular interactions that were not con-
sidered by in silico calculations but seem to be important for bio-
logical activity.
For the results of antimycobacterial assay see the right part of
Table 1. Substitution of chlorine in 0 with short alkylamino chain
in 1–4 led to the decrease of antimycobacterial activity against
M. tuberculosis. The reasonable activity appeared again in pentyla-
(Mycobacteria Other Than Tuberculosis). These cannot be consid-
ered as extra potent compounds, but the fact they inhibit the
growth of MOTTs resistant to PZA is interesting and implicates
there might be a different mode of action for these new structures.
Fourteen new compounds were prepared, characterized with ana-
lytical data and screened for antimycobacterial activity. This study
confirmed the great importance of lipophilicity for the antimycobac-
terial activity of different trisubstituted pyrazines. It also showed that
–CONH2 moiety was not necessary for the antimycobacterial activity
of N-substituted 6-amino-5-cyanopyrazine-2-carboxamides10, since
the presented N-substituted 3-aminopyrazine-2,5-dicarbonitriles
possessed the same or even better activity.
mino substituted compound 5 with MIC = 50
l
g/mL (232
l
mol/L)
Acknowledgments
and increased with the further prolongation of the alkyl chain.
Figure 2A illustrates the comparison of antimycobacterial activity
log (1/MIC) against M. tuberculosis and compound lipophilicity
expressed as logk. It is evident that the initial hypothesis proposed
based on Ref.10,11 was true and the activity is strongly influenced
by lipophilicity of the prepared compounds. The peak activity of
This study was supported by the Grant Agency of Charles Uni-
versity in Prague (B-CH/120509) and the Ministry of Education of
the Czech Republic (grants No. MSM0021620822 and SVV-2011-
263-001).
MIC = 12.5
for heptylamino substitution 7, while the activity of octylamino
derivative 8 dropped slightly to 25 g/mL (97 mol/L). This de-
lg/mL (51 lmol/L) against M. tuberculosis was obtained
Supplementary data
l
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.129.
crease might have been actually caused also by decreased solubil-
ity of compound 8 in testing media (precipitation occurred). It can
be speculated whether longer than C(8) alkyl chains could further
potentiate the antimycobacterial activity, but for these more lipo-
philic compounds the testing conditions would have to be changed
to prevent the precipitation during the dilution of samples.
Compounds 9–11 with cycloalkyamino substitution were com-
pletely inactive. This inactivity can be caused by lower flexibility
of N-substitution chain connected with poor aqueous solubility.
Substitution with secondary amines (tertiary amino moiety) led
to poorly active 12 or inactive 13, 14 compounds. This observation
testifies the importance of the hydrogen atom bound to the amino
group (introduction of tertiary amine moiety into the molecule de-
creases antimycobacterial activity). It can be speculated this
hydrogen atom could be important for non-covalent interactions
with possible cellular targets.
References and notes
1. Treatment of tuberculosis: guidelines – 4th ed. WHO document no. WHO/HTM/
TB/2009.420. 2010. ISBN 978 92 4 154783 3.
2. Zhang, Y. Front. Biosci. 2004, 9, 1136.
3. Zhang, Y.; Wade, M. M.; Scorpio, A.; Zhang, H.; Sun, Z. H. J. Antimicrob.
Chemother. 2003, 52, 790.
4. Zhang, Y.; Scorpio, A.; Nikaido, H.; Sun, Z. H. J. Bacteriol. 1999, 181, 2044.
5. Shi, W.; Zhang, W.; Jiang, X.; Yuan, H.; Lee, J. S.; Barry, C. E.; Wang, H. H.; Zhang,
W. H.; Zhang, Y. Science 2011, 333, 1630.
6. Sayahi, H.; Zimhony, O.; Jacobs, W. R., Jr.; Shekhtman, A.; Welch, J. T. Bioorg.
Med. Chem. Lett. 2011, 21, 4804.
7. Sriram, D.; Yogeeswari, P.; Reddy, S. P. Bioorg. Med. Chem. Lett. 2006, 16, 2113.
8. Chung, W. J.; Kornilov, A.; Brodsky, B. H.; Higgins, M.; Sanchez, T.; Heifets, L. B.;
Cynamon, M. H.; Welch, J. Tuberculosis (Edinb) 2008, 88, 410.
9. Ancizu, S.; Moreno, E.; Solano, B.; Villar, R.; Burguete, A.; Torres, E.; Pérez-
Silanes, S.; Aldana, I.; Monge, A. Bioorg. Med. Chem. 2010, 18, 2713.
10. Moreno, E.; Ancizu, S.; Perez-Silanes, S.; Torres, E.; Aldana, I.; Monge, A. Eur. J.
Med. Chem. 2010, 45, 4418.
11. Dolezal, M.; Kesetovicova, D.; Zitko, J. Curr. Pharm. Des. 2011, 17, 3506.
12. Dlabal, K.; Palat, K.; Lycka, A.; Odlerova, Z. Collect. Czech. Chem. Commun. 1990,
55, 2493.
13. Palek, L.; Dvorak, J.; Svobodova, M.; Buchta, V.; Jampilek, J.; Dolezal, M. Arch.
Pharm. 2008, 341, 61.
14. Zitko, J.; Dolezal, M.; Svobodova, M.; Vejsova, M.; Kunes, J.; Kucera, R.; Jilek, P.
Bioorg. Med. Chem. 2011, 19, 1471.
15. Jampilek, J.; Dolezal, M.; Kunes, J.; Satinsky, D.; Raich, I. Curr. Org. Chem. 2005, 9, 49.
16. General coupling method: 100 mg (0.608 mmol) of 3-chloropyrazine-2,5-
dicarbonitrile (0) was dissolved in dry THF together with 74 mg (0.729 mmol,
The general trend of dependence of antimycobacterial activity
against M. kansasii on lipophilicity is similar. Activity increases
with lipophilicity increase, that is, with chain elongation, to the
optimum—compound 6 that exerted the highest activity within
the series (logk = 0.91, MIC = 50 lg/mL (218 lmol/L)) and then de-
creases. It is important to note that although the activity of com-
pound 6 against M. kansasii can be estimated as moderate, PZA
alone is absolutely ineffective due to well-known natural resis-
tance. Other compounds 4–6 were slightly active against MOTTs

J. Zitko et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1598–1601
1601
1.2 equiv) triethylamine. One equivalent of corresponding amine was diluted
with approx. 2 mL of THF and added dropwise to the reaction mixture during
approx. Fifteen minutes while stirring. The mixture turned yellow and was
stirred for next 15 min at rt. The completion of the reaction was checked by TLC
chromatography. The mixture was filtered and the product was absorbed on
silica by solvent evaporation. The product was purified by flash chromatography
(hexane/ethyl acetate gradient elution with 1% (v/v) of acetic acid, silica).
17. Foks, H.; Janowiec, M. Pol. J. Pharmacol. Pharm. 1977, 29, 61.
22. Lipophilicity HPLC determination (capacity factor k/calculated logk): The HPLC
separation module Waters Alliance 2695 XE and Waters Photodiode Array
Detector 2996 (Waters Corp., Milford, MA, USA) were used. The
chromatographic column SymmetryÒ C18 5 m, 4.6 Â 250 mm, Part No.
WAT054275, (Waters Corp., Milford, MA, USA.) was used. The HPLC
separation process was monitored by Millennium32Ò Chromatography
Manager Software, Waters 2004 (Waters Corp., Milford, MA, USA). The
mixture of MeOH p.a. (70.0%) and H2O-HPLC—Mili-Q Grade (30.0%) was used
as a mobile phase. The total flow of the column was 1.0 mL/min, injection
18. Shirai, K.; Fukunishi, K.; Yanagisawa, A.; Takahashi, H.; Matsuoka, M. J.
Heterocyclic Chem. 2000, 37, 1151.
30 lL, column temperature 30 °C and sample temperature 10 °C. The detection
19. Analytical data of the most active compounds: 3-(hexylamino)pyrazine-2,5-
dicarbonitrile (6). Yellow crystalline comp. Yield: 86%; mp 82–84 °C; 1H NMR
(300 MHz, CDCl3) d 8.13 (1H, s, H6), 5.63 (1H, br s, NH), 3.55–3.45 (2H, m,
NCH2), 1.73–1.57 (2H, m, CH2), 1.46–1.27 (6H, m, CH2), 0.90 (3H, t, J = 6.9 Hz,
CH3); 13C NMR (75 MHz, CDCl3) d 154.5, 135.3, 130.6, 117.6, 115.0, 113.9, 41.6,
31.3, 28.8, 26.4, 22.5, 14.0; IR (KBr): 3342, 2955, 2926, 2855, 2223, 1571, 1258,
1195; Anal. Calcd. for C12H15N5: C 62.86, H 6.59, N 30.54; Found: C 62.86, H
6.75, N 30.45.
wavelength 210 nm was chosen. The KI acetonitrilic solution was used for the
dead time (tD) determination. Retention times (tR) were measured in minutes.
The capacity factors
k
were calculated using the Millennium32Ò
Chromatography Manager Software according to the formula k = (tR À tD)/tD,
where tR is the retention time of the solute, whereas tD denotes the dead time
obtained via an unretained analyte. Logk, calculated from the capacity factor k,
is used as the lipophilicity index converted to logP scale.
23. Evaluation of in vitro antimycobacterial activity: Microdilution panel method.
The antimycobacterial assay was provided by Regional Hospital in Pardubice
(Czech Republic). Five strains were used: M. tuberculosis H37Rv CNCTC My 331/
88, M. kansasii CNCTC My 235/80, M. avium CNCTC My 80/72 and M. avium
CNCTC My 152/73 (Czech National Collection of Type Cultures, National
Institute of Public Health, Prague, Czech Republic). Tested compounds were
dissolved in DMSO, diluted with Šula’s semisynthetic medium (Trios, Prague,
Czech Republic) and placed into microdilution panel. Tested species were
added in the form of suspension in isotonic saline solution. Compounds were
3-(heptylamino)pyrazine-2,5-dicarbonitrile (7). Yellow crystalline comp. Yield:
79%; mp 84-85 °C; 1H NMR (300 MHz, CDCl3) d 8.13 (1H, s, H6), 5.62 (1H, br s,
NH), 3.54-3.45 (2H, m, NCH2), 1.73-1.57 (2H, m, CH2), 1.45-1.21 (8H, m, CH2),
0.89 (3H, t, J=6.2 Hz, CH3); 13C NMR (75 MHz, CDCl3) d 154.5, 135.3, 130.6,
117.6, 115.0, 113.9, 41.6, 31.6, 28.8, 28.8, 26.7, 22.5, 14.0; IR (KBr): 3350, 2955,
2925, 2855, 2222, 1575, 1257, 1206; Anal. Calcd. for C13H17N5: C 64.17, H 7.04,
N 28.78; Found: C 64.02, H 7.12, N 28.69.
3-(octylamino)pyrazine-2,5-dicarbonitrile (8). Yellow crystalline comp. Yield:
78%; mp 82–83 °C; 1H NMR (300 MHz, CDCl3) d 8.13 (1H, s, H6), 5.62 (1H, br s,
NH), 3.49 (2H, q, J = 6.5 Hz, NCH2), 1.74–1.57 (2H, m, CH2), 1.48–1.18 (10H, m,
CH2), 0.88 (3H, t, J = 6.5 Hz, CH3); 13C NMR (75 MHz, CDCl3) d 154.5, 135.3,
130.6, 117.6, 115.0, 113.9, 41.6, 31.7, 29.1, 28.8, 26.7, 22.6, 14.0; IR (KBr): 3344,
2955, 2923, 2851, 2223, 1616, 1582, 1257, 1203; Anal. Calcd. for C14H19N5: C
65.34, H 7.44, N 27.22; Found: C 65.28, H 7.49, N 27.19.
tested at final concentrations of 200, 100, 50, 25, 12.5, 6.25 and 3,125 lg/mL.
The final concentration of DMSO did not exceed 1% (v/v). It was confirmed that
at this concentration DMSO itself did not affect the growth of mycobacteria.
PZA was used as a standard. The cultures were grown in Šula’s semisynthetic
medium at pH 5.5 and 37 °C. The antimycobacterial activity was determined
visually after 14 days (6 days for M. kansasii) of incubation as MIC [lg/mL], i.e.
20. Kerns, E. H.; Li, D. Drug-like properties: Concept, structure design and methods;
Elsevier: San Diego, CA, USA, 2008.
the lowest used concentration of tested substance which inhibited the growth
of mycobacteria. To eliminate the influence of different molar mass of the
21. CS ChemOffice Ultra ver. 10.0 (CambridgeSoft, Cambridge, MA, USA) and ACD/
Log P ver. 1.0 (Advanced Chemistry Development Inc., Toronto, Canada).
compounds, the MIC values were converted to
determination of SAR.
lmol/L before the