Development of 3,5-Dinitrophenyl-Containing 1,2,4-Triazoles and Their Trifluoromethyl Analogues as Highly Efficient Antitubercular Agents Inhibiting Decaprenylphosphoryl-β- d -ribofuranose 2′-Oxidase

Article abstract of DOI:10.1021/acs.jmedchem.9b00912

We report herein the discovery of 3,5-dinitrophenyl 1,2,4-triazoles with excellent and selective antimycobacterial activities against Mycobacterium tuberculosis strains, including clinically isolated multidrug-resistant strains. Thorough structure-activity relationship studies of 3,5-dinitrophenyl-containing 1,2,4-triazoles and their trifluoromethyl analogues revealed the key role of the position of the 3,5-dinitrophenyl fragment in the antitubercular efficiency. Among the prepared compounds, the highest in vitro antimycobacterial activities against M. tuberculosis H₃₇Rv and against seven clinically isolated multidrug-resistant strains of M. tuberculosis were found with S-substituted 4-alkyl-5-(3,5-dinitrophenyl)-4H-1,2,4-triazole-3-thiols and their 3-nitro-5-(trifluoromethyl)phenyl analogues. The minimum inhibitory concentrations of these compounds reached 0.03 μM, which is superior to all the current first-line anti-tuberculosis drugs. Furthermore, almost all compounds with excellent antimycobacterial activities exhibited very low in vitro cytotoxicities against two proliferating mammalian cell lines. The docking study indicated that these compounds acted as the inhibitors of decaprenylphosphoryl-β-d-ribofuranose 2′-oxidase enzyme, which was experimentally confirmed by two independent radiolabeling experiments.

Full text of DOI:10.1021/acs.jmedchem.9b00912

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Article
Development of 3,5-Dinitrophenyl-Containing 1,2,4-Triazoles and
their Trifluoromethyl Analogues as Highly Efficient Antitubercular
Agents Inhibiting Decaprenylphosphoryl-#-D-ribofuranose 2#-Oxidase
Galina Karabanovich, Jan Dušek, Karin Savková, Oto Pavliš, Ivona Pávková, Jan Korabecny,
Tomáš Ku#era, Hana Ko#ová Vl#ková, Stanislav Huszár, Zuzana Konyariková, Klára
Kone#ná, Ondrej Jandourek, Ji#ina Stola#íková, Jana Korduláková, Kate#ina Vávrová,
Petr Pavek, V#ra Klimešová, Alexandr Hrabalek, Katarína Mikušová, and Jaroslav Roh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00912 • Publication Date (Web): 08 Aug 2019
Downloaded from pubs.acs.org on August 9, 2019
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Development of 3,5-Dinitrophenyl-Containing
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,2,4-Triazoles and their Trifluoromethyl Analogues
as Highly Efficient Antitubercular Agents Inhibiting
Decaprenylphosphoryl-β-D-ribofuranose 2′-Oxidase
a
a
b
c
d
Galina Karabanovich, Jan Dušek, Karin Savková, Oto Pavliš, Ivona Pávková, Jan
Korábečný,d, e Tomáš Kučera, Hana Kočová Vlčková, Stanislav Huszár, Zuzana
d
a
b
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Konyariková, Klára Konečná, Ondřej Janďourek, Jiřina Stolaříková, Jana
b
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Korduláková, Kateřina Vávrová, Petr Pávek, Věra Klimešová, Alexandr Hrabálek,
b
a,
Katarína Mikušová, and Jaroslav Roh *
a
Charles University, Faculty of Pharmacy in Hradec Králové, Akademika Heyrovského
1203, 50005 Hradec Králové, Czech Republic
b
Comenius University in Bratislava, Faculty of Natural Sciences, Department of
Biochemistry, Mlynská dolina, Ilkovičova 6, 842 15 Bratislava, Slovakia
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c
Military Health Institute, Biological Defense Department, 561 64 Techonin, Czech
Republic
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University of Defence, Faculty of Military Health Sciences, Třebešská 1575, 50005
Hradec Králové, Czech Republic
e
Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500
05 Hradec Kralove, Czech Republic
f
Regional Institute of Public Health, Department of Bacteriology and Mycology,
Partyzánské náměstí 7, 70200 Ostrava, Czech Republic
ABSTRACT. We report herein the discovery of 3,5-dinitrophenyl 1,2,4-triazoles with
excellent and selective antimycobacterial activities against Mycobacterium tuberculosis
strains, including clinically isolated multidrug-resistant strains. Thorough structure-activity
relationship studies of 3,5-dinitrophenyl-containing 1,2,4-triazoles and their
trifluoromethyl analogs revealed the key role of the position of the 3,5-dinitrophenyl
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fragment in the antitubercular efficiency. Among the prepared compounds, the highest in
vitro antimycobacterial activities against M. tuberculosis H Rv and against seven
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clinically isolated multidrug-resistant strains of M. tuberculosis were found with S-
substituted 4-alkyl-5-(3,5-dinitrophenyl)-4H-1,2,4-triazole-3-thiols and their 3-nitro-5-
(trifluoromethyl)phenyl analogues. The minimum inhibitory concentrations of these
compounds reached 0.03 μM, which is superior to all the current first-line anti-tuberculosis
drugs. Furthermore, almost all compounds with excellent antimycobacterial activities
exhibited very low in vitro cytotoxicities against two proliferating mammalian cell lines.
The docking study indicated that these compounds acted as the inhibitors of
decaprenylphosphoryl-β-D-ribofuranose 2′-oxidase (DprE1) enzyme, which was
experimentally confirmed by two independent radiolabeling experiments.
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Introduction
Tuberculosis (TB), one of the most widespread and dangerous infectious diseases, is
among the world’s top 10 causes of death, and it claimed more than 1.6 million lives (3%
of all deaths) in 2017. This is comparable with the number of deaths caused by lung
cancer, diabetes, Alzheimer disease or road injuries.1
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The main threat to successful recovery from TB is the ability of Mycobacterium
tuberculosis (M. tuberculosis), the causative agent of TB, to survive under the conditions
of anti-TB therapy. To cure drug-susceptible TB strains, a four-drug combination
(isoniazid - INH, rifampicin - RIF, pyrazinamide - PZA, and ethambutol - EMB) is
administered for 6 months. Such therapy leads to recovery of at least 82 % of patients.1
To treat multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of TB, a
wide palette of drugs, such as aminoglycosides, polypeptides, fluoroquinolones,
clofazimine, para-aminosalicylic acid, cycloserine, terizidone, ethionamide and
prothionamide, are being used. Nevertheless, the treatment durations of such cases are
18-24 months, and only 55 % of patients are successfully cured. Such a low recovery rate
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is associated with the limited efficacy of the applied drugs, poor adherence to treatment
regimens and adverse effects that can develop during such long therapies.1
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New prospects in the treatment of MDR M. tuberculosis strains appeared with the
2
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introduction of bedaquiline and delamanid
for TB therapy (Figure 1). These two
medications are highly effective against drug-susceptible and MDR strains of M.
tuberculosis as well as against their latent forms. Importantly, they have unique
mechanisms of action that differ from those of other currently used drugs. Bedaquiline
and delamanid are still under evaluation in phase III clinical development, nonetheless,
they are already in the clinical use since 2012 and 2014, respectively, and are
recommended for the therapy of drug-resistant TB or in cases of low tolerability to
standard regimens.5, 6
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Br
N
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Me
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O N
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OH
OCF3
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Bedaquiline (TMC-207)
MIC 0.05 µM
Delamanid (OPC-67683)
MIC 0.023 µM
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NO2
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Pretomanid (PA-824)
MIC 0.05-0.7 µM
PBTZ-169
MIC 0.4 nM
Figure 1. Selected compounds that are in clinical development for TB treatment and their
MICs against M. tuberculosis H Rv.
3
7
Currently, there are fourteen new compounds or repurposed drugs in clinical phases of
6
development against TB, and these drugs include nitro group-containing compounds
7
such as nitroimidazole PA-824 (pretomanid), having multifactorial mechanism of action,
and benzothiazinone PBTZ-169 (macozinone), an inhibitor of mycobacterial
8
decaprenylphosphoryl-β-D-ribofuranose 2’-oxidase (DprE1) (Figure 1). It should be
noted that delamanid, pretomanid and PBTZ-169 have nitro group-dependent
mechanisms of action.9-11
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To avoid both recent and future problems with the treatment of TB caused by multidrug-
resistant strains of M. tuberculosis, it is necessary to continue to identify and develop new
compounds with high antimycobacterial activities and new mechanisms of action.12-17
In our previous studies, we identified 2-alkyl-5-[(3,5-dinitrobenzyl)sulfanyl]-2H-
tetrazoles (1) as potent antitubercular compounds with minimum inhibitory concentrations
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(
MIC values) of 1 µM against various strains of M. tuberculosis (Figure 2).18 Later, we
found that isosteres of those compounds, 2-alkyl/aryl-5-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-
oxadiazoles (2)19, 20 and their reverse analogues, 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-
1
,3,4-oxadiazoles (3),20, 21 showed outstanding activities against both drug-susceptible
and multidrug-resistant strains of M. tuberculosis with MIC values against M. tuberculosis
reaching 0.03 µM (0.011-0.026 µg/mL). Furthermore, lead compounds of series 2 showed
strong bactericidal effects on nonreplicating streptomycin (STR)-starved M. tuberculosis
strain 18b-Lux. We showed that the presence of a 3,5-dinitrophenyl group linked through
a methylsulfanyl group in compounds of series 1 and 2 is crucial for their high
antimycobacterial activity, and any change in its structure led to a significant decrease in
the antimycobacterial activity.18, 19, 22 In the case of lead compounds of structure 3, the
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replacement of a nitro group with a trifluoromethyl group did not substantially affect the
antimycobacterial activity.21
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In this study, we focused on 4H-1,2,4-triazole analogues of lead compounds 2 and 3 as
potential antitubercular agents. The main benefit of the 4H-1,2,4-triazole core is the
possibility to have substituents not only in positions 3 and 5, which would be isosteric to
1,3,4-oxadiazole, but also in position 4. The additional substituent in position 4 can be
used to optimize the physico-chemical properties but can also be used to tune the
pharmacodynamics
of
the
molecule.
Thus,
3,4-di(alkyl/aryl)-5-[(3,5-
dinitrobenzyl)sulfanyl]-4H-1,2,4-triazoles (4 and 5) and 4-alkyl-3-alkylsulfanyl-5-(3,5-
dinitrophenyl)-4H-1,2,4-triazoles (7-9) were designed and prepared as analogues of lead
compounds 2 and 3, respectively. In addition, a series of 3-alkylsulfanyl-4-(3,5-
dinitrobenzyl)-5-phenyl-4H-1,2,4-triazoles (11) with a 3,5-dinitrophenyl group connected
to position 4 were also prepared and studied (Figure 2).
To further investigate the role of both nitro groups in the antimycobacterial activity and
selectivity of action of these triazoles, the series of trifluoromethyl analogues, 3-alkyl/aryl-
4-benzyl-5-[(3-nitro-5-(trifluoromethyl)benzylsulfanyl]-4H-1,2,4-triazoles (6) and 3-
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alkylsulfanyl-4-benzyl-5-[3-nitro-5-(trifluoromethyl)phenyl]-4H-1,2,4-triazoles (10), were
prepared (Figure 2). The antimycobacterial activities of all the prepared compounds were
evaluated against the standard M. tuberculosis H Rv strain and against nontuberculous
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M. avium and M. kansasii strains. Furthermore, the antimycobacterial activities of
selected compounds were evaluated against seven clinically isolated MDR/XDR M.
tuberculosis strains. To further study the selectivity of their antimycobacterial effects, all
compounds with excellent activities were assessed for antibacterial and antifungal
activities, and their cytotoxicities were evaluated. Furthermore, genotoxicity of selected
compounds was studied. As all investigated compounds contain nitro group and showed
partial isostericity with known DprE1 inhibitors, their interaction with mycobacterial DprE1
enzyme was evaluated in silico and in vitro to elucidate their mechanism of
antimycobacterial action.
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Parent compounds
N
N
NO2
N
N
NO2
N
N
O N
2
R
N
R
S
R
S
S
N
O
O
R1 = alkyl/aryl
R2 = H, alkyl, aryl
R3 = alkyl
NO2
2
NO2
3
1
O N
2
Target compounds
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0
N
N
R3
N
N
NO2
N
N
N
N
O N
O N
2
2
R3
Ph
S
N
Ph
Ph
S
S
S
N
R2
N
N
NO2
R2
NO2
O N
2
O N
2
4
a-k
7b-d, 7f, 7g, 7m
8d-g
NO2
11d, 11f-h
N
N
CF3
N
N
NO2
N
N
N
N
O N
R3
CF3
R3
2
R1
S
R1
S
S
S
N
N
N
N
Ph
b, 6e-h
NO2
Ph
NO2
Ph
Ph
10c-h
O N
2
O N
2
6
5a-l
9a-g
1
2
3
R : a = CH (CH )
R : a = H
b = CH3
c = CH CH
R : a = CH3
3
2 5
b = cyclohexyl
b = CH (CH )
3
2 2
c = HO(CH2)5
d = CH (CH )
c = CH (CH )
3
2
3
2 7
d = CH (CH )
e = cyclohexyl
f = CH (CH )
g = 2,4-Cl PhCH
h = 4-BrPhCH2
i = 4-CH OPhCH
j = Ph
k = 3,4-Cl Ph
d = 4-CH OPhCH
3
2 10
3
2 5
3
2
e = Ph
f = 4-CH Ph
e = 4-BrPhCH2
f = 4-ClPhCH2
g = 3,4-Cl PhCH
3
3
2 11
g = 4-CH OPh
3
2
2
2
2
h = 4-ClPh
h = PhCH2
i = 3,4-Cl Ph
2
3
2
j = 4-BrPh
k = 4-HOPh
l = 4-NO Ph
2
l = tert-butyl
m = PhCH2
2
Figure 2. Structures of lead compounds 1,18, 22 219 and 321 and their 4H-1,2,4-triazole
analogues (4-10) studied in this work.
Results and discussion
Synthetic protocols consisted of scalable and reproducible methods. The synthesis of
4-alkyl/aryl-3-[(3,5-dinitrobenzyl)sulfanyl]-5-phenyl-4H-1,2,4-triazoles
(4a-k)
was
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
performed according to Scheme 1. The reaction of benzoyl chloride (12) with
thiosemicarbazide in THF gave 1-benzoylthiosemicarbazide (14a) in 90% yield, whereas
the reaction of benzohydrazide (13) with the corresponding alkyl/aryl isothiocyanates in
boiling ethanol gave 4-alkyl/aryl-1-benzoylthiosemicarbazides (14b-l). Resulting
thiosemicarbazides 14a-k were cyclized at 90 °C in aqueous potassium hydroxide to give
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,2,4-triazole-3-thiols 15a-k, which were obtained in 38-93% yields. However, 4-(tert-
butyl)-1-benzoylthiosemicarbazide (14l) did not cyclize to corresponding 1,2,4-triazole-3-
thiol, probably because of the presence of a bulky tert-butyl substituent. Alkylation of thiols
15a-k with 3,5-dinitrobenzyl chloride was performed in acetonitrile in the presence of
triethylamine and gave final triazoles 4a-k in moderate-to-high yields (65-98%).
Scheme 1. Synthesis of four series of final nitrobenzyl-containing triazole derivatives 4a-
k, 5a-l, 6b, 6e-h, 11d and 11f-ha
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9
O
N
N
N
N
NO2
a
H
H
c
d
N
N
SH
N
R2
N
R2
S
PhCOCl
N
R2
H
S
12
b
NO2
Method a: 14a
Method b: 14b-l
O
15a-k
4a-k
NH2
N
H
N
N
N
N
NO2
R3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SH
S
13
N
N
O
e
H
N
H
f
g
NO2
NO2
N
N
H
NO2
S
NO2
NO2
2
2
11d, 11f-h
21
X
N
N
N
N
R1
S
O
N
O
h
H
N
H
i
R1
SH
j, k
N
N
NH2
R1
R1
N
H
N
H
NO2
S
method j; X = NO : 5a-l
2
1
6a-l
17a-l
18a-l
method k; X = CF : 6b, 6e-h
3
aReagents and conditions: (a) thiosemicarbazide, THF, 5 °C to rt, 1 h, 90%; (b) alkyl/aryl
isothiocyanate, EtOH, reflux, 3-7 h, 21-96%; (c) KOH, H O, 90 °C, 3-7 h, 38-93%; (d) 3,5-
2
(
NO ) PhCH Cl, Et N, CH CN, 1-6 h, rt, 65-98%, (e) 3,5-(NO ) PhCH NCS (20), EtOH,
2
2
2
3
3
2 2
2
reflux, 7 h, 80%; (f) KOH, H O, 90 °C, 5 h, 85%; (g) Alkyl halide, Et N, CH CN, 4 h, rt, 76-
2
3
3
9
0%, (h) benzyl isothiocyanate, EtOH, reflux, 3-7 h, 54-98%; (i) KOH, H O, 90 °C, 3-5 h,
2
6
9-96%; (j) 3,5-(NO ) PhCH Cl, Et N, CH CN, reflux, 1-5 h, 73-96%; (k) 3-NO -5-CF -
2
2
2
3
3
2
3
PhCH Br (19), Et N, CH CN, rt, 1-2 h, 70-96%.
2
3
3
3-Alkyl/aryl-4-benzyl-5-[(3,5-dinitrobenzyl)sulfanyl]-4H-1,2,4-triazoles (5a-l) and 3-
alkyl/aryl-4-benzyl-5-[(3-nitro-5-(trifluoromethyl)benzyl)sulfanyl]-4H-1,2,4-triazoles (6b,
6e-h) were prepared using an approach similar to what was used for the synthesis of
target compounds 4a-k. In the first step, the reaction of the corresponding hydrazides
1
6a-l and benzyl isothiocyanate gave 4-benzyl-1-alkanoyl/aroylthiosemicarbazides 17a-
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5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
l, which were cyclized to corresponding 1,2,4-triazole-3-thiols 18a-l. Their alkylation with
3,5-dinitrobenzyl chloride in boiling acetonitrile in the presence of triethylamine resulted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
in the formation of final products 5a-l in 73-96% yields. Selected 1,2,4-triazole-3-thiols
18b, 18e, and 18f-h were alkylated with 3-nitro-5-(trifluoromethyl)benzyl bromide (19) in
acetonitrile in the presence of triethylamine to form final products 6b and 6e-h in good-to-
excellent yields (70-96%, Scheme 1).
The reaction of 3,5-dinitrobenzyl isothiocyanate (20) with benzohydrazide in boiling
ethanol led to the formation of crude 1-benzoyl-4-(3,5-dinitrobenzyl)thiosemicarbazide
(21), which was directly cyclized in aqueous KOH at 90 °C to form corresponding 1,2,4-
triazole-3-thiol (22). Thiol 22 was purified using column chromatography and then
alkylated with the corresponding alkyl halide in acetonitrile in the presence of
triethylamine to produce final 3-alkylsulfanyl-4-(3,5-dinitrobenzyl)-5-phenyl-4H-1,2,4-
triazoles 11d and 11f-h in good yields (Scheme 1).
The synthesis of 4-alkyl-3-benzylsulfanyl-5-(3,5-dinitrophenyl)-4H-1,2,4-triazoles (7b-d,
7f, 7g, and 7m) was performed via a three-step procedure starting from 3,5-
dinitrobenzohydrazide (23). In the first step, 3,5-dinitrobenzohydrazide (23) was
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converted to the 4-alkyl-1-(3,5-dinitrobenzoyl)thiosemicarbazide (24b-d, 24f, 24g, and
24m) by its reaction with the corresponding alkyl isothiocyanate. The following cyclization
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of thiosemicarbazides 24b-d, 24f, 24g, and 24m in aqueous KOH proceeded with
satisfactory yields (58-82%) of corresponding 1,2,4-triazole-3-thiols 25b-d, 25f, 25g, and
25m. The final step of the synthesis of final products 7b-d, 7f, 7g, and 7m was the
alkylation of thiols 25b-d, 25f, 25g, and 25m with benzyl bromide in acetonitrile in the
presence of triethylamine (Scheme 2).
Scheme 2. Synthesis of four series of final nitrophenyl-containing triazoles 7b-d, 7f, 7g,
7
m, 8d-g, 9a-g and 10c-h.a
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
N N
H
H
O N
2
O N
2
NH2
O N
N
N
2
R2
a
b
SH
N
N
N
R2
H
H
S
X
X
X
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
X = NO : 23
X = CF : 26
X = NO : 24b-d, 24f, 24g, 24m
X = CF : 27m
X = NO : 25b-d, 25f, 25g,
2
2
2
25m
X = CF : 28m
3
3
3
N N
O N
2
c
S
25b-d, 25f, 25g, 25m
N
R2
O N
2
7b-d, 7f, 7g, 7m
N N
O N
R3
2
d
25b
S
N
CH3
O N
2
8
d-g
N N
N
O N
N N
N
2
d
O N
2
R3
SH
S
X
X
X = NO : 25m
X = NO : 9a-g
2
X = CF : 10c-h
2
X = CF : 28m
3
3
aReagents and conditions: (a) Alkyl isothiocyanate, EtOH, reflux, 2-6 h, 63-81%; (b)
KOH, H O, 90 °C, 3-8 h, 58-82%; (c) Benzyl bromide, Et N, CH CN, rt, 12 h, 54-93%, (d)
2
3
3
Alkyl halide, Et N, CH CN, rt, 1-5 h, 42-95%.
3
3
Alkylation of 1,2,4-triazole-3-thiols 25b and 25m with the corresponding alkylating agent
led to the target 3-alkylsufanyl-5-(3,5-dinitrophenyl)-4-methyl-4H-1,2,4-triazoles (8d-g)
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9
and 3-alkylsufanyl-4-benzyl-5-(3,5-dinitrophenyl)-4H-1,2,4-triazoles (9a-g), respectively
(Scheme 2). The final 3-alkylsulfanyl-4-benzyl-5-[3-nitro-5-(trifluoromethyl)phenyl]-4H-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,2,4-triazoles
(10c-h)
were
prepared
starting
from
3-nitro-5-
(trifluoromethyl)benzohydrazide 26 using a same protocol that was used for the synthesis
of compounds 9a-g (Scheme 2).
3,5-Dinitrophenyl derivatives 7-9 and their trifluoromethyl analogues 10 showed
excellent antimycobacterial activity against drug-sensitive and multidrug-resistant
Mycobacterium spp in vitro. M. tuberculosis CNCTC My 331/88 (H Rv) and
37
nontuberculous mycobacterial strains – M. avium CNCTC My 330/88, M. kansasii CNCTC
My 235/80 and clinically isolated M. kansasii 6509/96 – were used to evaluate the
antimycobacterial activities of all final compounds 4a-k, 5a-l, 6b, 6e-h, 11d, 11f-h, 7b-d,
7f, 7g, 7m, 8d-g, 9a-g and 10c-h and 4-alkyl-1-(3,5-dinitrobenzoyl)thiosemicarbazide
intermediates 24c, 24g and 24m. Furthermore, selected compounds were assessed for
their in vitro antimycobacterial activities against seven clinically isolated MDR/XDR strains
of M. tuberculosis.
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1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A series of 4-alkyl/aryl-3-[(3,5-dinitrobenzyl)sulfanyl]-5-phenyl-4H-1,2,4-triazoles (4a-k)
were prepared to explore the influence of the substituent on the nitrogen at position 4 of
the 4H-1,2,4-triazole ring on the antimycobacterial activity. It was found that almost all
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
compounds bearing various alkyl, benzyl or aryl R -substituents displayed similar
antimycobacterial activities regardless of their structure or lipophilicity. The MIC values of
compounds 4a-k against drug-susceptible M. tuberculosis and nontuberculous M.
kansasii My 235/80 and clinically isolated M. kansasii 6509/96 ranged between 1 and 4
µM. Only one derivative in this series, compound 4g, with a bulky 2,4-dichlorobenzyl
substituent, showed very low antimycobacterial efficiency (MIC values higher than 32 µM)
(Table 1).
To study the role of the substituent at position 3 of the 4H-1,2,4-triazole on the
antimycobacterial properties of the compounds, a series of 3-alkyl/aryl-4-benzyl-5-[(3,5-
dinitrobenzyl)sulfanyl]-4H-1,2,4-triazoles (5a-l) were prepared. In general, compounds
5a-l were slightly less active than compounds 4a-k, with MIC values of 2-4 µM.
Compounds 4d, 4i, 5j and 5e were also evaluated for their activity against MDR/XDR-TB
strains and showed comparable efficacy found for drug-susceptible M. tuberculosis (MIC
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1
=
2-4 µM, Table 2). We found that the R -substituent at position 3 can affect the
antimycobacterial activity of the studied compounds by modifying their lipophilicities. The
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
least active compounds were those with the lowest lipophilicity, i.e., compounds 5c and
5
k with hydroxyl groups in their structures. Nevertheless, the structure of the lipophilic
1
alkyl or aryl R -substituent had a negligible influence on the antimycobacterial activities
of the studied compounds (Table 1) and we did not find any correlation between cLogP
and antimycobacterial activity in the series of compounds 4 and 5. Although the
antimycobacterial activities of compounds of series 4 and 5 were comparable to that of
INH, they were significantly lower than those of parent oxadiazole compounds 2.19
The comparison of the antimycobacterial activities of the parent 2-alkyl/aryl-5-[(3,5-
dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles (2) and newly prepared 1,2,4-triazole
derivatives of series 4 and 5 showed that the replacement of the 1,3,4-oxadiazole ring
with the 1,2,4-triazole ring is detrimental and led to deterioration of the antimycobacterial
activity. The MIC values of 1,3,4-oxadiazoles 2 reached 0.03 μM against drug-susceptible
and drug-resistant strains of M. tuberculosis, while 3,4-di(alkyl/aryl)-5-[(3,5-
dinitrobenzyl)sulfanyl]-4H-1,2,4-triazoles (4a-k and 5a-l) were active with MIC values of
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
-4 μM. From this point of view, the 1,3,4-oxadiazole core remains the best choice for the
core of 3,5-dinitrobenzylsulfanyl-substituted antitubercular agents.18-20, 22
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The significant decrease in the antimycobacterial activity upon exchanging one nitro
group for
a
trifluoromethyl group in the 3-alkyl/aryl-4-benzyl-5-[(3-nitro-5-
(trifluoromethyl)benzyl)sulfanyl]-4H-1,2,4-triazoles (6b and 6e-h) compared to those of
compounds 5 (Table 1) highlighted the importance of both nitro groups on the 3,5-
dinitrobenzylsulfanyl group for the high antimycobacterial efficacy of these triazoles. This
finding is in agreement with our previous results in which the trifluoromethyl analogues of
(3,5-dinitrobenzylsulfanyl) oxadiazoles 2 showed substantially lower antimycobacterial
activities.19
3-Alkylsulfanyl-4-(3,5-dinitrobenzyl)-5-phenyl-4H-1,2,4-triazoles (11d and 11f-h) had
the lowest antimycobacterial activity among the compounds with the 3,5-dinitrophenyl
groups in their structure. The MIC values of compounds 11d and 11f-h against M.
tuberculosis and both strains of M. kansasii ranged between 8 and 32 μM. Thus, the
attachment of the 3,5-dinitrobenzyl group to the nitrogen at position 4 of the 1,2,4-triazole
ring was unfavorable for the antimycobacterial activity of the studied compounds. This
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result is in agreement with our previous observation made in compounds with general
structure 1: the attachment of the 3,5-dinitrobenzyl group to the sulfur atom was beneficial
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
for the efficacy of these compounds, whereas the N-(3,5-dinitrobenzyl)-substituted
tetrazoles displayed considerably lower activity.18
A series of 4-alkyl-3-alkylsulfanyl-5-(3,5-dinitrophenyl)-4H-1,2,4-triazoles (7-9) were
prepared as the analogues of 5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles 3 and as the
reverse analogues of 4,5-di(alkyl/aryl)-3-[(3,5-dinitrobenzyl)sulfanyl]-4H-1,2,4-triazoles 4
and 5 with the 3,5-dinitrophenyl group shifted from the methylsulfanyl linker to being
directly bound to the 1,2,4-triazole. It should be noted that a series of differently
substituted 4-substituted-5-(3,5-dinitrophenyl)-4H-1,2,4-triazole-3-thiols, i.e., compounds
with unsubstituted thiol groups, were recently prepared and evaluated for their
antitubercular activity. However, these compounds showed very low antimycobacterial
activities (12.5-100 µg/mL).23
4-Alkyl-3-alkylsulfanyl-5-(3,5-dinitrophenyl)-4H-1,2,4-triazoles (7b-d, 7f, 7g, 7m, 8d-g
and 9a-g) showed the highest in vitro antimycobacterial activities among the compounds
studied in this work. The MIC values of several compounds from series 7-9 reached 0.03
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Journal of Medicinal Chemistry
Page 22 of 149
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
µM against drug-susceptible M. tuberculosis and against seven MDR/XDR strains of M.
tuberculosis. Notably, almost all compounds of series 7-9 had submicromolar MIC levels.
Similar MIC values were even observed against both strains of M. kansasii (Table 1).
These results were consistent with the results obtained for 5-(3,5-dinitrophenyl)-1,3,4-
oxadiazoles 3; the MIC values of oxadiazoles 3 also reached 0.03 µM against drug-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
susceptible and MDR/XDR strains of M. tuberculosis. The similarities among the MIC
values of compounds in series 8d-g and 9a-g demonstrated that the lipophilic R3-
substituent on the sulfur atom had a negligible influence on the antimycobacterial activity,
21
which is consistent with the same phenomenon observed for parent compounds 3. Only
less lipophilic methyl derivatives 8d and 9a showed slightly decreased activity, especially
against M. kansasii strains. Similarly to compounds 4 and 5, we did not find any
correlation between the cLogP of the lipophilic compounds of series 8 and 9 and their
2
antimycobacterial activity. Regarding the role of the R substituent on the nitrogen at
position 4 of the 1,2,4-trizole ring, the results indicated that compact substituents are
beneficial for antimycobacterial activity, as compounds 7b, 7c and 7m with methyl, ethyl
and benzyl substituents, respectively, had submicromolar MIC values against M.
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Page 23 of 149
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
2
tuberculosis and M. kansasii strains. The introduction of a bulky R substituent decreased
the antimycobacterial activities; the MIC values of 7d, 7f and 7g with hexyl, dodecyl and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
,4-dichlorobenzyl substituents, respectively, were 1-2 µM. Furthermore, compounds 8e
and 8f were the only compounds in this study to show significant activities against highly
resistant M. avium. However, the parent compounds of series 3 were more effective
21
against this mycobacterial species. Moderate in vitro antimycobacterial activities of 3,5-
dinitrobenzoylthiosemicarbazides 24c, 24g and 24m, i.e., the precursors of final triazoles
7c, 7g and 7m, highlighted the positive effect of the triazole heterocycle on the
antimycobacterial activity.
Replacement of the 1,3,4-oxadiazole ring in S-substituted 5-(3,5-dinitrophenyl)-1,3,4-
oxadiazole-2-thiols (3) with a 4H-1,2,4-triazole ring had a negligible effect on the
antimycobacterial activity; compounds of series 7-9 were highly active against drug-
susceptible and drug-resistant strains of M. tuberculosis and against both tested strains
21
of M. kansasii. The MIC values of parent compounds 3 and compounds of series 7-9
reached 0.03 µM, which are superior to those of all current first-line anti-TB drugs.
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Previously, we found that 5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles 3 tolerated the
21
replacement of one nitro group for a trifluoromethyl group. Similarly, 3-alkylsulfanyl-4-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzyl-5-[3-nitro-5-(trifluoromethyl)phenyl]-4H-1,2,4-triazoles
10c-h,
which
were
synthesized as the trifluoromethyl analogues of compounds 9c-g and 7m, showed slightly
lower but comparable activities to their 3,5-dinitrophenyl counterparts. Similarly to
compounds 7-9, trifluoromethyl derivatives of series 10 had the same MIC values against
drug susceptible and MDR/XDR strains (Table 2), indicating the distinct mechanism of
action from currently used anti-TB drugs.
Compounds of series 9 and 10 with excellent antimycobacterial activities showed high
lipophilicity with cLogP values mostly above five. This is not surprising as compounds
24
with antiTB activity are generally more lipophilic than others. From this point of view, it is
obvious that the classic “rule of five” does not apply to antimycobacterials. As examples worth
mentioning, bedaquiline (cLogP = 7.10), clofazimine (cLogP = 8.43) and delamanid (cLogP =
4
.84) recently marketed/or in phase III of clinical trials for the treatment of MDR/XDR
tuberculosis possess clogP values beyond the optimal values defined by Lipinski or Gleeson.25, 26
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7
8
9
Table 1. In vitro antimycobacterial activities of the final compounds from series 4-11 and
intermediates 24c, 24m and 24g expressed as MICs (μM).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
M. tuberculosis
CLogP My 331/88
M. avium
M. kansasii
M. kansasii
My 330/88
My 235/80
6509/96
14 / 21 days
7 / 14 / 21 days
μM
μM
μM
μM
4a
3.08
2.9
2/4
62/125
125/250
250/250
250/250
250/250
250/250
250/250
250/250
250/250
4/8/8
4/8/16
4/8/16
2/4/4
4b
4c
4d
4e
4/4
4/8/16
2/2/4
3.43
5.54
4.93
8.72
5.84
5.28
4.34
2/4
1/2
1/1/2
1/2/2
1/2
1/2/4
1/2/4
4f
2/4
2/2/4
2/4/4
4g
32/32
2/2
32/32/32
2/4/4
32/32/32
2/4/4
4h
4i
1/2
1/2/2
n.d.
>
0
1000/>100
4j
4.99
4/4
2/4/8
2/4/8
4
k
6.3
1/2
2/2
2/4
125/125
250/250
250/250
0.5/1/2
1/2/2
0.5/1/2
2/4/4
5a
5.23
4.7
5b
4/4/4
4/4/4
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Journal of Medicinal Chemistry
Page 26 of 149
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6

1000/100
5
c
2.72
7.88
4.42
16/32
4/4
8/16/32
2/4/4
16/32/32
2/4/8
0
5d
250/250
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>
0
1000/>100
5e
4/4
2/4/8
2/8/8
5f
4.92
4.47
5.13
5.77
5.28
4.04
4.17
5.85
5.56
6.06
5.61
6.27
2.91
3.44
5.55
8.73
4/4
250/250
250/250
250/250
250/250
250/250
250/250
250/250
250/250
250/250
250/250
250/250
250/250
16/16
4/4/4
4/4/4
5g
4/4
4/8/8
8/8/8
5h
4/4
4/8/8
8/8/8
5
5
5
5
i
2/2
1/2/2
2/4/4
j
2/4
1/2/4
2/4/4
k
l
16/16
4/4
8/16/16
2/4/4
8/16/16
2/4/8
6b
16/16
16/32
32/32
32/32
32/32
0.03/0.03
0.03/0.03
1/1
16/16/16
32/32/32
32/32/32
32/32/32
32/32/32
16/16/32
32/32/32
32/32/32
32/32/32
32/32/32
6
6
6
6
e
f
g
h
7b
0.25/0.25/0.25 0.06/0.125/0.25
0.03/0.03/0.03 0.03/0.03/0.03
7c
250/250
250/250
250/250
7d
1/1/1
2/4/4
1/1/1
2/4/4
7f
2/4
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Page 27 of 149
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
7
g
5.73
4.43
2.83
3.77
3.62
2/2
16/16
125/125
16/16
8/8
2/4/4
4/4/4
7m
0.03/0.03
0.25/0.5
0.25/0.5
0.125/0.125
0.03/0.03/0.03 0.03/0.03/0.03
8d
1/2/4
2/2/2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8e
0.5/0.5/1
0.25/0.5/0.5
0.25/0.5/1
0.125/0.25/0.5
8f
8/8
0.06/0.06
>500/>500
0.125/0.25/0.5 0.06/0.125/0.12
5
8g
4.21
2.86
3.92
9a
0.5/1
500/500
250/250
2/4/4
4/8/8
0.125/0.25/0.2
9b
0.06/0.125
0.25/0.25/0.5
5
0.06/0.06/0.12
5
9c
9d
9e
6.56
4.35
5.29
0.06/0.125
0.03/0.03
250/250
0.06/0.06/0.125
62/62
62/62
0.03/0.03/0.03 0.03/0.03/0.03
0.125/0.25
0.125/0.25/0.2 0.125/0.125/0.2
5
5
0.125/0.25
62/62
62/62
0.125/0.25/0.5 0.06/0.125/0.12
5
9f
5.14
5.73
0.25/0.25
0.25/0.25/0.25 0.06/0.125/0.12
5
9g
1
0c 7.7
0d 5.49
2/2
250/250
250/250
250/250
1/2/2
n.d.
1
0.5/0.5
1/1
0.5/0.5/0.5
0.5/1/1
0.5/1/1
0.5/1/2
10e 6.43
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Journal of Medicinal Chemistry
Page 28 of 149
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0f
6.28
0.5/0.5
2/2
250/250
250/250
250/250
250/250
250/250
250/250
0.5/0.5/0.5
4/4/4
0.5/0.5/0.5
4/4/8
10g 6.87
10h 5.57
1d 4.34
11f 5.13
1g 5.72
0.03/0.03
16/16
8/16
0.03/0.03/0.03 0.03/0.03/0.03
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
>32/>32
4/8/16
>32/>32
16/32/32
8/16/16
1
8/16
4/8/16

1000/100
11h 4.42
8/16
4/8/16
8/16/16
0
24c 0.23
24g 2.9
24m 1.47
32/32
8/8
1000/1000
16/32
32/32/32
16/16/16
32/32/32
16/16/16
8/8
62/62
32/32/32
16/16/16
>
250/>250/>25
INH
RIF
0.5/1
>250/>250
32/62
2/4/4
0
0.125/0.25/0.2
5
0.25/0.25
0.125/0.25/0.25
n.d., not determined;
Table 2. In vitro antimycobacterial activities of compounds 4d, 4i, 5e, 5j, 6b, 7b, 7c, 7m,
8f, 8g, 9b-g, 10d, 10g and 10h and common anti-TB drugs against MDR/XDR strains of
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Page 29 of 149
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
M. tuberculosis. The results are expressed as MIC (μM) after 14 and 21 days of incubation
and 14 days of incubation for anti-TB drugs.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MDR/XDR M. tuberculosis strains
Praha 1
1/2
Praha 4
2/4
Praha 131 9449/2007 234/2005
7357/1998 8666/2010
4d
2/4
1/2
2/2
1/2
1/2
4
5
5
6
i
2/4
2/4
2/4
2/4
2/4
2/2
2/2
e
j
2/4
2/4
2/4
4/4
4/4
2/4
2/4
2/4
2/4
4/4
2/4
4/4
2/4
2/4
b
16/16
0.03/0.03
0.03/0.03
0.03/0.03
16/16
0.03/0.03
0.03/0.03
0.03/0.03
16/16
0.03/0.03
0.03/0.03
0.03/0.03
16/16
0.03/0.03
0.03/0.03
0.03/0.03
16/16
0.03/0.03
0.03/0.03
0.03/0.03
16/16
0.03/0.03
0.03/0.03
0.03/0.03
16/16
0.03/0.03
0.03/0.03
0.03/0.03
7b
7c
7m
0
.125/0.12
0.125/0.12 0.125/0.12 0.125/0.12 0.125/0.12 0.125/0.12
8
f
0.06/0.125
0.06/0.06
5
5
5
5
5
5
8g
0.06/0.06
0.06/0.06
0.06/0.06
0.06/0.06
0.06/0.06
0.06/0.06
0
.125/0.12 0.125/0.12 0.125/0.12 0.06/0.125 0.06/0.125 0.06/0.125 0.06/0.125
9b
5
5
5
9c
0.125/0.25 0.125/0.25 0.125/0.25 0.125/0.25 0.125/0.25 0.125/0.25 0.125/0.25
0.03/0.03 0.03/0.03 0.03/0.03 0.03/0.03 0.03/0.03 0.03/0.03 0.03/0.03
9d
9e
0.125/0.25 0.125/0.25 0.125/0.25 0.125/0.25 0.125/0.25 0.125/0.25 0.25/0.25
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