Conjugation of a 5-nitrofuran-2-oyl moiety to aminoalkylimidazoles produces non-toxic nitrofurans that are efficacious in vitro and in vivo against multidrug-resistant Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2018.08.068

Within the general nitrofuran carboxamide chemotype, chimera derivatives incorporating diversely substituted imidazoles attached via an alkylamino linker were synthesized. Antimycobacterial evaluation against drug-sensitive M. tuberculosis H37Rv strain id

Full text of DOI:10.1016/j.ejmech.2018.08.068

European Journal of Medicinal Chemistry 157 (2018) 1115e1126
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Conjugation of a 5-nitrofuran-2-oyl moiety to aminoalkylimidazoles
produces non-toxic nitrofurans that are efficacious in vitro and in vivo
against multidrug-resistant Mycobacterium tuberculosis
,
Mikhail Krasavin a *, Alexei Lukin b, Tatiana Vedekhina b, Olga Manicheva c,
Marine Dogonadze c, Tatiana Vinogradova c, Natalia Zabolotnykh c, Elizaveta Rogacheva d,
Liudmila Kraeva d, Piotr Yablonsky c
a Saint Petersburg State University, Saint Petersburg, 199034, Russian Federation
b Lomonosov Institute of Fine Chemical Technologies, MIREA ꢀ Russian Technological University, Moscow, 119571, Russian Federation
c Saint Petersburg Research Institute of Phthisiopulmonology, 2-4 Ligovsky Prospekt, Saint Petersburg, 191036, Russian Federation
d Pasteur Institute of Epidemiology and Microbiology, 14 Mira Street, Saint Petersburg, 197101, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
Within the general nitrofuran carboxamide chemotype, chimera derivatives incorporating diversely
substituted imidazoles attached via an alkylamino linker were synthesized. Antimycobacterial evaluation
against drug-sensitive M. tuberculosis H37Rv strain identified five active druglike compounds which were
further profiled against patient-derived M. tuberculosis strains in vitro. One of the compounds displayed
Received 10 July 2018
Received in revised form
22 August 2018
Accepted 25 August 2018
Available online 29 August 2018
promising potent activity (MIC 0.8 mg/mL) against one of such strains otherwise resistant to such first-
and second-line TB therapies as streptomycin, isoniazid, rifampicin, ethambutol, kanamycin, ethion-
amide, capreomycin and amikacin. The compound was shown to possess low toxicity for mice
(LD50 ¼ 900.0 83.96 mg/kg) and to be similarly efficacious to etambutol, in the mouse model of drug-
sensitive tuberculosis, and to neurotoxic cycloserine in mice infected with MDR tuberculosis.
© 2018 Elsevier Masson SAS. All rights reserved.
Keywords:
Chimera drug design
Nitrofurans
Imidazoles
Antitubercular
Mycobacterium tuberculosis
Multidrug resistant
Selective antimycobacterial activity
Druglikeness
1. Introduction
especially from co-infection with HIV [3]. The latest breakthrough
in antitubercular drug development productivity (which man-
The constant emergence of drug-resistant microorganisms [1]
mandates that new molecular entities are brought up through the
drug discovery process into preclinical and clinical development.
However, the current situation with serious non-contained in-
fections in developing countries such as tuberculosis (caused by
Mycobacterium tuberculosis or MTb) is such that the demand for
new drug candidates in the development pipeline is not met with
adequate productivity in the discovery process [2]. The multidrug-
resistant (MDR) forms of tuberculosis are particularly hard to treat
with the existing therapies, which leads to increased death rates,
ifested itself in the approvals of Janssen's bedaquiline [4] and
Otsuka's delamanid [5]) is somewhat reassuring. However, other
classes of drugs should be validated for the development, even by
revisiting known antimicrobial chemotypes and attempting to
optimize them specifically to target MTb.
Nitrofurans (along with nitroimidazoles to which delamanid
belongs) are the so-called bioreducible compounds that have
demonstrated potential to treat various infectious diseases caused
by bacterial pathogens [6]. Their mechanism of action is thought to
involve reduction, by the bacterial cell wall enzyme, of the nitro-
group to produce free radical species that can react with the bac-
terial biomolecules and are, therefore, lethal to the microorganism
[7]. The major concern associated with nitrofurans and possibly
hindering their advancement into development is their being also a
structural alert from toxicology perspective. Indeed, if it is not
* Corresponding author. Laboratory of Chemical Pharmacology, Saint Petersburg
State University, 26 Universitetskyi Prospect, Peterhof, 198504, Russian Federation.
E-mail address: m.krasavin@spbu.ru (M. Krasavin).
https://doi.org/10.1016/j.ejmech.2018.08.068
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

1116
M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
ensured that a chemotherapeutic agent in question is selectively
taken up and metabolized by the bacteria, issues of toxicity and
mutagenicity may arise [8,9]. It is perhaps unsurprising that since
the introduction of nitrofurazone (1) [10] in the 1940s as well as the
other, mostly topical, compounds nifuroxazide (2) and nitro-
furantoin (3) [11] examples of nitrofuran compounds as advanced
as preclinical phase of development have remained scarce. How-
ever, the recent success of nitroimidazoles delamanid (vide infra)
and pretomanid (which is currently in phase III clinical study [12])
led to a belief that the nitrofuran drug class, too, can deliver effi-
cacious and non-toxic antitubercular drug candidates via a careful
optimization of compounds' periphery. The interest to revisiting
this well-established class of antimicrobial agents in antitubercular
context (i. e. in the area highly prone to drug resistance issues) is
additionally fueled by the latest clinical data meta-analysis on
furantoin demonstrating that the potential of microbial resistance
to nitrofurans is low [13].
Exploration of the nitrofuran periphery for the purposes of
selectively increasing antimycobacterial activity and reducing
toxicity to the host is potentially a vast and resource-intensive area
of research. Here certain guidelines for selecting specific groups
and moieties should be considered. While predictive computa-
tional models are being developed [14], medicinal chemist often
choose a more intuitive drug design strategy whereby the phar-
macophoric nitrofuran moiety is conjugated with or embedded
into structures that also have some documented antitubercular
efficacy associated with them. For instance, nitrofuranylamides
discovered by Lee in the form of the early hit (4) [15] and optimized
into more advanced compounds such as Lee562 (5) with demon-
strated safety and efficacy in vivo [16] attest both to acceptability of
the amide linkage and the presence of basic heterocyclic moieties in
the candidate compounds' periphery. N-Aminolactams conjugated
with a nitrofuran motif via a hydrazone linkage (6) helped reveal a
preferred N-cyclohexyl carboxamide periphery [17]. Nitrofuran-
crude material obtained in these reactions was taken on to the
deprotection step. The latter was brought about by 4 N solution of
HCl in 1,4-dioxane and the desired 2- and 1-aminoalkyl substituted
imidazoles 14 and 15, respectively, were obtained as di- and tri-
hydrochloride salts (see Experimental Section) in moderate to good
yields over two steps (see Table 1). The amine hydrochlorides 14 or
15, in presence of triethylamine (required to obtain the free-base
amine for acylation) was brought in contact with acyl imidazolide
prepared in situ from 5-nitrofuran-2-carboxylic acid. The yield in
the last acylation step obtained via the use of CDI was surprisingly
low throughout the range of amines 14 and 15 employed. However,
the employment of an alternative acylation strategy via in situ
preparation of 5-nitrofur-2-oyl chloride or the use of EDC in lieu of
CDI (as was described previously for the same type of acylation
reaction by Lee and co-workers [24]) did not improve the yield of
the reaction. Overall, the target conjugates of a substituted imid-
azole moiety with pharmacophoric 5-nitrofur-2-yl moiety via an
aminoalkyl linker 9 and 10 were obtained in four chemical opera-
tions involving only three isolation or purification steps (Scheme 1).
2.2. In vitro biological activity
The antimycobacterial activity of compounds 9a-e and 10a-g
was initially evaluated against the drug-sensitive H37Rv strain of
MTb. As it is evident from the data presented in Table 1, substantial
antimycobacterial activity was observed for compounds belonging
to either series. It is obvious that, to a large degree, introducing
various cyclic motifs as rigidity elements into the structure of the
linker appeared to increase the antimycobacterial potency.
Five compounds (9a-b, 9d, 10d and 10e) that displayed the
lowest values of minimum inhibitory concentration (MIC) were
analyzed for the critical characteristics determining druglikeness
and prospects for sufficiently high oral bioavailability [25]. As it is
evident from the calculated data summarized in Table 2, the com-
pounds which displayed the highest antimycobacterial activity, are
well within the limits of druglikeness (as defined by the Lipinski's
rule-of-five [26]). The notable feature of the five active leads
identified in this work is the narrow range of calculated total polar
surface area (TPSA) these compounds fall into. This is in line with
the significance of TPSA characteristic for bacterial cell wall
permeability noted by Tan and co-workers [27].
The five compound selected based on their antimycobacterial
activity against H37Rv strain were further evaluated for the same
activity in MDR strains available in the tuberculosis patient-derived
mycobacterial strain collection of Saint Petersburg Research Insti-
tute of Phthisiopulmonology. In general, the activity of these
compounds against the MDR strains was comparable to or signifi-
cantly lower than that observed on H37Rv strain. The notable
exception was compound 9d which displayed even somewhat
higher activity (compared to drug-sensitive strain) against 2712
strain which was isolated from a patient not responding to a
number of first- and second-line drugs (Table 3).
containing isostere
(þ)-calanolide A illustrates another productive design strategy [18].
Merging broadly antimicrobial 2-aminothiazole and anti-
7 of antimycobacterial natural product
a
mycobacterial nitrofuranyl moieties in the structure of compound 8
speaks for the power of the chimera design [19].
In this work, we explored a similar approach with respect to
combining, within a single molecule (9 or 10), the pharmacophoric
5-nitrofuran-2-oyl moiety and a substituted imidazole motif linked
together by a diverse set of aminoalkyl linkers (Fig. 1). This research
was particularly motivated by the recently established significance
of non-nitrated imidazole motifs in the antitubercular compound
design [20] and the validation of imidazole fragments in targeting
MTb [21]. Herein, we describe the synthesis of compounds 9e10
(via Zn(OTf)2-catalyzed amination-cyclization of propargylamides
followed by the introduction of the 5-nitrofuran-2-oyl moiety) and
the results of their profiling, as potential antitubercular agents
in vitro and in vivo.
2. Results and discussion
It should be noted that the most active compound (9d) char-
acterized herein further (vide infra) carries a number of attractive
features (a rigid achiral azetidine linker, low lipophility) which
makes the compound a promising drug development candidate
[28]. The same compound did not display any activity (Table 4)
against three (E. faecium, K. pneumoniae and P. aeruginosa) out of six
Gram-positive and Gram-negative bacteria belonging to the so-
called ESKAPE panel of pathogens (i. e. the ones most prone to
developing resistance to drugs) [29]. The activity against the other
three ESKAPE pathogens (S. aureus, A. baumanii and E. aerogenes)
can be noted albeit it was lower than the activity of ciprofloxacin
(employed as a positive control).
2.1. Chemistry
A variety of substituted imidazoles 14 or 15 containing the
aminoalkyl side chains required for conjugation to 5-nitrofur-2-yl
moiety via an amide linkage in position 2 or 1 of the imidazole
nucleus were accessed from the respective propagylamides 12
(prepared, in turn, from carboxylic acids 11 in good to excellent
yield, vide infra) according to Zn(OTf)2-catalyzed amination-
cyclization sequence as described by Beller [22] and us [23]. The
initial imidazole cycloadducts 13 (although observed by TLC and 1H
NMR analysis of the reaction mixtures) were not isolated and the

M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
1117
Fig. 1. Known antibacterial (1e3) and antimycobacterial (4e8) nitrofurans and the general structure of compounds 9e10 investigated in this work.
2.3. In vivo toxicology evaluation
a particular component or if resistance to any of them emerges. The
recent reports on emerging resistance to Cs [34] as well as its well-
documented neurotoxicity [35] prompted us to consider Cs as a
good candidate for component replacement in the above four-drug
combination. To our delight, the use of compound 9d (given orally)
in lieu of Cs produced a new drug combination which was similarly
efficacious to the original four-component combination in allevi-
ating symptoms of experimental tuberculosis in mice infected by
multidrug-resistant “2712” strain (Fig. 3).
The toxicology data were obtained for the frontrunner com-
pound (9d) identified in this study as described in Section 4.4. Their
analysis is presented in Table 5. The toxicometry assessment along
with necropsy data and observation of the animals during the
period of up to 24 h post intoxication allowed concluding that
compound 9d belongs to the low toxicity group (class 4) according
to classification by Hodge and Sterner [30].
2.4. In vivo efficacy testing
3. Conclusion
Having established the low toxicity profile of compound 9d in
mice, we were interested to see if the same compound would
demonstrate efficacy lowering infection symptoms in mice infected
with tuberculosis. Oral administration of compound 9d (20 mg/kg)
to mice infected with drug-sensitive H37Rv strain of Mtb over 42
days significantly reduced the key in vivo indicators of severity of
tubercular infection compared to no-treatment group (see Section
4.5 for experimental details and ESI e for statistical analysis). This
effect found to be practically identical to that produced by etam-
butol 20 mg/kg p. o. (Fig. 2).
The established oral efficacy of compound 9d in alleviating
symptoms of drug-sensitive tuberculosis in mice encouraged us to
consider it for possible replacement of the drug components in the
four-component treatment scheme which includes prothionamide
(Pt), levofloxacin (Lfx), p-aminosalicylic acid (PAS) and cycloserine
(Cs) [31] and to which the “2712” strain employed earlier for in vitro
testing (vide supra) was sensitive. It should be noted that it had
been demonstrated by drug susceptibility testing [32] that each
component in this four-component [33] treatment scheme is crit-
ical for its efficacy. However, it is of paramount importance to
identify possible replacements for the components of this drug
combination should patients exhibit adverse effects in response to
By employing a chimera drug design strategy - i. e. combining
the pharmacophoric antibacterial nitrofuran moiety with struc-
turally diverse aminoalkyl imidazole fragments present in various
reported antitubercular drugs e we have identified a lead com-
pound that demonstrated high potency (MIC 1.6 mg/mL) against
drug-sensitive Mtb H37Rv strain in vitro. The same compound was
found to have comparable activity in vitro against three multidrug-
resistant strains from the pathogenic mycobacterial strain collec-
tion of Saint Petersburg Research Institute of Phthisiopulmonology.
The major safety concern associated with the development of
bioreducible nitrofuran drugs e namely, their non-specific toxicity
e has been eliminated by in vivo toxicity testing in mice where the
lead compound demonstrated low toxicity profile. Finally, the same
compound proved similarly efficacious to the clinically used
etambutol in treating tuberculosis in mice infected with drug-
sensitive H37Rv strain. It also showed similar activity to neuro-
toxic cycloserine (emerging resistance to which has been recently
reported) when employed as a part of a four-drug combination
therapy. These results demonstrate that the lead compound iden-
tified and characterized in this study represents a promising
candidate for further development.

1118
M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
Table 1
Structures, yields and in vitro antimycobacterial activity of compounds 9a-e and 10a-g against drug-sensitive strain H37Rv of MTb investigated in this work.
Entry
1
Compound
Yield of 9 (10), %a
66
12 (% yield)
14 (15) (% yield)b
MIC (
mg/mL)
12a (88)
14a (55)
6.2
2
3
64
48
12b (97)
12b (97)
14b (47)
14c (36)
6.2
25
4
5
42
21
12c (92)
14d (44)
1.6
12d (92)
14e (72)
>100
6
7
47
62
12e (54)
12f (92)
15a (46)
15b (42)
>100
50
8
9
28
48
12g (85)
15c (44)
50
12h (98)
15d (42)
6.2

M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
1119
Table 1 (continued )
Entry
10
Compound
Yield of 9 (10), %a
57
12 (% yield)
14 (15) (% yield)b
MIC (
mg/mL)
12e (54)
15e (34)
3.1
11
12
38
49
12i (81)
15f (41)
100
100
12e (54)
15g (41)
a
Isolated yield of 9 (10) from 14 (15).
Isolated yield of 14 (15) over 2 steps from 12.
b
Scheme 1. Synthesis of compounds 9a-e and 10a-g investigated in this work.
Table 2
thin-layer chromatography was carried out on Silufol UV-254 silica
gel plates using appropriate mixtures of ethyl acetate and hexane.
Compounds were visualized with short-wavelength UV light. 1H
NMR and 13C NMR spectra were recorded on Bruker MSL-300
spectrometers in DMSO‑d6 using TMS as an internal standard.
Mass spectra were recorded using Shimadzu LCMS-2020 system
with electron impact (EI) ionization. All and reagents and solvents
were obtained from commercial sources and used without
purification.
Molecular characteristics of selected compounds 9 and 10 defining their druglike-
ness and oral bioavailability.
Molecular characteristic
9a
9 b
9 d
10 d
10e
Molecular weight
cLogPa
HBD/HBA
358.40
2.28
0/8
318.33
1.32
1/8
318.33
1.43
0/8
380.40
3.24
0/8
304.31
0.83
0/8
# rotatable bonds
6
6
5
5
3
TPSA, Ǻ2a
97.10
105.89
97.10
97.10
97.10
a
Calculated using online molecular property calculator available at www.
molinspiration.com.
4.1. Synthesis
4. Experimental protocols
4.1.1. General procedure for the preparation of propargylamides
12a-i (exemplified by the synthesis of acetic acid propargylamide
12e)
All reactions were conducted in oven-dried glassware in at-
mosphere of nitrogen. Melting points were measured with a Buchi
В-520 melting point apparatus and were not corrected. Analytical
To
a solution of acetic acid (4.0 g, 67 mmol) in CH2Cl2
(100 mL) N,N-carbonyldiimidazole (CDI, 11.88 g, 73 mmol) was

1120
M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
Table 3
Antimicrobial activity of selected compounds 9 and 10 evaluated against mutant MDR clinical isolates of M. Tuberculosis.
Mutant strain
Gene mutations
MTb family
MDR profilea
MIC (mg/mL)
rpoB
katG
inhA
ahpC
9a
9 b
9 d
10 d
10e
2712b
5023c
7106b
S531L
S531L
S531L
I335V
S315T
S315T
e
T10
wt
wt
Beijin
Beijin
Beijin
SHREKCapA
SHREK
SHREKCapAZ
1.6
6.2
12.5
12.5
12.5
12.5
0.8
3.1
3.1
3.1
6.2
6.2
25
25
12.5
wt
wt
a
S e streptomycin, H e isoniazid, R e rifampicin, E e ethambutol, K e kanamycin, Et e ethionamide, Cap e capreomycin, A e amikacin, Z e pyrazinamide.
Pulmonary tuberculosis.
Bone and joint tuberculosis.
b
c
Table 4
Antimicrobial activity of the lead compound (9d) against common Gram-positive and Gram-negative pathogens evaluated by the Kirby-Bauer disk diffusion method.
Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa Enterobacter aerogenes
9d
0
15
27
0
26
25
31
0
25
19
26
cyprofloxacin 25
a
Data shown are the diameter (mm) of bacterial growth inhibition zone around the circular area of dried compound 9d solution or disc containing a standard amount of
ciprofloxacin.
Table 5
Toxicology data obtained on oral administration of compound 9d to C56BL6 mice.
Dose (mg/kg)
Dead/total animals
za
db
zd
LD50
LD16
LD84
LD100
1100
1000
900
800
700
S
6/6
5/6
3/6
1/6
0/6
e
e
e
900.0 83.96c
287.5 83.96c
1037.5 83.96c
1100.0 83.96c
5.5
4.0
2.0
0.5
100
100
100
100
550
400
200
50
1200
a
50% of the animals killed by the two subsequent doses.
Interval between two subsequent doses.
SEM ¼ sqrt (k∙s∙d/n) ¼ sqrt (0.564∙750.0∙100/6) ¼ 83.96, where s ¼ LD84 - LD16 ¼ 750.0.
b
c
added and the mixture was stirred at r. t. for 30 min. Propargyl-
amine (6.75 g, 73 mmol) was added dropwise and the stirring
continued for 18 h. The reaction mixture was successively washed
with 5% aqueous citric acid (2 ꢁ 30 mL) and 10% aqueous K2CO3
(2 ꢁ 30 mL). The organic phase was dried over anhydrous Na2SO4,
filtered, concentrated in vacuo and further dried under high vac-
uum to provide analytically pure 12e (54%) as pale yellow solid: m.
4.1.1.4. 1-tert-Butoxycarbonyl-4-piperidylacetic acid propargylamide
(12d). Pale yellow solid, m. p. 90e92ꢂС, yield 92%; 1H NMR
(300 MHz, DMSO‑d6)
d ppm 8.17 (s, 1H), 3.93e3.82 (m, 4H),
3.00e2.96 (m, 1H), 2.68 (t, J ¼ 11.9 Hz, 2H), 2.05e1.99 (m, 2H),
1.90e1.77 (m, 1H), 1.62e1.52 (m, 2H), 1.38 (s, 9H), 1.08e0.91 (m,
2H); MS m/z 281.4 (MþHþ).
p. 70e72ꢂС. 1H NMR (300 MHz, DMSO‑d6)
d ppm 8.33 (br.s, 1H),
4.1.1.5. Phenylacetic acid propargylamide (12f). Pale yellow solid, m.
p. 80e82ꢂС, yield 92%; 1H NMR (300 MHz, CDCl3)
d ppm 7.42e7.24
3.83 (dd, J ¼ 5.5, 2.5 Hz, 2H), 3.08e3.02 (m, 1H), 1.82 (s, 3H); MS m/z
98.1 (MþHþ).
(m, 5H), 5.74 (s, 1H), 4.02 (dd, J1 ¼5.3 Hz, J2 ¼ 2.5 Hz, 2H), 3.61 (s,
2H), 2.20 (t, J ¼ 2.5 Hz, 1H); MS m/z 174.2 (MþHþ).
4.1.1.1. (3-N-tert-Butoxycarbonylpyrrolidyl)acetic
argylamide (12a). Yellow oil, yield 88%; 1H NMR (300 MHz,
DMSO‑d6)
acid
prop-
4.1.1.6. Tetrahydro-2-furoic acid propargylamide (12g). Pale yellow
solid, m. p. 54e56ꢂС, yield 85%; 1H NMR (300 MHz, CDCl3)
d ppm
d
ppm 8.28 (t, J ¼ 5.1 Hz, 1H), 3.84 (dd, J ¼ 5.1, 2.2 Hz, 2H),
3.42e3.38 (m, 1H), 3.34e3.25 (m, 1H), 3.18e3.11 (m, 1H), 3.02e2.99
(m, 1H), 2.86e2.77 (m, 1H), 2.45e2.35 (m, 1H), 2.20e2.15 (m, 2H),
1.97e1.85 (m, 1H), 1.54e1.45 (m, 1H), 1.37 (s, 9H); MS m/z 267.3
(MþHþ).
6.88 (br.s, 1H), 4.38 (dd, J ¼ 8.3, 5.8 Hz, 1H), 4.09e4.05 (m, 2H),
4.01e3.86 (m, 2H), 2.36e2.23 (m, 2H), 2.14e2.02 (m,1H), 2.00e1.83
(m, 2H); MS m/z 154.2 (MþHþ).
4.1.1.7. Benzoic acid propargylamide (12h). Pale yellow solid, m. p.
103e105ꢂС, yield 98%; 1H NMR (300 MHz, DMSO‑d6)
d ppm 8.90 (s,
4.1.1.2. 3-(N-tert-Butoxycarbonylamino)propanoic
argylamide (12b). Pale yellow solid, m. p. 111e113ꢂС, yield 97%; 1H
NMR (300 MHz, DMSO‑d6)
acid
prop-
1H, NH), 7.86 (d, J ¼ 7.2 Hz, 2H, Ph 2,6-H), 7.59e7.41 (m, 3H, Ph
3,4,5-H), 4.06 (dd, J1 ¼5.5 Hz, J2 ¼ 2.4 Hz, 2H, CH2), 3.10 (s, 1H, CH);
MS m/z 160.2 (MþHþ).
d
ppm 8.22 (d, J ¼ 3.5 Hz, 1H), 6.64 (br.s,
1H), 3.83 (d, J ¼ 3.1 Hz, 2H), 3.12 (dd, J ¼ 13.3, 6.8 Hz, 2H), 3.03 (s,
1H), 2.25 (t, J ¼ 7.2 Hz, 2H), 1.37 (s, 9H); MS m/z 227.3 (MþHþ).
4.1.1.8. Nicotinic acid propargylamide (12i). Pale yellow solid, m. p.
4.1.1.3. 1-tert-Butoxycarbonylazetidine-3-carboxylic
argylamide (12c). Pale yellow solid, m. p. 107e109ꢂС, yield 92%; 1H
NMR (300 MHz, DMSO‑d6)
acid
prop-
85e87ꢂС, yield 81%; 1H NMR (300 MHz, DMSO‑d6)
d ppm d 9.15 (s,
1H, NH), 9.00 (d, J ¼ 1.7 Hz, 1H, Py 2-H), 8.71 (dd, J1 ¼4.7 Hz,
J2 ¼ 1.3 Hz, 1H, Py 6-H), 8.25e8.15 (m, 1H, Py 4-H), 7.51 (dd,
J1 ¼7.8 Hz, J2 ¼ 4.8 Hz, 1H, Py 5-H), 4.08 (dd, J1 ¼5.4 Hz, J2 ¼ 2.4 Hz,
2H, CH2), 3.16 (t, J ¼ 2.3 Hz, 1H, CH); MS m/z 161.2 (MþHþ).
d
ppm 8.47 (t, J ¼ 4.9 Hz, 1H), 3.96e3.77
(m, 6H), 3.30e3.20 (m, 1H), 3.14 (t, J ¼ 2.5 Hz, 1H), 1.37 (s, 9H); MS
m/z 239.3 (MþHþ).

M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
1121
Fig. 2. In vivo indicators of antitubercular efficacy of compound 9d in comparison with etambutol in mice infected with drug-sensitive H37Rv strain.
a
A: Lung mass index (relative units); B: Tubercular foci index (relative units); C: cfu per lung mass unit.
4.1.2. General procedure for the preparation of compounds 14a-e
and 15a-g exemplified by the synthesis of 2,5-dimethyl-1-(4-
piperidyl)-1H-imidazole dihydrochloride (15a)
3.14e3.03 (m, 1H), 2.97e2.78 (m, 2H), 2.32 (2s, 3H), 2.09e1.97 (m,
1H), 1.72e1.58 (m, 1H), 1.26e1.12 (m, 1H), 0.58e0.51 (m, 2H),
0.49e0.43 (m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 144.7, 130.7,
To a solution of 12e (2.0 g, 21 mmol) in toluene (50 mL) tert-
butyl 4-aminopiperidine-1-carboxylate (4.9 g, 6.25 mmol) and
Zn(OTf)2 (1.6 g, 4.5 mmol) were added. The resulting mixture was
heated at reflux for 8 h and then cooled down to r. t. It was poured
into 10% aqueous К2СО3 (40 mL) and extracted with ethyl acetate
(3 ꢁ 50 mL). The combined organic extracts were dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The residue
was fractionated by column chromatography on silica gel using
0 / 2.5% gradient of methanol in chloroform to give, on evapora-
tion of the volatiles, orange oil. It was dissolved in 1,4-dioxane
(10 mL) and 4 M HCl in 1,4-dioxane (3 mL, 12.0 mmol) was added.
The mixture was stirred at r. t. and the brown crystalline precipitate
was collected by filtration. Yield 46%, m. p. 194e196ꢂС (decomp.).
116.1, 48.8, 48.6, 44.3, 36.0, 29.6, 27.4, 11.2, 9.9, 4.5, 4.4; MS m/z
220.3 (MþHþ).
4.1.2.2. 2-(2-Aminoethyl)-1-cyclobutyl-5-methyl-1H-imidazole dihy-
drochloride (14b). Pale gray solid, m. p. 218e220ꢂС, yield 47%; 1H
NMR (300 MHz, DMSO‑d6)
d ppm 14.86 (br.s, 1H), 8.72 (br.s, 3H),
7.37 (s, 1H), 5.07e4.93 (m, 1H), 3.44 (t, J ¼ 7.5 Hz, 2H), 3.30e3.19 (m,
2H), 2.69e2.53 (m, 4H), 2.33 (s, 3H), 1.92e1.74 (m, 2H); 13C NMR
(75 MHz, DMSO‑d6)
d ppm 143.0, 131.2, 116.7, 51.4, 36.8, 30.1, 23.9,
15.5, 11.3; MS m/z 180.3 (MþHþ).
4.1.2.3. 2-(2-Aminoethyl)-1-cyclooctyl-5-methyl-1H-imidazole dihy-
drochloride (14c). Pale gray solid, m. p. 177e179ꢂС, yield 36%; 1H
1H NMR (300 MHz, DMSO‑d6)
d ppm 14.58 (s, 1H), 9.76 (d,
NMR (300 MHz, DMSO‑d6)
d ppm 14.75 (br.s, 1H), 8.65 (br.s, 3H),
J ¼ 9.4 Hz, 1H), 9.47 (d, J ¼ 8.1 Hz, 1H), 7.32 (s, 1H), 4.76e4.62 (m,
1H), 3.41e3.31 (m, 2H), 3.20e3.03 (m, 2H), 2.72 (s, 3H), 2.60e2.52
(m, 1H), 2.38 (s, 3H), 2.13e2.02 (m, 2H), 1.89e1.52 (m, 1H); 13C NMR
7.39 (s, 1H), 4.44 (br.s, 1H), 3.52e3.44 (m, 2H), 3.32e3.16 (m, 2H),
3.01e2.88 (m, 1H), 2.56e2.51 (m, 1H), 2.33 (s, 3H), 2.18e2.08 (m,
2H), 1.96e1.84 (m, 2H), 1.80e1.63 (m, 6H), 1.55e1.44 (m, 2H); 13C
(75 MHz, DMSO‑d6)
d ppm 144.5, 130.3, 116.0, 52.8, 43.0, 26.4, 12.6,
NMR (75 MHz, DMSO‑d6) d ppm 169.6, 142.1, 129.9, 81.3, 73.5, 36.9,
11.3; MS m/z 180.3 (MþHþ).
35.5, 33.4, 32.3, 28.2, 25.8, 25.5, 25.2, 11.7; MS m/z 236.4 (MþHþ).
4.1.2.1. 1-(Cyclopropylmethyl)-5-methyl-2-(3-pirrolidylmethyl)-1H-
4.1.2.4. 2-(3-Azetidyl)-5-methyl-1-propyl-1H-imidazole
oacetate (14d). Dark brown oil, yield 44%; 1H NMR (300 MHz,
DMSO‑d6) ppm 9.64 (br.s, 1H), 9.09 (br.s, 1H), 7.51 (s, 1H),
4.72e4.58 (m, 1H), 4.47e4.38 (m, 2H), 4.35e4.27 (m, 2H),
difluor-
imidazole dihydrochloride (14a). Orange oil, yield 55%; 1H NMR
(300 MHz, DMSO‑d6)
d
ppm 14.85 (br.s, 1H), 9.90 (br.s, 1H), 9.73
d
(br.s, 1H), 7.41 (2s, 1H), 4.18e4.01 (m, 2H), 3.34e3.20 (m, 4H),

1122
M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
Fig. 3. In vivo indicatorsa of antitubercular efficacy of the combination therapy of prothionamide (Pt), levofloxacin (Lfx), p-aminosalycilic acid (PAS) and cycloserine (Cs) in
comparison with the combination Pt þ Lfx þ PAS þ compound 9d (replacing Cs) in mice infected with multidrug-resistant “2712” strain.
a
A: Lung mass index (relative units); B: Tubercular foci index (relative units); C: cfu per lung mass unit.
4.02e3.90 (m, 2H), 2.28 (s, 3H),1.69e1.55 (m, 2H), 0.89 (t, J ¼ 7.3 Hz,
3H); 13C NMR (75 MHz, DMSO‑d6)
3.94 (dd, J ¼ 14.5, 8.2 Hz, 1H), 3.83e3.77 (m, 2H), 3.62 (dd, J ¼ 14.5,
7.0 Hz, 1H), 3.07e2.99 (m, 2H), 2.72e2.61 (m, 1H), 2.55e2.43 (m,
2H), 2.13 (s, 3H), 2.20e2.02 (m, 1H), 1.96e1.89 (m, 2H), 1.86e1.78
(m, 1H), 1.58e1.45 (m, 2H), 1.26e1.06 (m, 2H); 13C NMR (75 MHz,
d
ppm 159.3 (q, J ¼ 36.7 Hz),
143.9, 131.1, 116.7, 116.0 (q, J ¼ 290.6 Hz), 49.4, 46.1, 27.2, 22.9, 10.7,
9.0; MS m/z 180.3 (MþHþ).
CDCl3)
d ppm 147.0, 128.7, 125.0, 72.6, 68.2, 49.1, 46.0, 37.7, 30.8,
30.7, 29.2, 26.0, 10.1; MS m/z 250.4 (MþHþ).
4.1.2.5. 1-(2-Methoxyethyl)-5-methyl-2-(4-piperidylmethyl)-1H-
imidazole dihydrochloride (14e). Dark brown solid, m. p.
124e126ꢂС, yield 72%; 1H NMR (300 MHz, DMSO‑d6)
d ppm 14.93
(br.s, 1H), 9.48 (br.s, 1H), 9.25 (br.s, 1H), 7.37 (s, 1H), 4.30 (t,
J ¼ 4.9 Hz, 2H), 3.60 (t, J ¼ 4.8 Hz, 2H), 3.23 (s, 3H), 3.28e3.16 (m,
2H), 2.97 (d, J ¼ 7.4 Hz, 2H), 2.85e2.69 (m, 2H), 2.28 (s, 3H),
2.33e2.17 (m, 1H), 1.76e1.65 (m, 2H), 1.61e1.45 (m, 2H); 13C NMR
4.1.2.8. 5-Methyl-2-phenyl-1-(3-pirrolidylmethyl)-1H-imidazole
dihydrochloride (15d). Brown solid, m. p. 204e206ꢂС, yield 42%; 1H
NMR (300 MHz, DMSO‑d6) d ppm 9.85e9.57 (m, 2H), 7.83e7.77 (m,
2H), 7.74e7.61 (m, 4H), 4.46e4.29 (m, 2H), 3.18e3.10 (m, 1H),
2.95e2.88 (m, 2H), 2.71e2.59 (m, 1H), 2.49e2.44 (m, 1H), 2.43 (s,
3H), 1.84e1.72 (m, 1H), 1.31e1.17 (m, 1H); 13C NMR (75 MHz,
(75 MHz, DMSO‑d6)
d ppm 145.4, 131.1, 115.6, 70.3, 58.9, 44.9, 43.0,
31.8, 30.5, 28.1, 9.7; MS m/z 238.3 (MþHþ).
DMSO‑d6)
d ppm 144.0, 132.4, 131.9, 130.3, 129.8, 123.6, 117.3, 46.9,
46.4, 43.8, 38.1, 27.9, 10.2; MS m/z 242.3 (MþHþ).
4.1.2.6. 1-(2-Aminoethyl)-1-benzyl-5-methyl-1H-imidazole dihydro-
chloride (15b). Dark gray solid, m. p. 227e229ꢂС, yield 42%; 1H
NMR (300 MHz, DMSO‑d6)
d ppm 14.90 (br.s, 1H), 8.72 (s, 3H),
7.51e7.47 (m, 2H), 7.43 (s, 1H), 7.40e7.27 (m, 3H), 4.53 (s, 2H), 4.45
4.1.2.9. 2,5-Dimethyl-1-(3-pirrolidyl)-1H-imidazole dihydrochloride
(t, J ¼ 6.8 Hz, 2H), 3.18e3.05 (m, 2H), 2.31 (s, 3H); 13C NMR (75 MHz,
(15e). Dark gray solid, m. p. 42e44ꢂС, yield 34%; 1H NMR
DMSO‑d6)
d ppm 146.3, 134.6, 130.9, 129.4, 129.3, 128.0, 116.2, 41.7,
(300 MHz, DMSO‑d6)
d ppm 14.69 (br.s, 1H), 10.25 (br.s, 1H), 10.03
37.8, 30.2, 9.6; MS m/z 216.3 (MþHþ).
(br.s, 1H), 7.35 (s, 1H), 5.25 (p, J ¼ 9.1 Hz, 1H), 3.81e3.67 (m, 2H),
3.52e3.42 (m, 2H), 3.29e3.19 (m, 1H), 2.73 (s, 3H), 2.59e2.52 (m,
1H), 2.39 (s, 3H), 2.37e2.29 (m, 1H); 13C NMR (75 MHz, DMSO‑d6)
4.1.2.7. 5-Methyl-1-(4-piperidylmethyl)-2-(2-tetrahydrofuranyl)-1H-
imidazole (15c). Pale yellow solid, m. p. 81e83ꢂС, yield 44%; 1H
d ppm 145.4, 130.5, 116.1, 54.2, 45.3, 44.3, 28.7, 12.7, 11.2; MS m/z
NMR (300 MHz, CDCl3)
d
ppm 6.65 (s, 1H), 4.89 (t, J ¼ 6.7 Hz, 1H),
166.2 (MþHþ).

M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
1123
4.1.2.10. 5-Methyl-1-[2-(4-piperidyl)ethyl]-2-(3-pyridyl)-1H-imid-
azole trihydrochloride (15f). Brown solid, m. p. 202e204ꢂС, yield
41%; 1H NMR (300 MHz, DMSO‑d6)
48%; 1H NMR (300 MHz, DMSO‑d6)
J ¼ 3.9 Hz, 1H), 7.39 (d, J ¼ 3.8 Hz, 1H), 6.51 (s, 1H), 4.31e4.16 (m,
1H), 3.56 (q, J ¼ 6.6 Hz, 2H), 2.89 (t, J ¼ 7.3 Hz, 2H), 2.19 (s, 3H),
2.06e1.97 (m, 2H), 1.80e1.65 (m, 7H), 1.59e1.47 (m, 5H); 13C NMR
d
ppm 9.05 (br.s, 1H), 7.75 (d,
d
ppm 9.38 (d, J ¼ 9.0 Hz, 1H),
9.14 (d, J ¼ 9.6 Hz, 1H), 9.05 (d, J ¼ 1.6 Hz, 1H), 8.95 (d, J ¼ 4.2 Hz,
1H), 8.41 (d, J ¼ 8.1 Hz, 1H), 7.84 (dd, J ¼ 7.9, 5.1 Hz, 1H), 7.71 (s, 1H),
4.19e4.09 (m, 2H), 3.13e3.05 (m, 2H), 2.67 (dd, J ¼ 22.1, 10.8 Hz,
2H), 2.43 (s, 3H), 1.65e1.54 (m, J ¼ 12.8 Hz, 4H), 1.48e1.40 (m, 1H),
(75 MHz, DMSO‑d6)
d ppm 156.1, 151.4, 148.3, 143.9, 126.1, 115.7,
115.6, 113.6, 73.1, 55.5, 38.0, 35.6, 34.6, 34.0, 27.5, 25.8, 25.6, 25.1,
11.2; HRMS (ESI), m/z calcd for C19H26N4О4 [MþHþ] 375.2027,
found 375.2040; HRMS (ESI), m/z calcd for C19H26N4О4 [MþNaþ]
397.1846, found 397.1864; HRMS (ESI), m/z calcd for C19H26N4О4
[MþKþ] 413.2586, found 413.2672.
1.35e1.21 (m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 150.1, 148.0,
141.4, 140.3, 132.3, 125.7, 121.3, 117.9, 43.2, 42.9, 35.3, 30.9, 28.1, 9.8;
MS m/z 271.4 (MþHþ).
4.1.2.11. 2,5-Dimethyl-1-(3-piperidyl)-1H-imidazole dihydrochloride
4.1.3.4. 5-Methyl-2-{1-[(5-nitrofuran-2-yl)carbonyl]azetidin-3-yl}-
(15g). Brown solid, m. p. 213e215ꢂС (decomp.), yield 41%; 1H NMR
1-propyl-1H-imidazole (9d). Pale yellow solid, m. p. 112e114ꢂС,
(300 MHz, DMSO‑d6)
d
ppm 14.73 (br.s, 1H), 10.01 (br.s, 2H), 7.33 (s,
yield 42%; 1H NMR (300 MHz, DMSO‑d6)
d
ppm 7.76 (d, J ¼ 3.9 Hz,
1H), 5.01e4.88 (m, 1H), 3.48e3.36 (m, 2H), 3.30e3.22 (m, 1H),
3.12e3.02 (m, 1H), 2.72 (s, 3H), 2.37 (s, 3H), 2.29e2.09 (m, 2H),
1H), 7.35 (d, J ¼ 3.9 Hz, 1H), 6.62 (s, 1H), 4.91e4.82 (m, 1H),
4.73e4.67 (m, 1H), 4.47e4.38 (m, 1H), 4.23e4.16 (m, 1H), 4.15e4.06
(m, 1H), 3.75 (t, J ¼ 7.5 Hz, 2H), 2.15 (s, 3H), 1.62e1.47 (m, 2H), 0.85
2.00e1.92 (m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 145.0,130.5,
116.0, 51.8, 44.7, 42.5, 26.7, 22.0, 12.7, 11.2; MS m/z 180.3 (MþHþ).
(t, J ¼ 7.4 Hz, 3H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 156.5, 151.8,
147.5, 146.4, 128.0, 124.8, 117.3, 113.3, 57.5, 54.0, 44.2, 26.2, 23.9, 11.1,
9.6; HRMS (ESI), m/z calcd for C15H18N4О4 [MþHþ] 319.1401, found
319.1394.
4.1.3. General procedure for the preparation of compounds 9a-e
and 10a-g exemplified by the synthesis of 4-(2,5-dimethyl-1H-
imidazol-1-yl)-1-[(5-nitrofuran-2-yl)carbonyl]piperidine (10a)
To a solution of 5-nitro-2-furoic acid (0.2 g, 1.27 mmol) in DMF
(10 mL) CDI (0.23 g, 1.4 mmol) was added and the mixture was
stirred at r. t. for 30 min. This solution was added dropwise to a
mixture of 15a (0.35 g, 1.4 mmol) and triethylamine (0.21 mL,
1.52 mmol) in DMF (15 mL) and the stirring continued for 18 h. The
resulting mixture was poured into water and extracted with ethyl
acetate. The organic phase was successively washed with 10%
aqueous K2CO3 (2 ꢁ 10 mL), dried over anhydrous Na2SO4, filtered
and concentrated in vacuo. The residue was suspended in diethyl
ether and filtered under vacuum to obtain a beige solid: m. p.
4.1.3.5. 4-[(1-Methoxyethyl-5-methyl-1H-imidazol-1-yl)methyl]-1-
[(5-nitrofuran-2-yl)carbonyl]piperidine (9e). Orange oil, yield 21%;
1H NMR (300 MHz, DMSO‑d6)
d
ppm 7.75 (d, J ¼ 3.7 Hz, 1H), 7.22 (d,
J ¼ 3.7 Hz, 1H), 6.57 (s, 1H), 4.42e4.30 (m, 1H), 4.13e4.04 (m, 1H),
4.00 (t, J ¼ 5.1 Hz, 3H), 3.48 (t, J ¼ 5.1 Hz, 3H), 3.40e3.34 (m, 1H),
3.21 (s, 3H), 2.88e2.77 (m, 1H), 2.58 (d, J ¼ 6.8 Hz, 2H), 2.13 (s, 3H),
1.84e1.73 (m, 2H), 1.31e1.15 (m, 2H), 1.11e1.02 (m, 1H); 13C NMR
(75 MHz, DMSO‑d6)
d ppm 156.6, 151.2, 148.2, 145.7, 127.2, 123.2,
116.5, 113.0, 71.2, 58.4, 46.6, 42.7, 42.6, 34.4, 32.4, 31.3, 9.5; HRMS
(ESI), m/z calcd for C18H24N4О5 [MþHþ] 377.1819, found 377.1815.
178e180ꢂС. Yield 47%; 1H NMR (300 MHz, DMSO‑d6)
d ppm 7.76 (d,
J ¼ 3.8 Hz, 1H), 7.32 (d, J ¼ 3.8 Hz, 1H), 6.45 (s, 1H), 4.61e4.46 (m,
1H), 4.42e4.26 (m, 2H), 3.55e3.38 (m, 2H), 3.12e2.93 (m, 1H), 2.34
(s, 3H), 2.21 (s, 3H), 2.14e1.98 (m, 2H), 1.96e1.84 (m, 2H); 13C NMR
4.1.3.6. N-[2-(2-Benzyl-5-methyl-1H-imidazol-1-yl)ethyl]-5-
nitrofuran-2-carboxamide (9f). Brown solid, m. p. 94e96ꢂС, yield
62%; 1H NMR (300 MHz, DMSO‑d6)
d
ppm 9.05 (t, J ¼ 5.7 Hz, 1H),
(75 MHz, DMSO‑d6)
d
ppm 156.8, 151.2, 147.7, 143.5, 126.7, 124.8,
7.74 (d, J ¼ 3.9 Hz, 1H), 7.38 (d, J ¼ 3.9 Hz, 1H), 7.29e7.16 (m, 5H),
117.0, 112.9, 53.0, 46.1, 42.1, 31.2, 30.5, 14.9, 11.2; HRMS (ESI), m/z
6.57 (s, 1H), 4.02 (s, 2H), 3.94 (t, J ¼ 6.8 Hz, 2H), 3.43e3.35 (m, 2H),
calcd for C15H18N4О4 [MþHþ] 319.1401, found 319.1408.
2.16 (s, 3H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 156.5, 151.4, 147.3,
145.7, 138.0, 128.4, 128.3, 127.2, 126.2, 124.5, 115.8, 113.4, 41.7, 39.0,
32.6, 9.3; HRMS (ESI), m/z calcd for C18H18N4О4 [MþHþ] 355.1401,
found 355.1394.
4.1.3.1. 1-(Cyclopropylmethyl)-5-methyl-2-({1-[(5-nitrofuran-2-yl)
carbonyl]pyrrolidin-3-yl}methyl)-1H-imidazole (9a). Brown solid,
m. p. 122e124ꢂС, yield 66%; 1H NMR (300 MHz, DMSO‑d6)
d ppm
7.78e7.74 (m, 1H), 7.33 (dd, J ¼ 11.3, 3.9 Hz, 1H), 6.53 (d, J ¼ 6.7 Hz,
1H), 4.10e3.89 (m, 1H), 3.84e3.60 (m, 3H), 3.57e3.41 (m, 1H),
3.28e3.19 (m, 1H), 2.812.59 (m, 3H), 2.25e2.04 (m, 4H), 1.85e1.59
(m, 1H), 1.14e0.96 (m, 1H), 0.55e0.44 (m, 2H), 0.36e0.25 (m, 2H);
4.1.3.7. N-[2-(2-Benzyl-5-methyl-1H-imidazol-1-yl)ethyl]-5-
nitrofuran-2-carboxamide (10b). Brown solid, m. p. 94e96ꢂС, yield
62%; 1H NMR (300 MHz, DMSO‑d6)
d
ppm 9.05 (t, J ¼ 5.7 Hz, 1H),
7.74 (d, J ¼ 3.9 Hz, 1H), 7.38 (d, J ¼ 3.9 Hz, 1H), 7.29e7.16 (m, 5H),
13C NMR (75 MHz, DMSO‑d6)
d ppm 155.4, 155.3, 151.3, 148.4, 145.3,
6.57 (s, 1H), 4.02 (s, 2H), 3.94 (t, J ¼ 6.8 Hz, 2H), 3.43e3.35 (m, 2H),
145.2, 126.6, 126.5, 124.3, 117.3, 117.2, 113.1, 52.8, 52.3, 47.0, 46.4,
46.3, 37.7, 35.0, 31.6, 29.4, 29.3, 29.1, 11.7, 11.7, 9.7, 3.7; HRMS (ESI),
m/z calcd for C18H22N4О4 [MþHþ] 359.1714, found 359.1720.
2.16 (s, 3H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 156.5, 151.4, 147.3,
145.7, 138.0, 128.4, 128.3, 127.2, 126.2, 124.5, 115.8, 113.4, 41.7, 39.0,
32.6, 9.3; HRMS (ESI), m/z calcd for C18H18N4О4 [MþHþ] 355.1401,
found 355.1394.
4.1.3.2. N-[2-(1-Cyclobutyl-5-methyl-1H-imidazol-2-yl)ethyl]-5-
nitrofuran-2-carboxamide (9b). Brown solid, m. p. 156e158ꢂС, yield
4.1.3.8. 4-[(5-Methyl-2-tetrahydrofuran-2-yl)-1H-imidazol-1-yl)
methyl]-1-[(5-nitrofuran-2-yl)carbonyl]piperidine (10c). Pale brown
solid, m. p. 91e93ꢂС, yield 28%; 1H NMR (300 MHz, DMSO‑d6)
64%; 1H NMR (300 MHz, DMSO‑d6)
d
ppm 8.98 (t, J ¼ 5.7 Hz, 1H),
7.75 (d, J ¼ 3.9 Hz,1H), 7.39 (d, J ¼ 3.9 Hz,1H), 6.50 (s,1H), 4.77e4.62
(m, 1H), 3.57 (q, J ¼ 7.0 Hz, 2H), 2.91 (t, J ¼ 7.4 Hz, 2H), 2.61e2.51 (m,
2H), 2.47e2.35 (m, 2H), 2.25 (s, 3H), 1.88e1.69 (m, 2H); 13C NMR
d
ppm 7.75 (d, J ¼ 3.9 Hz, 1H), 7.23 (d, J ¼ 3.9 Hz, 1H), 6.59 (s, 1H),
4.96 (t, J ¼ 6.7 Hz, 1H), 4.47e4.31 (m, 1H), 4.19e4.07 (m, 1H),
3.97e3.82 (m, 2H), 3.82e3.67 (m, 3H), 3.19e3.07 (m, 1H),
2.86e2.69 (m, 1H), 2.57e2.44 (m, 3H), 2.17 (s, 3H), 2.11e1.94 (m,
5H), 1.93e1.83 (m, 1H), 1.66e1.56 (m, 1H), 1.55e1.46 (m, 1H),
(75 MHz, DMSO‑d6)
d ppm 156.1, 151.4, 148.3, 144.9, 127.6, 125.3,
115.7, 113.6, 49.0, 37.8, 30.0, 27.8, 14.8, 11.2; HRMS (ESI), m/z calcd
for C15H18N4О4 [MþHþ] 319.1401, found 319.1395; HRMS (ESI), m/z
calcd for C15H18N4О4 [MþNaþ] 341.1220, found 341.1222.
1.36e1.21 (m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 156.7, 151.2,
148.0, 146.7,128.6,124.1, 116.6, 113.0, 71.7, 67.5, 47.6, 46.3, 42.2, 36.6,
29.9, 28.9, 25.7, 9.7; HRMS (ESI), m/z calcd for C19H24N4О5 [MþHþ]
389.1819, found 389.1813; HRMS (ESI), m/z calcd for C19H24N4О5
4.1.3.3. N-[2-(1-Cyclooctyl-5-methyl-1H-imidazol-2-yl)ethyl]-5-
nitrofuran-2-carboxamide (9c). Beige solid, m. p. 188e190ꢂС, yield

1124
M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
[MþNaþ] 411.1639, found 411.1639.
The minimal inhibitory concentration (MIC) of the compounds
was determined using the REMA (resazurin microtitre plate assay)
[36]. A 3-week M. tuberculosis culture was transferred into a dry,
sterile tube containing 8e9 3-mm glass beads. The tube was
placed on a Vortex shaker for 30e40 s and then 5 mL Middlebrook
7H9 Broth (Becton Dickinson, catalogue No. 271310) was intro-
duced. The turbidity of bacterial suspension was adjusted to 1.0
McFarland units (corresponding to approximately 3х108 bacteria/
mL) and diluted 20-fold with Middlebrook 7H9 Broth containing
OADC enrichment (Becton Dickinson, catalogue No. 245116). The
same culture medium was used to prepare the 1:100 M. tuber-
culosis (1% population) control. The stock solutions of the com-
pounds in DMSO (10 mg/mL) were diluted with Middlebrook 7H9
4.1.3.9. 5-Methyl-1-({1-[(5-nitrofuran-2-yl)carbonyl]pyrrolidin-3-yl}
methyl)-2-phenyl-1H-imidazole (10d). Brown solid, m. p.
129e131ꢂС, yield 48%; 1H NMR (300 MHz, DMSO‑d6)
d ppm 7.75
(dd, J ¼ 5.2, 4.1 Hz, 1H), 7.59e7.50 (m, 2H), 7.46e7.34 (m, 3H), 7.19
(dd, J ¼ 23.4, 3.9 Hz, 1H), 6.79 (d, J ¼ 3.5 Hz, 1H), 4.19e4.08 (m, 2H),
3.72e3.43 (m, 3H), 3.24e2.95 (m, 1H), 2.49e2.33 (m, 1H), 2.27 (s,
3H), 1.86e1.68 (m, 1H), 1.52e1.36 (m, 1H); 13C NMR (75 MHz,
DMSO‑d6)
d ppm 203.8, 155.3, 155.2, 151.2, 148.1, 148.1, 146.7, 146.6,
131.8,131.7,128.8,128.6,128.6,128.5,128.3,128.2,126.2,126.1,117.4,
117.3, 113.1, 113.0, 50.2, 49.5, 46.2, 45.8, 44.9, 44.8, 36.9, 29.0, 26.4,
9.9, 9.8; HRMS (ESI), m/z calcd for C20H20N4О4 [MþHþ] 381.1557,
found 381.1547.
Broth (containing OADC enrichment) to a concentration of 800
mg/
mL 200 L of the solution thus obtained was introduced into the
m
4.1.3.10. 2,5-Dimethyl-1-{1-[(5-nitrofuran-2-yl)carbonyl]pyrrolidin-
2nd row of a 96-well microtitre plate. This raw was used to
perform 2-fold serial dilutions using and 8-channel pipette to
obtain final concentrations of 1.6, 3.1, 6.2, 12.5, 25, 50, 100, 200 and
3-yl}-1H-imidazole (10e). Dark orange solid, m. p. 84e86ꢂС, yield
57%; 1H NMR (300 MHz, DMSO‑d6)
d
ppm 7.80 (dd, J ¼ 7.3, 3.9 Hz,
1H), 7.44 (dd, J ¼ 5.9, 4.0 Hz, 1H), 6.53 (br.s, 1H), 5.04e4.91 (m, 1H),
4.30e4.12 (m,1H), 4.04e3.94 (m,1H), 3.93e3.81 (m,1H), 3.80e3.65
(m, 1H), 2.45e2.31 (m, 5H), 2.23 (d, J ¼ 4.6 Hz, 3H); 13C NMR
400
m
g/mL concentrations of the compound in rows 2e9 (ac-
of bacterial suspension introduced for
counting for 100
m
L
testing). Row 10 e MTb suspension control, row 11 e same culture
diluted 10-fold (the 1% control). Row 12 was used as a blank
(75 MHz, DMSO‑d6)
d
ppm 155.7, 155.7, 151.4, 147.9, 147.9, 144.2,
144.1, 127.2, 127.1, 125.0, 124.8, 117.7, 117.7, 113.06, 53.8, 51.9, 49.6,
49.0, 46.4, 45.4, 30.5, 27.3, 14.6, 14.5, 10.9, 10.8; HRMS (ESI), m/z
calcd for C14H16N4О4 [MþHþ] 305.1244, found 305.1256; HRMS
(ESI), m/z calcd for C14H16N4О4 [MþNaþ] 327.1064, found 327.1080.
control for optical density reading (200
The bacterial suspension (100 L) was introduced into each well
except rows 11 (1% population control) and 12 (blank culture
medium), to the total volume of 200 L in each well. The plates
were incubated at 35 ꢂC for 7 days. At that point, 0.01% aqueous
solution (30 L) of resazurin (Sigma, product No. R7017) was
mL of the grown medium).
m
m
4.1.3.11. 4-[(5-Methyl-2-(3-pyridyl)-1H-imidazol-1-yl)ethyl]-1-[(5-
m
nitrofuran-2-yl)carbonyl]piperidine (10f). Brown solid, m. p.
introduced in each well and the incubation continued for 18 h at
35 ꢂC. The fluorescence reading was performed using FLUOstar
Optima plate reader operating at lex ¼ 520 nm and lem ¼ 590 nm.
The bacterial viability was determined by comparing the mean
values ( SD at p ¼ 0.05) of fluorescence in the control wells (row
12, blank and row 11, 1% control) and the wells containing the
compound tested. The MIC was determined as the compound
concentration at which the fluorescence reached a plateau or was
statistically (t criterion) similar to that of 1% control.
The mutant, patient-derived multidrug-resistant strains of
M. tuberculosis were obtained from the pathogenic mycobacterial
strain collection of Saint Petersburg Research Institute of Phthi-
siopulmonology. These strains were genotyped using microchip
technology and were confirmed to contain various combinations of
mutations in genes rpoB (resistance to rifampicin), katG, inhA and
ahpC (resistance to isoniazid) as shown in Table 3.
79e81ꢂС, yield 38%; 1H NMR (300 MHz, DMSO‑d6)
d ppm 8.78 (s,
1H), 8.62 (d, J ¼ 4.2 Hz, 1H), 7.99 (d, J ¼ 7.7 Hz, 1H), 7.73 (d,
J ¼ 3.6 Hz, 1H), 7.51 (dd, J ¼ 7.5, 5.0 Hz, 1H), 7.20 (d, J ¼ 3.7 Hz, 1H),
6.82 (s, 1H), 4.33e4.19 (m, 1H), 4.08e3.94 (m, 3H), 3.17e3.02 (m,
1H), 2.81e2.66 (m, 1H), 2.27 (s, 3H), 1.63e1.46 (m, 5H), 1.16e1.02
(m, 2H); 13C NMR (75 MHz, DMSO‑d6)
d ppm 156.6, 151.2, 149.2,
148.9, 148.1, 143.5, 135.7, 129.4, 127.7, 126.6, 123.7, 116.6, 113.0, 46.4,
42.4, 41.6, 36.4, 32.9, 32.0, 31.0, 9.5; HRMS (ESI), m/z calcd for
C
21H23N5О4 [MþHþ] 410.1823, found 410.1839; HRMS (ESI), m/z
calcd for C21H23N5О4 [MþNaþ] 432.1642, found 432.2658.
4.1.3.12. 3-(2,5-Dimethyl-1H-imidazol-1-yl)-1-[(5-nitrofuran-2-yl)
carbonyl]piperidine (10g). Pale brown solid, m. p. 65e67ꢂС, yield
49%; 1H NMR (300 MHz, DMSO‑d6)
7.29 (br.s, 1H), 6.50 (s, 1H), 4.51e4.30 (m, 1H), 4.29e4.03 (m, 2H),
3.70e3.57 (m, 2H), 2.36 (s, 3H), 2.24 (s, 3H), 2.26e2.10 (m, 1H),
2.05e1.94 (m, 1H), 1.91e1.82 (m, 1H), 1.74e1.59 (m, 1H); 13C NMR
d
ppm 7.76 (d, J ¼ 5.3 Hz, 1H),
(75 MHz, DMSO‑d6)
d
ppm 157.1, 151.3, 147.7, 143.8, 127.0, 124.6,
4.3. Counter-testing against non-mycobacterial strains
117.3, 113.0, 65.0, 53.0, 52.4, 49.4, 46.3, 46.0, 42.2, 29.2, 25.6, 24.2,
15.2, 14.8, 11.2; HRMS (ESI), m/z calcd for C15H18N4О4 [MþHþ]
319.1401, found 319.1395.
Testing of susceptibility of other microorganisms (Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes) to
compound 9d as well as ciprofloxacin (positive control) was per-
formed using the conventional Kirby-Bauer disk diffusion test [37]
under the Standart Operating Procedure of The European Com-
mittee on Antimicrobial Susceptibility Testing (EUCAST) [38]. Disks
4.2. Determination of antimycobacterial activity in vitro
Mycobacterium tuberculosis H37Rv strain (originated from the
Institute of Hygiene and Epidemiology in Prague, 1976) was ob-
tained on August 7th, 2013 from the Federal Scientific Center for
Expertise of Medical Products (RF Ministry of Health Care). The
containing 5
mg of ciprofloxacin were used. Compound 9d (1 mg)
dissolved in dimethyl sulfoxide (10
mL) and diluted to a volume of
€
lyophilized strain was seeded on LowensteineJensen growth
1 mL with deionized water. To a Petri dish containing Muller-Hilton
medium. The 3-weeks culture was suspended in physiological
solution containing glycerol (15%) and transferred into cryotubes
to be kept at ꢀ80ꢂС. Three weeks in advance of the experiment,
the culture was brought to ambient temperature and re-seeded
agar inoculated with a bacterial suspension (McFarland OD ¼ 0.5)
5 mL of this solution (containing 5 mg of compound 9d) was added.
After drying of the compound solution, the Petri dish was incubated
at 37 ꢂC for 18 h. The susceptibility to a drug was assessed by
measuring the bacterial growth inhibition zone diameter around
the disc with ciprofloxacin or the compound 9d dried solution
circular spot.
€
into LowensteineJensen growth medium. Thus, the 2nd genera-
tion of the original M. tuberculosis culture was used in present
study.

M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
1125
4.4. Toxicology studies
groups. The first series of experiments (H37Rv strain) included (A)
the no-treatment group (n ¼ 17), (B) the group receiving etambutol
(n ¼ 10) and (C) the group receiving compound 9d (n ¼ 10). Etam-
butol (20 mg/kg) and compound 9d (20 mg/kg) were administered
orally as suspension in water (0.2 mL/mouse) using a metal gavage
over 42 days (excluding weekends). The second series of experi-
ments (2712 strain) included (A) the no-treatment group (n ¼ 15),
(B) the group receiving the efficacious combination of prothiona-
mide (12.5 mg/kg), levofloxacin (12.5 mg/kg), p-aminosalicylic acid
(130 mg/kg) and cycloserine (12.5 mg/kg) to which 2712 strain was
found susceptible in vitro (n ¼ 6) and (C) the group receiving the
same combination of drugs except for cycloserine which was
substituted for compound 9d (28 mg/kg, i. e. 1/10 LD16). The drugs
were administered orally as suspension in water (0.2 mL/mouse)
using a metal gavage over 3 months (excluding weekends).
The experiment was conducted on 100 sexually mature male
C57BL6 mice (initial body weight range 19.0e22.0 g). The animals
were kept according to ETS 123 (European Convention for the
Protection of Vertebrate Animals Use for Experimental and Other
Scientific Purposes, Strasbourg 18. III.1986). Animals were quaran-
tined for 14 days during which period the animals were examined
on a daily basis. The mice were kept in plastic cages (15e20 animals
per cage) with sawdust flooring. Daylight and dark phases were
alternating each 12 h, the ambient temperature was maintained
within þ23…þ25 ꢂC range with humidity in 50e70% range. The air
in the cages was actively circulated by a ventilation system and was
disinfected by a UV lamp.
The animals were divided at random in groups of 6 mice for the
treatment and one group of 7 animals to be used as a control (no
treatment) group. Randomization criteria included the absence of
sickness signs and group homogeneity with respect to mouse body
weight. A given dose of compound 9d was given orally via a metal
gavage as a suspension in Tween-80 diluted with a calculated
amount of distilled water. Animals from the control group were
receiving 0.01% aqueous solution of Tween-80. Dosage precision
was regulated by the variable volume of the drug solution admin-
istered (normalized to the animal's body weight) at a constant
concentration of the solution. Compound 9d dose was varied in the
700…1100 mg/kg range and increased by 100 mg/kg between the
treatment groups. Each dose was administered to 6 animals.
The following parameters were recorded during the experi-
ment: animal lethality, time to death, poisoning symptoms and the
observable changes in the animals' general condition recorded
daily over 14 days of treatment. Animals were euthanized by cer-
vical dislocation. Both dead and euthanized animals were analyzed
for macroscopic parameters. Surviving and euthanized animals
were analyzed for the fraction of heart, lungs, liver, spleen and left
kidney relative to the total body weight (see Table S3).
The animals were euthanized by cervical dislocation. The ther-
apeutic efficacy in the treatment groups were compared with that
in the no-treatment group by lung mass indices (in relative units),
tubercular foci indices (in relative units) and by cfu per lung mass
unit count (determined after seeding a given quantity of the lung
€
homogenate on the LowensteineJensen medium). The data were
analyzed using the Statistica 7.0 software package employing and
evaluated for significance using the Student's t-test.
Acknowledgements
This research was supported by the Russian Science Foundation
(project grant 14-50-00069). Mass spectrometry studies were
performed at the Center for Chemical Analysis and Materials
Research of the Saint Petersburg State University Research Park.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.08.068.
The median lethal dose (LD50
) was calculated using the
following formula: LD50 ¼ LD100 e S (z,d)/m, where z is half of the
total number of animals that died after the following two doses, m
e number of animals receiving each dose. Standard error of mea-
surement was calculated using the following folmula: SEM
(LD50) ¼ sqrt (k∙SD/n), where n ¼ 6 (animals per group), D ¼ 100
(dose excalation increment), S ¼ LD84 e LD16. Statistical analysis
was performed using the Student's parametric t-test.
References
[1] J.M.A. Blair, M.A. Webber, A.J. Baylay, D.O. Ogbolu, L.J.V. Piddock, Nat. Rev.
Microbiol. 13 (2005) 42e51.
[2] J.R. Corey, M.E. Stryjewski, W. Weyenberg, U. Yasothan, P. Kirkpatrick, Nat.
Rev. Drug Discov. 8 (2009) 929e930.
€
[3] D.M. Barter, S.O. Agboola, M.B. Murray, T. Barnighausen, BMC Publ. Health 12
(2012) 980.
[4] D.H. Diacon, A. Pym, M. Grobusch, et al., N. Engl. J. Med. 360 (2009)
2397e2405.
4.5. In vivo efficacy testing
[5] M. Stehr, A.A. Elamin, M. Singh, Curr. Top. Med. Chem. 14 (2014) 110e129.
[6] R.C. Goldman, Future Med. Chem. 2 (2010) 1253e1271.
[7] L. Zhou, H. Ishizaki, M. Spitzer, K.L. Taylor, N.D. Temperley, S.L. Johnson,
P. Brear, P. Gautier, Z. Zeng, A. Mitchell, V. Narayan, E.M. McNeil, D.W. Melton,
T.K. Smith, M. Tyers, N.J. Westwood, E.E. Patton, Chem. Biol. 19 (2012)
883e892.
[8] V. Purohir, A.K. Basu, Chem. Res. Toxicol. 13 (2000) 673e692.
[9] M. Hofnung, P. Quillardet, V. Michel, E. Touati, Res. Microbiol. 153 (2002)
427e434.
The efficacy testing of compound 9d against experimental
tuberculosis model was conducted using 71 inbred C57black/6
mice according to the European Union Directive 2010/63/EU on the
protection of animals used for scientific purposes adopted on 22
September 2010. The tuberculosis was modeled by inoculation of
mice with a suspension of three-week culture of Mtb (106 cfu per
mouse). In the first series of experiments (n ¼ 37), the same drug-
sensitive H37Rv strain of Mtb as was used for in vitro compound
testing (see Section 4.2) was employed. In the second series of
experiments, the mice (n ¼ 34) were inoculated with the same
patient-derived strain 2712 of Mtb (obtained from the pathogenic
mycobacterial strain collection of Saint Petersburg Research Insti-
tute of Phthisiopulmonology) with an established multidrug
resistance and mutations profile as was used for in vitro compound
testing (see Table 3).
€
[10] O. Dann, E.F. Moller, Chem. Ber. 80 (1947) 23e36.
[11] R.E. Chamberlain, J. Antimicrob. Ther. 2 (1976) 325e336.
[12] C.K. Stover, P. Warrener, D.R. VanDevanter, D.R. Sherman, T.M. Arain,
M.H. Langhorne, S.W. Anderson, J.A. Towell, Y. Yuan, D.N. McMurray,
B.N. Kreiswirth, C.E. Barry, W.R. Baker, Nature 405 (2000) 962e966.
[13] A. Huttner, E.M. Verhaegh, S. Harbarth, A.E. Muller, U. Theuretzbacher,
J.W. Mouton, J. Antimicrob. Chemother. 70 (2015) 2456e2464.
[14] S. Ekins, R. Pottorf, R.C. Reynolds, A.J. Williams, A.M. Clark, J.S. Freundlich,
J. Chem. Inf. Model. 54 (2014) 1070e1082.
[15] R.P. Tangallapally, R. Yendapally, A.J. Daniels, R.E.B. Lee, R.E. Lee, Curr. Top.
Med. Chem. 7 (2007) 509e526.
[16] J.G. Hurdle, R.B. Lee, N.R. Budha, et al., J. Antimicrob. Chemother. 62 (2008)
1037e1045.
[17] M. Krasavin, V. Parchinsky, G. Kantin, O. Manicheva, M. Dogonadze,
Macroscopic pulmonary evaluation of euthanized mice (5 ani-
mals from each group) performed 10 days post inoculation
demonstrated the presence of tubercular foci in the lungs. The
animals for each series of experiments were randomized into three
€
T. Vinogradova, B. Karge, M. Bronstrup, Bioorg. Med. Chem. 25 (2017)
1867e1874.
[18] P. Zheng, S. Somersan-Karakaya, S. Lu, J. Roberts, M. Pingle, T. Warrier,
D. Little, X. Guo, S.J. Brickner, C.F. Nathan, B. Gold, G. Liu, J. Med. Chem. 57

1126
M. Krasavin et al. / European Journal of Medicinal Chemistry 157 (2018) 1115e1126
(2014) 3755e3772.
[29] J.A. Karlowsky, D.J. Hoban, M.A. Hackel, S.H. Lob, D.F. Sahm, J. Med. Microbiol.
66 (2017) 61e69.
[30] H.C. Hodge, L.H. Sterner, Clinical Toxicology of Commercial Products. Acute
Poisoning. 4th Edition. Baltimore, 1975, 427 pp.
[31] S.D. Ahuja, D. Ashkin, M. Avendano, R. Banerjee, M. Bauer, J.N. Bayona, et al.,
PLoS Med. 9 (2012) e1001300.
[32] In-house Data of Saint Petersburg Research Institute of Phthisiopulmonology.
[33] Treatment of Tuberculosis, Guidelines. 4th Edition. Geneva, World Health
Organization, 2010 (Chapter 7): Treatment of drug-resistant tuberculosis.
[34] J. Chen, S. Zhang, P. Cui, W. Shi, W. Zhang, Y. Zhang, J. Antimicrob. Chemother.
72 (2017) 3272e3276.
[35] S.R. Snavely, G.R. Hodges, Ann. Intern. Med. 101 (1984) 92e104.
[36] A. Martin, M. Camacho, F. Portaels, C.C. Palomino, Antimicrob. Agents Che-
mother. 47 (2003) 3616e3619.
[19] K. Ran, C. Gao, H. Deng, Q. Lei, X. You, N. Wang, Y. Shi, Z. Liu, W. Wei, C. Peng,
L. Xiong, K. Xiao, L. Yu, Bioorg. Med. Chem. Lett 26 (2016) 3669e3674.
[20] Y.-L. Fan, X.-H. Jin, Z.-P. Huang, H.-F. Yu, Z.-G. Zeng, T. Gao, L.-S. Feng, Eur. J.
Med. Chem. 150 (2018) 347e365.
[21] A. Trapero, A. Pacitto, V. Singh, M. Sabbah, A.G. Coyne, V. Mizrahi, T.L. Blundell,
D.B. Ascher, C. Abell, J. Med. Chem. 61 (2018) 2806e2822.
[22] A. Pews-Davtyan, M. Beller, Chem. Commun. 47 (2011) 2152e2154.
[23] A. Safrygin, E. Krivosheyeva, D. Dar’in, M. Krasavin, Synthesis 50 (2018) (in
press), https://doi.org/10.1055/s-0036-1591599.
[24] R.P. Tangallapally, R. Yendapally, R.E. Lee, K. Hevener, V.C. Jones,
A.J.M. Lenaerts, M.R. McNeil, Y. Wang, S. Franzblau, R.E. Lee, J. Med. Chem. 47
(2004) 5276e5283.
[25] G. Vistoli, A. Pedretti, B. Testa, Drug Discov. Today 13 (2008) 285e294.
[26] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 46
(2001) 3e26.
[37] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1966)
493e496.
[27] T.D. Davis, C.J. Gerry, D.S. Tan, ACS Chem. Biol. 9 (2014) 2535e2544.
[28] G.R. Bickerton, G.V. Paolini, J. Besnard, S. Muresan, A.L. Hopkins, Nat. Chem. 4
(2012) 90e98.
[38] Standard Operating Procedure: Procedure for establishing zone diameter
breakpoints and quality control criteria for new antimicrobial agents, EUCAST
SOP 9 (2018) 1.