Antimycobacterial activity of nitrogen heterocycles derivatives: Bipyridine derivatives. Part III [13,14]

Article abstract of DOI:10.1016/j.ejmech.2013.09.061

Three classes of fused bipyridine heterocycles were designed, synthesized and evaluated for their antimycobacterial activities. The method for preparation of fused bipyridine derivatives is straight and efficient. The primary antimycobacterial screening reveals that mono-indolizine mono-salts are displaying potency superior to the second-line antitubercular drugs Cycloserine and Pyrimethamine and, equal as the first line anti-TB Ethambutol. The data from Cycle-2 screening assay (MIC, MBC, LORA, intracellular (macrophage) drug screening, and MTT cell proliferation) confirm the promising anti-TB results from Cycle-1 for mono-indolizine mono-salts. These data indicate that mono-indolizine mono-salt 6d is a potent compound against both replicating and non-replicating Mycobacterium tuberculosis, is active against both extracellular and intracellular organisms, has a bacteriostatic mechanism of action and has basically no toxicity. We see no influence concerning the anti-TB activity of the fused-pyridine substituents.

Full text of DOI:10.1016/j.ejmech.2013.09.061

European Journal of Medicinal Chemistry 74 (2014) 664e670
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antimycobacterial activity of nitrogen heterocycles derivatives:
Bipyridine derivatives. Part III [13,14]
*
Ramona Danac, Ionel I. Mangalagiu
“Al. I. Cuza” University of Iasi, Organic and Biochemistry Department, Bd. Carol 11, 700506 Iasi, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Three classes of fused bipyridine heterocycles were designed, synthesized and evaluated for their anti-
mycobacterial activities. The method for preparation of fused bipyridine derivatives is straight and
efficient. The primary antimycobacterial screening reveals that mono-indolizine mono-salts are dis-
playing potency superior to the second-line antitubercular drugs Cycloserine and Pyrimethamine and,
equal as the first line anti-TB Ethambutol. The data from Cycle-2 screening assay (MIC, MBC, LORA,
intracellular (macrophage) drug screening, and MTT cell proliferation) confirm the promising anti-TB
results from Cycle-1 for mono-indolizine mono-salts. These data indicate that mono-indolizine mono-
salt 6d is a potent compound against both replicating and non-replicating Mycobacterium tuberculosis, is
active against both extracellular and intracellular organisms, has a bacteriostatic mechanism of action
and has basically no toxicity. We see no influence concerning the anti-TB activity of the fused-pyridine
substituents.
Received 31 May 2013
Received in revised form
2 September 2013
Accepted 4 September 2013
Available online 7 November 2013
Keywords:
Antimycobacterial
Bipyridine
Bis-fused pyridine
MIC
MBC
LORA
Ó 2013 Elsevier Masson SAS. All rights reserved.
MTT
1. Introduction
the aim of shortening and improving TB treatment. The other in-
volves a search for novel structures that M. tuberculosis has never
Throughout history, tuberculosis (TB), a contagious disease
caused by Mycobacterium tuberculosis, has claimed the lives of over
one billion people and currently infects one third of the world’s
population. According to the World Health Organization (WHO), TB
remains the leading infectious disease among humans, annually
being registered about 9 (nine) million new cases and about 1.5
million people are dying from TB [1]. In spite of major advances that
have been made in the drug discovery process over the last 40
years, no new drugs have been developed specifically against M.
tuberculosis [2]. More than ever, there is an urgent need to develop
new antitubercular drugs to combat the spread of TB, to reduce the
total duration of treatment and to provide more effective treatment
against multidrug resistance (MDR), extensive drug resistance
(XDR) and latent tuberculosis infection [3]. In addition, tuberculosis
is one of the opportunistic infections in AIDS patients, when
M. tuberculosis enters into contact with highly immunocompro-
mised HIV-infected populations, the mortality rate within a few
weeks of infection approaches 100% [4]. There are currently two
main strategies for the development of new anti-TB drugs [3,5]. One
of them is based on the synthesis of analogs of existing drugs, with
been challenged before. Many of these novel structures that
M. tuberculosis has never encountered with before, are fused
nitrogenv heterocycles, especially pyridine fused systems [6,7].
Particularly, the imidazo-pyridine class emerged as the most
promising by having potency similar to isoniazid against replicating
M. tuberculosis H37Rv [7].
In our previous research work we showed that some fused pyri-
dine and diazine derivatives posses antimicrobial activity [8e12],
antimycobacterial including [13,14]. In these studies, we noted that
particular fused pyridine and diazine moieties display appreciable
antimicrobial and anti-TB activity.
As continuation of our work in the class of fused nitrogen het-
erocycles and as part of our concern in the field of biologically
active compounds, we report here the synthesis, structure and
in vitro antimycobacterial activity of some fused 4,40-bipyridine
derivatives.
2. Results and discussion
2.1. Chemistry
The strategies adopted for synthesis of fused 4,40-bipyridine
derivatives, are depicted in Schemes 1 and 2. Synthesis of the
* Corresponding author. Tel.: þ40 232 201343; fax: þ40 232 201313.
E-mail address: ionelm@uaic.ro (I.I. Mangalagiu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.061

R. Danac, I.I. Mangalagiu / European Journal of Medicinal Chemistry 74 (2014) 664e670
665
Scheme 1. Reaction pathway to obtain fused 4,40-bipyridine derivatives 5e7.
compounds 5e7, Scheme 1, has been described previously by part
of our group [9,15].
are those of carbonyl ester groups which appear around 163.1 ppm
(9a) and 163.3 ppm (9b), followed by carbon of carbonyl amide group
which appears around 162.5 ppm (9b). Carbon of nitrile group ap-
pears at 112.3 ppm (9a). The signals of carbon atoms (C1, C2, C3, C9) in
the new pyrrolo ring appear at high chemical shifts (97e138 ppm),
which is also a strong evidence for the aromatized structures. All the
remaining signals from NMR spectra are in accordance with the
proposed structures.
In this respect, 4,40-bipyridinium mono-ylides 3aed (generated
in situ from the corresponding mono-salts 2aed, in alkaline me-
dium Et3N), react with ethyl propiolate via a Huisgen [3 þ 2] dipolar
cycloaddition, leading in the first step to dihydroindolizines in-
termediates (4aed), and finally to the fully aromatized mono-
indolizines 5aed. Following the same algorithm (obtaining of
salts 6aed, then cycloaddition), bis-indolizines 7aed were ob-
tained, Scheme 1.
To establish unequivocally the structure of these fused pyr-
roloepyridine derivatives, the X-ray data analysis was performed
in the case of compound (9a), Fig. 1.
Synthesis of the compounds 9a,b, Scheme 2, is described herein
and involves a similar strategy: obtaining the corresponding mono-
The X-ray structure fully confirms the proposed structure, this
molecule being a flat coplanar fused pyrroloepyridine system (the
torsion angles related to the two fused cycle are closely to 0ꢀ or 180ꢀ)
and with the ethyl group from carboxylate moiety slightly rotated out
of plane [torsion angle C16eO2eC15eO1 (ꢁ3.5ꢀ), C16eO2eC15eC11
(176.5ꢀ)]. Full information concerning X-ray structure could be found
in the Cambridge Crystallographic Data Centre, the CCDC 941837
structure.
salts 8aed [16,17], followed by a Huisgen [3
cycloaddition.
þ
2] dipolar
The structure of compounds was assigned by elemental and
spectroscopic analysis: IR,1H NMR,13C NMR, COZY, HMQC, HMBC and
1
X-ray diffraction for compound 9a. The H NMR spectra of 9a,b are
characterized by protons of the ester (a triplet at
and a quartet at
¼ 4.34e4.35 ppm) and amide (7.04 and 7.35 ppm for
compound 9b) groups. The 1H NMR spectrum of compound 9b shows
a low field absorption (
¼ 9.83 ppm) for the proton H5 of indolizine
d
¼ 1.37e1.45 ppm
d
d
2.2. Design, biological activity and mechanism of action
ring. The same deshielding was as well observed in the case of other
indolizines having a C]O group at position 3 [15,18], and it is caused
by the proximity of nearby carbonyl group. Interestingly, the same
proton H5 appears more shielded in NMR spectrum (8.42 ppm) of
compound 9a which has a linear eC^N group as substituent at po-
sition 3. In 13C NMR spectra the most important data are furnished by
carbonyl ester groups, carbonyl amide group (compound 9b) and
nitrile group (compound 9a) and those ones furnished by carbons
from pyrrolo ring (C1, C2, C3, C9). Thus, the most deshielded carbons
Taking into consideration the promising antimycobacterial
activity of some fused pyridine derivatives [6,7], as well as our
previous results in the field of anti-TB derivatives [13,14], we have
focused on the design of novel structures that incorporate a bis-
fused pyridine unit in the molecular scaffold. In this respect, we
chose 4,40-bipyridine heterocycle as main skeleton for synthesis of
mono-indolizines, bis-indolizines and mono-indolizine mono-salts
derivatives. In equal measure we were interested to study the
Scheme 2. Reaction pathway to obtain fused 4,40-bipyridine derivatives 9.

666
R. Danac, I.I. Mangalagiu / European Journal of Medicinal Chemistry 74 (2014) 664e670
Fig. 1. The ORTEP diagram of the compound (9a) including atom numbering scheme. Acquisition parameters. Radiation source: fine-focus sealed tube. Graphite monochromator.
Detector resolution: 16.15 pixels mmꢁ1
,
u
scans. Intensity data were collected using Mo-K
a
radiation (
l
¼ 0.71073 A) with the crystal cooled in a stream of nitrogen from a
ꢀ
ꢀ
ꢀ
Cryostream collar (200.00 K during data collection). Crystal and experimental data. Crystal system: monoclinic; space group Cc with a ¼ 12.6169(11) A, b ¼ 17.3233(11) A,
ꢀ
c ¼ 6.8645(6) A,
a
¼ 90.00ꢀ,
b
¼ 109.003(9)ꢀ,
g
¼ 90.00ꢀ, V ¼ 1418.57(19). Goodness of fit for all reflections 1.030.
influence of the fused-pyridine substituents concerning the anti-
mycobacterial activity, Scheme 3.
A selection of compounds, one representative per each series,
was evaluated for in vitro antimycobacterial activity against M.
tuberculosis, as a part of the TAACF TB screening program under di-
rection of the US National Institute of Health, the NIAID division.
Ethambutol, Cycloserine and Amikacin were used as reference
compounds in these assays. MIC was defined as the lowest con-
centration effecting a reduction in fluorescence of 90%, relative to
control. This value was determined from the doseeresponse curve
as the IC90 using a curve-fitting program. IC90 is defined as the
concentration effecting a reduction in fluorescence of 90% relative to
A
standard primary in vitro screen was conducted against
controls. Compounds with IC90 values ,10 mg/mL were considered
M. tuberculosis H37Rv (ATCC 27294), in BACTEC 12B medium using
the Microplate Alamar Blue Assay (MABA) [16,17]. Compounds that
are active in this assay are reconfirmed using a BACTEC 460 Radio-
metric System. Compounds demonstrating a percent inhibition of
bacterial growth ,90% in the primary screenwere tested in the next
stage of the procedure, against M. tuberculosis H37RV to determine
the Minimum Inhibitory Concentration (MIC) in the MABA.
Compounds were tested in 10 twofold dilutions, typically from
active antitubercular agents. The results are summarized in Table 1.
As shown in Table 1, compounds 5a, 7d, and 9b did not exhibit
antimycobacterial activity (having MIC >100 mg/mL when tested
in MABA), while compound 9a showed weak inhibitory activity
against M. tuberculosis (IC50 ¼ 33.35, IC90 ¼ 59.52). Notably, 6d
was identified as a potent inhibitor against M. tuberculosis, with a
MIC value of 12.50
mycobacterial activity was superior to the second-line antitu-
bercular drugs Cycloserine (MIC ¼ 25 g/mL) and Pyrimethamine
mg/mL in the MABA assay and its anti-
100
mg/mL to 0.195
mg/mL. Rifampicin, Pyrimethamine, Isoniazid,
m
Scheme 3. Design in the class of fused 4,40-bipyridine derivatives.

R. Danac, I.I. Mangalagiu / European Journal of Medicinal Chemistry 74 (2014) 664e670
667
Table 1
Antimycobacterial activity against M. tuberculosis H37Rv in the MABA assay.
MIC and LORA, respectively 75, 7.5 and 0.75
and MTT.
mg/mL for macrophage
MIC is reported as the lowest concentration (mg/mL) of drug that
Compd
Antimycobacterial activity %
inhibitiona
g/mL)
IC50
g/mL)
IC90
g/mL)
MIC
M)
(
m
(
m
(
m
(m
visually inhibited growth of the organism. In addition, the per-
centage of inhibition at MIC is provided. These data are briefly
presented in Table 2.
6d
100, 50, 25, 12.5(98.50),
6.25 (65.10), 3.13 (38.60),
0.79 (9.90)
100(97.20), 50(84.90), 25(21.50), 33.35
12.5 (15.30), 0.79 (8.60)
3.88
13.07
12.50
The MIC was 2.34 mg/mL and the percentage of inhibition at MIC
9a
59.52
>100
>100
>100
13.49
>100
>100
>100
>100
25.00
concentration was 51%, suggesting that compound 6d is potent
against replicating M. tuberculosis H37Rv (SRI 1345). The results ob-
tained against drug resistant strains exhibited similar values of MIC.
Minimal Bactericidal Concentration (MBC). The MBC is deter-
mined subsequent to MIC testing by sub-culturing diluted aliquots
from wells that fail to exhibit macroscopic growth. The MBC is
5a
100(-8.60), 6.5 (0.20),
3.13(3.90), 1.56 (3.0)
>100
7d
100(-4.50), 50(5.30), 12.5(0.80), >100
1.56 (ꢁ2.0)
9b
100(-17.0), 50(2.0), 12.5(4.0),
1.56 (2.80)
>100
defined as the lowest concentration (mg/mL) of compound exhib-
Cycloserine
100(97.70), 50(96.30), 25(93.90), 12.47
12.5(51)
iting 99.9% kill over the same time period used to determine MIC
(18e24 h). MBC values 16 times higher than MIC, typically indicate
antimicrobial tolerance. The established rejection value of ꢂ40
colonies for the MBC assay was based on the calculated concen-
tration of M. tuberculosis in the MIC plates. The results, reported as
Pyrimethamine 100(95.60), 50(69.70), 25(26.70) 37.35
Amikacin
74.96
0.08
100.00
0.16
5, 2.5, 1.25(98), 0.63(97.30),
0.31, 0.16(96), 0.08(79.80)
5, 2.5, 1.25(94), 0.63(93.90),
0.31(92.90)
100(90.00), 50, 25, 12.5(89),
6.25 (86.60),
3.13 (84.30), 1.56 (69.10)
5, 2.5, 1.25, 0.63, 0.31,
0.16(99.50),
0.08(97.60), 0.04(91.90),
0.02(61.20)
0.07
Isoniazid
0.18
0.29
0.31
Ethambutol
<1.56
32.79
12.50
m
g/mL concentration, are determined based on Colony Forming
Units (CFUs) enumerated from agar plates. Only agar plates with
countable colonies have reportable counts. If a compound lacks
bactericidal activity, many times the CFUs are too numerous to
count (TNTC) and are thus reported accordingly. As far for com-
pound 6d, the MBC value (Table 2) are above the established
rejection value of ꢂ40 colonies and the CFUs are too numerous to
count. These results suggest an antimicrobial tolerance.
Rifampicin
0.02
0.02
0.04
a
Antimycobacterial activity was typically determined at concentrations 100, 50,
g/mL.
25, 12.5, 6.25, 3.13, 1.56, 0.79, 0.4 and 0.2
m
Low-Oxygen Recovery Assay (LORA). Traditional screening of
drugs against M. tuberculosis only addresses or targets the organism
in an active replicating state. It is well known that M. tuberculosis
can reside in a state of non-replicating persistence (NRP) which has
not been adequately assessed in the development of new antimi-
crobials. The LORA assay tests drugs for activity against
M. tuberculosis in a state of NRP. LORA test was conducted in
duplicate and the following controls were included in each test
plate: i) medium only (sterility control); ii) organism in medium
(negative control); and iii) rifampin or isoniazid (positive control).
Results for the LORA assay are reported as the lowest concentration
(mg/mL) of drug that visually inhibited growth of the organism. The
data are summarized in Table 3.
The LORA assay test for compound 6d displays a value of 1.17 mg/
mL, suggesting that compound 6d was potent against non-
replicating M. tuberculosis.
(MIC ¼ 100
mg/mL), Table 1. Moreover, 6d have a MIC value equal
to the first line anti-TB Ethambutol.
The data from Table 1 illustrate that mono-indolizine mono-salt
derivative 6, displays a better antimycobacterial activity compara-
tive with bis-indolizine 7, suggesting a possible influence of
bromine anion concerning antimycobacterial activity.
Compound 6d found promising in primary TB assay, was then
subjected to the secondary assay for evaluation of antimycobacterial
activity. These assays included MIC, Minimal Bactericidal Concen-
tration (MBC), Low-Oxygen Recovery Assay (LORA), intracellular
(macrophage) drug screening, and MTT cell proliferation.
Minimum Inhibitory Concentration (MIC). The MIC screening
was conducted for M. tuberculosis H37Rv (SRI 1345), isoniazid (INH)-
resistant M. tuberculosis (SRI 1369), rifampin (RMP)-resistant
M. tuberculosis (SRI 1367), and ofloxacin (OFX)-resistant
M. tuberculosis (SRI4000). MBC, LORAand intracellulardrugscreening
assays were conducted using only M. tuberculosis H37Rv (SRI 1345).
The MIC for each compound was determined by testing ten, two-fold
The Intracellular (macrophage) Drug Screening, and MTT cell
proliferation (MTT). This assay evaluates intracellular drug effec-
tiveness. Briefly, the purpose of this assay is to assess the inhibition
of M. tuberculosis H37Rv phagocytised by J774.A1 macrophages
when exposed to a battery of test compounds. J774.A1 is an
dilutions in the following concentration ranges: 75 to 0.146 mg/mL for
Table 2
MIC and MBC results.
Compd
MIC H37Rv
g/mL)
Inha, %
MBC H37Rv
g/mL)
MIC INH-Rb
g/mL)
Inh, %
MIC RMP-Rc
g/mL)
Inh, %
MIC OXF-Rd
g/mL)
Inh, %
(
m
(
m
(
m
(
m
(m
6d
Rifampin
2.34
0.049
51
63
NA
1.56
4.69
0.02
60
56
1.17
NAe
65
NA
1.17
0.05
61
80
(pos control)
Isoniazid
(pos control)
NA
NA
NA
NA
NA
0.02
92
NA
NA
Inha e Percent inhibition at MIC concentration.
INH-Rb e Isoniazid resistance.
RMP-Rc e Rifampin resistance.
OFX-Rd e Oxofloxacin resistance.
NAe e Not applicable: compound not used in assay.
NA e Not applicable: colony counts above the established rejection value of ꢂ40.
Inh e inhibition.

668
R. Danac, I.I. Mangalagiu / European Journal of Medicinal Chemistry 74 (2014) 664e670
Table 3
LORA, Macrophage and MTT results.
Compd
LORA
g/mL)
Macrophage log reduction
(low conc.)
Macrophage log reduction
(mid conc.)
Macrophage log reduction
(high conc.)
MTT % viability
(low conc.)
MTT % viability
(mid conc.)
MTT % viability
(high conc.)
(
m
6d
Rifampin
1.17
0.13
1.19
0.98
2.65
0.81
3.11
1.49
86
94
71
93
<10
85
adherent macrophage cell line originating in mice routinely used
for the determination of inhibition of intracellular pathogens. The
macrophages are subsequently lysed and plated onto Middlebrook
7H10 agar plates for bacterial enumeration. The number of myco-
bacteria surviving treatment is compared to the number of myco-
bacteria not receiving treatment in order to determine the
intracellular activity of the test compounds.
Each compound was tested in duplicate, using 3 concentrations,
and rifampin was used as the positive control drug. The three
concentrations chosen were based on the MIC data generated in
HTS primary screen. The mid concentration bracketed the reported
MIC with the lower concentration ten-fold below the mid and the
higher concentration ten-fold above the mid. Intracellular drug
activity is reported as log reduction value calculated as reduction in
M. tuberculosis concentration from zero hour to 7 days post-
infection. The data are presented in Table 3. A drug cytotoxicity
control plate assay (MTT proliferation) was also conducted in par-
allel using uninfected macrophages to confirm that concentrations
utilized for testing were not toxic to the macrophages. Drug cyto-
toxicity is reported as cell proliferation, or percentage of viability.
These data are summarized in Table 3.
As we may notice from Table 3, the log reduction value for
compound 6d, is almost equal to control drug rifampin at low
concentration and almost double at mid and high concentrations.
These results suggest that 6d is a potent compound against intra-
cellular M. tuberculosis.
We may also notice from Table 3 that the MTT viability for
compound 6d (at low and intermediate concentrations) is close to
control drug rifampin, suggesting that compound 6d has basically
no toxicity at these concentrations. However, taking into consid-
eration that at high concentrations the MTT viability for compound
6d is lower than 10%, we may only to presume that the activity of
compound 6d is not due to cytotoxicity of compound. Further
studies remain to be done in this respect.
illustrates that compound 6d has basically no toxicity at low and
intermediate concentrations, suggesting that the activity is not due
to cytotoxicity of compound. The mono-indolizine mono-salts
derivatives have shown
a better antimycobacterial activity
comparative with bis-indolizines, suggesting a possible influence of
bromine anion concerning antimycobacterial activity. We see no
influence of the substituents on the fused-pyridine rings concerning
the antimycobacterial activity.
4. Experimental protocols
4.1. Chemistry
All the reagents and solvents employed were used without
further purification. Melting points were determined using an
electrothermal apparatus (MELTEMP II) and were uncorrected. The
NMR spectra were recorded on a Bruker Avance 400 DRX spec-
trometer operating at 400 MHz. The following abbreviations were
used to designate chemical shift multiplicities:
s
¼
singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet. The IR spectra
were recorded on a FTIR Shimadzu Prestige 8400s spectropho-
tometer. The microanalyses were in satisfactory agreement with
the calculated values: C, ꢃ0.15; H, ꢃ0.10; N, ꢃ0.30. X-ray analysis
was recorded with an Agilent Xcalibur Eos diffractometer equipped
ꢀ
with a Mo (K
a
radiation,
l
¼ 0.71073 A) fine-focus sealed X-ray tube
and a graphite monochromator. The following crystal structure has
been deposited at the Cambridge Crystallographic Data Centre and
allocated the deposition number: (9a), CCDC 941837. Copies of the
data can be obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK, (fax: þ44 (0)1223 336033 or
email:deposit@ccdc.cam.ac.uk).
4.1.1. General procedure for preparation of compounds (9)
The cycloimmonium salt (8a, b) (1 mmol, 1 equiv., 0.28 g 8a,
0.34 g 8b), and ethyl propiolate (1.1 mmol, 1.1 equiv., 0.11 g) were
added to 10 mL of anhydrous acetone and the obtained suspension
was stirred at room temperature. Then, a solution of triethylamine
(TEA) (3 mmol, 3 equiv., 0.30 g) in anhydrous acetone (3 mL) was
added drop-wise over 2 h (magnetic stirring) and the resulting
mixture was then stirred over the night at rt. Water (10 mL) is added
and after stirring for another 15 min, the solid was collected by
filtration to give a powder which was washed with 10 mL methanol.
The product was crystallized from ethanolechloroform (1:1, v/v).
3. Conclusions
In conclusion, we describe herein the design, synthesis, and
evaluation of antimycobacterial activities of some fused 4,40-bipyr-
idine heterocycles. Three classes of bipyridine heterocycles (namely,
mono-indolizines, bis-indolizines and mono-indolizinemono-salts)
were synthesized and their structure was assigned byelemental and
spectral analysis, X-ray analysis including. The primary cycle high
throughput screening reveals that mono-indolizines and bis-
indolizines did not exhibit any significant anti-TB activity, while
the mono-indolizine mono-salts are displaying potency superior to
the second-line antitubercular drugs Cycloserine and Pyrimeth-
amine and, equal to the first line anti-TB Ethambutol. The data from
Cycle-2 screening assay (MIC, MBC, LORA, intracellular (macro-
phage) drug screening, and MTT cell proliferation) confirm the
promising antimycobacterial results from Cycle-1 for mono-
indolizine mono-salts. The MIC and LORA assay illustrate that
mono-indolizine mono-salt 6d is a potent compound against both
replicating and non-replicating M. tuberculosis while the MBC assay
indicates a bacteriostatic mechanism of action. The intracellular
(macrophage) drug screening demonstrates that compound 6d is an
effective drug against intracellular M. tuberculosis. The MTT assay
4.1.1.1. Ethyl 3-cyano-7-(pyridine-4-yl)indolizine-1-carboxylate (9a).
Yellow crystals, mp 158e160 ꢀC; Anal. C17H13N3O2: C, H, N; IR (KBr):
n
/cmꢁ1: 2209 (C^N), 1687 (C]Oester), 1206, 1068 (CeO); 1H NMR
(400 MHz, CDCl3): dppm: 1.45 (t, J ¼ 7.2 Hz, 3H, CH3), 4.35 (q,
J ¼ 7.2 Hz, 2H, CH2), 7.31 (dd, J6,5 ¼ 7.2 Hz, J6,8 ¼ 1.6 Hz, 1H, H6), 7.60
0
0
0
0
(d, J3 ,2 ¼ 5.6 Hz, 2H, H3 , H5 ), 7.82 (s, 1H, H2), 8.42 (d, J5,6 ¼ 7.2 Hz,
1H, H5), 8.66 (s, 1H, H8), 8.72 (d, J2 ,3 ¼ 5.6 Hz, 2H, H2 , H6 ). 13C NMR
0
0
0
0
(100 MHz, CDCl3): dppm: 14.5 (CH3), 60.5 (CH2), 97.0 (C3), 107.3 (C1),
0
0
112.3 (CN), 113.6 (C6), 118.1 (C8), 121.1 (C3 ,C5 ), 125.7 (C2), 126.1 (C5),
0
0
0
135.7 (C7), 137.6 (C9), 144.8 (C4 ), 150.7 (C2 ,C6 ), 163.1 (COO).
4.1.1.2. Ethyl 3-carbamoyl-7-(pyridine-4-yl)indolizine-1-carboxylate
(9b). Yellow crystals, mp 275e277 ꢀC; Anal. C17H15N3O3: C, H, N; IR

R. Danac, I.I. Mangalagiu / European Journal of Medicinal Chemistry 74 (2014) 664e670
669
(KBr):
n
/cmꢁ1: 3401 (NHamide),1686 (C]Oester),1605(C]Oamide, NH)),
starting concentration and serially diluted two-fold across the
plate. The plates were then inoculated (0.1 mL/well) with a target
concentration of 1.0 ꢄ 106 CFU/mL M. tuberculosis and incubated at
37 ꢀC for 7 days in approximately 90% humidity. Following incu-
bation, the plates were read visually and individual wells scored for
turbidity, partial clearing or complete clearing. Testing was con-
ducted in duplicate and the following controls were included in
each test plate: i) medium only (sterility control); ii) organism in
medium (negative control); and iii) rifampin or isoniazid (positive
1396 (CeN);1HNMR(400 MHz,DMSO-d6): dppm:1.37(t,J¼ 7.2Hz,3H,
CH3), 4.34 (q, J ¼ 7.2 Hz, 2H, CH2), 7.04 (s, 1H, NHb), 7.35 (s, 1H, NHa),
0
0
0
7.53(dd, J6,5 ¼7.6 Hz, J6,8 ¼ 1.6Hz,1H, H6), 7.80 (d, J3 ,2 ¼ 5.6 Hz, 2H, H3 ,
0
0
0
0
0
H5 ), 8.14 (s,1H, H2), 8.56 (s,1H, H8), 8.72 (d, J2 ,3 ¼ 5.6 Hz, 2H, H2 , H6 ),
9.83 (d, J5,6 ¼ 7.6 Hz,1H, H5).13C NMR (100 MHz, DMSO-d6): dppm: 14.4
(CH3), 59.5 (CH2),104.7 (C1),112.2 (C6),115.8 (C8),117.9 (C3),120.6 (C2),
0
120.8 (C3’,C5’), 128.3 (C5), 133.2 (C7), 136.9 (C9), 144.4 (C4 ), 150.5
0
0
(C2 ,C6 ), 162.5 (COONH2), 163.3 (COO).
control). The MIC is reported as the lowest concentration (
drug that visually inhibits growth of the organism.
mg/mL) of
4.2. Microbiology
Compounds were evaluated for antimycobacterial activity
against M. tuberculosis, as a part of the TAACF TB screening program
under direction of the US National Institute of Health, the NIAID
division. Antimycobacterial activities of the compounds were per-
formed by Center of Tuberculosis Antimicrobial Acquisition and
Coordinating Facility (TAACF) at Southern Research Institute.
4.2.2.2. Minimal Bactericidal Concentration (MBC). MBC is deter-
mined subsequent to MIC testing by sub-culturing diluted aliquots
from wells that fail to exhibit macroscopic growth. The sample al-
iquots were inoculated onto Middlebrook 7H10 agar plates and
subsequently incubated for 16e21 days at 37 ꢀC. Once growth was
readily apparent, the bacterial colonies were enumerated. MBC is
defined as the lowest concentration (mg/mL) of compound exhib-
4.2.1. The primary cycle high throughput screening (HTS) e Cycle 1
determination of 90% inhibitory concentration (IC90), 50% inhibitory
concentration (IC50) and Minimum Inhibitory Concentration (MIC)
Compounds were tested for in vitro antitubercular activity
against M. tuberculosis H37Rv (ATCC 27294), in BACTEC 12B
medium using a broth microdilution assay, the Microplate Alamar
Blue Assay (MABA) [19,20]. The M. tuberculosis HTS assay used
black, clear-bottom, 384-well microtiter plates and 7H12 broth.
Compounds stocks of 10 mM in 100% DMSO were diluted in media,
adjusted with DMSO to maintain the final 1% DMSO concentration
throughout, to create ten serial-dilution compound source plates at
100ꢄ the targeted test concentration, ranging from 10 mM to
0.0195 mM. The compounds were diluted in assay media to 2ꢄ final
iting 99.9% kill over the same time period used to determine MIC
(18e24 h). MBC values 16 times higher than MIC typically indicate
antimicrobial tolerance.
4.2.2.3. Low-Oxygen Recovery Assay (LORA). Traditional screening
of drugs against M. tuberculosis only addresses or targets the
organism in an active replicating state. It is well known that
M. tuberculosis can reside in a state of non-replicating persistence
(NRP) which has not been adequately assessed in the development
of new antimicrobials. Briefly, microplates were prepared in the
same manner as MIC testing format. Instead of incubating aerobi-
cally, the plates are placed under anaerobic conditions using a
MACS MIC automated jar gassing system and incubated for 7 days
at 37 ꢀC. The plates were subsequently transferred to an ambient
gaseous condition (5% CO2) for 7 days after which the plates are
read visually and individual wells scored for turbidity, partial
clearing or complete clearing. Testing was conducted in duplicate
and the following controls were included in each test plate: i)
medium only (sterility control); ii) organism in medium (negative
control); and iii) rifampin or isoniazid (positive control). Results are
test concentration and 25
mL of these diluted compounds were
transferred to 384-well plates. Amikacin was included in the pos-
itive control wells in every assay plate at two concentrations, 0.17
and 3.20 mM. The low concentration was the approximate MIC and
it is an indicator of proper assay performance of each plate. The
high concentration completely inhibits growth and was used in lieu
of uninoculated medium (background) to calculate percent inhi-
bition by the test compounds for each plate. Positive and negative
control wells were included in each plate. Plates were placed in
stacks of two and incubated for 7 days at 37 ꢀC with approximately
95% humidity. After 7 days of incubation, autofluorescence of any
test compounds was determined by pre-reading the high dose plate
by a bottom read for fluorescence using a Perkin Elmer Envision
plate reader at 535 nm excitation and 590 nm emission. The IC90 is
defined as the concentration effecting a reduction in fluorescence
of 90% relative to controls. Any compound with IC90 value of
reported as the lowest concentration (mg/mL) of drug that visually
inhibits growth of the organism.
4.2.2.4. Intracellular (macrophage) drug screening, and MTT cell
proliferation. Briefly, the murine J774 cell line was propagated in
RPMI 1640 supplemented with L-glutamine and fetal bovine serum
(FBS). Cells were maintained in tissue culture flasks at 37 ꢀC in the
presence of 5% CO2. For infection studies, J774 cells were transferred
to 12-well tissue culture chambers in 1 mL volumes at a density of
2.0 ꢄ 105 in the presence of 10% FBS. After overnight incubation, the
medium was replaced with fresh medium containing 1% FBS to stop
macrophage division while maintaining cell viability. Twenty-four
hours later, the macrophage monolayer was enumerated with an
ocular micrometerfortotalnumberofcells perwell todetermine the
infection ratio. The medium was removed and replaced with 1 mL of
fresh medium with 1% FBS containing M. tuberculosis at a multi-
plicity of infection (MOI) of 5 Mycobacteria/macrophage. The cells
are infected for 4 h and then nonphagocytosed Mycobacteria were
washed from the monolayers and fresh medium was added. Drugs
were then added, using 3 concentrations, and infection allowed to
proceed for 7 days. At0 and 7 days, the macrophages werelysed with
sodium dodecyl sulfate, treated with DNAase, diluted and plated
onto7H10 agar todetermine thecell numberorcolonyforming units
(CFU). Each drug concentration was tested in duplicate and rifampin
was used as the positive control drug (at 3 concentrations: 0.01, 0.1
ꢅ10
mg/mL is considered ‘active’ for antitubercular activity. The
Minimum Inhibitory Concentration (MIC) is defined as the lowest
concentration effecting a reduction in fluorescence of 90% relative
to controls.
4.2.2. In vitro assessments for antimicrobial activity-preclinical
anti-tubercular testing e Cycle 2
4.2.2.1. Minimal inhibitory concentration (MIC). The broth micro-
dilution assay format following guidelines established by the
Clinical and Laboratory Standards Institute (CLSI) is routinely utilized
for MIC testing. Briefly, testing was conducted using 96-well,
U-bottom microplates with an assay volume of 0.2 mL/well. First,
the test media, Middlebrook 7H9 broth supplemented with OADC
Enrichment (BD BioSciences; Sparks, MD), was added (0.1 mL/well)
to each well. The test compounds, solubilized in appropriate sol-
vent and subsequently diluted in test media, were subsequently
added (0.1 mL/well) to appropriate wells at twice the intended
and 1.0
mg/mL). A drug cytotoxicity control plate assay (MTT

670
R. Danac, I.I. Mangalagiu / European Journal of Medicinal Chemistry 74 (2014) 664e670
[2] L.V. Sacks, R.E. Behrman, Tuberculosis 88 (2008) 93e100.
[3] Z. Ma, C. Lienhardt, H. Mclleron, A.J. Nunn, X. Wang, Lancet 5 (2010) 1e10.
[4] W.J. Burman, B.E. Jones, Am. J. Respir. Crit. Care Med. 164 (2001) 7e12.
[5] C. Crabb, Bull. World Health Organ. 80 (2002) 518e519.
[6] B. Dulla, B. Wan, S.G. Franzblau, R. Kapavarapu, O. Reiser, J. Iqbal, M. Pal,
Bioorg. Med. Chem. Lett. 22 (2012) 4629e4635.
[7] G.C. Moraski, L.D. Markley, M. Chang, S. Cho, S.G. Franzblau, C.H. Hwang,
H. Boshoff, M.J. Miller, Bioorg. Med. Chem. 20 (2012) 2214e2220.
[8] R. Gradinaru, A. Luca, I. Cretescu, R. Danac, Rev. Chim. (Bucharest) 61 (2010)
903e906.
proliferation) was also conducted in parallel using uninfected
macrophages to confirm that concentrations utilized for testing
were not toxic to the macrophages.
Bacteria. MIC screening was conducted for M. tuberculosis H37Rv
(SRI 1345), isoniazid (INH)-resistant M. tuberculosis (SRI 1369),
rifampin (RMP)-resistant M. tuberculosis (SRI 1367), and ofloxacin
(OFX)-resistant M. tuberculosis (SRI 4000). MBC, LORA and intra-
cellular drug screening assays were conducted using only
M. tuberculosis H37Rv (SRI 1345).
[9] A. Rotaru, R. Danac, I. Druta, G. Drochioiu, I. Cretescu, Rev. Chim. (Bucharest)
56 (2005) 179e183.
[10] A. Rotaru, M. Ungureanu, R. Danac, A. Poeata, I. Druta, Ann. Pharm. Fr. 62
(2004) 428e430.
[11] R. Butnariu, I.I. Mangalagiu, Bioorg. Med. Chem. 17 (2009) 2823e2829.
[12] M. Caprosu, R. Butnariu, I.I. Mangalagiu, Heterocycles 65 (2005) 1871e
1879.
[13] D. Mantu, C. Luca, C. Moldoveanu, G. Zbancioc, I.I. Mangalagiu, Eur. J. Med.
Chem. 45 (2010) 5164e5168.
[14] C. Moldoveanu, G. Mangalagiu, G. Drochioiu, M. Caprosu, M. Petrovanu,
I.I. Mangalagiu, Stiint. Univ. “Al.I.Cuza” Iasi 11 (2003) 367e374 (Chem. Abstr.
142 (2004) 56249).
Acknowledgments
Authors are also thankful to CNCSIS Bucuresti, Romania, grant
PN-II-DE-PCE-2011-3-0038, no. 268/05.10.2011, for financial sup-
port. Part of this work (biological tests) was supported by National
Institutes of Health and the National Institute of Allergy and In-
fectious Diseases, Contract No. HHSN272201100012I.
[15] A. Rotaru, I. Druta, E. Avram, R. Danac, Arkivoc 13 (2009) 287e299.
[16] R. Kannappan, C. Bucher, E. Saint-Aman, J.C. Moutet, A. Milet, M. Oltean,
E. Metay, S. Pellet-Rostaing, M. Lemaire, C. Chaix, New J. Chem. 34 (2010)
1373e1386.
Appendix A. Supplementary data
[17] D.S.N. Rao, F. Donald, Helv. Chim. Acta 81 (1998) 902e915.
[18] A. Rotaru, R. Danac, I. Druta, J. Heterocycl. Chem. 41 (2004) 893e897.
[19] L.A. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004e1009.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.061.
[20] J. Secrist, S. Anathan, C. Kwong, J. Maddry, R. Reynolds, A. Poffenberger,
M. Michael, L. Miller, J. Krahenbuhl, L. Adams, A. Biswas, S.G. Franzblau,
D. Rouse, D. Winfield, J. Brooks, I. Orme, Antimicrob. Agents Chemother. 45
(2001) 1943e1946.
References
[1] World Health Organization Tuberculosis Programme Available from: Global
tuberculosis report/WHO/HTM/TB/2012.6.