Antimycobacterial activity of nitrogen heterocycles derivatives: 7-(pyridine-4-yl)-indolizine derivatives. Part VII8–12

Article abstract of DOI:10.1080/14756366.2017.1375483

A series of 13 compounds having a monoindolizine mono-salt skeleton was designed and synthesised in order to evaluate their antimycobacterial activity. The synthesis is efficient, involving only three steps: two alkylations and one 3 + 2 dipolar cycloaddition. The antimicrobial activity against Mycobacterium tuberculosis H37Rv grown under aerobic conditions was evaluated, eight compounds showing a very good antimycobacterial activity. SAR correlation reveals a certain influence of the R substituent from the para position of benzoyl moiety at position 3 of indolizine. The most active five compounds passed the second stage of anti-TB testing, the assay demonstrating that they are potent against both replicating and non-replicating Mtb, have a bactericidal mechanism of action, are active against drug-resistant Mtb strains, present a moderate to good activity against nontuberculous mycobacteria, a good intracellular activity, and a moderate to high cytotoxicity. For one compound showing a promising anti-TB profile, a complete ADMET study has been performed.

Full text of DOI:10.1080/14756366.2017.1375483

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Antimycobacterial activity of nitrogen heterocycles
derivatives: 7-(pyridine-4-yl)-indolizine derivatives.
Part VII8–12
Anda-Mihaela Olaru, Violeta Vasilache, Ramona Danac & Ionel I. Mangalagiu
To cite this article: Anda-Mihaela Olaru, Violeta Vasilache, Ramona Danac & Ionel I. Mangalagiu
(2017) Antimycobacterial activity of nitrogen heterocycles derivatives: 7-(pyridine-4-yl)-indolizine
derivatives. Part VII8–12, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1, 1291-1298,
DOI: 10.1080/14756366.2017.1375483
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1375483
© 2017 The Author(s). Published by Informa
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Date: 30 October 2017, At: 03:15

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 1291–1298
https://doi.org/10.1080/14756366.2017.1375483
RESEARCH PAPER
Antimycobacterial activity of nitrogen heterocycles derivatives: 7-(pyridine-4-yl)-
indolizine derivatives. Part VII8–12
Anda-Mihaela Olarua, Violeta Vasilacheb, Ramona Danaca and Ionel I. Mangalagiua
b
aFaculty of Chemistry, “Alexandru Ioan Cuza” University of Iasi, Iasi, Romania; “Alexandru Ioan Cuza” University of Iasi, CERNESIM Research
Centre, Iasi, Romania
ABSTRACT
ARTICLE HISTORY
Received 11 July 2017
Revised 21 August 2017
Accepted 21 August 2017
A series of 13 compounds having a monoindolizine mono-salt skeleton was designed and synthesised in
order to evaluate their antimycobacterial activity. The synthesis is efficient, involving only three steps: two
alkylations and one 3 þ 2 dipolar cycloaddition. The antimicrobial activity against Mycobacterium tubercu-
losis H37Rv grown under aerobic conditions was evaluated, eight compounds showing a very good anti-
mycobacterial activity. SAR correlation reveals a certain influence of the R substituent from the para
position of benzoyl moiety at position 3 of indolizine. The most active five compounds passed the second
stage of anti-TB testing, the assay demonstrating that they are potent against both replicating and non-
replicating Mtb, have a bactericidal mechanism of action, are active against drug-resistant Mtb strains, pre-
sent a moderate to good activity against nontuberculous mycobacteria, a good intracellular activity, and a
moderate to high cytotoxicity. For one compound showing a promising anti-TB profile, a complete ADMET
study has been performed.
KEYWORDS
Antimycobacterial;
indolizine; pyridinium salts;
ADMET
Introduction
skeleton, but different substituents on adjacent phenyl rings, in
order to have a better understanding of the acting mode of these
compounds and to be able to see any influence the substituents
have on the antimycobacterial activity. The results presented
herein refer to the synthesis and antimycobacterial evaluation of a
new series of thirteen compounds.
Tuberculosis is one of the major causes of disability and death
worldwide1, remaining one of the top 10 causes of deaths in 20152.
More than 95% of TB deaths occur in low- and middle-income
countries, according to the World Health Organisation2. In 2015,
10.4 million people became ill with TB, and 1.4 million people died
from the disease. An additional 0.4 million deaths resulted from TB
disease among people living with HIV2. Globally in 2015, an esti-
mated 480,000 people developed multidrug-resistant TB (MDR-TB)
and around 100,000 people developed rifampicin-resistant TB2. The
emergence of MDR-TB and, more recently, extensively drug-resist-
ant (XDR) TB has intensified the need for new TB drugs. Major
efforts are done for the discovery and development of new TB
drug targets and candidate drugs, and evaluation of novel TB drugs
Methods
General
€
Melting points were recorded on a A. Kruss Optronic Melting Point
Meter KSPI and are uncorrected. Proton and carbon nuclear mag-
netic resonance (dH, dC) spectra were recorded on a DRX-500
Bruker (Bruker, Bremen, Germany) (500 MHz). All chemical shifts
are quoted on the d-scale in ppm. Coupling constants are given in
Hz. IR spectra were recorded on a FTIR Shimadzu spectrometer.
Thin layer chromatography (TLC) was carried out on Merck silica
gel 60F254 plates. Visualisation of the plates was achieved using a
UV lamp (kmax ¼ 254 or 365 nm).
and optimal drug combinations in preclinical and clinical studies2,3
.
There are currently two important strategies used for discovery of
new anti-TB drugs4,5. One involves the synthesis of analogous of
the existing drugs, and the other refers to the search for novel
structures. Between the various classes of organic compounds,
fused N-heterocyles, especially pyridine fused systems, showed
promising anti-TB activity against replicating Mycobacterium tuber-
culosis (Mtb) H37Rv, similar to isoniazid6,7
In our previous work, we showed that several new classes of
compounds with fused heterocyclic structure possess antimicrobial
.
General procedure for synthesis of quaternary salts 6a–m
The monoindolizine 5 (1 mmol, 1 equiv., 0.37 g 5a, 0.40 g 5 b,
activity8–14, antimycobacterial including8–12. Recently, we reported 0.45 g 5c, 0.38 g 5d, 0.40 g 5e) and bromacetophenone derivative
compound 1 with monoindolizine mono-pyridinium salt structure (p or/and m substituted, 2 mmol, 2 equiv.) was suspended in
showing a promising antimycobacterial activity against both repli- anhydrous acetone (20 ml) and magnetically stirred over night at
cating and non-replicating Mtb8.
reflux. The resulting precipitate was collected by filtration and
then washed with acetone. All products were purified by crystal-
lisation (CHCl3:MeOH 1:1, v:v).
These results prompted us to extend our study to a series of
compounds having the same monoindolizine mono-pyridinium
CONTACT Ramona Danac
rdanac@uaic.ro
Faculty of Chemistry, “Alexandru Ioan Cuza” University of Iasi, Iasi, Romania; Ionel I. Mangalagiu
ionelm@uaic.ro
Faculty of Chemistry, “ Alexandru Ioan Cuza” University of Iasi, Iasi, Romania
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

1292
A.-M. OLARU ET AL.
4-(3-Benzoyl-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-oxo-2-(p-toly-
1643, 1248, 1198. Anal. Calcd. for C32H26BrClN2O5: C, 60.63; H, 4.13;
l)ethyl)pyridin-1-ium bromide (6a). Orange powder (0.52 g, 89% yield), N, 4.42; Found: C, 60.65; H, 4.10; N, 4.48.
1
mp ¼ 279–282 ꢀC. H-NMR (400 MHz, DMSO-d6): d 1.36 (t, J ¼ 7.2 Hz,
4-(3-(4-Chlorobenzoyl)-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-(4-
3H, H12), 2.47 (s, 3H, CH3), 4.37 (q, J ¼ 7.2 Hz, 2H, H11), 6.53 (s, 2H, fluorophenyl)-2-oxoethyl)pyridin-1-ium bromide (6e). Orange powder
1
H22), 7.50 (d, J ¼ 8.0 Hz, 2H, H26, H28), 7.63 (t, J ¼ 7.2 Hz, 2H, 2 ꢁ H16), (0.55 g, 89% yield), mp 307–310 ꢀC. H-NMR (400 MHz, DMSO-d6): d
7.70 (s, 1H, H2), 7.71 (t, J ¼ 7.2 Hz, 1H, H17), 7.84 (d, J ¼ 7.2 Hz, 2H, 2 x 1.37 (t, J ¼ 7.2 Hz, 3H, H12), 4.38 (q, J ¼ 7.2 Hz, 2H, H11), 6.51 (s, 2H,
H15), 7.96 (dd, J ¼ 7.6 Hz, J ¼ 1.6 Hz, 1H, H6), 8.01 (d, J ¼ 8.0 Hz, 2H, H22), 7.55 (t, J ¼ 8.8 Hz, 2H, H26, H28), 7.70 (d, J ¼ 8.4 Hz, 2H,
H25, H29), 8.79 (d, J ¼ 6.8 Hz, 2H, 2 ꢁ H19), 8.93 (as, 1H, H8), 9.14 (d, 2 ꢁ H16), 7.76 (s, 1H, H2), 7.88 (d, J ¼ 8.4 Hz, 2H, 2 ꢁ H15), 7.98 (ad,
J ¼ 6.8 Hz, 2H, 2 ꢁ H20), 9.92 (d, J ¼ 7.6 Hz, 1H, H5). 13 C-NMR J ¼ 7.2 Hz, 1H, H6), 8.20 (dd, J ¼ 8.4 Hz, J ¼ 5.6 Hz, 2H, H25, H29), 8.81
(125 MHz, DMSO-d6): d 14.3 C12, 21.3 CH3, 60.1 C11, 65.5 C22, 108.1 C1, (d, J ¼ 6.4 Hz, 2H, 2 ꢁ H19), 8.97 (as, 1H, H8), 9.11 (d, J ¼ 6.4 Hz, 2H,
113.7 C6, 118.8 C8, 123.0 C3, 124.6 2 ꢁ C19, 127.9 C2, 128.3 C25, C29, 2 ꢁ H20), 9.92 (d, J ¼ 7.2 Hz, 1H, H5). 13C-NMR (125 MHz, DMSO-d6):
128.6 2 ꢁ C15, 128.7 2 ꢁ C16, 129.1 C5, 129.6 C26, C28, 131.0 C24, 132.1 d 14.3 C12, 60.2 C11, 65.5 C22, 108.2 C1, 113.8 C6, 116.3 (d, C26, C28,
C17, C7, 137.8 C9, 138.7 C14, 145.4 C27, 146.6 2 ꢁ C20, 152.3 C18, 162.6 J¼ 22 Hz), 118.8 C8, 122.9 C3, 124.7 2 ꢁ C19, 127.9 C2, 128.7 2 ꢁ C16,
C10, 184.9 C13, 190.1 C23. IR (KBr, ꢀ(cmꢂ1): 3399, 3032, 3974, 1707, 129.2 C5, 130.3 (d, C24, J¼ 3.0 Hz), 130.7 2 ꢁ C15, 131.4 (d, C25, C29,
1643, 1622, 1642, 1205. Anal. Calcd. for C32H27BrN2O4: C, 65.87; H, J¼ 10.0 Hz), 132.2 C7, 137.0 C17, 137.3 C14, 137.9 C9, 146.6 2 ꢁ C20,
4.54; N, 4.66; Found: C, 65.93; H, 4.50; N, 4.75.
152.3 C18, 162.6 C10, 165.7 (d, C27, J¼ 253 Hz), 183.6 C13, 189.4 C23.
4-(3-Benzoyl-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-(3-methoxy-
IR (KBr, ꢀ(cmꢂ1): 3024, 3926, 1713, 1624, 1348, 1204. Anal. Calcd.
phenyl)-2-oxoethyl)pyridin-1-ium (6b). Yellow powder (0.53 g, 89% for C31H23BrClFN2O4: C, 59.87; H, 3.73; N, 4.50; Found: C, 59.93; H,
yield), mp 255–256 ꢀC. 1H-NMR (400 MHz, DMSO-d6): d 1.37 (t, 3.70; N, 4.54.
J ¼ 7.2 Hz, 3H, H12), 3.89 (s, 3H, OCH3), 4.37 (q, J ¼ 7.2 Hz, 2H, H11),
4-(3-(4-Bromobenzoyl)-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-oxo-
6.58 (s, 2H, H22), 7.40 (dd, J ¼ 8.4 Hz, J ¼ 2.4 Hz, 1H, H27), 7.59–7.64 2-(p-tolyl)ethyl)pyridin-1-ium bromide (6f). Orange powder (0.61 g,
1
(m, 4H, H26, H29, 2 ꢁ H16), 7.68–7.73 (m, 3H, H2, H17, H25), 7.84 (d, 92% yield), mp 283–284 ꢀC. H-NMR (400 MHz, DMSO-d6): d 1.37 (t,
J ¼ 7.2 Hz, 2H, 2 ꢁ H15), 7.96 (dd, J ¼ 7.6 Hz, J ¼ 1.6 Hz, 1H, H6), 8.80 J ¼ 7.2 Hz, 3H, H12), 2.47 (s, 3H, CH3), 4.37 (q, J ¼ 7.2 Hz, 2H, H11),
(d, J ¼ 6.8 Hz, 2H, 2 ꢁ H19), 8.92 (as, 1H, H8), 9.15 (d, J ¼ 6.8 Hz, 2H, 6.51 (s, 2H, H22), 7.50 (d, J ¼ 8.0 Hz, 2H, H26, H28), 7.74 (s, 1H, H2),
2 ꢁ H20), 9.90 (d, J ¼ 7.6 Hz, 1H, H5). 13C-NMR (125 MHz, DMSO-d6): 7.79 (d, J ¼ 8.4 Hz, 2H, 2 ꢁ H16), 7.83 (d, J ¼ 8.4 Hz, 2H, 2 ꢁ H15),
d 14.2 C12, 55.6 OCH3, 60.1 C11, 65.7 C22, 108.1 C1, 112.9 C29, 113.6 7.96 (ad, J ¼ 7.6 Hz, 1H, H6), 8.01 (d, J ¼ 8.0 Hz, 2H, H25, H29), 8.78
C6, 118.8 C8, 120.5 C27, 120.6 C25, 123.0 C3, 124.6 2 ꢁ C19, 127.8 C2, (d, J ¼ 7.2 Hz, 2H, 2 ꢁ H19), 8.94 (as, 1H, H8), 9.12 (d, J ¼ 6.4 Hz, 2H,
128.6 2 ꢁ C15, 128.7 2 ꢁ C16, 129.1 C5, 130.4 C26, 134.8 C24, 132.0 2 ꢁ H20), 9.90 (d, J ¼ 7.2 Hz, 1H, H5). 13 C-NMR (125 MHz, DMSO-d6):
C17, 132.1 C7, 137.7 C9, 138.6 C14, 146.5 2 ꢁ C20, 152.2 C18, 159.5 d 14.4 C12, 21.4 CH3, 60.3 C11, 65.6 C22, 108.3 C1, 113.9 C6, 118.9
C28, 162.6 C10, 184.9 C13, 190.6 C23. IR (KBr, ꢀ(cmꢂ1): 3032, 2976, C8, 122.9 C3, 124.7 2 ꢁ C19, 126.1 C17, 128.1 C2, 128.4 C25, C29,
1697, 1624, 1342, 1198. Anal. Calcd. for C32H27BrN2O5: C, 64.11; H, 129.3 C5, 129.7 C26, C28, 130.9 2 ꢁ C16, 131.1 C24, 131.8 2 ꢁ C15,
4.54; N, 4.67; Found: C, 64.23; H, 4.45; N, 4.70.
132.4 C7, 137.8 C14, 138.0 C9, 145.6 C27, 146.7 2 ꢁ C20, 152.4 C18,
4-[3-(4-Chlorophenyl)-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-(4-
162.7 C10, 183.9 C13, 190.2 C23. IR (KBr, ꢀ(cmꢂ1): 3419, 3021, 2930,
methoxyphenyl)-2-oxoethyl)pyridine-1-ium bromide (6c). Yellow pow- 1705, 1682, 1622, 1344, 1206. Anal. Calcd. for C32H26Br2N2O4: C,
1
der (0.59 g, 93% yield), mp 288 ꢀC. H-NMR (400 MHz, DMSO-d6): d 58.03; H, 3.96; N, 4.23; Found: C, 58.07; H, 3.93; N, 4.25.
1.36 (t, J ¼ 7.2 Hz, 3H, H12), 3.91 (s, 3H, OCH3), 4.37 (q, J ¼ 7.2 Hz,
4-[3-(4-Bromophenyl)-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-(4-
2H, H11), 6.49 (s, 2H, H22), 7.20 (d, J ¼ 8.8 Hz, 2H, H26, H28), 7.68 (d, methoxyphenyl)-2-oxoethyl)pyridine-1-ium bromide (6g). Orange
J ¼ 8.4 Hz, 2H, 2 ꢁ H16), 7.73 (s, 1H, H2), 7.86 (d, J ¼ 8.4 Hz, 2H, powder (0.56 g, 82% yield), mp 277–278 ꢀC. 1H-NMR (400 MHz,
2 ꢁ H15), 7.96 (dd, J ¼ 7.2 Hz, J ¼ 2.0 Hz, 1H, H6), 8.08 (d, J ¼ 8.8 Hz, DMSO-d6): d 1.36 (t, J ¼ 7.2 Hz, 3H, H12), 3.92 (s, 3H, OCH3), 4.37 (q,
2H, H25, H29), 8.78 (d, J ¼ 7.2 Hz, 2H, 2 ꢁ H19), 8.92 (d, J ¼ 1.2 Hz, 1H, J ¼ 7.2 Hz, 2H, H11), 6.45 (s, 2H, H22), 7.20 (d, J ¼ 8.8 Hz, 2H, H26, H28),
H8), 9.12 (d, J ¼ 6.8 Hz, 2H, 2 ꢁ H20), 9.88 (d, J ¼ 7.2 Hz, 1H, H5). 13 C- 7.76 (s, 1H, H2), 7.79 (d, J ¼ 8.4 Hz, 2H, 2 ꢁ H16), 7.84 (d, J ¼ 8.4 Hz, 2H,
NMR (125 MHz, DMSO-d6): d 14.3 C12, 55.9 OCH3, 60.3 C11, 65.4 2 ꢁ H15), 7.97 (dd, J ¼ 7.2 Hz, J ¼ 1.6 Hz, 1H, H6), 8.08 (d, J ¼ 8.8 Hz,
C22, 108.3 C1, 113.9 C6, 114.5 C26, C28, 118.9 C8, 122.9 C3, 124.7 2H, H25, H29), 8.78 (d, J ¼ 7.2 Hz, 2H, 2 ꢁ H19), 8.96 (as, 1H, H8), 9.10
2 ꢁ C19, 126.3 C24, 128.0 C2, 128.8 2 ꢁ C16, 129.3 C5, 130.8 2 ꢁ C15, (d, J ¼ 6.8 Hz, 2H, 2 ꢁ H20), 9.92 (d, J ¼ 7.2 Hz, 1H, H5). 13 C-NMR
C25, C29, 132.3 C7, 137.0 C17, 137.4 C14, 138.0 C9, 146.7 2 ꢁ C20, (125 MHz, DMSO-d6): d 14.3 C12, 55.8 OCH3, 60.2 C11, 65.3 C22, 108.2
152.3 C18, 162.7 C10, 164.3 C27, 183.7 C13, 189.0 C23. IR (KBr, C1, 113.8 C6, 114.4 C26, C28, 118.8 C8, 122.8 C3, 124.9 2 ꢁ C19, 126.0
ꢀ(cmꢂ1): 3395, 3022, 2936, 1707, 1680, 1642, 1242, 1206, 1173. C17, 126.2 C24, 127.9 C2, 129.2 C5, 130.7 C25, C29, 130.8 2 ꢁ C16, 131.6
Anal. Calcd. for C32H26BrClN2O5: C, 60.63; H, 4.13; N, 4.42; Found: 2 ꢁ C15, 132.2 C7, 137.6 C14, 137.9 C9, 146.6 2 ꢁ C20, 152.2 C18, 162.6
C, 60.70; H, 4.10; N, 4.45.
C10, 164.2 C27, 183.7 C13, 188.9 C23. IR (KBr, ꢀ(cmꢂ1): 3406, 3018,
4-(3-(4-Chlorobenzoyl)-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-(3-
2932, 1713, 1680, 1622, 1346, 1205. Anal. Calcd. for C32H26Br2N2O5:
methoxyphenyl)-2-oxoethyl)pyridin-1-ium (6d). Yellow powder C, 56.66; H, 3.86; N, 4.13; Found: C, 56.69; H, 3.85; N, 4.16.
1
(0.46 g, 73% yield), mp 252–254 ꢀC. H-NMR (400 MHz, DMSO-d6): d
4-(3-(4-Bromobenzoyl)-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-(3-
1.36 (t, J ¼ 7.2 Hz, 3H, H12), 3.88 (s, 3H, OCH3), 4.37 (q, J ¼ 7.2 Hz, methoxyphenyl)-2-oxoethyl)pyridin-1-ium (6h). Yellow powder
1
2H, H11), 6.52 (s, 2H, H22), 7.40 (dd, J ¼ 8.4 Hz, J ¼ 2.4 Hz, 1H, H27), (0.67 g, 99% yield), mp 256–259 ꢀC. H-NMR (400 MHz, DMSO-d6): d
7.58 (as, 1H, H29), 7.62 (t, J ¼ 8.0 Hz, 1H, H26), 7.68–7.71 (m, 3H, 1.37 (t, J ¼ 7.2 Hz, 3H, H12), 3.89 (s, 3H, OCH3), 4.38 (q, J ¼ 7.2 Hz,
2 ꢁ H16, H25), 7.75 (s, 1H, H2), 7.87 (d, J ¼ 8.4 Hz, 2H, 2 ꢁ H15), 7.97 2H, H11), 6.55 (s, 2H, H22), 7.40 (dd, J ¼ 8.4 Hz, J ¼ 2.4 Hz, 1H, H27),
(dd, J ¼ 7.6 Hz, J ¼ 2.0 Hz, 1H, H6), 8.80 (d, J ¼ 6.8 Hz, 2H, 2 ꢁ H19), 7.59 (as, 1H, H29), 7.62 (t, J ¼ 8.0 Hz, 1H, H26), 7.70–7.74 (m, 2H, H2,
8.92 (d, J ¼ 0.8 Hz, 1H, H8), 9.11 (d, J ¼ 6.8 Hz, 2H, 2 ꢁ H20), 9.91 (d, H25), 7.79 (d, J ¼ 8.4 Hz, 2H, 2 ꢁ H16), 7.83 (d, J ¼ 8.4 Hz, 2H,
J ¼ 7.2 Hz, 1H, H5). 13 C-NMR (125 MHz, DMSO-d6): d 14.3 C12, 55.6 2 ꢁ H15), 7.97 (dd, J ¼ 7.6 Hz, J ¼ 2.0 Hz, 1H, H6), 8.80 (d, J ¼ 6.8 Hz,
OCH3, 60.2 C11, 65.8 C22, 108.3 C1, 113.0 C29, 113.8 C6, 118.9 C8, 2H, 2 ꢁ H19), 8.94 (d, J ¼ 1.2 Hz, 1H, H8), 9.13 (d, J ¼ 6.8 Hz, 2H,
120.5 C25, 120.7 C27, 123.0 C3, 124.7 2 ꢁ C19, 128.0 C2, 128.8 2 ꢁ H20), 9.89 (d, J ¼ 7.6 Hz, 1H, H5). 13 C-NMR (125 MHz, DMSO-d6):
2 ꢁ C16, 129.3 C5, 130.4 C26, 130.7 2 ꢁ C15, 132.3 C7, 134.9 C24, d 14.3 C12, 55.6 OCH3, 60.2 C11, 65.8 C22, 108.3 C1, 113.0 C29, 113.8
137.0 C17, 137.0 C14, 138.0 C9, 146.6 2 ꢁ C20, 152.4 C18, 159.6 C28, C6, 118.8 C8, 120.5 C27, 120.7 C25, 122.9 C3, 124.7 2 ꢁ C19, 126.0
162.7 C10, 183.7 C13, 190.6 C23. IR (KBr, ꢀ(cmꢂ1): 3030, 2920, 1701, C17, 128.0 C2, 129.2 C5, 130.4 C26, 130.8 2 ꢁ C16, 131.7 2 ꢁ C15,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1293
132.3 C7, 134.8 C24, 137.7 C14, 137.9 C9, 146.6 2 ꢁ C20, 152.3 C18, with various substituents on both phenyl rings. Having in mind
159.6 C28, 162.7 C10, 183.8 C13, 190.6 C23. IR (KBr, ꢀ(cmꢂ1): 3025, the observation that a (p)substituted-benzoyl moiety is usefully
2930, 1701, 1642, 1248, 1200, 1171. Anal. Calcd. for C32H26Br2N2O5: pharmacophoric unit for the antimycobacterial activity10,12,16, but
C, 56.66; H, 3.86; N, 4.13; Found: C, 56.68; H, 3.83; N, 4.16.
as well the structure of model compound 1, we considered the
synthesis of new derivatives having as para substituents at ben-
4-(3-(4-Chlorobenzoyl)-1-(ethoxycarbonyl)indolizine-7-yl)-1-(2-(2,4-
hydroxyphenyl)-2-oxoethyl)pyridin-1-ium (6m). Yellow powder zoyl rest from position 3 of indolizine: H, Cl, Br, Me and OMe. On
1
(0.35 g, 56% yield), mp 265–267 ꢀC. H-NMR (400 MHz, DMSO-d6): d the second phenyl ring, we chose as substituents in para: Me,
1.37 (t, J ¼ 7.2 Hz, 3H, H12), 4.37 (q, J ¼ 7.2 H, 2H, H11z), 6.43 (s, 2H, OMe, F, Cl, Br and NO2 groups. In order to allow structure–activity
H22), 7.02 (d, J ¼ 8.4 Hz, 1H, H28), 7.49 (s, 1H, H25), 7.52 (d, relationship (SAR) comparisons with (p)substituted-benzoyl salts,
we synthesized as well compounds having the OMe group as sub-
J ¼ 8.4 Hz, 1H, H29), 7.69 (d, J ¼ 8.0 Hz, 2H, 2 ꢁ H16), 7.73 (s, 1H, H2),
stituent in meta, and one compound having two OH groups in
7.87 (d, J ¼ 8.0 Hz, 2H, 2 ꢁ H15), 7.96 (d, J ¼ 7.2 Hz, 1H, H6), 8.76 (d,
meta and para positions.
J ¼ 6.0 Hz, 2H, 2 ꢁ H19), 8.93 (s, 1H, H8), 9.12 (d, J ¼ 6.0 Hz, 2H,
The synthesis of the new 4,40-bipyridine derivatives was in line
2 ꢁ H20), 9.89 (d, J ¼ 7.2 Hz, 1H, H5), 9.70 (s, 1H, OH), 10.41 (s, 1H,
with the strategy reported previously by us8,15 and is presented in
Scheme 1. Thus, 4,40-bipyridine mono salts 2a–e (obtained by 4,40-
bipyridine alkylation17) were used for the in situ generation of
ylides 3a–e which reacted with ethyl propiolate in [3 þ 2] cyclo-
addition, leading to the intermediate compound 4a–e, and finally
to the completely aromatized monoindolizines 5a–e. Another
alkylation of indolizines 5a–e using x-bromoacetophenones led to
the compounds 6a–m (compounds 6a–h and 6m are new entities,
while compounds 6i–l were previously synthetized in our group15
(Scheme 1).
OH). 13C-NMR (125 MHz, DMSO-d6): d 14.3 C12, 60.2 C11, 65.2 C22,
108.2 C1, 115.0 C25, 115.5 C28, 113.8 C6, 118.8 C8, 121.8 C29, 122.9
C3, 124.6 2 ꢁ C19, 125.1 C24, 128.0 C2, 128.8 2 ꢁ C16, 129.2 C5, 130.7
2 ꢁ C15, 132.3 C7, 137.0 C17, 137.4 C14, 137.9 C9, 146.6 2 ꢁ C20,
145.7 C26, 152.1 C18, 152.3 C27, 162.7 C10, 183.7 C13, 189.6 C23. IR
(KBr, ꢀ(cmꢂ1): 3420, 3030, 2974, 1693, 1609, 1526, 1204, 1084.
Anal. Calcd. for C31H24Cl2N2O6: C, 62.95; H, 4.09; N, 4.74; Found: C,
62.98; H, 4.03; N, 4.76.
Microbiology
Alkylation towards 2a–e is high yielding while cycloadditions
led to indolizine 5a–e in ꢃ50% yield. All compounds were fully
characterised using elemental and spectral (NMR and IR) analysis.
Compounds were evaluated for antimycobacterial activity against
M. Tuberculosis, as a part of the TAACFTB screening program under
direction of the US National Institute of Health, the NIAID division.
Antimycobacterial activities of the compounds were performed by
Center of Tuberculosis Antimicrobial Acquisition and Coordinating
Facility (TAACF) at Southern Research Institute. All protocols con-
cerning the antimycobacterial evaluation of tested compounds
can be found in the Supplementary Appendix.
Activity against mycobacterium tuberculosis
The antimicrobial activity of compounds 6 against Mycobacterium
tuberculosis H37Rv grown under aerobic conditions was evaluated
as part of the TAACF TB screening program under direction of the
US National Institute of Health, the NIAID division. The standard
primary in vitro screen was assessed by determining the minimum
inhibitory concentration at which growth was completely inhibited
(MIC), and the concentrations that resulted in 50% and 90% inhib-
Results and discussion
Design and synthesis
Our strategy included the synthesis of compounds with the same ition of growth (IC50 and IC90 respectively)18–21. As can be seen in
4-(indolizine-7-yl)-pyridin-1-ium scaffold as in compound 1, but Table 1, eight compounds showed activity against Mtb H37Rv, five
Et3N
CH2COC6H4R(p)
CHCOC6H4R(p)
N
CH2
N
N
N
N
N
Et3NH]+Br
Br
3a-e
2a-e
O
N
XCH2COC6H4R'(p or m)
N
N
COOEt
N
-2[H]
R
5a. R= H
5b.
5c. R= Br
5d. R=Me
4a-e
O
OEt
15
O
R= Cl
OEt
5e.
R=OMe
O
20 19
5
6
16
14
X
4
6a. R= H; R'= Me(p); X= Br
6h. R= Br; R'= OMe(m); X= Br
7
3
18
22
13
N
N
R
6b. R
= H; R'= OMe(m); X= Br
6i.
21
17
R=Cl; R'=Me(p); X= Br
6j. R=Br; R'=Cl(p); X= Br
6k.
O
23
8
6c. R= Cl; R'= OMe(p); X= Br
9
2
1
29
6d. R= Cl; R'= OMe(m); X= Br
24
25
R=Me; R'=NO2(p); X= Br
6l. R=OMe; R'=Br(p); X= Br
COOCH2CH3
6e. R
= Cl; R'= F(p); X= Br
28
R'
10
11
12
6f. R= Br; R'= Me(p); X= Br
6m.
R=Cl; R'=OH (m and p); X= Cl
27
26
6a-l
6g. R= Br; R'= OMe(p); X= Br
Scheme 1. Synthesis pathway to obtain indolizineyl-pyridinium quaternary salts 6a–l.

1294
A.-M. OLARU ET AL.
Table 1. Results of antimycobacterial activity of compounds 6 against M. tuberculosis H37Rv grown under aerobic conditions.
Compound
MIC (lM)
IC50 (lM)
IC90 (lM)
Compound
MIC (lM)
IC50 (lM)
IC90 (lM)
6a
6b
6c
14
>50
15
13
39
12
15
>50
14
6g
6h
6i
50
14
8
35
13
7.2
46
14
8.2
6d
6e
6f
6g
15
>100
20
50
0.0065
14
>100
13
35
0.0036
16
>100
19
46
0.0071
6j
6k
6l
6m
>200
>200
>200
>100
0.0065
>200
200
>200
>100
0.0036
>200
>200
>200
>100
0.0071
Rifampicin
Rifampicin
Bold and italic values indicate the best ones from the series.
Table 2. Revaluation of antimycobacterial activity of compounds 6a, 6c, 6d, 6h
and 6i against M. tuberculosis H37Rv grown under aerobic conditions.
O
MIC aerobic
condition
(lM)
MIC aerobic
condition
(lM)
N
IC90
IC50
(lM) (lM)
IC90
Compound
IC50 (lM) (lM) Compound
O
6a
6c
6d
22
9.5
14
12
6.7
12
23
9.5
6h
6i
27
16
0.0071
15
9.8
26
17
COOEt
N
17 Rifampicin
0.0043 0.0092
1
Br
MeO
Table 3. The bactericidal activity (MBC) of compounds 6a, 6c, 6d, 6h and 6i.
IC -3.88 µM; IC - 13.07 µM; MIC- 3.91 µM
50
90
Concentration
dependent
Time
dependent
Figure 1. The structure of the reported compound 1 having anti-TB activity8.
Compound
MIC (lM)
MBC (lM)
6a
6c
6d
6h
6i
14
15
15
14
8
14
Y
Y
Y
N
Y
N
N
N
Y
3.75
3.75
14
O
8
N
N
N
R
R'
R'= Me, OMe
Br
R= Cl, Br, H
O
Table 4. Results of antimycobacterial activity of compounds 6a, 6c, 6d, 6h and
6i against M. tuberculosis H37Rv under low oxygen.
COOCH CH
2
3
MIC< 50 mM
Low oxygen
Normal oxygen
Compounds biologically active against Mtb H37Rv
Figure 2. SAR conclusions (using anti-TB potential) of the current series.
Compound
MIC (lM) IC50 (lM) IC90 (lM) MIC (lM) IC50 (lM) IC90 (lM)
6a
6c
15
57
12
6.5
14
18
100
75
9.2
8.1
42
23
of them with a MIC <15 mM. Compound 6i showed the best value
of MIC, IC50 and IC90 from all tested compounds (Table 1), being
superior to the model compound 1 (Figure 1).
6d
6h
6i
41
100
63
0.018
200
7.4
7.9
1.9
0.0027
30
17
37
10
0.0067
64
43
38
140
0.021
>200
17
16
6.5
0.0057
>200
27
24
28
0.011
Rifampicin
Metronidazole
Interestingly, the active compounds are only the ones having
para substituent R¼ –Cl, –Br and –H and R’ ¼ –Me(para) or
–OMe(para or meta). Replacing para R group with –Me or –OMe
and/or R’ group with –F(p), –Cl(p), –Br(p), –NO2(p) or –OH (m and
p) led to a dropping of antimycobacterial activity (MIC >100 mM)
(Figure 2). We thus hypothesised that the activity of these com-
pounds bearing a p-halogen-benzoyl or a benzoyl moiety at pos-
ition 3 of indolizine and a p-methylbenzoyl or methoxy(p or
m)benzoyl moiety is somehow connected with a specifically inter-
actions with putative binding sites.
Compounds 6a, 6c, 6d, 6h and 6i that showed promising anti-
TB activity in the primary assay, were subjected to the advanced
antimycobacterial susceptibility profiling including MIC, IC50 and
IC90 (repeated at lower starting concentrations), MIC under low
oxygen, minimal bactericidal concentration (MBC), testing on
drug-resistant Mtb, intracellular activity and cytotoxicity.
MIC, IC50 and IC90 determinations were repeated using similar
assays for compounds 6a, 6c, 6d, 6h and 6i and the obtained val-
ues (Table 2) were comparable with the previous ones (Table 1).
The bactericidal activity of compounds was assessed against Mtb
H37Rv grown in aerobic conditions. Viable cell counts are meas-
ured over 3 weeks of exposure to determine the rate of kill. MBC
was defined as the minimum concentration required to achieve a
2-log kill in 21 d. For compounds with >1-log kill, an assessment
of time- and/or concentration dependence was determined from
the kill kinetics (DMSO was used as a positive control for growth).
>200
predominates over that of time; therefore, these compounds dis-
play concentration-dependent effects that are significantly associ-
ated with an optimal free drug maximum concentration to MIC
ratio22. For compound 6h the effect of time is greater, displaying
a time-dependent effect, and bacterial outcome is associated with
free drug concentrations remaining above the MIC for a defined
portion of the dosing interval22. The MIC value used in this experi-
ment was taken from the first MIC assay presented herein.
Encouraging, all five tested compounds are bactericidal against
replicating cultures with MBCs equal or smaller then MICs.
Traditional screening of drugs against Mtb only addressed or
targets the organisms in an active replicating state. It is now
widely accepted that Mtb can reside in a state of non-replicating
persistence which has not been adequately assessed in the devel-
opment of new antimicrobials. Therefore, we determined the anti-
mycobacterial activity (MIC, IC50 and IC90) of the compounds 6
against Mtb H37Rv grown under hypoxic conditions using the low
oxygen recovery assay (LORA)23–25. Bacteria are first adapted to
low oxygen conditions and then exposed to compounds under
hypoxia for 10 d followed by incubation under aerobic conditions
(outgrowth) for 28 h. Parallel, oxygen-deprived bacteria were also
inoculated into compound assay plates and incubated under aer-
For compounds 6a, 6c, 6d and 6i, the effect of concentration obic conditions for 5 d. The growth in both assays was measured

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1295
Table 5. MIC, IC50 and IC90 of compounds 6 against M. tuberculosis resistant at different treatments and non-tuberculous mycobacteria.
INH-R1
INH-R2
RIF-R1
M. avium
Compound
MIC (lM)
IC50 (lM)
IC90 (lM)
MIC (lM)
IC50 (lM)
IC90 (lM)
MIC (lM)
IC50 (lM)
IC90 (lM)
MIC (lM)
6a
6c
6d
6h
6i
C1
C2
C3
33
14
28
>50
22
0.022
>200
2.9
13
6.9
14
18
13
0.012
>200
1.9
35
14
36
>50
23
0.031
>200
3.0
19
16
30
>50
20
0.013
>200
3.5
12
6.2
15
16
9.8
0.0070
>200
2.4
22
19
35
43
20
0.016
>200
4.3
14
10
22
24
15
5.3
0.092
2.7
8.3
6.5
12
13
7.7
1.1
0.063
1.6
13
10
24
26
15
3.8
0.094
2.6
50
50
200
200
50
0.0983
–
–
RIF-R2
FQ-R1
M. abscessus
MIC (lM)
IC50 (lM)
IC90 (lM)
MIC (lM)
IC50 (lM)
IC90 (lM)
MIC (lM)
IC50 (lM)
IC90 (lM)
6a
6c
6d
6h
6i
C1
C2
C3
31
13
16
>50
22
>50
0.31
2.9
12
8.1
12
15
12
>50
0.27
1.7
29
12
14
>50
22
>50
0.30
3.0
31
16
32
>50
27
0.024
0.16
110
15
8
21
26
11
0.013
0.13
49
32
16
32
>50
28
0.033
0.15
110
>200
23
>200
>200
25
3.6
–
–
24
16
81
>200
14
2.3
–
37
21
150
>200
23
3.6
–
–
–
INH: isoniazid-resistant strains; RIF: rifampicin-resistant strains; FQ: fluoroquinolone-resistant strains; C1: rifampicin control; C2: isoniazid control; C3: levofloxacin
control.
Bold and italic values indicate the best ones from the series.
Table 6. Results of cytotoxicity evaluation.
against M. abscessus, while compounds 6a, 6c and 6i showed simi-
lar moderate activity (MIC ¼ 50 mM) against M. avium.
Cytotoxicity IC50
IC50 intracell
IC90 intracell
Compound
(lM)
(lM)
(lM)
The cytotoxixity of compounds towards eukaryotic cells was
determined using the THP-1 human monocytic cell line, by calcu-
lating the concentration of compound causing 50% loss in viability
(IC50)27 (Table 6). The cytotoxicity of tested compounds proved to
be moderate to high, all compounds having a selectivity index
(SI) < 1 (SI ¼ IC50/MIC). These findings were somewhat disappoint-
ing, since structural elements of these compounds are part of dif-
ferent used drugs.
6a
6c
2.9
4.1
5.2
7
11
15
34
>50
43
6d
6h
3.9
3.8
13
6i
3.8
10
48
Control compounda,b
0.020a
0.18b
0.27b
aStaurosporine control.
bIsoniazid control.
Since the overall efficacy of any anti TB drug will be
improved by its ability to traffic into the macrophage phago-
some containing replicating bacteria, we evaluated the intracel-
Table 7. Results of plasma protein binding assay for compound 6i.
Test Mean plasma Mean plasma
Compound species fraction unbound (%) fraction bound (%) Recovery
lular activity of compounds24
. This was measured by using
6i
Human
Human
Human
0.35
20.7
0.51
99.7
79.3
99.5
48.7
93
96.7
THP-1 cell line infected with Mtb. Infected cells were exposed
to compounds for 72 h and viable bacterial counts were meas-
ured using luminescence as a measure of intracellular growth.
The IC50 and IC90 were defined as the compounds concentra-
tions that produced 50% and 90% inhibition of bacterial
growth, respectively.
All tested compounds exhibited a good intracellular activity
(IC50 ¼ 7–13 mM), even if the results are inferior to the control
Isoniazid (Table 6).
Propanolol
Warfarin
using luminescence (Table 3). Rifampicin was included in each
plate and metronidazole was included in each run as positive con-
trols for aerobic and anaerobic killing of Mtb, respectively23–25
.
As can be seen in Table 4, all tested compounds showed a bet-
ter antimycobacterial activity in anaerobic conditions than the
Taking into considerations the promising anti-TB activity of
control Metronidazole. Interestingly, for compounds 6a, 6c, 6d compound 6i, a complete absorption, distribution, metabolism,
and 6i, MIC values in anaerobic conditions were smaller than the excretion and toxicity (ADMET) study has been performed for it.
values obtained in aerobic conditions. Usually, the antimycobacte-
rial agents targeting the cell wall are inactive in anaerobic condi-
tions26; therefore, we presume that tested compounds 6 hit other
cellular targets of Mtb.
A good antimycobacterial activity of compounds 6a, 6c, 6d
and 6i is maintained against five resistant isolates of Mtb strains
under aerobic conditions18–21, especially for compounds 6c and 6i
(see MIC, IC50 and IC90 values in Table 5). Strains tested were two
isoniazid resistant strains (INH-R1 and INH-R2), two rifampicin
resistant strains (RIF-R1 and RIF-R2) and a fluoroquinolone resistant
strain (FQ-R1).
First, plasma protein binding (PPB) for compound 6i was deter-
mined by equilibrium dialysis using a semi-permeable membrane
which separates two compartments containing protein (human
plasma) and buffer28,29. The experiments used propranolol as
internal binding standard and warfarin as a high-binding control.
Molecules can penetrate freely, but proteins cannot pass through
the membrane. Compound 6i was strongly bound to the plasma
proteins (Table 7).
Usually high PPB is associated with a lower clearance rate
resulting in a greater half-time in vivo compared with low protein
binding compounds. Despite the fact that drugs with low protein
binding are believed to be more efficacious because of higher free
drug concentration, there are studies concluding that the binding
of a drug to plasma proteins has little effect on the in vivo efficacy
The antimycobacterial activity against nontuberculous myco-
bacteria (NTM) Mycobacterium avium and Mycobacterium abscessus
was as well evaluated under aerobic conditions18,21,27. As can be
seen in Table 5, only compounds 6c and 6i showed activity
of that drug29,30
.

1296
A.-M. OLARU ET AL.
The Caco-2 cell layer permeability assay is widely used as a quantified by LC-MS/MS as a measure of enzyme activity38–40. For
more predictive in vitro model of absorption through the intestinal compound 6i, enzyme activity was calculated and IC50 generated
epithelium31. Therefore, the permeability (measured in both direc- (Table 9).
tions) of compound 6i was assessed using a Caco-2-cell mono-
Compound 6i showed a high inhibition of CYP3A4-catalyzed
layer. For A–B permeability, compound 6i was added to the apical testosterone and a moderate inhibition of midazolam 1’-hydroxyl-
ation, this profile suggesting a high potential for drug-drug inter-
actions. No other CYPs were directly inhibited by 6i, IC50s of 6i on
these CYPs being >5 mM.
Compound 6i was tested for microsomal stability using pooled
human liver S9 microsomes. Microsomes are incubated with the
test compound at 37 ꢀC in the presence of the co-factor NADPH;
the reaction was terminated, the supernatant recovered and test
compound quantified by LC-MS/MS. The stability of compound is
expressed as a function of time41,42 (Table 10).
side of the Caco-2 monolayer and the transport to the basal side
monitored. For B–A permeability, test compound was added to
the basal side of the Caco-2 monolayer and the transport of the
compound to the apical side monitored. The amount of com-
pound present in each compartment was quantified by LC-MS/MS.
Each experiment included the control compounds atenolol (low
permeability, paracellular transport), propranolol (high permeabil-
ity, passive transcellular transport) and talinolol (P-gp efflux con-
trol)32–36 (Table 8).
Compound 6i proved to be a very highly cleared compound.
Compounds with this profile are generally considered that they
are likely to be rapidly cleared in vivo resulting in a short duration
of action and, it should be cleared strongly in vivo by CYP
metabolism43.
The cytotoxicity of compound 6i was also tested towards
eukaryotic cell using the human liver cells (HepG2), and
Staurosporine as control (IC50¼ 0.0086 mM). The IC50 was deter-
mined as the concentration of compound causing a 50% loss of
viability44–47. Compound 6i showed an IC50 value of 7.0 mM, similar
with its MIC value (8.0 mM), which maintains its cytotoxicity profile.
Compound 6i can be considered poorly permeable with a
A!B Papp < 2, and shows a low active efflux (Re ¼ 1.7). This led us
to suppose that the mechanism of absorption of compound 6i is
almost
a paracellular one with basically no involvement of
transporter proteins. However, recent studies proved no significant
correlation between antimycobacterial activity and Caco-2 perme-
ability, indicating that permeability is not a predictor of activity
inside of mycobacterium31.
Drug metabolism via the cytochrome P450 system has
emerged as an important determinant in the occurrence of several
drug-drug interactions that can result in drug toxicities, reduced
pharmacological effect, and adverse drug reactions37. Therefore,
compound 6i was tested for inhibition of six cytochrome P450
enzyme isoforms: CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and
CYP3A4. For each assay, human liver microsomes are incubated
with a probe substrate for each CYP isoform in the presence of
compound. The formation of metabolites for each isoform was
Conclusion
In summary, we have employed the 4-(indolizine-7-yl)-pyridin-1-
ium scaffold as core for the synthesis of 13 compounds in order
to test their antimycobacterial activity. The reaction pathway is
efficient and straight applicable, involving two N-alkylations of the
4,4,-bipyridine and, a Huisgen [3 þ 2] dipolar cycloaddition of
resulting ylides to ethyl propiolate. The primary antimycobacterial
screening reveals that eight of the 13 tested compounds had a
good activity against Mycobacterium tuberculosis H37Rv under aer-
obic conditions. SAR correlation reveals a certain influence of the
R substituent from the para position of benzoyl moiety at position
3 of indolizine, the most active being compounds with R¼ –H, –Cl,
–Br. The most active five compounds (namely 6a, 6c, 6d, 6h, 6i)
passed the second stage of anti TB testing, these including MIC,
IC50 and IC90 (repeated at lower starting concentrations), MIC
under low oxygen, MBC, testing on drug-resistant Mtb strains and
Table 8. Permeability evaluation of compound 6i.
Mean A!B
Mean B!A
a
a
Compound
Papp (10ꢂ6cm/s)
Papp (10ꢂ6cm/s)
Efflux ratiob
6i
0.76
0.13
12.7
1.3
0.37
25
1.7
2.8
2
Atenolol
Propanolol
Talinolol
0.077
3.3
43
aPapp is the apparent permeability rate coefficient ¼ (dQ/dt)/(C0A) where dQ/dt
is rate of permeation, C0 is the initial concentration of compound and A is the
area of monolayer.
bEfflux ratio (Re) is Papp (B!A)/Papp (A!B). An Re > 2 indicated a potential sub-
strate for P-glycoprotein or other active transporters.
Table 9. Cytochrome P450 inhibition results.
IC50 (lM)
Compound
CYP3A4-Midazolam
CYP3A4-Testosterone
CYP2C9
CYP2D6
CYP2C8
CYP2B6
CYP2C19
6i
0.48
0.033
–
–
–
–
–
0.081
0.022
–
–
–
–
–
>5
–
0.16
–
–
–
>5
–
–
>5
–
–
–
0.14
–
>5
–
–
–
–
–
0.72
>5
–
–
–
–
Ketoconazolea
Sulfaphenazolea
Quinidinea
0.032
Montelukasta
Tranylcyprominea
Ticlopidinea
–
–
–
7.5
–
–
–
aControl compounds.
Table 10. In vitro microsomal stability assay.
a
a
NADPH-dependent CLint
NADPH-dependent
NADPH-free CLint
(lL/min/mg)
NADPH-free
b
b
Compound
C (lM)
Test species
(lL/min/mg)
T1/2 (min)
T1/2 (min)
6i
1
1
1
Human
Human
Human
369
123
24.3
6.3
18.7
94.9
<12.8
<12.8
<12.8
>180
>180
>180
Verapamil
Dextromethorphan
aMicrosomal intrinsic clearance ¼ ln(2)/(T1/2[microsomal protein]).
bHalf-life ¼ 0.693/ꢂk, where k is the rate constant.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1297
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Acknowledgements
Part of this work (biological tests) was supported by National
Institutes of Health and the National Institute of Allergy and
Infectious Diseases, Contract no. HHSN27220110009I. Special
thanks for Jim P. Boyce, senior officer to Division of Microbiology
and Infectious Diseases, NIAID. The authors also thank the
POSCCE-O 2.2.1, SMIS-CSNR 13984-901, Project no. 257/28.09.2010,
CERNESIM, for the NMR experiments.
18. Ollinger J, Bailey MA, Moraski GC, et al. A dual read-out
assay to evaluate the potency of compounds active against
Mycobacterium tuberculosis. PLoS One 2013;8:e60531.
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test anti-tuberculosis drugs using fluorescence imaging. J
Antimicrob Chemother 2012;67:1948–60.
20. Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, et al.
Sensitive detection of gene expression in mycobacteria
under replicating and non-replicating conditions using opti-
mized far-red reporters. PLoS One 2010;5:e9823.
Disclosure statement
No potential conflict of interest was reported by the authors.
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