Antimycobacterial activity of new N1-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.05.053

N¹-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives were design, synthesized and tested for their in vitro antimycobacterial activity. The new compounds showed a moderate antimycobacterial activity

Full text of DOI:10.1016/j.bmcl.2016.05.053

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3287–3290
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimycobacterial activity of new N1-[1-[1-aryl-3-[4-(1H-imidazol-1-
yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives
a
a
b
c
Daniele Zampieri a, , Maria Grazia Mamolo , Luciano Vio , Maurizio Romano , Nataša Skoko ,
⇑
Marco Baralle c, Valentina Pau d, Alessandro De Logu d
a Department of Chemistry and Pharmaceutical Sciences, Piazzale Europa 1, University of Trieste, 34127 Trieste, Italy
b Department of Life Sciences, Via Valerio 28/1, University of Trieste, 34127 Trieste, Italy
c ICGEB, International Centre for Genetic Engeneering and Biootechnology, Padriciano 99, 34149 Trieste, Italy
d Department of Life and Enviromental Sciences, Via Porcell 4, University of Cagliari, 09124 Cagliari, Italy
a r t i c l e i n f o
a b s t r a c t
N1-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives
were design, synthesized and tested for their in vitro antimycobacterial activity. The new compounds
showed a moderate antimycobacterial activity against the tested strain of Mycobacterium tuberculosis
H37Ra and a significant antimycobacterial activity against several mycobacteria other than tuberculosis
strains.
Article history:
Received 29 March 2016
Revised 16 May 2016
Accepted 18 May 2016
Available online 19 May 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Antimycobacterial activity
Pyridine-2-carboxamidrazone derivatives
Mycobacteria other than tuberculosis
(MOTT)
Tuberculosis (TB) is a chronic infection and one of the most
widespread disease in the world and it’s the second cause of death
from a single infectious agent. The M. tuberculosis is the causative
pathogen and the re-emergence of the extensively multidrug-resis-
tant strains of M. tuberculosis (MDR) together with the high inci-
dence of severe disseminated infections produced by
mycobacteria other than tuberculosis (MOTT) necessitates the
development of new antimycobacterial therapeutic agents. In this
context, imidazole derivatives have shown antimycobacterial
activity associated with good antifungal activity.1,2 In general, the
common goal of the azole antifungal agents is the inhibition of
cytochrome P450-dependent lanosterol 14-a-demethylase (P450,
CYP51). Interestingly, it has been shown that a CYP51-like gene
of M. tuberculosis (MT) H37Rv strain may also be exploited as a
target for novel drugs.3 This gene encodes a mycobacterial sterol
Figure 1. Structure of new derivatives 1a–n.
14-
methyl sterols, and that forms complexes with antifungal azole
inhibitors of P450DM
a-demethylase (MT P45014DM), whose substrates are 14-a-
.
nation of the two moieties because the first is essential for the
antifungal activity of a number of azole drugs, and the latter is
important for the antimycobacterial activity of a number of deriva-
tives (Fig. 1).4–10
To obtain compounds with both antifungal and antimycobacte-
rial properties, we design and synthesized the series of derivatives
1a–n in which, the imidazol-1-yl and the pyridine-2-carboxam-
idrazone nuclei are present simultaneously. We tested the combi-
The N1-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]pro-
pyl]-pyridine-2-carboxamidrazone derivatives 1a–n (Table 1, sup-
plementary data) were prepared by reacting pyridine-2-
carboxamidrazone 3, prepared from 2-cyanopyridine and hydrazine
⇑
Corresponding author. Tel.: +39 (0)405587858.
E-mail address: dzampieri@units.it (D. Zampieri).
http://dx.doi.org/10.1016/j.bmcl.2016.05.053
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

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D. Zampieri et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3287–3290
Scheme 1. Synthetic route for the synthesis of compounds 1a–n. Reagents and conditions: (a) BF3-AcOH 72 h rt; (b) EtOH 36 h 0 °C; (c) EtOH 48 h rt.
hydrate in accordance with a slightly modified method previously
described,11 with the corresponding 3-aryl-1-[4-(1H-imidazol-1-
yl)phenyl]-propenones 2a–n, which in turn were obtained,
accordingly by a conventional procedure,12 by treating 1-(4-imida-
zol-1-yl-phenyl)-ethanone 4 with aromatic aldehydes in the pres-
ence of the boron trifluoride-acetic acid complex (Scheme 1).
Compounds 1a–n were preliminary tested against a Gram pos-
itive bacterial strain (S. aureus ATCC 25923), a Gram negative strain
(E. coli ATCC 25922) and a fungal strain of Candida albicans 685. We
performed the zone of inhibition assays for all the synthesized
compounds in comparison with Cefalotin and Amphotericin B, as
standard references.
Table 2
Antibacterial and antifungal activities of the tested compounds 1a–n; zone inhibition
test
Cpd.
R
S. aureus ATCC
25923
E. coli ATCC
25922
C. albicans
685
Inhibition diameter (millimeter) after 24 ha
1a
1b
1c
1d
1e
1f
H
14
19
19
20
20
22
20
17
7
5
8
6
6
7
8
6
5
8
13
8
7
11
9
8
2-Br
3-Br
4-Br
2-Cl
3-Cl
4-Cl
2,4-
Cl2
1g
1h
As shown in Table 2, all the tested compounds (at dose of 20 lg)
revealed an antibacterial activity, in particular towards the Gram
positive strain of S. aureus, where all the chlorinated derivatives
showed a inhibition zone ranging from 17 to 22 mm in comparison
1i
1j
1k
2-F
3-F
4-F
14
16
20
7
7
6
5
11
6
to Cefalotin (dose 30 lg; zone 31 mm), used as a standard
1l
1m
1n
INH
2-NO2 13
3-NO2 13
4-NO2 13
6
5
5
—
11
—
4
10
5
—
—
16
reference. Regarding the antifungal activity (Table 2), all the com-
pounds showed a weak inhibition towards C. albicans 865, clinical
isolate strain, with diameter values ranging from 4 to 13 mm at
—
31
—
Cefalotinb
Amph. Bc
20
l
g dose (Amphotericin B; 5
The derivatives 1a–n were tested against a strain of M. tubercu-
losis H37Ra and in all cases were observed to result in MIC values
ranging from 4.4 to 36 M. Furthermore all the synthesized com-
lg and 16 mm).
a
b
c
20
30
5 lg disk.
l
l
g dose cpd 1a–n.
g disk.
l
pounds were tested against seven strains of MOTT and several
derivatives showed interesting antimycobacterial activity (Table 3).
In particular, the 2,4-dichloro derivative 1h showed the best MIC
values towards M. intracellulare and M. avium (33.3
l
M) and
(MIC = 8.8
l
M for all the nitro derivatives 1l-n) compared to refer-
M).
against M. kansasii (MIC = 4.2 g/ml; INH=>467, 116 and 29.2
l
lM
ence standard drug isoniazid (MIC = 116 l
respectively). The entire series 1a–n revealed a potent antimy-
cobacterial activity towards M. gordonae, with the best values for
Towards M. marinum and M. Bovis, once again, the bromo
derivative 1d, it proved to be the more active compound of the ser-
the bromo derivatives 1c, d (MIC = 2.0
lM), and M. scrofulaceum
ies (MIC = 8.2 lM).

D. Zampieri et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3287–3290
3289
Table 3
Antimycobacterial activities (Minimum Inhibition Concentration: MIC) and cytotoxicity of the tested compounds 1a–n
Cpd.
MICa
(lM)
CC50 Vero
cells (lM)
M. tuberculosis
M. intracellulare
M. gordonae
M. scrofulaceum
M. bovis
M. kansasii
M. marinum
M. avium
ATC 25197
NCTC 10425
NCTC 10267
NCTC 10803
NCTC 10772
NCTC 10268
CIP 6423
NCTC 08559
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
INHc
19.5
32.7
16.3
16.3
36.0
18.0
9.0
9.0
17.6
4.7
78.0
131
131
131
144
72.0
72.0
33.3
74.7
74.7
74.7
140
>140
>140
>467
9.7
8.2
2.0
2.0
18.0
4.5
4.5
9.0
4.7
2.3
18.7
8.8
8.8
8.8
116
19.5
16.3
32.7
16.3
18.0
18.0
18.0
16.7
18.7
9.3
18.7
8.8
8.8
8.8
116
39.0
32.7
16.3
8.2
18.0
18.0
18.0
9.0
18.7
18.7
18.7
17.6
17.6
35.1
7.3
19.5
16.3
16.3
4.1
18.0
9.0
9.0
4.2
18.7
9.3
9.3
19.5
16.3
16.3
8.2
78.0
65.4
131
63.5
29.0
51.3
131
298 (18)b
53.2
18.0
18.0
18.0
33.3
18.7
18.7
18.7
35.1
35.1
35.1
116
72.0
72.0
72.0
33.3
74.7
74.7
74.7
70.3
140
68.6
78.8
27.8
62.1
26.5
78.8
48.0
28.7
9.3
8.8
4.4
4.4
17.6
8.8
35.1
29.2
140
116
23.1
>7000
0.9
a
b
c
Minimum Inhibition Concentration, duplicate.
Selectivity index (CC50/MIC).
Isoniazid.
All the synthesized compounds were also tested for their cyto-
toxicity against Vero cells ( M) with the derivative 1d showing
maximum selectivity index (CC50/MIC) of 18.
Regarding the antitubercular activity towards M. tuberculosis
H37Ra strain, the derivatives that present a substituent in the
meta-position showed the lower MIC’s values within the entire
panel of compounds presented in this study (MIC = 4.7 and
zone group; in fact we already proved that, at the contrary of picol-
inic acid hydrazide and its isoster isoniazid, the position of
nitrogen atom in the piridinecarboxamidrazone derivatives, it does
not seem to be crucial for the antimycobacterial activity.5,6
l
In particular, the final results suggest that: (i) notwithstanding
the presence of the imidazole ring, none of the synthesized com-
pounds possess a relevant antifungal property as supported also
by results obtained from the CYP51 spectral analysis; (ii) a highly
lipophilic scaffold, as starting calchone, is necessary for the activity
against bacteria and mycobacteria in order to efficiently interact
and/or penetrate into the cell wall and, possibly, increase the
antimycobacterial activity; (iii) the ortho substitution, in general,
lead to compounds gifted with worst antimycobacterial activity;
on the contrary, the ortho and para positions seems to be more
favourable for the mycobacteria inhibition; (iv) the 2,4-dichloro
and the 4-bromo substituted derivatives (1h and 1d, respectively)
exhibited the most interesting activity against MOTT strains and
the latter showed, also, the best cytotoxic profile; the nitro deriva-
tives 1m,n were observed to result the lower MIC value towards M.
tuberculosis H37Ra strain; (v) it could be speculated that the hydro-
philic pyridine-2-carboxyamidrazone scaffold, being structurally
related to isoniazid, is the effective drug which acts inhibiting
the mycolic acid synthesis and promotes the mycobacterial killing.
In conclusion, the newly synthesized compounds 1a–n exhib-
ited a weak antifungal activity, a good antibacterial activity against
E. coli and S. aureus and, in particular, a remarkable high activity
towards several MOTT strains with MIC values, in almost all cases,
better than reference drug isoniazid. The antimycobacterial activ-
ity against the tested strain of M. tuberculosis H37Ra is appreciable.
Future studies will extend the strains panel for a more compre-
hensive and detailed knowledge of these promising compounds.
Hence, these derivatives could serve as promising lead molecules
for further generation of more potent antitubercular agents.
4.4 lM, 1j, m). On the other hand, the poorest results with higher
MIC values, were obtained by the ortho-substituted derivatives (1b,
1e, 1i, 1l) of our new pyridine-2-carboxamidrazone derivatives,
suggesting that the position of the substituents on phenyl ring
can modulate the antimycobacterial activity. In conclusion, the
activity against MOTT strains is remarkable, when compared to
that of reference standard drug isoniazid, in particular towards
M. intracellulare, M. kansasii and M. avium (1h), M. gordonae (1c,
d and 1j) and M. scrofulaceum (1j and 1l-n), while the antitubercu-
lar activity towards M. tuberculosis H37Ra is moderate except for
nitro derivatives 1m, n that showed the best MIC value (4.4 lM)
of the entire series.
Subsequently, in order to investigate a possible interaction of
the compounds with the common azole-drug-like target, a spectral
analysis with the CYP51 (sterol 14a-demethylase) from Mycobac-
terium tuberculosis was performed. None of the novel compounds
(1a–n) were able to bind the enzyme as efficiently as the reference
standard, econazole (supplementary data). This observation can
explain the low antifungal activity of our derivatives and suggests
a different target for their antimycobacterial activity. Our mole-
cules, probably, act as isoniazid-like drugs, being sufficient lipophi-
lic (LogPcalc. range = 3.09–4.21) they can be able to penetrate into
the waxy layer of the mycobacterial cell wall inhibiting the mycolic
acid synthesis and induce the cell death. This could also explain the
good antibacterial activity against the tested strains of G+ and
GÀ bacteria.
By our experience, from previously results obtained by our
group,5–10 we focused only on lipophilic and electron withdrawing
substituents, which increase the lipophilicity of our molecules and
can ensure a possible interaction with the mycobacteria cell wall,
known to be highly hydrophobic, waxy, due to the presence of
the mycolate-arabinogalactan-peptidoglycan layer. For this reason,
some substituents like hydroxyl or amino were deliberately omit-
ted due to their polar/hydrophilic profile. Furthermore, we have
decided to retain only the 2-pyridine nucleus, linked to the amidra-
Acknowledgments
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Authors are thankful to Dr. Fabio Hollan (Dept. of Chemistry and
Pharm. Sciences – University of Trieste) for providing the MS anal-
ysis and it’s our wish to dedicate this work in honor of the memory
of Professor Luciano Vio.

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D. Zampieri et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3287–3290
4. Banfi, E.; Mamolo, M. G.; Vio, L.; Cinco, M.; Fabris, C.; Predominato, M. J.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.05.
053.
8. Mamolo, M. G.; Vio, L.; Banfi, E. Il Farmaco 1996, 51, 65.
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