Design and synthesis of 5-(5-nitrothiophen-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazole derivatives with improved solubility and potential antituberculosis activity

Article abstract of DOI:10.1111/cbdd.13386

We report the design-synthesis of several nitrothiophene containing molecules as antituberculosis agents. The molecules were designed on the basis of previously reported nitrofuran molecules in our laboratory, and the α,β-unsaturated linker was modified t

Full text of DOI:10.1111/cbdd.13386

PROFESSOR MARIAM S DEGANI (Orcid ID : 0000-0002-
455-2642)
Article type : Research Letter
and tested for in vitro antituberculosis activity and
some molecules were found to be highly active
comparable to standard drugs. Further, the aqueous
solubility was determined using visual inspection
method and the designed molecules were found to
be more soluble than their chalcone counterparts.
Cytotoxicity studies were performed and the
molecules were found to be non-cytotoxic.
Electroanalytical studies proved nitro reduction as
the mechanism of action for these molecules. Thus,
this study provides potential nitrothiophene
containing hits with improved solubility and
reduced chances of toxicity.
3
Design and synthesis of 5-(5-
nitrothiophen-2-yl)-3-phenyl-4,5-
dihydro-1H-pyrazole derivatives with
improved solubility and potential
antituberculosis activity
a#
a#
Mihir Khambete , Harish Kundaikar , Archana Keywords: Nitrothiophene, Antituberculosis,
a
a
a
Solubility, Cytotoxicity, Electroanalysis
Raju , Sachin Lonkar , Mariam Degani *, Mukti
b
Kanta Ray
a
Department of Pharmaceutical Sciences and
Technology, Institute of Chemical Technology,
Matunga (E), Mumbai 400019, India.
Mycobacterium tuberculosis (Mtb), an obligate
bacterial pathogen, is the causative agent of
tuberculosis (TB). Despite elucidation of the genomic
b
Radiation Medicine Centre-BARC, Tuberculosis sequence of Mtb and the emergence of a multiplicity of
Immunology & Immunoassay Development Section, targets, residence of Mtb within the macrophages
coupled with its largely impermeable lipid cell wall
barrier, remain key issues challenging the development
of successful therapies. Additionally, development of
resistance, polytherapy for prolonged duration, and
Tata Memorial Hospital- Annexe Bldg, Parel, Mumbai
–
400012, India
1
serious side effects with some of the agents pose
barriers for the successful therapy. Hence, there is a
need to develop new potent inhibitors against latent
and resistant bacteria acting by novel mechanisms,
which will have minimal chances of development of
cross resistance with current drugs. Nitroheterocycles
are an important class of drugs that have been well
studied in modern chemotherapy. Various nitrofuran
derivatives like nitrofurantoin, nitrofurazone, and
furazolidone have been used for the treatment of burns
#
Authors have contributed equally
*Corresponding author: Mariam S. Degani,
ms.degani@ictmumbai.edu.in, msdegani@gmail.com
and urinary tract infections. Delamanid,
nitrodihydroimidazooxazole derivative , has been
approved for medical use in Europe, Japan and South
a
2
We report the design-synthesis of several
nitrothiophene
containing
molecules
as
Korea while Pretomanid,
derivative , is currently in Phase III clinical trials.
a
nitroimidazopyran
3
antituberculosis agents. The molecules were
designed on the basis of previously reported
nitrofuran molecules in our laboratory and the -
unsaturated linker was modified to cyclized linker in
order to overcome the challenge of low solubility
and possible toxicity. The stereoelectronic
properties such as HOMO, LUMO and HOMO-LUMO
gap (HLG) along with other properties such as
aqueous solvation energies and QPLogS values were
studied. The designed molecules were synthesized
Previously in our laboratory, a series of 4-(5-
nitrofuran-2-yl)prop-2-en-1-one derivatives as potent
antituberculosis agents were reported. In this work,
monocyclic nitrofuran ring was explored and it was
proposed
that
these
compounds
possessed
characteristic localized negative potential regions close
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to both the oxygen atoms of the nitro group. Screening
of these compounds for their antimycobacterial activity
led to identification of several derivatives with high
Table 1: Anti-TB activity (MIC), cytotoxicity (CC50),
HOMO and LUMO for the designed compounds 4a-4g.
4
antituberculosis activity . Therefore, these nitrofuran
derivatives were considered as starting point for this
study (Figure 1).
Code
R
Activity
µM)
CC50
(µM)
HOMO
(eV)
LUMO
(eV)
(
Various nitrothiophene derivatives have been
5–7
reported in literature as anti-infective agents
.
4a
-H
5.71
2.09
>292
-0.21
-0.09
-0.09
Further, inclusion of the sulphur atom significantly
changes the shape, size and lipophilicity of the five
membered heterocycle. Considering this, the first part
of the design involved the replacement of the furan ring
with the thiophene ring.
4b
4c
4d
4e
4-F
254.89
-0.21
4-CH3
2.71
5.06
>278.41
209.26
221.28
235.78
-0.22
-0.22
-0.21
-0.20
-0.09
-0.09
-0.09
-0.09
2-Cl
The other design aspect involved the addition of 5-
membered heterocyclic ring in the structure, which was
based on various modifications carried out on several
3-Cl
10.15
4.67
4
f
3,4-
dimethoxy
conformationally rigid biphenyl analogs of Pretomanid
8
(
PA-824) that suffered from poor solubility . Using the
concept of substituting the rigid aromatic ring with the
heterocyclic ring by Sutherland et al and combining it
9
4g
3,4,5-
trimethoxy
2.14
208.59
-0.20
-0.09
with the knowledge of pyrazoline containing
10,11
antituberculosis compounds
The α, β-carbonyl
INH
EMB
-
-
0.73
7.64
ND
ND
ND
ND
ND
ND
bridge in the structures reported by our laboratory
were replaced by cyclized pyrazoline ring along with
nitrothiophene to yield a series of 5-(5-nitrothiophen-2-
yl)-3-phenyl-4,5-dihydro-1H-pyrazole (Figure 2). Various
mono, di, and tri substitutions were included to obtain
the preliminary structure-activity relationships for the ND: Not determined
designed compounds.
All the synthesized compounds of this series showed
potential activity around or below 10 µM. The
Thereafter, these compounds were considered for
synthesis and bioassay against Mtb. The compounds 3a– unsubstituted derivative, 4a showed an activity of 5.71
g were synthesized using acid catalyzed Claisen– µM. Substitution by an electron withdrawing group such
3
Schmidt condensation. These intermediates were then
as 4-fluoro at the para position led to increased activity
as observed in compound 4b. The ortho-chloro
converted into corresponding cyclized derivatives by
microwave assisted organic synthesis using hydrazine substituted compound retained the activity whereas the
hydrate with ethanol as solvent. The purity of the meta-chloro substituted derivative 4d showed reduction
compounds was checked by chromatographic in activity. The 3,4,5-trimethoxy derivative showed an
techniques and the representative structure were
confirmed by spectral data (FT-IR, H NMR, C NMR,
and MS).
excellent activity which was higher as compared to that
of the dimethoxy derivative. Earlier work carried out in
1
13
4,13
our laboratory
using the Density Functional Theory
(
DFT) postulates indicated that the presence of
The synthesized compounds (4a–4g) were screened
characteristic localized negative potential regions near
both the oxygen atoms of the nitro group. Further,
higher negative values of LUMO energy distribution
were observed for the most active compounds. It was
indicative of the electron accepting capacity of these
compounds, suggesting that these compounds are
prodrugs and must be activated by TB-nitroreductase.
Taking this into consideration, the stereoelectronic
properties like HOMO, LUMO, and HLG were calculated
for the designed nitrothiophene derivatives. HOMO and
LUMO values were found to be ranging from -0.20 to -
against Mtb H Rv using two-fold dilution to determine
37
the actual minimum inhibitory concentration (MIC) by
12
employing the Resazurin microtiter assay (REMA) . MIC
was determined by visual inspection (color change from
blue to pink) (Table 1). Isoniazid (INH) and ethambutol
(
EMB) were used as the positive control.
0
.21 and from -0.11 to -0.09 eV respectively. Therefore,
this observation corroborated the above mentioned
postulates proposed by our research group.
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useful in providing additional information on the
mechanism of action. Taking into consideration these
studies, nitrothiophene molecule 4c and metronidazole,
as a standard, were selected for electroanalytical
studies. Cyclic voltammogram of metronidazole showed
a peak at -500 mV indicating reduction of nitro group
and another peak around -1400 mV corresponding to
diazole moiety (Figure 3). Nitrothiophene derivatives
with pyrazoline spacer showed a similar pattern.
Therefore it can be concluded that these derivatives act
via reduction of nitro group and the reduction potential
indicates possible involvement of Ddn.
The nitroheterocycles suffer from the disadvantage
of mutagenicity, which was responsible for arresting the
development of well-known nitroheterocyclic lead CGI-
14
15–17
17341 . Earlier QSAR studies
indicated that the
important features responsible for mutagenicity were
high log P values, large fused planar hydrophobic rings
and presence of 2 or more nitro groups among others.
Additionally, the analysis of the MIC, CC , and HOMO
50
and LUMO values of earlier derivatives indicated that
the HLG of −0.09 to −0.13 eV is sufficient to retain
activity without causing cytotoxicity. Thus, taking into
consideration all these structural findings, it was
postulated that the designed molecules were less likely
Thus, in the present work, 5-(5-nitrothiophen-2-yl)-
3-phenyl-4,5-dihydro-1H-pyrazole were designed as
to be cytotoxic. To confirm this hypothesis, CC50 values antituberculosis agents considering the previously
were evaluated using HEPG2 cell lines, and it was found
that the designed compounds showed low cytotoxicity
values.
reported nitrofuran derivatives. The quantum
properties such as HOMO, LUMO, and HLG were
calculated and were correlated with the activity and
mutagenicity of the designed derivatives. The free
energy of solvation for the intermediate chalcones and
the designed derivatives was calculated, and it was
observed that the designed derivatives exhibit more
aqueous solubility than the intermediates. Further,
these molecules were successfully synthesized and
characterized using spectroscopic techniques. In vitro
activity testing was performed against Mtb H Rv using
REMA method. The derivatives 4b and 4g were found to
possess activity comparable with that of INH and more
than that of EMB. Cytotoxicity studies were performed
and the molecules were found to be non-cytotoxic. This
study has emphasized the potential of combining DFT
calculations with synthetic approaches and biological
evaluation in developing improved nitroaromatics as
antituberculosis agents. Mechanistic investigations
carried out using electroanalytical method showed that
nitro reduction is responsible for the antituberculosis
activity of these designed molecules. Further detailed
structure-activity relationship and in-depth mechanistic
studies are under progress in our laboratory.
For a compound to be orally active, aqueous
solubility is one of the most important properties. A
solubility value of 50 µg/mL is considered as the
acceptable limit for most of the preclinical drug
development programs. The aqueous solubility was
calculated for the designed molecules along with their
respective chalcone intermediates using the standard
37
18
Poisson–Boltzmann solver in Jaguar .The solvation free
energies for the molecules under study varied from
−10.787 to −12.387 kcal/mol. Solvation energy gives a
measure of compound solubility. Higher negative values
of solvation free energy are indicative of higher aqueous
solubility. The free energy of solvation for cyclized
molecules was found to be higher than that of the
intermediate chalcones, which indicates that the
designed cyclized derivatives have more aqueous
19
solubility. QikProp predictions for the molecules under
study revealed that QPlogS for the molecules was −3.53
to −5.13 (supporting information). The higher the
negative values, higher the solubility, and therefore,
cyclized molecules were found to be more soluble than
the
non-cyclized
chalcone
intermediates.
Experimentally, the aqueous solubility was determined
using visual inspection and a similar trend was
observed, showing up to 2 to 3 times increase in
solubility.
Acknowledgements
Author MK would like to thank UGC-JRF and DST-
INSPIRE for funding the work reported in the
manuscript. Author MK would also like to thank Mr. Lalit
Khare for his help in the preparation of this manuscript.
The bicyclic nitroimidazole Pretomanid is a pro-drug
with a very complex mechanism of action active against
both replicating and hypoxic, non-replicating
Mycobacterium tuberculosis. It is a pro-drug activated
Conflict of Interest
Authors declare no conflicts of interest from a financial
or commercial standpoint.
by
deazaflavin
(cofactor
F420)
dependent
nitroreductase (Ddn). Consequently, the reduction of
the nitro group in the PA-824 and metronidazole is
considered to be a key step in the metabolic activation.
Figure captions:
There is
a
marked similarity between the
electrochemical reactions at the electrode/solution
interface, and the enzymatic redox reactions. Thus,
knowledge of the electrochemical mechanism of
reduction of such type of compounds would be very
Figure 1. Design of molecules
Figure 2. Designed derivatives to explore preliminary
SAR
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Scheme 1. Synthetic scheme for the designed
compounds
Wan, B.; Wang, Y.; Ma, Z.; Denny, W. a.
Synthesis and Structure−Activity Studies of
Biphenyl Analogues of the Tuberculosis Drug (6
Figure 3. Cyclic voltammogram for (a) Metronidazole (b)
molecule 4c
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Sutherland, J. J.; O’Brien, L. a.; Weaver, D. F. A
Comparison of Methods for Modeling
Quantitative Structure-Activity Relationships. J.
Med. Chem. 2004, 47 (22), 5541–5554.
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