Synthesis, computational studies, antimycobacterial and antibacterial properties of pyrazinoic acid-isoniazid hybrid conjugates

Article abstract of DOI:10.1039/c9ra03380g

Benzotriazole and microwave mediated syntheses led to a new set of hybrid conjugates of pyrazinoic acid with isoniazid via amino acid linkers in excellent yields with retention of chirality. Microbiological screening of the synthesized conjugates revealed an exceptionally high activity against some of the pathogenic bacterial strains at low concentrations. Promising antimycobacterial properties were observed against tuberculous and non-tuberculous mycobacteria. Robust molecular models (2D-QSAR and 3D-pharmacophore) support the observed biological properties. Safety profile of the synthesized conjugates against human normal cell (RPE1) was evaluated by MTT technique.

Full text of DOI:10.1039/c9ra03380g

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Synthesis, computational studies,
antimycobacterial and antibacterial properties of
Cite this: RSC Adv., 2019, 9, 20450
pyrazinoic acid–isoniazid hybrid conjugates†
a
b
Bibhuti B. Mishra,c Mohamed Elagawany,ad
*
*
Siva S. Panda,
Adel S. Girgis,
Venkatasai Devarapalli,a William F. Littlefield,a Ahmed Samir,e Walid Fayad,f
Nehmedo G. Fawzy,b Aladdin M. Srourg and Riham M. Bokhtiaah
Benzotriazole and microwave mediated syntheses led to a new set of hybrid conjugates of pyrazinoic acid
with isoniazid via amino acid linkers in excellent yields with retention of chirality. Microbiological screening
of the synthesized conjugates revealed an exceptionally high activity against some of the pathogenic
bacterial strains at low concentrations. Promising antimycobacterial properties were observed against
tuberculous and non-tuberculous mycobacteria. Robust molecular models (2D-QSAR and 3D-
pharmacophore) support the observed biological properties. Safety profile of the synthesized conjugates
against human normal cell (RPE1) was evaluated by MTT technique.
Received 6th May 2019
Accepted 22nd June 2019
DOI: 10.1039/c9ra03380g
rsc.li/rsc-advances
Nearly 10.0 million people (range, 9.0–11.1 million) developed TB
disease in 2017: among which 5.8 million were men, 3.2 million
women and 1.0 million were children (WHO, 2018).1
Introduction
Infectious diseases continue to pose a major threat to human
health caused by pathogenic microorganisms, such as viruses,
bacteria, fungi or parasites. According to the World Health
Organization (WHO), despite all the progress made to control or
cure infectious diseases, the major challenges persist in the new
drug development with high efficacy/selectivity for these
diseases. TB (tuberculosis) is one of the top 10 causes of death
worldwide and the leading cause of mortality/morbidity from
a single infectious agent (above HIV/AIDS). Millions of people
continue to fall sick with TB each year. In 2017, TB caused an
estimated 1.3 million deaths (range, 1.2–1.4 million) among HIV-
negative people and there were an additional 300 000 deaths
from TB (range, 266 000–335 000) among HIV-positive people.
TB disease is caused by a bacterial pathogen Mycobacterium
tuberculosis, that existed for millennia and continues to be the
leading cause of infectious disease deaths globally.2 It is known
to cause pulmonary infection and become extremely pervasive
within the lungs and between subjects. The current directly
observed treatment, short course (DOTS) tuberculosis therapy
requires a minimum of six months of treatment consisting of
two months of the intensive phase of treatment with the rst-
line drugs including isoniazid (INH), rifampin (RIF), pyr-
azinamide (PZA) and ethambutol (EMB), followed by another
four months of therapy with INH and RIF alone. Among the rst
line antibiotics, PZA is a highly effective bactericidal drug which
is known to penetrate into TB lesions and remain active in the
highly acidic environment of the TB granuloma. The parent
prodrug is metabolized via pyrazinamidase (PZase) to pyr-
azinoic acid; which is the active form of the drug. However, the
exact mechanism of action is not clear. Like PZA, INH is also
a prodrug that requires the activity of mycobacterial catalase
KatG. This interaction activates a series of reactions that even-
tually lead to the inhibition of mycolic acid synthesis, building
block of mycobacterial cell wall. Inappropriate treatment, poor
drug quality, and inadequate drug intake or treatment, all
contribute to the emergence of drug resistant bacterial strains
classied as multidrug-resistant (MDR) and extremely drug
resistant (XDR) strains.3 Currently, existing drugs are of limited
efficacy against drug resistance TB strains besides their known
side effects.4
aDepartment of Chemistry & Physics, Augusta University, Augusta, GA 30912, USA.
E-mail: sspanda12@gmail.com
bDepartment of Pesticide Chemistry, National Research Centre, Dokki, Giza 12622,
Egypt. E-mail: girgisas10@yahoo.com
cDepartment of Immunology and Microbial Disease, Albany Medical College, 47 New
Scotland Avenue, Albany, NY-12208, USA
dDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Damanhour
University, Damanhour, Egypt
eMicrobiology Department, Faculty of Veterinary Medicine, Cairo University, Cairo,
Egypt
fDrug Bioassay-Cell Culture Laboratory, Pharmacognosy Department, National
Research Centre, Dokki, Giza, 12622, Egypt
gDepartment of Therapeutic Chemistry, National Research Centre, Dokki, Giza 12622,
Egypt
hDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig
University, Zagazig, 44519, Egypt
Mycobacterium bovis is another Mycobacterium strain that
causes TB disease in animals and humans. In addition to
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra03380g
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Mycobacterium tuberculosis and Mycobacterium bovis, there are
many non-tuberculous mycobacteria like Mycobacterium mar-
inum and Mycobacterium fortuitum also cause various infections
in humans. Although many therapeutic agents were developed
for non-tuberculous mycobacterial infections, no standard
treatment protocol was approved due to their natural multi-
drug resistance.5
Drug resistance is a major global health concern for
a plethora of bacterial and fungal infections.6 Gram-positive
and Gram-negative bacterial strains are involved in the
majority of infectious diseases.7,8 The rapid emergence of MDR
infections necessitates the design and synthesis of novel anti-
microbial molecules. The development of novel antimicrobial
drugs is key to the treatment of infectious diseases, especially
for which combinatorial drug treatments are needed.
Scheme 1 Synthesis of POA hybrids through reaction of isoniazid and
amino acid (Route I).
Sub-optimal therapy caused by inefficient cellular penetra-
tion is a major issue associated with drug resistance. Many
therapeutic agents owing to their charge, hydrophilic property,
and/or size and stability under physiological conditions are not
suitable for treating intracellular bacterial infections that
require the drug to penetrate the cell membrane of the host and
also the pathogen. To compensate for the reduced penetration
and bioavailability, high drug doses are oen used that causes
strong adverse effects for vital organs. Synthetic vectors can
offer a solution for these problems associated with drug
delivery, overcoming the limitations such as bioavailability and/
or cytotoxicity. Among such synthetic vectors, cell-penetrating
peptides and amino acids have been successfully utilized
because of their drug carrier ability across cell membranes.9
Recently, several approaches have been reported to combat the
MDR problem.10–13
The present study deals with the synthesis of novel pyr-
azinoic acid–isoniazid conjugates connected through an amino
acid linker using molecular hybridization approach and inves-
tigating their properties against a variety of microbial strains
including, aerobic bacteria, tuberculous and non-tuberculous
mycobacteria. Conjugation of effective drug fragments for
developing novel agents is a well successful approach in drug
design.14 Microwave methodology is utilized to synthesize the
targeted conjugates since microwave protocol is well known in
medicinal chemistry for synthesizing drug like molecules in
high yield and purity.15 Molecular modeling techniques are
effective tools for understanding and validation of the biolog-
ical data.
were utilized but none of them was successful to isolate the
conjugates 5a–g in good yields.
Slight modication was considered for the synthetic strategy,
where the pyrazinoic acid (POA, 6) was activated as benzo-
triazolyl derivative 8 obeying the reported procedure16 and
coupled with free aminꢀo acids (9a–g) in a mixture of acetonitrile
and water (7 : 3) at 20 C for 2 h in the presence of 1.5 equiva-
lents of trimethylamine to give compounds 10a–g. In our
previous communication,14 we reported that we failed to
prepare benzotriazole derivatives of POA–amino acid conju-
gates 10a–g. This time we were able to optimize the reaction
condition and prepare the POA–aminoacyl benzotriazoles (11a–
g) in pure form by treating 10a–g with benzotriazole (7) in
ꢀ
presence of thionyl chloride at ꢁ15 C for 5–6 h. Compounds
11a–g were irradiated under microwave condition with 4 in the
presence of trimethylamine at 100 ꢀC for 1 h to obtain our
desired conjugates 12a–g in good yields without losing the
chiral integrity (Scheme 2). The conrmation of chiral integrity
was established by HPLC studies by comparing the spectra of L-
phenylalanine and DL-phenylalanine containing conjugates.
To better understand the role and effect of amino acids in
biological properties, we have also synthesized hybrid of pyr-
azinoic acid and isoniazid 13 without any linker (Scheme 3). As
Results and discussion
Chemistry
Synthesis of the targeted hybrids was initially studied by acti-
vating the Boc-protected amino acids (1a–g) with 1H-benzo-
triazole. The boc-protected aminoacyl benzotriazoles (3a–g)
were treated with isoniazid (4) in the presence of trimethyl-
amine in acetonitrile at 20 ꢀC for 2 h. According to the TLC
prole, the reaction was completed but the formed compounds
5a–g could not be isolated in good yield because of the hydro-
Scheme 2 Synthesis of POA hybrids through reaction of POA–ami-
philic nature of isoniazid (4) (Scheme 1). Different conditions noacyl benzotriazoles with isoniazid (Route II).
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observed for the synthesized compounds 10g/12g versus 10e/
12e. This can be attributed to the effect of indolyl heterocycle as
indole analogues are well known for potential activities. The
synthesized bis-conjugate 16 seems higher antimicrobial agent
against S. aureus, K. pneumonia, and P. vulgaris than the hybrid
analog 13 (MIC ¼ 16.51, 16.51, 0.12; 32.89, 32.89, 263.13 mM,
respectively). It also reveals close property to that of 13 against
E. faecalis (MIC ¼ 65.78, 66.05 mM for compounds 13 and 16,
respectively). Overall hybrid conjugates with amino acids are
more active than the hybrid conjugates without amino acid as
a linker. In our future studies, it is intended to explore more
possible linkers as well as use dipeptides and tripeptides to
establish an evidence-based SAR and to develop more potent
antibacterial agents.
Antimycobacterial properties. Antimycobacterial properties
of the synthesized compounds 10a–g, 12a–g, 13 and 16 were
screened against clinical isolate tuberculous (Mycobacterium
bovis) and non-tuberculous (Mycobacterium marinum, Mycobac-
terium fortuitum) strains using the standard technique.16,22
Antituberculosis properties of the conjugates were also studied
against highly virulent Mycobacterium tuberculosis at Albany
Medical College, Albany, NY. All the experimental data were
compared with the reference standards PZA, INH. Based on the
mycobacterial observations (Table 2, Fig. 1 and 2), the following
SAR are observed.
Mycobacterium marinum. Some of the synthesized conjugates
reveal high anti-mycobacterial properties against M. marinum
(about three folds potency; MIC ¼ 26.7, 25.6 mM of 12d and 12f,
respectively) than the standard references used (MIC ¼ 81.2,
72.9 mM of PZA, INH, respectively). Other conjugates synthe-
sized show potency comparable (slight enhancement) to that of
the standard references (MIC ¼ 46.6–58.4 mM for conjugates
12a–c, 12e and 12g). Compounds 10a and 10f show biological
properties (MIC ¼ 84.3, 73.7 mM, respectively) close to that of
the standard references used. The synthesized conjugates 12a–g
are more potent than their corresponding intermediates 10a–g.
The hybrid 13 (MIC ¼ 41.1 mM) seems an effective agent than its
precursors (PZA and NIH) and the bis-conjugate 16 (MIC ¼ 82.6
mM). The data clearly indicates the role of amino acids in the
hybrid conjugates and probably synergetic effect of the two
parent molecules. Interestingly, the aromatic amino acid resi-
dues show enhanced activity than the others.
Mycobacterium fortuitum. Compound 12f seems superior
among all the synthesized analogs (MIC ¼ 25.6 mM) with
a potency about 5.6, 3.2 folds than that of the standard refer-
ences used (MIC ¼ 145.8, 81.2 mM of INH, PZA, respectively).
Many of the synthesized compounds (MIC ¼ 46.6–58.4 mM of
conjugates 12a–e and 12g) also reveal promising biological
properties relative to the standards. All the synthesized conju-
gates 12a–g are of high potency than their corresponding
intermediates 10a–g. Hybrid conjugate 13 is of higher potency
than the bis-conjugate 16. Again we observed tryptophan plays
some important role in enhancing the activity.
Scheme 3 Synthesis of POA-INH hybrid.
isoniazid is the pro-drug of isonicotinic acid, we prepared the
bis conjugate of isonicotinic acid to compare the biological
property of the hybrid 13 and bis conjugate 16 of isoniazid
(Scheme 4). Conjugates 13 and 16 were previous reported and
synthesized by using various catalysts and reagents.17–20
However, we were able to complete the synthesis by utilizing the
aforementioned benzotriazole methodology under microwave
irradiation in good yield and purity.
Biological studies
Aerobic antibacterial properties. Antibacterial properties
were investigated for the synthesized compounds along with
parent drugs pyrazinamide (PZA), isoniazid (INH) and a stan-
dard drug ciprooxacin against a variety of Gram-positive
(Staphylococcus aureus, Enterococcus faecalis) and Gram-
negative (Klebsiella pneumonia, Proteus vulgaris, Pseudomonas
aeruginosa) clinical isolate bacteria utilizing the standard tech-
nique.21 From the results obtained (Table 1) the following
observations can be stated.
All the tested conjugates (10a–g and 12a–g) show high anti-
bacterial properties at sub-micromolar values (MIC ¼ 0.44–0.07
mM) with potency higher than that of the standard reference used
(MIC of ciprooxacin ¼ 1.21 mM) against Enterococcus faecalis.
Similar observations were also shown for most of the tested
conjugates against Proteus vulgaris (except compounds 10a and
12f) and Pseudomonas aeruginosa (except compounds 10b, 10f, and
12a) with MIC values ¼ 0.14–0.27, 0.13–0.51 mM, respectively
relative to the standard reference ciprooxacin (MIC ¼ 6.04, 12.07
mM, respectively). It has also been noticed that all the synthesized
conjugates 12a–g show higher potency than their precursors 10a–g
against Klebsiella pneumonia. Similar inhibition properties were
noticed towards Enterococcus faecalis [compounds 12b is an
exception revealing antimicrobial properties close to its precursor
10b (MIC ¼ 0.33, 0.13 mM, respectively)].
Establishing SAR (structure–activity relationships) with this
information is difficult. However, we noticed some evidence like
utilization of tryptophan enhances the antimicrobial properties
against all the tested microorganisms than phenylalanine as
Mycobacterium bovis. Only compounds 13 and 16 (MIC ¼
82.2, 82.6 mM, respectively) show promising antimycobacterial
properties (about 1.8–2.0 folds) than the standard references
(MIC ¼ 145.8, 162.4 mM of INH, PZA, respectively). The results
Scheme 4 Synthesis of INH bis-conjugate.
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Table 1 Antimicrobial properties of the tested compounds against aerobic bacteria
Minimum inhibitory concentration (MIC), mg mLꢁ1 (mM)
Staphylococcus
Entry Compd
aureus
Enterococcus faecalis Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa
1
1024 (8317.43) 0.12 (0.97)
64 (519.84)
8 (58.33)
8 (64.98)
4 (32.49)
2
3
64 (466.67)
0.4 (1.21)
16 (116.67)
0.4 (1.21)
64 (466.67)
64 (466.67)
2 (6.04)
2 (6.04)
4 (12.07)
4
5
1024 (4315.96) 0.03 (0.13)
64 (269.75)
0.03 (0.13)
8 (33.72)
0.03 (0.13)
4 (17.92)
512 (2293.58)
16 (67.44)
0.03 (0.13)
0.03 (0.13)
0.06 (0.27)
6
7
8 (33.72)
0.06 (0.25)
0.06 (0.24)
0.12 (0.51)
0.12 (0.47)
16 (62.67)
0.03 (0.12)
64 (250.69)
8
9
64 (235.92)
16 (58.98)
0.12 (0.44)
0.03 (0.11)
64 (235.92)
16 (58.98)
0.06 (0.22)
0.06 (0.22)
0.12 (0.44)
16 (58.98)
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Table 1 (Contd.)
Minimum inhibitory concentration (MIC), mg mLꢁ1 (mM)
Staphylococcus
Entry Compd
aureus
Enterococcus faecalis Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa
10
64 (206.24)
0.03 (0.10)
0.03 (0.08)
64 (206.24)
0.06 (0.19)
0.06 (0.17)
0.12 (0.39)
64 (179.58)
11
2 (5.61)
4 (11.22)
12
13
16 (46.73)
16 (44.90)
0.12 (0.35)
0.03 (0.08)
8 (23.37)
8 (22.45)
0.06 (0.18)
0.06 (0.17)
0.12 (0.35)
0.12 (0.34)
14
15
16
64 (170.93)
16 (40.98)
16 (40.98)
0.03 (0.08)
0.12 (0.31)
0.03 (0.08)
4 (10.68)
8 (20.49)
8 (20.49)
0.06 (0.16)
0.06 (0.15)
16 (40.98)
0.12 (0.32)
0.12 (0.31)
32 (81.97)
17
18
16 (37.26)
8 (32.89)
0.03 (0.07)
16 (65.78)
4 (9.31)
0.06 (0.14)
64 (263.13)
0.12 (0.28)
0.03 (0.12)
8 (32.89)
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Table 1 (Contd.)
Minimum inhibitory concentration (MIC), mg mLꢁ1 (mM)
Staphylococcus
Entry Compd
aureus
Enterococcus faecalis Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa
19
4 (16.51)
16 (66.05)
4 (16.51)
0.03 (0.12)
0.06 (0.25)
indicate the hybrid conjugates with amino acid linker lost the cell lines are 96.4–80.1% at 100 mM suggest that the synthesized
activity.
conjugates are likely to be safe to the human normal cell (ESI
Mycobacterium tuberculosis. The PZA activity was not observed Table S1†).
at 30 mg mLꢁ1 concentration at pH ¼ 7.0. However, at this
concentration, signicant growth inhibition was observed for
the synthesized conjugates even though the parent PZA is
known to exhibit M. tuberculosis activity in vitro at acidic pH <
6.0. INH showed >70% growth inhibition at 30 mg mLꢁ1
concentration, while compound 13 hybrid compounds exhibit
same growth inhibition at concentration as low as 3 mg mLꢁ1
.
Moreover, the hybrid conjugates 12a–g exhibited >95% growth
inhibitory activity at 30 mg mLꢁ1 and >80% growth inhibition at
10 mg mLꢁ1 concentration. Notably, PZA–INH hybrid
compounds exhibit potent bactericidal effect at physiological
PH, 7.0 (Fig. 1 and 2). We clearly notice the effect of hybrid
conjugates over the corresponding intermediates 10a–g. We
believe the combination along with amino acid enhance the
activity of the parent drugs.
Antiproliferative properties. Antiproliferative properties of
the synthesized compounds were screened against RPE1
(normal human immortalized retinal epithelial) cell line to
determine the toxicity/selectivity towards normal cell by the
standard MTT technique.23 The proliferation properties of RPE1
Fig. 1 Bacterial growth was measured by recording luciferase counts
in an IVIS-spectrum imaging system (data are presented as mean ꢂ
SD), one-way ANOVA Tukey's multiple comparison test. **, p < 0.001.
Table 2 Anti-mycobacterial properties of the tested compounds
Minimum inhibitory concentration (MIC), mg
mlꢁ1 (mM)
Entry
Compd
M. marinum
M. fortuitum
M. bovis
1
2
3
4
5
6
7
8
PZA
INH
10a
10b
10c
10d
10e
10f
10g
12a
12b
12c
12d
12e
12f
12g
13
10 (81.2)
10 (72.9)
20 (84.3)
>20 (>89.6)
>20 (>84.3)
>20 (>78.3)
>20 (>73.7)
20 (73.7)
>20 (>64.5)
20 (56.1)
20 (58.4)
20 (56.1)
10 (26.7)
20 (51.2)
10 (25.6)
20 (46.6)
10 (41.1)
20 (82.6)
10 (81.2)
20 (145.8)
20 (84.3)
>20 (>89.6)
>20 (>84.3)
>20 (>78.3)
>20 (>73.7)
>20 (>73.7)
>20 (>64.5)
20 (56.1)
20 (58.4)
20 (56.1)
20 (53.4)
20 (51.2)
10 (25.6)
20 (46.6)
10 (41.1)
20 (82.6)
20 (162.4)
20 (145.8)
>20 (>84.3)
>20 (>89.6)
>20 (>84.3)
>20 (>78.3)
>20 (>73.7)
>20 (>73.7)
>20 (>64.5)
>20 (>56.1)
>20 (>58.4)
>20 (>56.1)
>20 (>53.4)
>20 (>51.2)
>20 (>51.2)
>20 (>46.6)
20 (82.2)
8
10
11
12
13
14
15
16
17
18
Fig. 2 % growth inhibition was shown compared to untreated controls
(data are presented as mean ꢂ SD). Student's two-tailed t test. *, p <
0.05; **, p < 0.001.
16
20 (82.6)
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Computational studies
Mycobacterium tuberculosis. Three-descriptor model was
observed for the 2D-QSAR study of the tested compounds
against M. tuberculosis utilizing the log value of % growth
inhibition at 30 mg mLꢁ1 (ESI Tables S8–S10†). Minimum e–e
repulsion for bond H–N and maximum e–e repulsion for bond
H–C (semi-empirical descriptors, t ¼ 9.543, 7.207, respectively)
can be determined by the mentioned eqn (2). Maximum
coulombic interaction for bond C–O is also a semi-empirical
descriptor (t ¼ ꢁ14.963). Total interaction energy between two
given atomic species can be calculated by eqn (4).25
2D-QSAR. 2D-QSAR (quantitative structure activity relation-
ships) is a successful technique used intensively in medicinal
chemistry. It is capable to express the biological properties in
terms of mathematical equation(s) of descriptors (physico-
chemical) parameters.24 The biologically active compounds
against Mycobacterium marinum, Mycobacterium fortuitum and
Mycobacterium tuberculosis were considered for 2D-QSAR
studies utilizing CODESSA-Pro soware that allows under-
standing the parameters governing bio-properties and also
validates the bio-observations.
Mycobacterium marinum. The two-descriptor model was
determined to describe the properties of the biologically active
agents against Mycobacterium marinum (ESI Table S2†).
Maximum e–n attraction for bond C–N is a semi-empirical
descriptor with level of signicance ¼ ꢁ4.762. This descriptor
participates negatively in the 2D-QSAR model. In other words,
the highest value of the descriptor the more anti-mycobacterial
potency of the compound as observed in the high effective agent
synthesized 12d (MICobserved ¼ 26.7, MICestimated ¼ 30.8 mM)
relative to 16 (MICobserved ¼ 82.6, MICestimated ¼ 82.3 mM) that
possess descriptor values ¼ 330.7008, 329.2578, respectively
(ESI Tables S3 and S4†). Nuclear-electron attraction energy
between two given atoms is determined by eqn (1).25
Etot(AB) ¼ EC(AB) + Eexc(AB)
(4)
where, A and B are two atomic species. EC(AB) and Eexc(AB) are
electrostatic interaction and exchange energies between the two
atomic species, respectively.
Validation of the 2D-QSAR models. Due to the short training
set utilized in the 2D-QSAR study, internal validation seems the
most appropriate technique.23 Goodness of the QSAR models
are established by the statistical parameters [including squared
correlation coefficient (R2) and its leave-one-out (R2cvOO), F
(Fisher criteria) and s2 (standard deviation)] (R2 ¼ 0.908, 0.984,
0.965; R2cvOO ¼ 0.829, 0.962, 0.892; F ¼ 39.543, 240.314,
82.181; and s2 ¼ 0.003, 0.001, 0.004 for the BMLR-QSAR models
of M. marinum, M. fortuitum, and M. tuberculosis, respectively).
Estimated properties of the effective agents by the BMLR-QSAR
models are comparable to their observed values preserving their
potency among each other and to the standard references used
(Fig. 3–8; ESI Tables S3, S6 and S9†).
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢀ
ꢂ
X X
ZB
EneðABÞ ¼
Pmn
m;n˛A
m
n
(1)
RiB
B
where A, B are two atomic species. Pmn is the density matrix
elements over atomic basis {mn}. ZB is the charge of atomic
nucleus B, RiB is the distance between the electron and atomic
3D-pharmacophore
modelling.
3D-pharmacophore
modeling is an important computational technique explains
the biological/pharmacological properties of compound(s)
through alignment of the structural elements with chemical
features in 3D-array.26 The biologically active compounds were
undertaken by Discovery Studio 2.5 soware searching for the
3D-pharmacophoric modeling in which alignment of the
synthesized conjugates describes the observed biological
properties.26
Mycobacterium marinum. 3D-pharmacophoric modeling of
the biologically active compounds against Mycobacterium mar-
inum reveals 3D-array of three chemical features [two hydrogen
bonding acceptors (HBA-1, HBA-2) and one hydrogen bonding
donor (HBD)] (ESI Fig. S1 and S2†). The estimated/predicted
properties of the tested compounds due to alignment in the
3D-pharmacophore are presented in ESI Table S11.† From the
observed data it has been noticed that, the N-2 of isonicotinic
acid hydrazide is aligned with the pharmacophoric HBD in
compounds 12d and 12g, which are effective conjugates
synthesized against M. marinum (MICobserved ¼ 26.7, 46.6;
MICestimated ¼ 33.4, 38.1 mM for 12d and 12g, respectively).
However, slight displacement is observed for compounds 12a,
b and 12e, “which are also promising agents relative to the
standard reference used (INH)” where, the N-1 of isonicotinic
acid hydrazide of these compounds is aligned with the phar-
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢀ
ꢂ
ZB
RiB
nucleus B.
m
n
is the electron-nuclear attraction integrals
on atomic basis {mn}.
Maximum e–e repulsion for bond C–C is also a semi-
empirical descriptor participates negatively in the BMLR-QSAR
model. Electron–electron repulsion between two given atoms
can be calculated by eqn (2).25
X X
EeeðABÞ ¼
PmnPlshmn|lsi
(2)
m;n˛Al;s˛B
where A, B are two atomic species. Pmn, Pls are the density matrix
elements over atomic basis {mnls}. hmn|lsi is the electron
repulsion integrals on atomic basis {mnls}.
Mycobacterium fortuitum. Two semi-empirical descriptor
model is observed for the biologically active agents against M.
fortuitum (ESI Table S5†). Both of the descriptors positively
participate in the BMLR-QSAR model, i.e. the higher descriptor
value the lower biological properties (ESI Tables S6 and S7†).
Electron–electron repulsion energy for a given atomic species
can be calculated by eqn (3).25
X X X
EeeðAÞ ¼
PmnPlshmn|lsi
(3)
BsAm;n˛Al;s˛B
where A, B are two atomic species. Pmn, Pls are the density matrix
elements over atomic basis {mnls}. hmn|lsi is the electron
repulsion integrals on atomic basis {mnls}.
macophoric HBD (MICobserved ¼ 56.1, 58.4, 51.2; MICestimated
¼
46.9, 43.7, 39.3 mM for 12a, b and 12e, respectively). The
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Fig.
3 BMLR-QSAR model plot of correlations representing the
Fig.
5
BMLR-QSAR model plot of correlations representing the
observed vs. predicted log(MIC, mM) values for the tested compounds
against Mycobacterium marinum.
observed vs. predicted log(MIC, mM) values for the tested compounds
against Mycobacterium fortuitum.
Mycobacterium tuberculosis. Three chemical features
(hydrogen bonding donor, acceptor and a hydrophobic) were
observed for the biologically active compounds against M.
tuberculosis in the 3D-pharmacophoric study utilizing their %
growth inhibition at 30 mg mLꢁ1. All the tested conjugates (12a–
c, 12e, and 12g) show alignment of the isonicotinyl N-1 and
amino acid carbonyl with the hydrogen bonding donor and
acceptor, respectively while the pyridinyl heterocycle is aligned
with the hydrophobic function. Compound 13 also reveals
a similar alignment with a slight difference where the carbonyl
of pyrazinoic acid is aligned with the hydrogen bonding donor.
Compounds 10a, c, d, e show alignment of the amino acid
carbonyl and its alkyl fragment with the hydrogen bonding
acceptor and hydrophobic, respectively while the pyrazinamide
nitrogen with the hydrogen bonding donor function. Slight
division was observed for compounds 10b, g where the pyrazinyl
heterocycle N-1 is aligned with the hydrogen bonding acceptor
function. Most of the estimated properties due to the pharma-
cophoric model preserve the potencies of the tested compounds
(ESI Table S12, Fig. S5 and S6†).
estimated properties of the tested compounds are validated
with the experimental observations which help to establish the
SAR.
Mycobacterium fortuitum. Three chemical features [two
hydrogen bonding donors (HBD-1, HBD-2) and one hydrogen
bonding acceptor (HBA)] were exhibited by the 3D-
pharmacophore due to the tested bioactive agents against
Mycobacterium fortuitum (ESI Fig. S3 and S4†). Compound 12g,
which is one of the high potent hits synthesized against M.
fortuitum reveals alignment of isonicotinic acid hydrazide N-1
with HBD-1 (MICobserved ¼ 46.6, MICestimated ¼ 44.7 mM).
However, N-1 and N-2 of the hyrazide moiety of compounds 12d
and 12a are aligned with HBD-2 and HBD-1, respectively
(MICobserved ¼ 53.4, 56.1, MICestimated ¼ 50.6, 52.7 mM for 12d
and 12a, respectively). Meanwhile, the nitrogen atom of pyr-
azinecarboxamide function of compounds 12e and 12b are
aligned with HBD-1 and HBD-2, respectively (MICobserved ¼ 51.2,
58.4, MICestimated ¼ 48.0, 56.9 mM for 12e and 12b, respectively)
(ESI Table S11†).
Fig. 4 Observed and estimated activity MIC values for the tested Fig. 6 Observed and estimated activity MIC values for the tested
compounds against Mycobacterium marinum according to the BMLR- compounds against Mycobacterium fortuitum according to the
QSAR model.
BMLR-QSAR model.
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Table 3 ADMET descriptor values for the tested compounds
Entry
Compd
A.S.a
I.A.b
BBBc
PPBd
1
2
3
4
5
6
7
8
PZA
INH
10a
10b
10c
10d
10e
10g
12a
12b
12c
12d
12e
12g
13
5
4
4
4
4
4
4
3
4
4
4
4
4
3
4
4
1
0
0
0
0
0
0
0
1
1
1
1
1
1
0
0
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
3
0
1
0
0
0
0
2
2
0
0
0
1
2
2
2
2
9
10
11
12
13
14
15
16
16
a
A.S. (aqueous solubility) level: 0, 1, 2, 3, 4, 5 and 6 corresponding to
extremely low, very low, low; good, optimal, too soluble and unknown,
b
respectively.
I.A. (intestinal absorption) level: 0, 1, 2 and 3
corresponding to good, moderate, poor and very poor, respectively.
c
BBB (blood brain barrier penetration) level: 0, 1,2,3, 4 corresponding
d
Fig.
7 BMLR-QSAR model plot of correlations representing the
to very good, high, medium, low and very low, respectively. PPB
observed vs. predicted log(% growth inhibition at 30 mg mLꢁ1) values
for the tested compounds against Mycobacterium tuberculosis.
(plasma protein binding) level: 0, 1, 2 corresponding to <90%, >90%
and >95%, respectively.
From all the above 3D-pharmocophoric model observations it is
obvious that, alignment of various nitrogen atoms with the phar-
macophoric functions, is the main controlling parameter revealing
bio-properties. These are common observations between the two
techniques utilized in the present computational study (2D-QSAR
and 3D-pharmacophore) where, the 2D-QSAR studies reveal
important descriptors dealing with nitrogen atoms governing bio-
properties (max. e–n attraction for bond C–N for the QSAR model
of M. marinum, max. e–e repulsion for atom N, avg. electroph. react.
index for atom N for the QSAR model of M. fortuitum and min. e–e
repulsion for bond H–N for the QSAR model of M. tuberculosis).
the tested compounds show non-hepatotoxicity (Table 3). These
computational observations indicate that many of the con-
structed compounds are good hits to be handled by more
sophisticated biological/pharmacological studies for opti-
mizing promising bio-active agents. The predicted ADMET
properties are encouraging and we will further study the best
conjugates on animal models to generate experimental data. In
addition, our synthesized conjugates follow the Lipinski rule of
ve, which is used to evaluate drug likeness.
Conclusions
In conclusion, a series of novel hybrid conjugates of pyrazinoic
acid, isoniazid was synthesized using optimized benzotriazole,
and microwave assisted methodology. The synthesized conju-
gates showed strikingly high activity against various bacterial and
mycobacterial strains relative to the parent drugs. The presence
of amino acid in the hybrid conjugates also participates in
enhancing the activity. The biological data was supported by
different computational techniques (3D-pharmacophore and 2D-
QSAR). The robust models could be utilized further for devel-
oping more potent drug-like molecules. In our future studies, we
will explore the possibilities of other linkers as well as dipeptides
and tripeptides to unfold the structural activity relationship. In
our future study, we will further investigate the molecular
mechanism of action by using animal models.
ADMET (absorption, distribution, metabolism, excretion and
toxicity) studies
Computed ADMET studies were undertaken by Discovery Studio
2.5 soware. From the results obtained it has been noticed that
most of the tested compounds show optimal aqueous solubility
and good to moderate intestinal absorption. Blood brain barrier
penetration (BBB) is ranging from low to very low level. Many of
Experimental section
Chemistry
Fig. 8 Observed and estimated activity % growth inhibition values at
30 mg mLꢁ1 for the tested compounds against Mycobacterium
tuberculosis according to the BMLR-QSAR model.
General methods. Melting points were determined on
a
capillary point apparatus equipped with
a
digital
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thermometer. Reactions were monitored using thin layer
chromatography (TLC) on 0.2 mm silica gel F254 plates (Merck).
The chemical structures of nal products and intermediates
were characterized by nuclear magnetic resonance spectra (1H
NMR, 13C NMR) and determined on a Bruker NMR spectrom-
eter (500 MHz, 125 MHz). 13C NMR spectra are fully decoupled.
Chemical shis were reported in parts per million (ppm) using
a deuterated solvent peak or tetramethylsilane as an internal
standard. The chemical structures of nal products were
conrmed on a high-resolution Biosystems QStar Elite time-of-
ight electrospray mass spectrometer. HRMS was performed on
an Agilent 6210 instrument using time-of-ight mass spec-
trometry (TOF-MS) with electrospray ionization (ESI). HPLC
studies carried out on Agilent 6120 LCMS instrument using
Chirobiotic T column.
(Pyrazine-2-carbonyl)-L-phenylalanine (10e). Yellow amor-
1
ꢀ
phous, yield 0.15 g (98%), mp 165–166 C; H NMR (500 MHz,
DMSO-d6) d 13.01 (bs, 1H), 9.17 (s, 1H), 8.88 (s, 1H), 8.87 (s, 1H),
8.74 (s, 1H), 7.27 (s, 4H), 7.21–7.16 (m, 1H), 4.99 (dd, J ¼ 12.6,
7.8 Hz, 1H), 3.30–3.20 (m, 2H). 13C NMR (125 MHz, DMSO-d6)
d 172.4, 162.6, 147.9, 144.1, 143.5, 137.5, 129.1, 128.3, 126.5,
53.5, 36.1. HRMS m/z for C14H13N3O3 [M + H]+ calcd 272.1029.
Found: 272.1032.
(Pyrazine-2-carbonyl)-DL-phenylalanine (10f). Yellow amor-
phous, yield (94%), mp 155–157 ꢀC; 1H NMR (500 MHz, DMSO-
d6) d 9.15 (s, 1H), 8.88 (s, 1H), 8.86 (s, 1H), 8.74 (s, 1H), 7.25 (s,
4H), 7.23–7.14 (m, 1H), 4.81–4.73 (m, 1H), 3.29–3.18 (m, 2H).
13C NMR (125 MHz, DMSO-d6) d 172.4, 162.7, 147.9, 144.1,
143.5, 137.5, 129.1, 128.3, 126.5, 53.5, 36.1. HRMS m/z for
C
14H13N3O3 [M + H]+ calcd 272.1029. Found: 272.1030.
General procedure: synthesis of pyrazinoic acid–amino acid
conjugates 10a–g. A 50 mL round bottom ask containing
a small stir bar was charged with benzotriazole activated pyr-
azinoic acid 8 (0.5 g, 2.22 mM) and amino acid 9a–g (2.22 mM)
dissolved in a mixture acetonitrile and water (7 : 3) along with
trimethylamine (3.33 mM). The reaction mixture was stirred at
room temperature for 3–4 h and TLC monitored the progress of
the reaction. Aer completion of the reaction, the acetonitrile
was evaporated under reduced pressure and the residue was
treated with 4 N HCl solution. The precipitate formed was
ltered, washed with water and dried under vacuum to get the
desired products 10a–g.
(Pyrazine-2-carbonyl)-L-leucine (10a). White amorphous, yield
(78%), mp 155–156 ꢀC; 1H NMR (500 MHz, CDCl3) d 9.40 (s, 1H),
8.78 (s, 1H), 8.58 (s, 1H), 8.12 (d, J ¼ 8.4 Hz, 1H), 4.89–4.54 (m,
1H), 1.91–1.76 (m, 3H), 1.01 (d, J ¼ 4.8 Hz, 6H). 13C NMR (125
MHz, CDCl3) d 176.7, 163.0, 147.3, 144.3, 144.1, 142.9, 50.8,
41.3, 25.0, 22.9, 21.8. HRMS m/z for C11H15N3O3 [M + H]+ calcd
238.1186. Found: 238.1189.
(Pyrazine-2-carbonyl)-L-tryptophan (10g). Yellow amorphous,
yield (93%), mp 127–129 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 13.01 (bs, 1H), 10.85 (s, 1H), 9.17 (s, 1H), 8.88 (s, 1H), 8.72 (d, J
¼ 7.7 Hz, 1H), 8.70 (s, 1H), 7.52 (d, J ¼ 8.0 Hz, 1H), 7.32 (d, J ¼
8.0 Hz, 1H), 7.16 (s, 1H), 7.04 (t, J ¼ 7.4 Hz, 1H), 6.92 (t, J ¼
7.1 Hz, 1H), 4.82–4.75 (m, 1H), 3.36 (s, 2H). 13C NMR (125 MHz,
DMSO-d6) d 172.6, 162.4, 147.7, 143.9, 143.3, 136.0, 127.1, 123.6,
120.9, 118.1, 111.3, 109.4, 52.8, 26.5. HRMS m/z for C16H14N4O3
[M + H]+ calcd 311.1138. Found: 311.1145.
General procedure: synthesis of pyrazinoic acid–aminoacyl
benzotriazolides 11a–g. 1H-Benzotriazole 7 (4.0 mM) was dis-
solved in anhydrous methylene chloride (30 mL). Thionyl
chloride (1.2 mM) was added and stirred for 30 min. Reduced
the reaction mixture temperature to ꢁ15 ꢀC and then corre-
sponding pyrazinoic acid–amino acid conjugates (10a–g, 1.0
mM) was added. The reaction mixture was stirred for 4–5 h at
ꢁ15 ꢀC. Upon completion of the reaction, the reaction mixture
was ltered and the ltrate was evaporated under reduced
pressure. The residue was treated with 10% solution of sodium
carbonate and the precipitate obtained was ltered, washed
with water and dried to yield the desired product 11a–g.
(S)-N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-4-methyl-1-oxopentan-
2-yl)pyrazine-2-carboxamide (11a). White amorphous, yield
(88%), mp 144–146 ꢀC; 1H NMR (500 MHz, CDCl3) d 9.37 (s, 1H),
8.78 (s, 1H), 8.59 (s, 1H), 8.46 (d, J ¼ 8.2 Hz, 1H), 8.23 (d, J ¼
8.2 Hz, 1H), 8.12 (d, J ¼ 8.2 Hz, 1H), 7.64 (t, J ¼ 7.8 Hz, 1H), 7.51
(t, J ¼ 7.8 Hz, 1H), 6.28–6.21 (m, 1H), 2.09–1.84 (m, 3H), 1.12 (d,
J ¼ 6.6 Hz, 3H), 0.99 (d, J ¼ 6.6 Hz, 3H). 13C NMR (125 MHz,
CDCl3) d 171.6, 163.2, 147.7, 146.1, 144.5, 143.9, 142.8, 131.2,
130.8, 126.5, 120.4, 114.4, 51.8, 41.8, 25.5, 23.2, 21.4. HRMS m/z
for C17H18N6O2 [M + H]+ calcd 339.1564. Found: 339.1570.
(S)-N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-3-methyl-1-oxobutan-2-
yl)pyrazine-2-carboxamide (11b). White amorphous, yield (77%),
mp 133–135 ꢀC; 1H NMR (500 MHz, CDCl3) d 9.41 (s, 1H), 8.80 (s,
1H), 8.63 (s, 1H), 8.57 (d, J ¼ 8.2 Hz, 1H), 8.29 (d, J ¼ 8.2 Hz, 1H),
8.16 (d, J ¼ 8.2 Hz, 1H), 7.69 (t, J ¼ 7.5 Hz, 1H), 7.55 (t, J ¼ 7.5 Hz,
1H), 6.18 (dd, J ¼ 8.7, 5.2 Hz, 1H), 2.74–2.64 (m, 1H), 1.19 (d, J ¼
6.8 Hz, 3H), 1.10 (d, J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 170.9, 163.3, 147.7, 146.1, 144.6, 144.0, 142.8, 131.1, 130.8,
126.6, 120.5, 114.4, 57.7, 31.9, 19.9, 17.3. HRMS m/z for
(Pyrazine-2-carbonyl)-L-valine (10b). White amorphous, yield
(88%), mp 160–162 ꢀC; 1H NMR (500 MHz, CDCl3) d 9.40 (s, 1H),
8.78 (s, 1H), 8.59 (s, 1H), 8.26 (d, J ¼ 8.8 Hz, 1H), 4.80 (dd, J ¼
8.8, 4.5 Hz, 1H), 2.45–2.37 (m, 1H), 1.07 (t, J ¼ 6.0 Hz, 6H). 13
C
NMR (125 MHz, CDCl3) d 175.7, 163.1, 147.3, 147.2, 144.3, 144.2,
144.2, 142.9, 57.2, 31.3, 19.2, 17.7. HRMS m/z for C10H13N3O3 [M
+ H]+ calcd 224.1029. Found: 224.1031.
(Pyrazine-2-carbonyl)-L-alloisoleucine (10c). White amorphous,
yield (81%), mp 164–166 ꢀC; 1H NMR (500 MHz, CDCl3) d 9.40 (s,
1H), 8.78 (s, 1H), 8.59 (s, 1H), 8.27 (d, J ¼ 8.7 Hz, 1H), 4.84 (dd, J
¼ 8.8, 4.6 Hz, 1H), 2.14–2.08 (m, 1H), 1.66–1.55 (m, 1H), 1.38–
1.27 (m, 1H), 1.05 (d, J ¼ 6.8 Hz, 3H), 0.99 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (125 MHz, CDCl3) d 175.8, 162.9, 147.3, 144.3, 142.9, 56.6,
37.9, 25.1, 15.7, 11.6. HRMS m/z for C11H15N3O3 [M + H]+ calcd
238.1186. Found: 238.1182.
(Pyrazine-2-carbonyl)-L-methionine (10d). White amorphous,
yield (76%), mp 136–138 ꢀC; 1H NMR (500 MHz, CDCl3) d 10.27
(bs, 1H), 9.40 (s, 1H), 8.80 (s, 1H), 8.60 (s, 1H), 8.40 (d, J ¼ 8.0 Hz,
1H), 4.99 (dd, J ¼ 12.6, 7.5 Hz, 1H), 2.64 (t, J ¼ 7.3 Hz, 2H), 2.43–
2.33 (m, 1H), 2.27–2.16 (m, 1H), 2.12 (s, 3H). 13C NMR (125 MHz,
CDCl3) d 175.4, 163.1, 147.3, 144.2, 143.0, 51.6, 31.5, 30.1, 15.5.
HRMS m/z for C10H13N3O3S [M + H]+ calcd 256.0750. Found:
256.0755.
C
16H16N6O2 [M + H]+ calcd 325.1407. Found: 325.1411.
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N-((2S,3S)-1-(1H-Benzo[d][1,2,3]triazol-1-yl)-3-methyl-1-oxopentan-
General procedure for the synthesis of pyrazinoic acid–
2-yl)pyrazine-2-carboxamide (11c). White amorphous, yield (69%), isoniazid hybrid conjugates 12a–g. A dried heavy-walled Pyrex
mp 186–188 ꢀC; 1H NMR (500 MHz, CDCl3) d 9.40 (s, 1H), 8.80 (s, tube containing a small stir bar was charged with benzotriazole
1H), 8.62 (s, 1H), 8.56 (d, J ¼ 8.5 Hz, 1H), 8.29 (d, J ¼ 8.2 Hz, 1H), intermediate 11a–g (0.7 mM) and isoniazid 4 (0.7 mM) dissolved
8.16 (d, J ¼ 8.2 Hz, 1H), 7.69 (t, J ¼ 7.5 Hz, 1H), 7.55 (t, J ¼ 7.5 Hz, in THF (3 mL) along with trimethylamine (1.1 mM). The reaction
1H), 6.18 (dd, J ¼ 8.7, 5.2 Hz, 1H), 2.50–2.38 (m, 1H), 1.74–1.64 (m, mixture was exposed to microwave irradiation (50 W) at
1H), 1.43–1.31 (m, 1H), 1.15 (d, J ¼ 6.8 Hz, 3H), 0.95 (t, J ¼ 7.4 Hz, a temperature of 70 ꢀC for 1 h. Each mixture was allowed to cool
3H). 13C NMR (125 MHz, CDCl3) d 170.9, 163.2, 147.7, 146.1, 144.6, through an inbuilt system until the temperature had fallen below
143.9, 142.8, 131.1, 130.8, 126.6, 120.5, 114.4, 57.4, 38.4, 24.4, 16.2, 30 ꢀC (ca. 10 min). The reaction mixture was quenched with ice
11.3. HRMS m/z for C17H18N6O2 [M + H]+ calcd 339.1564. Found: cold water and the solid obtained was ltered and washed with
339.1573.
(S)-N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-4-(methylthio)-1-oxobutan-
10% Na2CO3 and water to give the desired compounds 12a–g.
(S)-N-(1-(2-Isonicotinoylhydrazineyl)-4-methyl-1-oxopentan-2-yl)
2-yl)pyrazine-2-carboxamide (11d). White amorphous, yield (74%), pyrazine-2-carboxamide (12a). White amorphous, yield (85%),
1
1
ꢀ
mp 171–173 ꢀC; H NMR (500 MHz, CDCl3) d 9.42 (s, 1H), 8.82 (s, mp 160–162 C; H NMR (500 MHz, DMSO-d6) d 10.74 (s, 1H),
1H), 8.69 (d, J ¼ 7.9 Hz, 1H), 8.64 (s, 1H), 8.29 (d, J ¼ 8.2 Hz, 1H), 10.39 (s, 1H), 9.22 (s, 1H), 8.91 (s, 1H), 8.82–8.67 (m, 3H), 8.70 (d, J
8.18 (d, J ¼ 8.2 Hz, 1H), 7.71 (t, J ¼ 7.5 Hz, 1H), 7.57 (t, J ¼ 7.5 Hz, ¼ 8.7 Hz, 1H), 7.78 (d, J ¼ 4.3 Hz, 2H), 4.80–4.72 (m, 1H), 1.86–
1H), 6.34 (dd, J ¼ 16.5, 45.2 Hz, 1H), 2.77 (t, J ¼ 7.2 Hz, 2H), 2.69– 1.64 (m, 3H), 0.96 (d, J ¼ 4.7 Hz, 3H), 0.94 (d, J ¼ 4.7 Hz, 3H). 13
C
2.59 (m, 1H), 2.45–2.34 (m, 1H), 2.12 (s, 3H). 13C NMR (125 MHz, NMR (125 MHz, DMSO-d6) d 170.9, 163.9, 162.6, 150.4, 147.8,
CDCl3) d 170.6, 163.2, 147.8, 146.1, 142.8, 131.2, 131.0, 126.7, 120.5, 144.2, 143.5, 139.3, 121.3, 50.0, 41.2, 24.3, 22.9, 21.6. HRMS m/z
114.4, 52.7, 32.4, 30.3, 15.5. HRMS m/z for C16H16N6O2S [M + H]+ for C17H20N6O3 [M + H]+ calcd 357.1670. Found: 357.1678.
calcd 357.1128. Found: 357.1125.
(S)-N-(1-(2-Isonicotinoylhydrazineyl)-3-methyl-1-oxobutan-2-yl)
(S)-N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-
pyrazine-2-carboxamide (12b). White amorphous, yield (87%),
1
ꢀ
2-yl)pyrazine-2-carboxamide (11e). Light yellow amorphous, yield mp 175–177 C; H NMR (500 MHz, DMSO-d6) d 10.74 (s, 1H),
(90%), mp 180–182 ꢀC; 1H NMR (500 MHz, DMSO-d6) d 9.65 (d, J 10.44 (s, 1H), 9.23 (s, 1H), 8.92 (s, 1H), 8.82–8.72 (m, 3H), 8.50
¼ 6.4 Hz, 1H), 9.11 (s, 1H), 8.91 (s, 1H), 8.80 (s, 1H), 8.29 (d, J ¼ (d, J ¼ 10.6 Hz, 1H), 7.77 (d, J ¼ 4.6 Hz, 2H), 4.59–4.52 (m, 1H),
8.2 Hz, 1H), 8.22 (d, J ¼ 8.2 Hz, 1H), 7.82 (t, J ¼ 7.4 Hz, 1H), 7.65 2.25–2.15 (m, 1H), 1.04 (d, J ¼ 6.6 Hz, 3H), 0.98 (d, J ¼ 6.6 Hz,
(t, J ¼ 7.4 Hz, 1H), 7.36 (d, J ¼ 7.0 Hz, 2H), 7.25 (t, J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) d 170.1, 164.39, 162.8, 150.9,
2H), 7.21–7.14 (m, 1H), 6.20–6.12 (m, 1H), 3.49 (d, J ¼ 6.3 Hz, 148.5, 144.4, 144.0, 139.7, 121.8, 57.0, 40.5, 40.4, 40.2, 40.0, 39.8,
2H). 13C NMR (125 MHz, DMSO-d6) d 170.5, 163.6, 148.1, 145.4, 39.7, 39.5, 31.7, 19.6, 18.7. HRMS m/z for C16H18N6O3 [M + H]+
143.9, 143.6, 136.8, 131.2, 130.6, 129.1, 128.4, 126.8, 120.3, calcd 343.1513. Found: 343.1524.
113.9, 54.7, 36.1. HRMS m/z for C20H16N6O2 [M + H]+ calcd
N-((2S,3S)-1-(2-Isonicotinoylhydrazineyl)-3-methyl-1-oxopentan-
373.1408. Found: 373.1412.
2-yl)pyrazine-2-carboxamide (12c). White amorphous, yield
N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-2-yl) (80%), mp 168–170 ꢀC; 1H NMR (500 MHz, DMSO-d6) d 10.75 (s,
pyrazine-2-carboxamide (11f). Light yellow amorphous, yield 1H), 10.46 (s, 1H), 9.23 (s, 1H), 8.92 (s, 1H), 8.82–8.72 (m, 3H),
(87%), mp 172–174 ꢀC; 1H NMR (500 MHz, DMSO-d6) d 9.66 (d, J 8.52 (d, J ¼ 9.2 Hz, 1H), 7.78 (d, J ¼ 4.0 Hz, 2H), 4.63–4.53 (m,
¼ 6.5 Hz, 1H), 9.12 (s, 1H), 8.92 (s, 1H), 8.81 (s, 1H), 8.29 (d, J ¼ 1H), 2.09–1.92 (m, 1H), 1.66–1.50 (m, 1H), 1.30–1.12 (m, 1H),
8.2 Hz, 1H), 8.23 (d, J ¼ 8.2 Hz, 1H), 7.82 (t, J ¼ 7.4 Hz, 1H), 7.65 1.10–0.85 (m, 6H). 13C NMR (125 MHz, DMSO-d6) d 170.2, 164.4,
(t, J ¼ 7.4 Hz, 1H), 7.36 (d, J ¼ 7.0 Hz, 2H), 7.25 (t, J ¼ 6.8 Hz, 162.8, 150.9, 148.5, 144.4, 144.0, 139.8, 121.8, 56.1, 55.1, 38.2,
2H), 7.21–7.14 (m, 1H), 6.21–6.13 (m, 1H), 3.50 (d, J ¼ 6.4 Hz, 37.7, 26.2, 25.0, 15.7, 14.9, 12.0, 11.4. HRMS m/z for C17H20N6O3
2H). 13C NMR (125 MHz, DMSO-d6) d 170.5, 163.6, 148.1, 145.4, [M + H]+ calcd 357.1670. Found: 357.1680.
143.9, 143.6, 136.8, 131.2, 130.6, 129.1, 128.4, 126.8, 120.3,
113.9, 54.6, 36.0. HRMS m/z for C20H16N6O2 [M + H]+ calcd 2-yl)pyrazine-2-carboxamide (12d). White amorphous, yield
373.1408. Found: 373.1410.
(78%), mp 145–147 ꢀC; 1H NMR (500 MHz, DMSO-d6) d 10.75 (s,
(S)-N-(1-(2-Isonicotinoylhydrazineyl)-4-(methylthio)-1-oxobutan-
(S)-N-(1-(1H-Benzo[d][1,2,3]triazol-1-yl)-3-(1H-indol-3-yl)-1- 1H), 10.40 (s, 1H), 9.20 (s, 1H), 8.89 (s, 1H), 8.87 (d, J ¼ 8.3 Hz,
oxopropan-2-yl)pyrazine-2-carboxamide (11g). Light yellow 1H), 8.80–8.71 (m, 3H), 7.77 (d, J ¼ 4.7 Hz, 2H), 4.77 (dd, J ¼ 14.5,
amorphous, yield (86%), mp 169–171 ꢀC; 1H NMR (500 MHz, 7.1 Hz, 1H), 2.66–2.53 (m, 2H), 2.19–2.11 (m, 2H), 2.07 (s, 3H). 13
C
DMSO-d6) d 10.87 (s, 1H), 9.47 (d, J ¼ 6.8 Hz, 1H), 9.12 (s, NMR (125 MHz, DMSO-d6) d 170.1, 164.0, 162.9, 150.4, 147.8,
1H), 8.91 (s, 1H), 8.79 (s, 1H), 8.29 (d, J ¼ 8.2 Hz, 1H), 8.21 (d, 144.3, 143.4, 139.3, 121.3, 51.0, 31.8, 29.4, 14.6. HRMS m/z for
J ¼ 8.2 Hz, 1H), 7.82 (t, J ¼ 7.4 Hz, 1H), 7.69–7.61 (m, 2H), C16H18N6O3S [M + H]+ calcd 375.1234. Found: 375.1233.
7.30 (s, 1H), 7.05 (t, J ¼ 7.4 Hz, 1H), 6.94 (d, J ¼ 7.4 Hz, 1H),
(S)-N-(1-(2-Isonicotinoylhydrazineyl)-1-oxo-3-phenylpropan-2-yl)
7.16 (s, 1H), 7.04 (t, J ¼ 7.4 Hz, 1H), 6.92 (t, J ¼ 7.1 Hz, 1H), pyrazine-2-carboxamide (12e). Light yellow amorphous, yield
6.20 (dd, J ¼ 13.3, 6.4 Hz, 1H), 5.76 (s, 1H), 3.64–3.58 (m, 2H). (95%), mp 177–179 ꢀC; 1H NMR (500 MHz, DMSO-d6) d 10.81 (s, 1H),
13C NMR (125 MHz, DMSO-d6) d 172.8, 162.4, 147.7, 143.8, 10.51 (s, 1H), 9.12 (s, 1H), 8.87 (s, 1H), 8.83–8.69 (m, 4H), 7.79 (s, 2H),
143.3, 143.6, 136.0, 131.1, 130.5, 127.1, 123.5, 121.0, 120.3, 7.38–7.14 (m, 5H), 5.01–4.98 (m, 1H), 3.29–3.15 (m, 2H). 13C NMR
118.1, 113.7, 111.3, 109.4, 52.8, 26.5. HRMS m/z for (125 MHz, DMSO-d6) d 170.0, 163.9, 162.5, 150.4, 147.8, 144.1, 143.4,
C
22H17N7O2 [M + H]+ calcd 412.1516. Found: 412.1524.
20460 | RSC Adv., 2019, 9, 20450–20462
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139.3, 137.3, 129.3, 128.2, 126.5, 121.3, 52.8, 37.5. HRMS m/z for Gram-negative (Klebsiella pneumonia, Proteus vulgaris, Pseudo-
C20H18N6O3 [M + H]+ calcd 391.1513. Found: 391.1510.
monas aeruginosa) clinical isolate bacteria utilizing the standard
N-(1-(2-Isonicotinoylhydrazineyl)-1-oxo-3-phenylpropan-2-yl) technique.21
pyrazine-2-carboxamide (12f). Light yellow amorphous,
Antimycobacterial studies. Antimycobacterial properties of
yield (94%), mp 143–145 ꢀC; 1H NMR (500 MHz, DMSO-d6) the synthesized compounds and standards were screened against
d 10.81 (s, 1H), 10.52 (s, 1H), 9.12 (s, 1H), 8.88 (s, 1H), 8.84– clinical isolate tuberculous (Mycobacterium bovis) and non-
8.69 (m, 4H), 7.79 (s, 2H), 7.38–7.11 (m, 5H), 4.99–4.89 (m, tuberculous (Mycobacterium marinum, Mycobacterium fortuitum)
1H), 3.29–3.15 (m, 2H). 13C NMR (125 MHz, DMSO-d6) strains using the standard technique21,22 and against Mycobacte-
d 170.1, 164.0, 162.6, 150.5, 147.8, 144.1, 143.5, 139.3, 137.4, rium tuberculosis by the following described method. Highly
129.3, 128.2, 126.5, 121.4, 52.9, 37.5. HRMS m/z for virulent M. tuberculosis Erdman strain expressing luciferase (Mtb-
C
20H18N6O3 [M + H]+ calcd 391.1513. Found: 391.1508.
(S)-N-(3-(1H-Indol-3-yl)-1-(2-isonicotinoylhydrazineyl)-1-oxopropan- mented with oleic acid, dextrose, albumin, catalase (OADC) and
Lux Erdman) was grown in 7H9 Middlebrook media supple-
2-yl)pyrazine-2-carboxamide (12g). Light yellow amorphous, yield 0.05% tween 80 at 37 ꢀC. PH of the culture media was maintained
(78%), mp 243–245 ꢀC; 1H NMR (500 MHz, DMSO-d6) d d 10.80 (s, at neutral pH (pH ¼ 7.0). All the antibiotics were dissolved in
1H), 10.52 (s, 1H), 9.25 (s, 1H), 8.84 (s, 1H), 8.70–8.59 (m, 3H), 7.74– 100% DMSO at a nal concentration of 10 mg mLꢁ1 106 bacteria
7.60 (m, 6H), 7.35–7.08 (m, 4H), 4.64–4.59 (m, 1H), 3.24–3.17 (m, per mL were incubated with increasing concentration of antibi-
2H). 13C NMR (125 MHz, DMSO-d6) d 170.6, 165.4, 162.8, 150.1, otics for 7 days in a round bottom 96 well plate without shaking
148.1, 146.0, 144.0, 144.0, 136.7, 134.0, 127.4, 123.4, 121.9, 120.0, in a nal volume of 300 ml. Bacterial growth was determined by
119.8, 118.6, 112.3, 110.0, 55.8, 27.8. HRMS m/z for C22H19N7O3 [M measuring the luciferase expression (expressed in counts) in
+ H]+ calcd 430.1622. Found: 430.1638.
a PerkinElmer IVIS-spectrum imaging system kept at the BSL-3
dried facility of Albany Medical College, Albany, NY. Antibiotics
N0-Isonicotinoylpyrazine-2-carbohydrazide (13).
A
heavy-walled Pyrex tube containing a small stir bar was charged concentrations manifesting >85% growth inhibition as measured
with benzotriazole activated pyrazinoic acid 8 (0.7 mM) and isoni- by a decline in luciferase counts was considered as effective
azid 4 (0.7 mM) dissolved in THF (3 mL) along with trimethylamine concentration for antimycobacterial activity.
(1.1 mM). The reaction mixture was exposed to microwave irradia-
Antiproliferative properties. Antiproliferative properties of
tion (50 W) at a temperature of 70 ꢀC for 1 h. The mixture was the synthesized compounds were tested against RPE1 (normal
allowed to cool through an inbuilt system until the temperature had human immortalized retinal epithelial) cell line at 100 mM to
fallen below 30 ꢀC (ca. 10 min). Upon completion the reaction determine the toxicity/selectivity towards normal cell by the
mixture was quenched with ice cold water and the solid obtained standard MTT technique.23
was ltered and washed with 10% Na2CO3 and water to give the
desired compound. White amorphous, yield (88%), mp 208–
Computational studies
210 C;17 1H NMR (500 MHz, DMSO-d6) d 10.96 (bs, 1H), 10.91 (s,
ꢀ
2D-QSAR modelling. 2D-QSAR modeling was conducted by
the standard technique utilizing CODESSA-Pro soware.24
3D-pharmacophore modeling. 3D-pharmacophore modeling
was undertaken by the standard technique utilizing Discovery
Studio 2.5 soware.26
1H), 9.22 (s, 1H), 8.94 (s, 1H), 8.82–8.77 (m, 3H), 7.82 (d, J ¼ 4.7 Hz,
2H). 13C NMR (125 MHz, DMSO-d6) d 164.0, 162.3, 150.5, 148.1,
144.1, 143.8, 143.7, 139.4, 121.3. HRMS m/z for C11H9N5O2 [M + H]+
calcd 244.0829. Found: 244.0831.
N0-Isonicotinoylisonicotinohydrazide (16). A dried heavy-
walled Pyrex tube containing a small stir bar was charged with
benzotriazole activated isonicitinic acid 15 (0.7 mM) and isoni-
azid 4 (0.7 mM) dissolved in THF (3 mL) along with trimethyl-
amine (1.1 mM). The reaction mixture was exposed to microwave
irradiation (50 W) at a temperature of 70 ꢀC for 1 h. The mixture
was allowed to cool through an inbuilt system until the temper-
ature had fallen below 30 ꢀC (ca. 10 min). Upon completion, the
reaction mixture was quenched with ice-cold water and the solid
obtained was ltered, washed with 10% Na2CO3 and water to give
the desirꢀed compound 16. White amorphous, yield (91%), mp
212–214 C;18–20 1H NMR (500 MHz, DMSO-d6) d 10.95 (bs, 2H),
8.80 (d, J ¼ 4.6 Hz, 2H), 7.82 (d, J ¼ 4.6 Hz, 2H). 13C NMR (125
MHz, DMSO-d6) d 164.3, 150.5, 139.3, 121.3. HRMS m/z for
C12H10N4O2 [M + H]+ calcd 243.0877. Found: 243.0886.
ADMET studies
Computed ADMET studies were conducted by the standard
technique utilizing Discovery Studio 2.5 soware.23
Conflicts of interest
The authors have declared no conict of interest.
Acknowledgements
We thank the Center for Undergraduate Research and Scholar-
ship (CURS), Translational Research Program (TRP), and Schol-
arly Activity Award at Augusta University for nancial support.
Notes and references
Biological studies
Aerobic antibacterial studies. Antibacterial properties were
investigated for the synthesized compounds against a variety of
Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and
1 World Health Organization (WHO), The world health report:
2018 — Monitoring Health for the SDGs, http://www.who.int/
gho/publications/world_health_statistics/2018/en/.
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