Novel pyrazine based anti-tubercular agents: Design, synthesis, biological evaluation and in silico studies

Article abstract of DOI:10.1016/j.bioorg.2020.103610

TB continues to be a leading health threat despite the availability of powerful anti-TB drugs. We report herein the design and synthesis of various hybrid molecules comprising pyrazine scaffold and various formerly identified anti-mycobacterial moieties. Thirty-one compounds were screened in vitro for their activity against Mycobacterium tuberculosis H37Rv strain using MABA assay. The results revealed that six compounds (8a, 8b, 8c, 8d, 14b and 18) displayed significant activity against Mtb with MIC values ≤6.25 μg/ml versus 6.25 μg/ml for pyrazinamide. The most active compounds were then assessed for their in vitro cytotoxicity against PBMC normal cell line using MTT assay and showed SI > 200. Several in silico studies have been carried out for target fishing of the novel compounds such as shape-based similarity, pharmacophore mapping and inverse docking. Based on this multi-step target fishing study, we suggest that pantothenate synthetase could be the possible target responsible for the action of these compounds. The most active compounds were then successfully docked into the active site of pantothenate synthetase enzyme with favorable binding interactions. In addition, in silico prediction of physicochemical, ADMET and drug-like properties were also determined indicating that compounds 8b, 8c and 8d are promising candidates for the development of new anti-TB agents with enhanced activity and better safety profile.

Full text of DOI:10.1016/j.bioorg.2020.103610

Journal Pre-proofs
Novel Pyrazine Based Anti-tubercular Agents: Design, Synthesis, Biological
Evaluation and In Silico Studies
Nayera W. Hassan, Manal N. Saudi, Yasser S. Abdel-Ghany, Azza Ismail,
Perihan A. Elzahhar, Dharmarajan Sriram, Rasha Nassra, Marwa M. Abdel-
Aziz, Soad A. El-Hawash
PII:
S0045-2068(19)31273-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.103610
YBIOO 103610
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 August 2019
2 December 2019
20 January 2020
Please cite this article as: N.W. Hassan, M.N. Saudi, Y.S. Abdel-Ghany, A. Ismail, P.A. Elzahhar, D. Sriram, R.
Nassra, M.M. Abdel-Aziz, S.A. El-Hawash, Novel Pyrazine Based Anti-tubercular Agents: Design, Synthesis,
Biological Evaluation and In Silico Studies, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.103610
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.

Novel Pyrazine Based Anti-tubercular Agents: Design, Synthesis,
Biological Evaluation and In Silico Studies
Nayera W. Hassan1, Manal N. Saudi1, Yasser S. Abdel-Ghany1, Azza Ismail1, Perihan
A. Elzahhar1, Dharmarajan Sriram2, Rasha Nassra3, Marwa M. Abdel-Aziz4, Soad A.
El-Hawash1*
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria 21521, Egypt.
2Medicinal Chemistry and Drug Discovery Research Laboratory, Pharmacy Group, Birla
Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar,
Telangana 500078, India
3Department of Medical Biochemistry, Faculty of Medicine, Alexandria University,
Alexandria, Egypt.
4The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo
11759, Egypt.
ABSTRACT
TB continues to be a leading health threat despite the availability of powerful anti-TB
drugs. We report herein the design and synthesis of various hybrid molecules comprising
pyrazine scaffold and various formerly identified anti-mycobacterial moieties. Thirty-one
compounds were screened in vitro for their activity against Mycobacterium Tuberculosis
H37Rv strain using MABA assay. The results revealed that six compounds (8a, 8b, 8c,
8d, 14b and 18) displayed significant activity against Mtb with MIC values ≤ 6.25 µg/ml
versus 6.25 µg/ml for pyrazinamide. The most active compounds were then assessed for
their in vitro cytotoxicity against PBMC normal cell line using MTT assay and showed SI
> 200. Several in silico studies have been carried out for target fishing of the novel
compounds such as shape-based similarity, pharmacophore mapping and inverse
docking. Based on this multi-step target fishing study, we suggest that pantothenate
synthetase could be the possible target responsible for the action of these compounds.
The most active compounds were then successfully docked into the active site of
1

pantothenate synthetase enzyme with favorable binding interactions. In addition, in silico
prediction of physicochemical, ADMET and drug-like properties were also determined
indicating that compounds 8b, 8c and 8d are promising candidates for the development
of new anti-TB agents with enhanced activity and better safety profile.
Keywords: Antimycobacterial evaluation; Pyrazine analogs; Hydrazide/hydrazones;
Target fishing; Inverse docking; Drug-likeness.
*Corresponding Authors:
Soad A. El-Hawash, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Alexandria University, Alexandria, Egypt.
Email: Soad.elhawash@alexu.edu.eg
Soadhawash@yahoo.com
2

1. Introduction
Tuberculosis (TB) is one of the most fatal infectious diseases caused by the bacillus
Mycobacterium tuberculosis (Mtb) [1]. It has been and still remains a high priority public
health threat throughout the world, significantly in developing nations [2]. According to
the latest World Health Organization (WHO) report, it is an ailment affecting
approximately 10 million people each year and is enlisted among the top causes of death
worldwide [3]. For the past five years, it preceded HIV/AIDS and has been the principal
reason for death from a sole infectious agent [3]. The spread of drug-resistant TB (DR-
TB) is a global ill health problem that hinders the success of TB management programs.
The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-
resistant TB (XDR-TB) has exacerbated the global TB scenario substantially [4].
Nowadays, the standard chemotherapy recommended by the WHO comprises 2 months
of intensive, directly observed treatment (DOT) with four first-line drugs isoniazid (INH),
rifampin (RIF), pyrazinamide (PZA) and ethambutol (EMB), followed by a minimum of 4
months of INH and RIF. MDR-TB necessitates a further 2 years of treatment with
combination therapy including three of second-line drugs, such as quinolones,
aminoglycosides, D-cycloserine in addition to any first-line drug to which the isolate is
susceptible [1, 5]. The success of the prevailing anti-TB drugs enticed the general view
that TB could be effectively held in reserve and even eradicated. In spite of half a century
of anti-TB therapy, one-third of the world’s population asymptomatically still harbor a
dormant or latent form of Mtb with a lifetime risk of disease reactivation [6]. Therefore, a
key challenge facing the anti-TB drug discovery is identifying new drugs with good
sterilizing activity that can eliminate ‘persisters’, remaining after the growing bacteria have
been abolished by bactericidal agents [7]. The obstinate nature of persistent infection
and upsurge in MDR-TB and XDR-TB are the main challenges for effective treatment of
TB with many of the currently existing anti-TB drugs [8].
Thus, there is an imperative need for finding new anti-TB drugs with enhanced properties
such as better activity against DR strains, less toxicity, shorter duration of therapy and
prompt mycobactericidal mechanism of action [9].
3

PZA is a critical first-line drug for TB treatment as it constitutes, along with INH and RIF,
the keystone of contemporary TB therapy [10]. It plays a unique role in reducing therapy
duration from 9–12 months to 6 months. This is because, in conjunction with RIF, PZA
is the solitary active drug to retain the so-called sterilizing activity as it kills a population
of dormant non-growing tubercle bacilli of low metabolic activity in acidic pH
environments. Other anti-TB drugs lose their activity under these acidic conditions [11,
12]. In spite of PZA utilization in clinical practice since the 1950s, its complex mechanism
of action remains inapprehensible among the anti-TB drugs [13]. Many efforts have been
attempted to elucidate the exact mechanism of action behind the sterilizing effect of PZA.
Conversion of PZA to pyrazinoic acid (POA) could play the key role in its action, since
PZA-resistant strains lack the enzyme responsible for the conversion to POA (namely
pyrazinamidase, PZase) [14, 15]. Based chiefly on biochemical clues, several separate
proteins were proposed to be targeted by POA. POA was suggested to inhibit fatty acid
synthesis via fatty acid synthase-I (FAS-I) [16] and trans-translation, a process that
releases ribosomes paused during translation, by interaction with ribosomal protein
S1/RpsA [17]. However, queries have been raised regarding these suggested
mechanisms [18, 19]. As well, it was hypothesized that POA, a structural analog of
quinolinic acid, suppreses the catalytic activity of quinolinic acid phosphoribosyl
transferase (QAPRTase) and, hence, de novo NAD biosynthesis [20]. POA was also
suggested to bind to aspartate decarboxylase (PanD) triggering its degradation by Mtb,
thus blocking biosynthesis of the crucial bacterial Coenzyme A [21]. Recently, it was
found that extremely high concentrations (mM) of POA could inhibit guanosine
pentaphosphate synthase (GpsI) which is implicated in nucleic acid and guanosine
pentaphosphate (ppGpp) metabolism [22]. Nonetheless, many reports disproved the
previously stated mechanisms of action [23-27].
In addition, the pyrazine ring manifested its status in the field of antimycobacterial agents
[28-32]. Numerous active anti-tubercular agents containing pyrazinamide nucleus are
frequently encountered in literature. These agents either contained a linker -such as the
carbohydrazide (A) or chalcone (B) linker or were directly attached (C) to various
aromatic/heterocyclic rings; as shown in Figure 1.
4

O
O
O
N
N
N
N
N
N
N
R
NH2
N
N
N
H
H
O
(A)
PZA
Morinamide
R = 2-Furyl, 4-ClC6H4, 4-NO2C6H4
O
S
N
N
O
N
O
N
NO2
N
(B)
(C)
Figure 1. Reported pyrazine based anti-tubercular agents
Based on the aforementioned facts, the present work illustrates the synthesis of a series
of compounds by introducing carbohydrazide linker to afford pyrazine-2-carbohydrazide
derivatives (6a-c, 8a-d). This selection was based on the fact that many anti-TB
hydrazones act as prodrugs [33-35]. In addition, further structural modification of the
carbohydrazide linker has been proposed by inverting the linker such that the carbonyl
group is directly attached to the bioactive heterocyclic moiety rather than the pyrazine
ring (10, 11, 12a-e) to evaluate the influence of the different linkers on the biological
activity against Mtb.
Furthermore, in an effort to decrease the flexibility of the linker, the carbohydrazide
function was incorporated into a ring structure (13a-e) to investigate the effect of such
structural modification on the anticipated biological activity. Additionally, pyrazine bearing
chalcones have been also proposed (14a,b) owing to their widely-recognized anti-TB
activity since 1950's [36]. Moreover, a series of hybrid molecules was designed by directly
attaching the pyrazine ring with various bioactive heterocyclic moieties (15-24) to
investigate their anti-TB activity and the necessity of linkers for the activity. The selected
heterocyclic moieties (pyrazoline, isoxazole, pyrimidine and dihydropyridine) have
attracted continuing interest and were found to be promising anti-TB structural units [37-
40]. Finally, in a trial to investigate the effect of hetero atom on the anticipated biological
5

activity, a representative carbocyclic analog (25) was synthesized to evaluate its anti-TB
effect (Figure 2).
Figure 2. Outline for the design of the target compounds.
It is noteworthy mentioning that an interesting and fruitful approach for exploring
biologically relevant space to develop new bioactive molecules is “Biology-oriented
synthesis or BIOS”. It entails efficient building on existing clinically-useful agents to
generate novel bioactive compounds with enhanced biological activities [41]. We
employed this approach for the synthesis of the target hydrazones 6 and 8, reckoning on
pyrazinoic acid, the active form of pyrazinamide (PZA) (Figure 2). On the other hand, a
diversity-guided strategy, known as diversity-oriented synthesis or DOS, was engaged in
order to attain high level of functional and structural versatility that can increase the
chances of finding molecules with exciting biological characters [42]. This was
productively achieved via using a simple starting material such as acetyl pyrazine, that
6

was converted into a set of structurally diversified pyrazine derivatives (10-25) in few
synthetic steps as depicted in Figure 2.
The present work is therefore directed towards the design and synthesis of various novel
pyrazine scaffold containing compounds to evaluate their anti-TB activity. Cytotoxicity
assay, physicochemical and pharmacokinetic parameters were also performed. A target
fishing study is carried out to elucidate the possible mechanism of action.
2. Results and Discussion
2.1. Chemistry
The synthetic routes adopted for the preparation of the target compounds are outlined in
Schemes 1-4.
The starting material, 2-acetyl pyrazine (2) was prepared from pyrazine-2-carbonitrile via
Grignard reaction according to reported procedures [43, 44].
Scheme 1 describes the preparation of the intermediate pyrazine-2-carboxylic acid
hydrazide (5) through alkaline hydrolysis of pyrazine-2-carbonitrile (1) to obtain pyrazine-
2-carboxylic acid (3) followed by esterification and hydrazinolysis to give 5 in a good yield.
The target hydrazones 6a-c and 8a-d were prepared by the condensation of the
hydrazide 5 with the appropriate ketones or pyrazole aldehydes (7a-d) in ethanol
1
according to the procedure described in literature [45]. The H-NMR spectra for
compounds 6a-c showed one characteristic upfield singlet corresponding to methyl
protons at δ 2.42-2.57 ppm and a D2O exchangeable singlet corresponding to NH proton
at δ 11.15-11.24 ppm. The latter values coincided with reported chemical shifts of NH for
similar compounds of 9-12 ppm for the E-isomer [46]. Interestingly, spectrum of
compound 6a displayed additional D2O exchangeable singlet corresponding to OH at δ
15.39 ppm. This could be attributed to the existence of amide-iminol tautomerism at a
ratio 1:2 which indicated that iminol tautomer was the predominant one. The careful
1
inspection of the H-NMR spectra of compounds 8a-d indicated the stereoselectivity of
the condensation step as depicted by the presence of only one imino-hydrogen (N=CH)
signal for each N-acylhydrazone derivative at higher chemical shifts at δ 8.80-8.93 ppm,
which was attributed to the (E)-stereoisomer [47].
7

In scheme 2, the key intermediate cyanoacetic acid hydrazide 9 was prepared by
condensing 2 with cyanoacetic acid hydrazide in boiling absolute ethanol, applying the
reported reaction conditions of related compounds [48]. The target 2-imino-2H-
chromene-3-carbohydrazide derivative 10 was obtained by reacting 9 with
salicylaldehyde in presence of piperidine as a catalyst. Acidic hydrolysis of 10 afforded
1
the corresponding 2-oxo-2H-chromene-3-carbohydrazide derivative 11. The H-NMR
spectrum of 9 showed one D2O exchangeable signal assigned for deshielded NH proton
at δ 11.34 ppm in addition to an upfield singlet assigned for methylene protons at δ 4.33
ppm, while that of 10 revealed the disappearance of the methylene protons and showed
an extra D2O exchangeable signal due to the imino proton at δ 9.33 ppm. The 1H-NMR
spectrum of 11 lacked the signal for =NH present in the precursor and its IR spectrum
displayed an absorption band characteristic to lactone C=O at 1700 cm-1. Gewald reaction
of compound 9 yielded the target compounds 12a-e, which then underwent cyclization in
a mixture of triethylorthoformate and acetic anhydride to give 13a-e. The 1H-NMR spectra
for 12a-e exhibited a broad D2O exchangeable singlet at δ 7.36-8.02 ppm, corresponding
1
to NH2 protons. The H-NMR spectra for 13a-e lacked signals for NH and NH2 protons
and indicated a characteristic singlet at δ 8.54-8.71 ppm corresponding to –CH=N proton.
Scheme 3 illustrates the synthesis of the target chalcones 14a, b via the condensation of
1
2 with aromatic aldehydes. H-NMR spectrum of compound 14a,b revealed characteristic
signals for olefinic protons in the range of
δ 6.93-8.14 ppm. Interestingly, the
magnitude of the olefinic vicinal coupling constant between the two protons (J=16 Hz) in
the1H-NMR spectrum of compound 14b indicated the existence of the E configuration. In
addition, the IR spectra of those compounds showed expected shift of C=O stretching
frequency to 1661-1662 cm-1 due to increased conjugation. Compound 14a was refluxed
with hydrazine hydrate or phenyl hydrazine in ethanol to give the respective pyrazole
derivatives 15a,b, while its reaction with hydrazine hydrate in acetic acid afforded the
ethanone derivative 16. Additionally, 14a was treated with hydroxylamine hydrochloride
to give the corresponding isoxazole derivative 17. Moreover, reaction of 14a with
thiosemicarbazide gave the corresponding carbothioamide derivative 18 which was
1
further reacted with phenacyl bromide to afford phenylthiazole derivative 19. H-NMR
spectra of compounds 15a, b, 16, 18 and 19 showed an Habx pattern due to coupling of
8

the pyrazoline ring protons. The existence of the methylenic protons (Ha and Hb)
separately in two peaks, clearly postulated the magnetic non-equivalence of these two
1
protons. H-NMR spectrum of 17 lacked the protons' signals of the Habx system and
showed a characteristic singlet at δ 6.88 ppm assigned for the isoxazole-C4-H. It is worth
1
noting that H-NMR spectrum of 18 showed two D2O exchangeable singlets at δ 8.16
ppm and δ 8.26 ppm assigned to the two non-equivalent NH2 protons which could be
attributed to the existence of intra-molecular hydrogen bonding. Furthermore, synthesis
of the target compounds 20 and 21, was achieved by reacting 14a with urea and thiourea,
1
respectively. H-NMR spectrum of 20 displayed two D2O exchangeable singlets at δ 7.10
and 7.13 ppm assigned for two NH protons which confirmed the presence of the
pyrimidinone ring in tautomerized form. 1H-NMR spectrum of 21 showed a characteristic
singlet at δ 8.47 ppm assigned for pyrimidine-C5-H, in addition to two D2O exchangeable
singlets assigned for NH and SH at δ 8.63 and 13.32 ppm, respectively. Other protons'
signals characteristic to the molecule appeared as paired signals which confirmed the
existence of thione-thiol tautomers at a ratio 2:3.
In scheme 4, one pot reaction conditions [49] were adopted to obtain the target
compounds 22-25. Condensation of 2 and the appropriate aromatic aldehyde with
malononitrile, ethyl cyanoacetate or cyanothioacetamide in presence of ammonium
acetate in ethanol was carried out to afford 22, 23 and 24, respectively. Similarly, the
cyclohexene carboxylate derivative 25 was obtained by refluxing a mixture of 2, 3,4-
dimethoxybenzaldehyde and ethyl acetoacetate in 10% ethanolic sodium hydroxide. 1H-
NMR spectra for compounds 22a,b revealed one D2O exchangeable singlet at δ 6.84 and
6.91 ppm assigned for NH2 protons, respectively. Their IR spectra showed characteristic
1
absorption bands due to C≡N function at 2211 and 2214 cm-1 respectively. H-NMR
spectra of 23a,b and 24a,b revealed the appearance of D2O exchangeable singlet due to
1
NH proton in the range of δ 12.53-13.54 ppm. H-NMR spectrum of 25 elicited a
characteristic triplet quartet pattern due to the ethyl ester protons.
9

2.2. Biological Evaluation
2.2.1. Prediction of activity spectra for substances (PASS) analysis
Before performing in vitro anti-TB assay, we applied the Prediction of Activity Spectra for
Substances (PASS) to explore the biological potential of the synthesized compounds and
to prioritize them for further in vitro studies. The PASS is an in silico tool used for
predicting biological activity spectra for natural and synthetic substances, which is based
on a robust analysis of structure–activity relationships knowledge base for more than 260
000 compounds with known biological activities including drugs, drug candidates and
pharmaceutical leads. The PASS predicts the probable biological potential of the
compound based on its structure and reveals the predicted activities as the probability of
activity (Pa) and inactivity (Pi). The higher Pa value, the lower is the predicted probability
of obtaining false results in biological testing [50, 51]. Predictive anti-TB values for all
newly synthesized compounds are summarized in table 1. The results obtained revealed
that most of the tested compounds exhibited anti-TB activity with Pa values in the range
of 0.19-0.801. Interestingly, Pa values estimated for anti-TB activity for compounds 6b,c
were > 0.7, while compounds 6a, 8a-d, 9, 11, 18 were between 0.5 and 0.7; thus, these
compounds could be likely to reveal this activity in in vitro assays. PASS analysis of
compounds 13b-e, 16, 19, 22b, 23b and 25 didn't show anti-TB activity, thus compounds
13b,d,e, 16, 19 and 25 were excluded from further in vitro biological screening, while
compounds 13c, 22b and 23b were retained for further biological investigation in order to
achieve SAR study. As for the rest of tested compounds, the estimated Pa values were
found to be in the range of 0.190-0.482. The in silico results obtained were further
confirmed in vitro by MABA assay.
10

Table 1: Predictive values of anti-TB activity calculated with PASS software
Compound ID
Predicted anti-TB activity
Pa
Pi
6a
6b
6c
8a
8b
8c
8d
9
10
0.692
0.767
0.801
0.593
0.660
0.600
0.699
0.645
0.432
0.635
0.461
0.436
0.414
0.347
0.415
0.211
-
0.004
0.003
0.003
0.006
0.005
0.006
0.004
0.005
0.023
0.005
0.017
0.022
0.027
0.050
0.027
0.155
-
11
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
14a
14b
15a
15b
16
-
-
-
-
-
-
0.466
0.211
0.251
0.190
-
0.016
0.155
0.118
0.186
-
17
18
19
0.309
0.592
-
0.067
0.010
-
20
21
0.289
0.482
0.219
-
0.230
-
0.257
0.190
-
0.173
0.014
0.151
-
0.133
-
0.103
0.183
-
22a
22b
23a
23b
24a
24b
25
11

2.2.2. In vitro anti-mycobacterial assay against H37RV
The selected compounds were screened for their in vitro activity against Mtb H37Rv strain
using the microplate alamar blue assay (MABA) [52]. PZA, INH and EMB were used as
reference drugs. The results were represented as minimum inhibitory concentration
(MIC) values and presented in table 2. The tested compounds were found to be effective
inhibitors of the growth of Mtb. Compounds 8a-d, 14b and 18 exhibited promising
inhibition with MIC values ≤ 6.25 µg/ml, a value postulated by the global program for the
discovery of new anti-TB drugs as an upper threshold for the evaluation of new Mtb
therapies [53]. Compound 8b displayed significant anti-TB activity with MIC value of 0.78
µg/ml which equals two times the activity of EMB (MIC = 1.56 µg/ml) and eight times the
activity of PZA (MIC = 6.25 µg/ml). Intriguingly, its MIC value operated within the same
order of magnitude as that of the powerful anti-TB reference drug INH, yet, it is still of
lower potency. In addition, compounds 8c,d were found to be as active as EMB and
showed four times the activity of PZA. As for compound 8a, it showed good activity (MIC
= 3.12 µg/ml) being two times as active as PZA. Furthermore, compounds 6b,c and 12c
exhibited moderate anti-Mtb activity with MIC value of 12.5 µg/ml which is still comparable
to that of PZA. Finally, compounds 6a, 14a and 23a,b possessed weak anti-Mtb activity
with MIC value of 25 µg/ml, showing one fourth the activity of PZA.
Table 2. Anti-mycobacterial activity of the newly synthesized compounds against Mtb
H37Rv strain (ATCC 27294)
Compound No.
MIC (µg/ml)a
CLogPc
6a
6b
6c
8a
8b
8c
8d
9
10
25
-0.76
0.19
0.19
2.17
2.89
2.51
1.85
-0.55
0.31
0.71
-0.37
0.45
2.15
0.55
12.5
12.5
3.12
0.78
1.56
1.56
>25
>25
>25
>25
>25
12.5
>25
11
12a
12b
12c
12d
12

12e
13a
13c
14a
14b
15a
15b
17
18
20
21
22a
22b
23a
23b
24a
>25
>25
>25
25
0.38
-0.17
1.26
1.22
1.22
1.07
3.03
1.52
0.19
1.00
1.36
1.53
1.69
0.45
0.46
0.67
0.68
-0.68
-0.67
0.12
6.25
>25
>25
>25
3.12
>25
>25
>25
>25
25
25
>25
>25
6.25b
0.1
24b
PZA
INH
EMB
1.56
a: MIC values tested at neutral pH.
b: MIC value from testing at pH = 5.6 (acidic) [54].
c: LogP (lipophilicity) calculated using "Osiris DataWarrior" software.
2.2.3. Structure Activity Relationship (SAR)
Careful inspection of the structures of the tested compounds revealed that hybrid
molecules with carbohydrazide linker between pyrazine and 1,3-diarylpyrazole moieties
(8a-d) displayed the most potent anti-Mtb activity (MIC = 3.12, 0.78, 1.56 and 1.56 µg/ml,
respectively). In addition, presence of a substitution at position-4 of phenyl group at
pyrazole-C-3 enhanced the activity. The nature of substituents greatly affected the
activity (Br > Cl=CH3). Compound with bromo substituent (8b) displayed the highest
potency (MIC = 0.78 µg/ml) which might be attributed to the increase of lipophilicity.
Replacement of 1,3-diarylpyrazole by pyridinyl or pyrazinyl moieties (6a-c) decreased the
activity (MIC = 24.26, 12.48 and 12.48 µg/ml, respectively). Moreover, compounds 10,
11, 12a, b, d, e did not show considerable activity. This clearly revealed that inversion of
the carbohydrazide linker was an unfavorable structural modification. However,
compound 12c showed moderate anti-TB activity (MIC = 12.5 µg/ml). This might be
attributed to the presence of lipophilic chloro substituent at the phenyl ring of the
13

dihydrothiazole moiety. Restricting the linker flexibility by incorporation into a ring
structure (13c) greatly decreased the anti-TB activity (MIC = >25 µg/ml). Furthermore,
hybridization between pyrazine and thiophene moiety through three atoms linker resulted
in compound 14b which displayed good anti-TB activity (MIC = 6.25 µg/ml), while
hybridization with 3,4-dimethoxyphenyl moiety resulted in compound 14a with weak
activity. This indicated the importance of the heteroatom on the anticipated biological
activity. On the other hand, direct attachment of pyrazine with various bioactive
heterocycles (compounds 15, 17 and 20-24) did not show any enhancement of activity.
However, compound 18 exhibited significant activity (MIC = 3.12 µg/ml). Such activity
might be attributed to the presence of the pharmacophore carbothioamide moiety. It is
worth mentioning that Mtb is uniquely surrounded by a thick and waxy cell wall, thus,
efficient anti-TB drugs should have a reasonable lipophilicity to penetrate the cell wall
[55]. Comparison of the logP data, presented in table 2, of the most active compounds
with those of the reference drugs revealed that compounds 6b,c, 8a-d, 12c and 14b were
more lipophilic in nature. Also, by correlating their calculated logP and MIC values, it is
evident that the most potent compound 8b was the most lipophilic.
2.2.4. In vitro cytotoxicity assay
In order to eliminate the possibility that the anti-TB activity of the tested compounds arises
from general toxicity and also to highlight their safety profile on normal cell, the
compounds exhibiting significant anti-TB activity (8a-d, 14b and 18) with MIC values ≤
6.25 µg/ml were further screened for cell viability assay using MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay against peripheral blood
mononuclear cells (PBMC). The MTT assay is a quantitative colorimetric assay for
assessing cell metabolic activity of enzymes that reduce tetrazolium dye to its insoluble
formazan, giving a purple color. Its main application is to assess the viability (cell
counting) and the proliferation of cells. It can also be used to determine cytotoxicity of
medicinal agents and toxic materials, since those agents would stimulate or inhibit cell
viability and growth [56]. The cytotoxicity results of the tested compounds, expressed as
inhibitory values (IC50 in µg/ml), are presented in table 3. The selectivity index (SI) of
each compound was also determined as the ratio between in vitro cytotoxicity (IC50 in
14

µg/ml) and anti-TB activity (MIC in µg/ml), as shown in table 3. SI is used to estimate the
therapeutic window of a drug and to identify drug candidates for further studies. According
to Orme et al. [57], candidates for new anti-TB drugs must have an index equal to or
higher than 10, with MIC lower than 6.25 mg/ml and a low cytotoxicity. SI values of
compounds equal to or greater than 10 are considered nontoxic. The results revealed
that the tested compounds exhibited very low cytotoxicity (IC50 value ranging from 589.1
to 1174.6 µg/ml). This also indicated that the active derivatives target Mtb to a greater
extent compared to normal human cells.
Table 3: Cytotoxicity and selectivity index (SI) for compounds (8a-d, 14b and 18) against
PBMCs
Compound
Cytotoxicity
(IC50 in µg/ml)
613.4
MIC (µg/ml)
Selectivity index (SI)
ID
8a
8b
8c
8d
14b
18
3.12
0.78
1.56
1.56
6.25
3.12
196.6
1085.7
411.9
752.9
122.8
188.8
846.9
642.6
1174.6
767.8
589.1
2.3. Bioactivity profiling and molecular modeling
2.3.1. Shape-based similarity
In an attempt to elucidate the mechanism of action and identify the putative molecular
target behind the antimycobacterial activity of the most active compounds, we followed a
sequential scheme combining both ligand-based and structure-based approaches. The
first step involved the use of similarity-based method via computation of atom pair
Tanimoto coefficient (in ChemMine tools) as a means of comparison and prioritization for
structurally-related compounds [58]. This similarity index was measured between the
most active compound 8b and a set of eight pyrazine-based compounds with reported
antimycobacterial activity. It is noteworthy to mention that each of these compounds was
either tested experimentally or successfully docked into a number of mycobacterial
targets as shown in table 4. In addition, these targets were selected according to the
reported target identification pipeline for Mtb [59], in addition to the reported possible
targets for PZA (RpsA) [17] and PZA analogs (FAS-1) [60]. These targets represent
15

various roles in the metabolic pathways of Mtb, which could be utilized for the
development of new anti-TB agents. For example, FAS-1, InhA, FabH and pantothenate
synthase (PS) are involved in fatty acid biosynthesis [61] while RpsA is a vital protein
involved in protein translation [17, 62]. Decaprenylphosphoryl-β-D-ribose-2'-epimerase
(DprE1) is a potential target essential for cell growth and survival [63] and isocitrate lyase
(ICL) is an important enzyme in the glyoxylate cycle during carbohydrate starvation in Mtb
[64].
Results revealed that compounds C, F and G showed the highest similarity indices in the
range of 0.297-0.346. These compounds were reported to exert their antimycobacterial
activity via acting on isocitrate lyase (ICL), β-ketoacyl-(acyl-carrier-protein) synthase III
(FabH) and pantothenate synthetase enzymes, respectively. The lowest tanimoto
coefficients were recorded for compounds A and B representing ribosomal protein S1
(RpsA) and fatty acid synthase (FAS I). Hence, they will be excluded from the next step.
Table 4: Atom pair Tanimoto similarity values with compound 8b using ChemMine
tools.
AP Tanimoto
Similarity with
8b
Potential
Anti-TB
target
Code
Structure
Reference
A
B
0.067
0.061
RpsA
FAS1
[17]
[60]
C
0.346
ICL
[65]
D
E
0.236
DprE1
InhA
[66]
[67]
0.204
16

F
0.297
0.34
FabH
[68]
[69]
[70]
Pantothenate
synthetase
G
H
0.204
DprE1
2.3.2. Pharmacophore mapping
The pharmacophore model was built using molecular operating environment (MOE)
software version 2016.0802 [71]. It was derived from the most active six compounds in
this study (8a-d, 14b and 18). The generated model was then tested against compounds
C – H, which passed the similarity searching step.
The pharmacophore Elucidator module of the software MOE was used to obtain the
pharmacophore model. It operates first by exhaustively generating pharmacophore
queries for a set of flexibly aligned molecules, followed by assigning scores to them based
on a joint measure of their cover (how many compounds were used to generate the model
out of the initial set), overlap (how well they overlay the ligands) and accuracy (how well
they group the dataset into actives and inactives). The selected model (cover = 6, overlap
= 4.11, accuracy = 1) is represented in Figure 3.
17

(B)
(A)
Figure 3. (A) Pharmacophore model (F1, orange spherical mesh, aromatic or Pi ring
centers), (F2 and F3, green spherical meshes, hydrophobic region) and (F4, cyan
spherical mesh, hydrogen bond acceptor projection). (B) Overlay of training set
compounds (8a-d, 14b and 18) on the pharmacophore model.
The generated model showed that the following structural features were shared with the
six compounds; location of potential aromatic or Pi ring centers (F1, orange spherical
mesh), two locations of potential hydrophobic regions (F2 and F3, green spherical
meshes) and potential location of hydrogen bond acceptor (F4, cyan spherical mesh).
Using another ligand-based approach to focus the target selection process, mapping of
structures C – H, resulting from the previous step and acting on different five TB targets,
on this pharmacophore model was performed. Three of them (E, F and G) showed good
fitting with reasonable root-mean-square distance (RMSD) between the model feature
and their matching ligand target points in the range of 0.68-0.84 A (Table 5, Figure
SM1A). This might assume that our target compounds can bind to the proposed
receptors with a similar set of interactions. Hence, it can be concluded that pantothenate
18

synthetase, InhA and FabH are the candidate TB targets for the action of our compounds
up to this stage.
Furthermore, flexible alignment tool was utilized to probe similarity between the 3D
structures of the most active compounds. The top-ranked alignment with the least strain
energy (U= 113.12, F= -127.01, S= -13.89) is depicted in Figure SM1B, where a good
overlay between the six compounds accounts for their activity. As well, proper alignment
(U= 100.64, F= -166.65, S= -66.01) could be observed between the most active
compound 8b and the top-ranked compound G from the mapping results (representing
pantothenate synthetase) (Figure SM1C).
Table 5. Pharmacophore mapping results represented as root-mean-square distance
(RMSD) values.
Code
RMSD (A)
0.6867
Compound E
Compound F
Compound G
0.8433
0.6795
2.3.3. Inverse docking study
Inverse docking is a structure-based target fishing tool which entails the screening of a
small-molecule ligand for its binding complementarity against plethora of clinically
pertinent macromolecular targets. The output is a set of targets ranked relying on a
certain ‘score’. The top-scored targets represent potential candidates for binding with the
ligand [72].
In this context, in order to find a clue for the mechanism of action of the target compounds,
the final step involved an inverse docking study that was conducted on the most active
compounds (8a-d, 14b and 18) against five selected Mtb targets namely; DprE1 (PDB ID:
4FDO), pantothenate synthetase (PDB ID: 3IVX), FabH (PDB ID: 1U6S), InhA (PDB ID:
4TZK) and isocitrate lyase (PDB ID: 1F8M). The X-ray crystal structures were obtained
from the protein data bank (PDB). Docking studies were performed using Molecular
operating environment software (MOE 2016.0802).
19

Pose retrieval was carried out in order to validate the docking procedure. This was done
by redocking the co-crystallized ligands and calculating root mean square deviation
(RMSD). The predictive capability of a software is confirmed if the deviation is less than
1.5 Å. The redocking of co-crystal ligands to their respective molecular targets
reproduced the docking poses with acceptable RMSD values of less than 1.5 Å.
Superimposition of the co-crystal ligand of pantothenate synthetase enzyme (PDB ID:
3IVX) and its docked pose is shown as an example in Figure SM2.
The docking results were interpreted on the basis of docking score, number of hydrogen
bonds formed and hydrophobic interactions of the ligand with the protein and were
summarized in table SM1. Careful inspection of the docking results revealed that all the
target compounds showed good interactions within the active sites of the target proteins
(with overall scores spanning from -4.83 to -8.51 Kcal/mol) with the exception of
compound 18 docked in the active site of isocitrate lyase enzyme. In order to rationalize
the correlation between the in vitro anti-TB activity and docking results, a cross
observational analysis was performed. The most potent three compounds (8b, 8c and
8d) in the in vitro anti-TB activity were cross observed with their docking ranks on all the
studied target proteins. The results are displayed in table 6. Based on the results, the
docking pose ranking of the three compounds showed marginal deviation in favor of
pantothenate synthetase (PDB ID: 3IVX) and FabH (PDB ID: 1U6S) equally with docking
ranks of 1st, 2nd and 3rd when compared with their docking pose rankings of the other
studied targets.
Table 6: Cross observational analysis of most active three compounds with their docking
ranks against the five studied targets
Studied targets
(PDB ID)
4FDO
Ranking positions based on docking scores
8b
2
8c
4
8d
1
3IVX
2
3
1
1U6S
1
3
2
4TZK
4
3
1
1F8M
4
2
1
20

Finally, the following conclusion can be derived from this sequential scheme; the
pantothenate synthetase inhibitor, compound G behaved better than the FabH inhibitor
compound F both in terms of Tanimoto coefficient and pharmacophore mapping steps.
Moreover, it exhibited better docking scores in the inverse docking study (-7.90 to -8.51
for pantothenate synthetase versus -7.16 to -7.75 Kcal/mol for FabH for the most active
compounds 8b-d). Hence, the study suggested that pantothenate synthetase enzyme
(PDB ID: 3IVX) might be the plausible target accounting for the in vitro anti-TB activity of
our compounds.
2.3.4. Docking into pantothenate synthetase active site:
The docking poses were selected within the top-scored conformations with the best
binding interactions detected by MOE search algorithm and scoring function. In addition,
binding energy scores, formation of hydrogen bonds with the neighboring amino acid
residues and the relative positioning of the docked poses in comparison with the co-
crystallized ligand were the factors determining the binding affinities to the binding pocket
of the enzyme.
Generally speaking, the target compounds (8a-d, 14b and 18) interacted via hydrogen
bonding and hydrophobic contacts with the key amino acid residues that were also
involved in the binding of the native ligand such as His47, Gly158Lys160, Ser197 (for 8b),
His44, His47, Gly158 (for 14b) and His44, His47, Gly158, Ser197 (for 18). In addition,
they established some additional interactions that account for further stabilization of the
formed complexes as shown in table SM2, Figures 4-6 for compounds 8b, 14b and 18,
and Figures SM3-SM5 for compounds 8a,c,d. Moreover, it was evident from the overlay
of the compounds over the co-crystallized ligand FG6 that they occupied the same spatial
area of the binding pocket of the enzyme.
In details, the most energetically profitable pose of compound 8b extended smoothly in
the active site through the formation of two hydrogen bonds between two nitrogens of the
acid hydrazide part and Ser197. The carbonyl oxygen also engaged in hydrogen bond
with Lys160. Binding was also strengthened by the formation of two arene-hydrogen
interactions between the phenyl ring and Gly46 and Gly158. Two arene-hydrogen
21

contacts were also observed between p-bromophenyl and pyrazine rings and His47 and
Arg198 residues, respectively (Figure 4). As for the chalcone 14b, one of the pyrazine
nitrogens was hydrogen bonded to His47 while the sulfur atom formed two hydrogen
bonds with Val187 and Met195. As well, one of the hydrogens of ,-unsaturated
carbonyl moiety attached via hydrogen bond to Met195. Moreover, the thiophene ring
interacted with the surrounding hydrophobic residues (His44, Gly46, Gly158 and Thr186)
through arene-hydrogen interactions (Figure 5). Regarding docking of the pyrazoline
thioamide derivative 18, it was well-fitted in the active site cavity and showed almost the
same binding pattern of the native ligand. The complex formed was stabilized by a
number of interactions with some important residues in close proximity. For example,
pyrazine ring was held in the active site via two arene-hydrogen interactions with His44
and Gly158. The amino group was hydrogen bonded to His47 and Tyr82. Furthermore,
sulfur atom was hydrogen-bonded to His44 and Ser197. Hydrogen bonding was also
observed between one of the aromatic hydrogens of the dimethoxy phenyl ring and Pro38
(Figure 6).
Figure 4. Docking and binding pattern of compound 8b (in orange) into pantothenate
synthetase active site (PDB 3IVX) and its overlay over FG6 (in yellow) in 3D (left
panel). Ligand interactions in 2D (right panel).
22

Figure 5. Docking and binding pattern of compound 14b (in pink) into pantothenate
synthetase active site (PDB 3IVX) and its overlay over FG6 (in yellow) in 3D (left panel).
Ligand interactions in 2D (right panel).
Figure 6. Docking and binding pattern of compound 18 (in pink) into pantothenate
synthetase active site (PDB 3IVX) and its overlay over FG6 (in yellow) in 3D (left panel).
Ligand interactions in 2D (right panel).
2.4. In silico prediction of drug-likeness, physicochemical properties
and pharmacokinetic profile
ADME, as well as toxicological profile, are important for the ultimate success or failure of
a possible drug candidate. Adverse effects in animal models or even clinical trials can be
reduced by filtering drug candidates by their ADME properties in early stages. Thus, in
23

silico ADMET prediction will permit the parallel optimization of compound efficacy and
drug suitability, which is expected to not only improve the overall quality of drug
candidates and therefore the probability of their success, but also to lower the overall
expenses due to a reduced downstream attrition rate [73].
In the present work, compounds (8a-d and 18) that displayed significant anti-TB activity
with MIC values ≤ 3.12 µg/ml, were subjected to physicochemical properties prediction
using Data Warrior software from Osiris property explorer [74], in order to filter and
analyze the overall potential of these compounds to qualify for a drug, also comparing
them to some current anti-TB drugs. The results are recorded in table 7.
Table 7: In silico physicochemical properties of the most active compounds
Comp M.Wta CLog
. ID
Sc
HBAd HBDe Lipinski' TPSAf %AB NRO
Pb
s
Sg
TBh
violation
8a
8b
8c
8d
18
368.39
447.29
382.42
447.84
343.41
137.14 -1.02 8568
123.11 -1.20 3591
3
2.17
2.89
2.51
1.85
8.38
0.72
3.05
0.36
7
7
7
10
7
4
1
1
1
1
1
2
1
0
0
0
0
0
0
0
85.06 79.65
85.06 79.65
85.06 79.65
130.88 63.84
117.95 68.30
68.01 85.53
68.87 85.23
5
5
5
6
4
1
1
0.91 94.35
INH
PZA
4
a M.Wt: Molecular weight.
b LogP: Logarithm of compound partition coefficient between n-octanol and water.
c S: Compound's solubility in mg/L.
d HBA: Number of hydrogen bond acceptors.
e HBD: Number of hydrogen bond donors.
f TPSA: Topological polar surface area.
g %ABS: Percentage of absorption after oral administration.
h NROTB: Number of rotatable bonds.
The results revealed that the tested compounds were shown to possess one H-bond
donor and acceptable range of H-bond acceptors (7-10). The compounds also displayed
logP values in the range from 0.91 to 2.89. and molecular weights of less than 500.
Hence, the physicochemical parameters of the tested molecules were encouraging with
zero Lipinski violation.
24

Number of rotatable bonds is important for conformational changes and ultimately for the
binding to receptors or channels. Tested compounds possessed (4–6) rotatable bonds
and therefore exhibiting moderate conformational flexibility. The results showed that all
tested compounds demonstrated TPSA values within allowable range (85.06–130.88)
and generally displayed % ABS range of 63.84–79.65%, which indicated their good
bioavailability by oral administration. Furthermore, compounds (8a, 8c and 18) were
found to fulfill the requirements of solubility of more than 0.0001 mg/L [75, 76] and could
be considered as drug candidates for oral absorption.
Although Lipinski's “Rule of 5” describes the molecular properties important for a drug’s
pharmacokinetics in the human body, it does not predict the toxicity profile of the
compounds. Therefore, mutagenicity, tumorigenicity, irritation effect, and risk of
reproductive effect were also predicted for toxicity study using Osiris DataWarrior. The
toxicity results as well as computed drug-likeness scores are recorded in table 8.
Table 8: Toxicity prediction and drug-likeness scores of the most active compounds
Drug-
likeness
score
5.91
4.12
5.82
0.78
2.59
-5.78
-0.67
Toxicity
Mutagenic
Comp. ID
Tumorigenic Reproductive Irritant
effect
8a
8b
8c
8d
18
INH
PZA
None
None
None
None
None
High
None
None
None
None
None
High
None
None
None
None
High
High
High
None
None
None
None
None
High
High
High
High
The results indicated that all tested compounds lacked mutagenic, tumorigenic, irritant,
reproductive effect properties, except for compound 18 which demonstrated high risk of
reproductive effect. In addition, the tested compounds showed good safety profile
compared to the tested reference drugs. Regarding drug-likeness scores, the
investigated compounds should be considered as drug-like as they have positive values
in the range 0.78-5.91.
Furthermore, Pre-ADMET calculator [77] was used to calculate ADME properties of the
active compounds. The value of each of the following parameters was calculated and
25

compared with the optimal values of the following properties: Caco2 cell permeability
coefficient (permeability through cells derived from human colon adenocarcinoma),
Madin-Darby canine kidney (MDCK) cell permeability coefficient, blood–brain barrier
(BBB) coefficient and plasma protein binding (PPB). The results are given in table 9.
Table 9: In silico ADME profiling of the most active compounds
Comp. ID
8a
Caco2a
20.59
23.01
23.51
16.20
21.83
19.49
16.43
MDCKb
137.10
0.03
17.18
0.07
121.28
0.81
119.55
HIAc
96.50
96.71
96.46
97.73
98.03
87.10
87.45
BBBd
0.08
0.06
0.07
0.11
0.07
0.26
0.15
PPBe
91.24
100.00
89.90
97.71
78.05
1.60
8b
8c
8d
18
INH
PZA
0.711
a Caco2: Permeability through cells derived from human colon adenocarcinoma;
Caco2 values < 4 nm/sec (low permeability), values from 4 to 70 nm/sec (medium
permeability) and values > 70 nm/sec (high permeability).
b MDCK: Permeability through Madin–Darby canine kidney cells; MDCK values < 25
nm/sec (low permeability), values from 25 to 500 nm/sec (medium permeability) and
values > 500 nm/sec (high permeability).
c HIA: Percentage human intestinal absorption; HIA values from 0 to 20% (poorly
absorbed), values from 20 to 70% (moderately absorbed) and values from 70 to
100% (well absorbed).
d BBB: Blood–brain barrier penetration; BBB values < 0.1 (low CNS penetration),
values from 0.1 to 2 (medium CNS absorption) and values > 2 (high CNS
absorption)
e PPB: Plasma protein binding; PPB values < 90 % (poorly bound) and > 90%
(strongly bound).
The in silico ADME results indicated that all tested compounds showed medium cell
permeability in the Caco2 cell model with values from 16.20 to 23.51 nm/sec. Additionally,
compounds 8a and 18 displayed medium cell permeability in the MDCK cell model, while
compounds 8b, 8c and 8d showed low permeability in this model.
Furthermore, the predicted oral bioavailability was excellent as all tested molecules
exerted high HIA values from 96.46 to 98.23 % indicating very well absorbed compounds.
Additionally, all tested compounds displayed low BBB penetration capability (0.06-0.08)
except for compound 8d (0.11). In addition, compounds 8c and 18 were found to be
26

weakly-bound to plasma proteins, whereas the rest were more than 90% bound.
Moreover, CYP2D6 enzyme is responsible for metabolism as well as excretion of
approximately 25% of clinically used drugs. The tested compounds showed no inhibition
of this enzyme, thus they may be metabolized and excreted successfully.
3. Conclusion
In this study, various pyrazine derivatives have been developed as new anti-TB
chemotypes. The synthesized compounds were subjected to in silico activity prediction
using PASS analysis. Thirty-one compounds showed anti-TB activity with reasonable Pa
values and were further screened in vitro against Mtb H37RV strain using MABA assay.
Six compounds (8a, 8b, 8c, 8d, 14b and 18) displayed significant activity against Mtb with
MIC values ≤ 6.25 µg/ml. Compound 8b exhibited anti-TB activity higher than the two
references PZA and EMB (MIC = 0.78 µg/ml). Compounds 8c and 8d showed higher
potency than PZA and equal to that of EMB (MIC = 1.56 µg/ml). In addition, compounds
8a and 18 were more active than PZA (MIC = 3.12 µg/ml), while compound 14b was
equipotent to PZA. Structure-activity correlation for the anti-mycobacterial activity of
these active derivatives has been analyzed and revealed that the presence of a linker
between the pyrazine ring and other heterocyclic moieties was necessary for biological
activity, while direct attachment led to loss of activity. Moreover, the in vitro cytotoxicity
of the most active compounds 8a-d, 14b and 18 against PBMC normal cell line was
evaluated using MTT assay and no cytotoxicity was detected for the tested concentrations
and the estimated IC50 values were in the range of 589.1-1174.6 µg/ml, indicating
satisfactory safety profile and selectivity (SI ≥ 200). In an attempt to get some clues on
the potential target behind the antimycobacterial activity of the most active compounds,
a target fishing approach was adopted. It started with calculating similarity index between
our most active compound 8b and some reported pyrazine derivatives with verified
molecular targets. Secondly, a pharmacophore mapping study was conducted using a
pharmacophore model from our most active compounds and the reported pyrazine
derivatives that passed the first step. Finally, an inverse docking study was employed.
All the three steps combined suggested pantothenate synthetase as a potential target.
Docking of the most active compounds in the active site of the enzyme demonstrated
favorable binding modes and interaction patterns. In silico prediction of physicochemical,
27

ADMET and drug-like properties were assessed and revealed that most of the tested
compounds were found to be within those considered adequate for a drug candidate.
Hence, these compounds could represent a promising template for the development of
antimycobacterial agents with improved activity.
4. Experimental
4.1. Chemistry
Melting points were recorded on electrotherm capillary tube Stuart melting point
apparatus SMP10 and are all uncorrected. Follow up of the reactions' rates were
performed by thin-layer chromatography (TLC) on silica gel precoated aluminum sheets
(Type 60 GF254; Merck; Germany) and the spots were visualized by exposure to iodine
vapors or UV-lamp at λ 254nm for few seconds. Infrared spectra (IR) were recorded using
KBr discs on a PerkinElmer IR spectrophotometer, Faculty of Pharmacy, Alexandria
University and on Schimadzu FT-IR Affinity-1 spectrometer at the microanalytical unit,
13
Faculty of Pharmacy, Cairo University. Nuclear magnetic resonance (1H NMR and C
NMR) spectra were recorded on a Jeol spectrophotometer (500 MHz), Faculty of Science,
Alexandria University and on a Bruker spectrophotometer (400 MHz), Faculty of
Pharmacy, Cairo University using deuterated dimethylsulfoxide (DMSO-d6) and
deuterated chloroform (CDCl3) as solvents, followed by recording the spectra again after
addition of D2O for detection of D2O exchangeable peaks. The data were recorded as
chemical shifts expressed in δ (ppm) relative to tetramethylsilane (TMS) as internal
standard. Signal splitting are expressed by the following abbreviations: s = singlet, d =
doublet, t = triplet, q = quartet and m = multiplet. Microanalytical data (C, H, N and S)
were performed on FLASH 2000 CHNS/O analyzer, Thermo Scientific at the regional
center for mycology and biotechnology (RCMB), Al-Azhar University. Compounds 2, 3, 5
and 7a-d were prepared according to reported procedures [28, 43, 78-81].
4.1.1. General procedure for synthesis of compounds 6a-c and 8a-d:
To a stirred solution of the acid hydrazide 5 (2 mmol, 0.27 g) in absolute ethanol (10 ml),
the appropriate heteroaromatic ketone or substituted pyrazolaldehyde (2 mmol) was
added in the presence of catalytic amount of glacial acetic acid (5 drops), the mixture was
28

refluxed for 12-16 h. The product obtained was filtered, washed with diethyl ether, dried
and recrystallized from ethanol.
4.1.1.1. (E)-N'-[1-(2-Pyrazinyl)ethylidene]pyrazine-2-carbohydrazide (6a).
White powder, yield 87%. m.p.278~280 °C. IR (KBr, cm-1): 3379.29 (enolic OH), 3321.42
(NH), 1701.22 (C=O), 1508.33 (C=N). 1H NMR (400 MHz, DMSO-d6): δ 2.57 (s, 3H, CH3),
8.84 (d, J = 4 Hz, 1H, pyrazine-C6'-H), 8.88 (d, J = 4 Hz, 1H, pyrazine-C5'-H), 8.95 (d, J =
2.3 Hz, 1H, pyrazine-C6-H), 8.97-8.98 (m, 1H, pyrazine-C5-H), 9.15 (s, 1H, pyrazine-C3'-
H), 9.31 (s, 1H, pyrazine-C3-H), 11.24 (s, 1/3 H, NH, D2O exchangeable), 15.39 (s, 2/3 H,
enolic OH, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6): δ 22.4 (CH3), 142.8
(pyrazine-C3'), 144.2 (pyrazine-C5'), 144.4 (pyrazine-C3, C5), 145.8 (C=N), 146.1
(pyrazine-C6, C6'), 148.6 (pyrazine-C2,2'), 160.1 (C=O). EI-MS: m/z (242.12) (M+•). Anal.
Calcd (%) for C11H10N6O (242.24): C, 54.54; H, 4.16; N, 34.69. Found: C, 54.31; H, 4.29;
N, 34.84.
4.1.1.2. (E)-N'-[1-(3-Pyridinyl)ethylidene]pyrazine-2-carbohydrazide (6b).
White powder, yield 81%. m.p.218~220 °C. IR (KBr, cm-1): 3336.86 (enolic OH), 3321.42
(NH), 1693.50 (C=O), 1516.05 (C=N). 1H NMR (400 MHz, DMSO-d6): δ 2.45 (s, 3H, CH3),
7.51 (t, J = 4 Hz, 1H, pyridine-C5-H), 8.25 (d, J = 8 Hz, 1H, pyridine-C4-H), 8.65 (d, J = 4
Hz, 1H, pyridine-C6-H), 8.83 (brs, 1H, pyrazine-C6-H), 8.96 (brs,1H,pyrazine-C5-H), 9.05
(brs, 1H, pyridine-C2-H), 9.29 (s, 1H, pyrazine-C3-H), 11.15 (s, 1H, NH, D2O
exchangeable). 13C NMR (100 MHz, DMSO-d6): δ 14.3 (CH3), 124.0 (pyridine-C5), 133.7
(pyridine-C3), 134.3 (pyridine-C4), 143.9 (pyrazine-C5), 144.3 (pyrazine-C3), 145.0
(pyrazine-C2), 148.1 (pyrazine-C6), 148.4 (pyridine-C2), 150.9 (pyridine-C6), 154.7 (C=N),
159.7 (C=O). Anal. Calcd (%) for C12H11N5O (241.25): C, 59.74; H, 4.60; N, 29.03. Found:
C, 59.98; H, 4.61; N, 29.41.
29