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Synthesis and molecular docking study of 6-chloropyrazine-2-carboxylic

acid derivatives
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The 2nd International Conference on Chemistry and Material Science (IC2MS) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 833 (2020) 012002 doi:10.1088/1757-899X/833/1/012002

Synthesis and molecular docking study of 6-chloropyrazine-2-

carboxylic acid derivatives

Nur Pasca Aijijiyah1, Muhammad Riza Ghulam Fahmi2, Sri Fatmawati1, Mardi
Santoso1,*
1
Department of Chemistry, Faculty of Science, Institut Teknologi Sepuluh Nopember,
Kampus ITS Sukolilo Surabaya 60111, Indonesia
2
Ma Chung Research Center for Photosynthetic Pigments, Universitas Ma Chung,
Villa Puncak Tidar N-01, Malang 65151,Indonesia

*Corresponding author : tsv09@chem.its.ac.id

Abstract. One of the most lethal and frequent infectious diseases worldwide is tuberculosis.
Multi and extensively tuberculosis drug-resistant constitutes a serious problem and emphasizes
the need for novel anti-tubercular agents. Accordingly, various pyrazine-2-carboxamides were
synthesized and evaluated as potential anti-tuberculosis agents. The synthesis involved reaction
of pyrazinoic acids with thionyl chloride to yield acyl chlorides which on treatment with various
anilines gave various pyrazine-2-carboxamides. Based on structure-activity relationships
extracted from previously published, this paper reported the synthesis and molecular docking
study of 6-chloropyrazine-2-carboxamides. Synthesis involved reaction of 6-chloropyrazinoic
acid with 2,4,6-trichlorobenzoyl chloride instead of thionyl chloride which listed under the
Chemical Weapons Convention as it may use for the production of chemical weapons. Structure
identification of 6-chloropyrazine-2-carboxamides was carried out by 1H NMR, 13C NMR, FTIR,
and high-resolution mass spectroscopy. It is predicted that 6-chloro-N-octylpyrazine-2-
carboxamide has better bioactivity against Mycobacterium tuberculosis, based on molecular
docking study.

1. Introduction
Mycobacterium tuberculosis (MTB) bacteria leads to tuberculosis disease wich primarily attacks the
lungs [1]. In 2018, the World Health Organization reported that Indonesia is a country with a high TB
burden [2]. The Ministry of Health Indonesia (2019) reported that there were 511,873 cases of TB in
2018 [3]. The genetic mutation of the TB bacteria causes resistance against first-line TB drugs called
multi-drug resistant TB. This disease becomes a more serious problem with the formation of extensively
drug-resistant TB wich bacteria MTB resistance against first-line and several second-line TB drugs [4].
Nowadays, TB treatment uses first-line drugs (isoniazid and rifampicin) and second-line drugs such as
amikacin (injectable drugs) and levoflaxin (fluoroquinolones derivatives) [5]. However, this treatment
is not enough to treat TB patients with resistance to TB drugs. [6]. The Food and Drug Administration
has assigned two kinds of antituberculosis drugs for the medication of the patient with drug-resistant
TB, namely bedaquiline (TMC207) and nitroimidazole bicyclic (PA-824), but both of them are reported
to have a side effect that causes abnormal heart rhythms and sudden cardiac death [7]. Accordingly, the
development of new antituberculosis drugs are needed.

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IOP Conf. Series: Materials Science and Engineering 833 (2020) 012002 doi:10.1088/1757-899X/833/1/012002

As one of the first-line TB drugs, pyrazinamide (pyrazine-2-carboxamide) considered having an

important role in the short course therapy of TB that can synergize with rifampicin and has a good
sterilizing effect[8]. However, genetic mutations in TB bacteria cause resistance to this drug [9]. In the
past 10 years, various pyrazine-2-carboxamide derivatives were prepared and evaluated their role as anti
Mycobacterium tuberculosis H37Rv. The structure-activity relationship (SAR) of the pyrazine-2-
carboxamide derivatives shows that substitution with phenyl and alkyl group at the nitrogen of amide
group, and chloro at C6 of pyrazine ring increased the antituberculosis activity against Mycobacterium
tuberculosis H37Rv [10–14]. Pyrazine-2-carboxamides as amides can be synthesized through reactions
involving several reagents such as thionyl chloride [15-16], 1,1’-carbonyldiimidazole (CDI) [14] and
hydroxybenzotriazole (HOBt) [7]. Nevertheless, these reagents have several disadvantages. Thionyl
chloride is a chemical listed under the Chemical Weapons Convention (CWC) as it may use for the
production of chemical weapons [17]. Hydroxybenzoyltriazole (HOBt) has explosive property [18],
while the reaction of carboxylic acid and 1,1’-carbonyldiimidazole (CDI) give an intermediate which
less reactive than the acyl chloride [19]. 2,4,6-Trichlobenzoyl chloride (TCBC) is a reagent used in the
synthesis of esters and thioesters compound [20–23]. The reaction of TCBC and carboxylic acid produce
the mixed anhydride which has a sterically hindered aromatic moieties that can regioselectively give the
corresponding ester [24]. Herewith, we report the synthesis of 6-chloropyrazine-2-carboxylic acid
derivatives (1a-c), and their prediction as anti-tuberculosis agent based on molecular docking study.

2. Experimental Section

2.1. General information

6-Chloropyrazine-2-carboxylic acid, p-ethylaniline, cycloheptylamine, and n-octylamine were
purchased from Tokyo Chemical Industry. Triethylamine (TEA), tetrahydrofuran (THF), sodium
hydroxide, sodium carbonate, and magnesium sulfate were purchased from Merck. 2,4,6-
Trichlorobenzoyl chloride (TCBC) and p-dimethylaminopyridine (DMAP) were recieved from Sigma-
Aldrich. The chromatography analysis and purification were carried out using Merck with 60 GF254
silica gel plates type for thin-layer chromatography (TLC) and 60 PF254 silica gel containing gypsum
type for radial chromatography. The compounds were detected with ultraviolet lights at 245 and 366
nm. Melting temperature measurement was performed on a Fisher John apparatus. The proton and
carbon NMR were analyzed using Jeol resonance at 400 MHz, using tetramethylsilane and CDCl3 as
standard and solvent respectively. The high resolution mass data (HRMS) were obtained using Waters-
Xevo QTof instrument, while Infrared data were recorded using FTIR Shimadzu 8400S.

2.2. General method for synthesis of 6-chloropyrazine-2-carboxylic acid derivatives (1a-c)

In the round-bottom flask, the 6-Chloropyrazine-2-carboxylic acid (1 eqiv), TCBC (1 eqiv), and TEA
(1 eqiv) were mixtured in THF (80 mL) solvent and stirred for 20 mins at room temperature. DMAP (1
eqiv) was added to the mixture while the corresponding amine (0.25 eqiv) was added droplet. After that,
the mixture was further stirred with heating at 50-55 oC for 1 hour and was allowed to stand at rt after
the completion of the reaction and then filtrated off. The cold water was added to the obtained filtrate
and extracted several times using dichloromethane. The organic layer was combined and washed
subsequently with solutions of 5% HCl, 5% NaOH, 5% Na2CO3 and water. After that, the organic layer
was dried over MgSO4 and evaporated under vacuum. The crude product was purified using
chromatogram with n-hexane:ethyl acetate (10:1 or 20:1) eluent to produce 6-chloropyrazine-2-
carboxylic acid derivatives (1a-c).

2.3. Molecular docking studies

Molecular docking study of 6-chloropyrazine-2-carboxylic acid derivatives (1a-c) was performed on
MVD using MolDock SE Algorithm.The geometry of the compound (1a-c) structures was optimized
using Semi-empiris (AM1) technique on Hyperchem 8.0.7 software. The protein used for docking was
M. Tuberculosis InhA (PDB ID: 4DRE) where the three-dimensional (3D) structure was taken from the

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The 2nd International Conference on Chemistry and Material Science (IC2MS) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 833 (2020) 012002 doi:10.1088/1757-899X/833/1/012002

RCSB Protein Databank. The protein and reference ligand (NAI) were prepared using Molegro Virtual
Docking (MVD) 5.0. The preferred docking pose of each compound was performed by 50 runs of
Moldock search by the Lamarckian genetic algorithm (LGA) and the results were visualized using
MVD.

3. Result and Discussion

3.1. Chemistry

(2) (1a) R = p-ethylphenyl

(1b) R = cycloheptyl
(1c) R = octyl

Scheme 1. Synthesis of 6-chloropyrazine-2-carboxylic acid derivatives (1a-c).

6-Chloropyrazine-2-carboxylic acid derivatives (1a-c) were synthesized based on the synthesis of 2-

phenylethyl esters [23]. The reaction of 6-chloropyrazine-2-carboxylic acid with TEA and TCBC in
THF at room temperature produced a mixed anhydride (2), then treated with DMAP and the
corresponding amine for 1 hour at 50-55 oC (Scheme 1). The mixture was filtered after cooling, extracted
with dichloromethane, washed subsequently with solutions of 5% hydrochloric acid, 5% sodium
hydroxide, 5% sodium carbonate, water, and then being evaporated. After that, the residue was purified
to produce 6-chloropyrazine-2-carboxylicacid derivatives (1a-c) in 71-75% yields.
1
H NMR spectra of synthesis compounds (1a-c) showed broad singlet signals of NH protons at 7.55-
9.34 ppm, which confirmed that carboxylic acid derivatives (1a-c) have formed. The aromatic proton
shows six protons at 7.23-9.39 ppm for 1a compounds, two protons at 8.72-9.27 ppm for 1b, and at two
protons at 8.73-9.28 ppm for 1c compound. The characteristic of ethyl proton (1a), N-cycloheptyl
protons (1b), and N-octyl proton (1c) appear at 1.24-2.65 ppm with 5 protons, 13 protons at1.54-4.16
ppm, and 17 protons at 0.86-3.46 ppm, respectively. 13C NMR spectra showed carbon signals of
carbonyl groups at 159.31-161.66 ppm. It was supported by the IR spectra which showed sharp
absorption bands around 1653-1693 cm-1 for the carbonyl groups and 3273-3369 cm-1 for the NH groups.
The HRMS data shows m/z at 262.0606 (35Cl) and 264.0656 (37Cl) ([M+H]+) for compound 1a, m/z at
254.0967 (35Cl) and 256.0997 (37Cl) ([M+H]+) for compound 1b, and m/z at 270.1252 (35Cl) and
272.1357 (37Cl) ([M+H]+) for compound 1c.

3.2. Molecular docking studies

Molecular docking or computational studies has an important role in the development of new drugs
through the design of structure-based drugs. The docking approach can help the researchers to find out
the behavior of a drug in the active site of the proteins through the interaction between them at the atomic
level [25]. Interaction analysis of the synthesis compounds (1a-c) at the InhA protein (PDB ID: 4DRE)
binding site of M. tuberculosis was carried out with MVD using MolDock SE Algorithm. The result
revealed that compound (1c) has the lowest rerank score which means that it has the best binding affinity.
Compound (1a) and (1b) have the hydrogen bonding and π-π interactions, while compound (1c) only
has the π-π interactions with Gly14, Phe41, Leu63, and Gly40 which reported in Tabel 1 and can be
seen in Figure 1.

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The 2nd International Conference on Chemistry and Material Science (IC2MS) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 833 (2020) 012002 doi:10.1088/1757-899X/833/1/012002

Table 1. Rerank score and interaction of compound (1a-c) within the InhA
protein binding site of M. tuberculosis.
Compound Rerank score Interaction
H-Bond: Val65
(1a) -79.8858
π-π:Ile16, Ile122, Asp64, and Val65
H-Bond: Gly96
(1b) -79.1557
π-π: Gly14, Val65, Phe41, Ile95, and Gly95
H-Bond: -
(1c) -85.7435
π-π:Gly14, Phe41, Leu63, and Gly40

Figure 1. The docking pose of compound (1c) at the InHA protein binding site of M. tuberculosis.

4. Conclusion
The synthesis of 6-chloropyrazine-2-carboxylic acid derivatives (1a-c) has been successfully carried out
with the presence of TCBC. Amidation of 6-chloropyrazine-2-carboxylic acid gave compounds (1a-c)
in 71-75% yields. The molecular docking studies result revealed that 6-chloro-N-octylpyrazine-2-
carboxamide (1c) has the best activity against Mycobacterium tuberculosis H37Rv strain.

Acknowledgments
The authors are very grateful to the Indonesian Government for the PenelitianTesis Master Grant No.
796/PKS/ITS/2019.

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