2-Aminoaryl-3,5-diaryl pyrazines: Synthesis, biological evaluation against Mycobacterium tuberculosis and docking studies

Received: 14 December 2019

DOI: 10.1002/ardp.201900368

Revised: 16 April 2020

Accepted: 22 April 2020

|

F U L L P A P E R

2‐Aminoaryl‐3,5‐diaryl pyrazines: Synthesis, biological

evaluation against Mycobacterium tuberculosis

and docking studies

Nagaraja Reddy Gangarapu^1,2| Archakam Ranganatham³| Eeda Koti Reddy⁴

Shivaraj Yellappa¹| Kothapalli Bannoth Chandrasekhar²

|

¹Department of Chemistry, Bangalore

University, Bengaluru, Karnataka, India

Abstract

²Department of Chemistry, Jawaharlal Nehru

Rationally designed Mycobacterium tuberculosis (Mtb) inhibitors were synthesized

under Buchwald conditions using Pd₂(dba)₃/xantphos and the compounds were in-

vestigated for their biological activity against the Mtb standard strain H37Rv and

two other clinically isolated multidrug‐resistant strains with different drug re-

sistance patterns. Compounds 5e, 6e, 7e, and 8e exhibited excellent antituberculosis

activity against H37Rv with a minimum inhibitory concentration (MIC) value of

15 μg/ml. Compounds 5a, 6c, 7b, 8a, 8b, and 8d also displayed their potency with a

MIC value in the range of 15–25 μg/ml. In addition to the Mtb studies, compounds

4e, 5e, 7e, and 8e were tested for cytotoxicity on HEK‐293 cells and compounds

7e and 8e were identified to have low toxicities of up to 200 and 300 μM, respec-

tively. The synthesized compounds docked with the 2FUM protein of Mtb and the

docking studies revealed that compounds 5e, 6e, 7e, and 8e can bind strongly in the

active site of the enzyme and showed binding energies of −9.62, −10.7, −11.48, and

−12.06 kcal/mol, respectively. Compound 7e forms four hydrogen bonds, whereas

compound 8e forms five hydrogen bonds with amino acids, respectively. Based on

these results, compounds 7e and 8e might be considered potential lead compounds

with good anti‐Mtb potency.

Technological University, Ananthapuramu,

Andhra Pradesh, India

³Laboratory Division, National Tuberculosis

Institute, Bangalore, Karnataka, India

⁴Department of Science and Humanities,

Division of Chemistry, Vignan's Foundation for

Science, Technology and Research‐VFSTR

(Deemed to be University), Guntur,

Andhra Pradesh, India

Correspondence

Shivaraj Yellappa, Department of Chemistry,

Bangalore University, Jnanabharathi Campus,

Bengaluru, Karnataka 560056, India.

Email: shivaraj_y@rediffmail.com

K E Y W O R D S

antituberculosis activity, cytotoxic activity, synthesis

|

1

INTRODUCTION

raised among the individuals. Tuberculosis is still a major disease

worldwide, the incidence of which continues to increase in de-

Tuberculosis (TB) is the most conspicuous disease among all

pathogenic diseases. Globally, more than two million lives are lost

every year due to TB and about 11 million new patients have been

identified.^[1]The medicine for tuberculosis is insufficient to ad-

dress the many challenges of treatment, whereas the tuberculosis

patients were decreased by an average of 1.5% every year. The

aim of the World Health Organization (WHO) is to eradicate this

infectious disease by 90% and loss of lives by 95%.^[2]The treat-

ment of TB is very complicated and more awareness needs to be

veloping countries.^[3]Even though medical treatment is currently

available to cure tuberculosis, there are some disadvantages such

as the timeline for treatment, the need to focus on multidrug

therapy, and the emergence of drug resistance, HIV coinfection,

and persistence of Mycobacterium tuberculosis (Mtb) bacilli.^[4,5]The

process of multidrug medical treatment takes 6 months, consisting

of isoniazid (INH), rifampicin (RMP), pyrazinamide (PZA), and

ethambutol (EMB) for 2 months, followed by fully susceptible

isolates of RMP and INH for 4 months.^[6–8]A key question is how

|

Arch Pharm. 2020;e1900368.

1 of 14

https://doi.org/10.1002/ardp.201900368

2 of 14

GANGARAPU ET AL.

|

RESULTS AND DISCUSSION

robust our approaches are toward the discovery of new TB drugs,

and the measures that could be taken to reduce the length of

protracted clinical development of new drugs. Several derivatives

with antitubercular activity have been described such as benzi-

midazole ureas (1)^[9]from Vertex Pharma, PZA derivatives^[10](2)

from AstraZeneca Pharma, quinoline derivatives from GSK

Pharma,^[11]and the nitrobenzothiazinone derivative (BTZ043),^[12]

and azaindole derivatives^[13](Figure 1) of known molecules have

been reported as promising drug candidates in preliminary studies.

New strategies have to be developed for tuberculosis disease by

screening libraries of small molecules for biologically active mo-

lecules. Nowadays, treatment with bedaquiline and PA‐824 is the

new antitubercular regime for chemotherapy. Bedaquiline^[14]is a

new drug for anti‐TB, which was approved by the WHO in 2012,

and used in combination with other TB drugs to treat pulmonary

TB in adults having multidrug‐resistant TB (MDR‐TB). However,

bedaquiline is a highly lipophilic drug (logP 7.25) with a tendency

to cause liver toxicity. Bedaquiline‐containing treatments are more

cost effective compared with injectable‐containing treatments. As

part of research to develop new molecules for anti‐TB, our team

developed a series of 3,5‐disubstituted 2‐aminopyrazine deriva-

tives and evaluated their anti‐TB activity.^[15]2‐Aminopyrazine

derivatives have biological importance because of their

anticancer,^[16]CHK1 inhibitor,^[17]Trypanosoma brucei inhibitor,^[18]

antimalarial,^[19]GSK3β inhibitor,^[20]PDK inhibitor,^[21]and antic-

onvulsant activities. To extend our research,^[22]we synthesized

novel 3,5‐disubstituted 2‐aminopyrazine derivatives and evaluated

their antimycobacterial activity. The binding nature, physico-

chemical properties (ADME), and cytotoxicity studies for

synthesized compounds were also analyzed.

2

|

2.1

Synthetic studies

The synthesis of 2‐aminoaryl 3,5‐disubstituted pyrazine mole-

cules^[23]is shown in Schemes 1 and 2. The compound 3,5‐dibromo‐

2‐aminopyrazine (2) was synthesized from the reaction of

2‐aminopyrazine (1) by treatment with bromosuccinimide in di-

methyl sulfoxide (DMSO) and Na₂CO₃as a base for 12 hr at room

temperature to yield 3,5‐dibromo‐2‐aminopyrazine (2). The inter-

mediate 2 was subjected to Suzuki–Miyaura cross‐coupling reac-

tion with different boronic acids (3) to obtain compound 4. The

N‐arylation of compound 4 was screened using different palladium

catalysts such as Pd(OAc)₂/BINAP, Pd(OAc)₂/Xphos, Pd(OAc)₂/t‐

BuXphos, Pd(OAc)₂/xantphos, and Pd₂(dba)₃/xantphos with

different bases like t‐BuOK, t‐BuONa, Na₂CO₃, Cs₂CO₃,

K₃PO₄·3H₂O, and K₂CO₃, and different solvents like 1,4‐dioxane,

toluene, dimethylformamide (DMF), dimethylacetal (DMA), and

dimethoxyethane (DME). Among all the screened conditions,

compounds 5–8 synthesized using Pd₂(dba)₃/xantphos and

Cs₂CO₃as a base in DMA at 120°C under microwave irradiation

for 2 hr gave a good yield (70–85%; Table 1).

|

2.2

Docking results

In this study, FUM protein was used as a standard and it plays a vital

role in the growth inhibition of mycobacteria. Fumarase (Fum) is an

enzyme of the canonical tricarboxylic acid cycle and is dispensable in

many organisms. Transposon mutagenesis studies in Mtb, however,

FIGURE 1 Biologically active

antituberculosis molecules

3 of 14

GANGARAPU ET AL.

|

SCHEME 1 Synthesis of 2‐aminoaryl‐3,5‐

disubstituted pyrazines

indicate that Fum is required for optimal growth. Here, the genera-

tion and characterization of a genetically engineered Mtb strain in

which Fum expression is conditionally regulated are reported. These

results identify Mtb Fum as a potentially species‐specific drug target

whose inactivation may kill Mtb through a covalently irreversible

form of metabolic toxicity. The intention behind the selected FUM

protein to prove the derivative showed good anti‐TB activity as well

as anticancer agent which will be expedited in further, the mechan-

ism of PZA was explained in detail.

amino acids and inhibitory concentrations of 711.2 and 4.85 µM,

respectively.

|

2.3

Results of biological activity

This study aims to discuss pyrazine derivatives and their activity

against Mtb and other drug‐resistant mycobacterial strains. The ex-

perimental process for testing anti‐TB drug susceptibility was based

on the H37Rv tuberculosis standard strain and another two clinically

isolated multidrug‐resistant strains with different drug‐resistant

patterns; the minimum inhibitory concentration (MIC) experiment

was carried out using different drug concentrations on the

Lowenstein–Jensen (LJ) medium.

Accordingly, the synthesized compounds were subjected to in

silico docking studies with the tuberculosis protein (Figures 2 –4).

These studies show various kinds of interactions such as hydrogen

bonding, electrostatic interaction, and van der Waals bonding. All

the compounds have binding energies ranging between −12.06 and

−6.7 kcal/mol. However, among the synthesized compounds,

8e presented excellent interaction with the protein and strong

binding energy of −12.06 kcal/mol. Compound 8e showed an in-

hibitory concentration of 7.88 µM with five hydrogen bonds. The

hydrogen bond interaction within the active site of amino acids

VAL95, GLY59, ASP156, PHE19, and LEU17 in the protein se-

quence. Correspondingly, compounds 5e, 6e, and 7e displayed

strong binding interaction with the protein with binding energies

of −9.62, −10.7, and −11.48 kcal/mol, respectively. These deriva-

tives exhibited inhibitory concentrations of 88.1, 14.41, and

3.87 µM, respectively. Compounds 6e and 7e exhibited four hy-

drogen bonding interactions within the active site of amino acids

ASP156, GLU59, LEU17, and VAL95. However, compounds 3a, 4a,

5a, and 6a displayed strong binding interaction with the protein

with binding energies of −8.17, −9.15, −8.74, and −8.85 kcal/mol,

respectively (Table 2). The remaining synthesized molecules have

shown moderate to good binding energy interactions. Moreover,

the reference drug RMP displays three strong H‐bond interactions

within the active site of amino acids VAL95, PHE19, and ASP156

with a binding energy of −9.16 kcal/mol. Other standard drugs

such as PZA and INH showed good binding energies of −4.29 and

3.16 kcal/mol with three and one H‐bond interaction with different

The experimental results of antimycobacterial activity are

shown in Table 3. It shows that all the tested compounds showed

good biological activity in the used test strains of Mtb. All the

compounds, in particular, were found to be effective against

mycobacteria at a concentration ranging from 4.0 to 40 µg/ml in

drug‐containing LJ medium. RMP and INH, used as positive drug

controls, at concentrations of about 40 and 0.2 µg/ml, respec-

tively, were prepared as per the standard guidelines of drug

susceptibility testing (DST) by the Revised National Tuberculosis

Control Program (RNTCP), Government of India, at the National

Tuberculosis Institute (NTI), Bangalore. The bioactivities of the

derivatives were assayed using a MIC experiment. All the syn-

thesized compounds showed good inhibition range from 15 to

50 µg/ml. In the structure–activity relationship (SAR) studies, the

compounds with a dipyridyl substitution at the amino group such

as 5e, 6e, 7e, and 8e displayed a very good MIC value of 15 µg/ml.

Similarly, the compounds with a monopyridyl substitution on

the amine group such as 7b and 8b exhibited moderate to good

MIC values of 15 and 25 µg/ml, respectively. The other pyridyl

substitution derivatives such as 5b and 6b showed good MIC

values in the range of 25–50 µg/ml. The compound with

6‐chloropyrimidine substitution on amino group (6c) presented a

4 of 14

GANGARAPU ET AL.

|

SCHEME 2 Substrate scope of the 2‐aminoaryl‐3,5‐disubstituted pyrazines

5 of 14

GANGARAPU ET AL.

|

TABLE 1 Optimization for the synthesis

of compounds 5–8

Catalyst (0.1 equiv)/ligand

Sl. No. (0.2 equiv)

Base (2.0 equiv) Solvent

Temp (°C) % of yield

1

Pd₂(OAc)₂

Na₂CO₃

K₂CO₃

1,4‐Dioxane 110

NA

2

Pd₂(OAc)₂/Xphos

10

13

20

25

50

34

35

57

20

62

78

85

50

3

Pd₂(OAc)₂/t‐BuXphos

Pd₂(OAc)₂/xantphos

Pd₂(dba)₃/xantphos

1,4‐Dioxane 110

4

5

6

Cs₂CO₃

t‐BuOK

K₃PO₄·3H₂O

Cs₂CO₃

7

8

9

10

11

12

13

14

Toluene

DMF

110

120

DMA

DME

Note: All reactions were carried out under microwave heating.

Abbreviations: DMA, dimethylacetal; DME, dimethoxyethane; DMF, dimethylformamide.

very good inhibitory value of 15 µg/ml. The other pyrimidine

substitution derivatives 5c, 7c, and 8c showed good MIC values in

the range of 25–50 µg/ml. The compounds with the substitution of

4‐iodo‐3,5‐dimethylphenyl derivatives (5a, 6a, 7a, and 8a) also

exhibit good MIC values in the range of 15–50 µg/ml. The other

compounds were also ideal with MIC values in the range of

25–50 µg/ml. SAR studies revealed that the compounds bearing a

dipyridyl group on the amine group could potentially become drug

candidates for TB and further studies of clinical trials and in vivo

experiments will be initiated in future.

FIGURE 2 Binding interactions, van der Waals interactions, and covalent hydrogen bond interactions in compound 6e

6 of 14

GANGARAPU ET AL.

|

2.4

DST results

Selection of control strains of Mtb and its DST pattern with the

standard anti‐TB drugs (positive controls) used in the RNTC Program.

Three strains were used to test the anti‐TB activity based on dif-

ferent drug‐resistant patterns. All these cultures were collected from

the culture bank of NTI and the standard DST results were obtained

through NTI laboratory. In addition, standard DST results were ob-

tained through RNTCP guidelines, which were used as comparative

standards for this study.

|

2.5

Cytotoxicity studies

The in vitro cytotoxicities of most potent antitubercular analogues

(5e, 6e, 7e, and 8e) were tested in human embryonic kidney

cells (HEK‐293) by the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐

diphenyltetrazolium bromide) assay method (Figure 5). Its main

application is to assess the viability (cell counting) and the pro-

liferation of cells. It can also be used to determine the cytotoxicity of

medicinal agents and toxic materials. The compounds were tested

using three different concentrations (50, 100, and 200 µM). Com-

pounds 5e and 6e displayed toxicity when the concentration was

above 100 µM and, for the other molecules, 7e and 8e were not toxic

up to 200 µM concentration.

|

2.6

ADMET results

|

2.6.1

In silico predictions of the synthesized

compounds

The newly synthesized compounds were evaluated for their drug‐like

behavior through in silico predictions. The ADMET (absorption, dis-

tribution, metabolism, excretion, and toxicity) profiles of the compounds

are given in Table 4. Some of the important parameters are molecular

weight (MW), partition coefficient (logP), number of hydrogen bond do-

nors and acceptors (HBD and HBA), topological polar surface area

(TPSA), number of rotatable groups (rotor), CaCO₂permeability (logPapp;

cm/s), Cyp2C19 substrate, Cyp2c19 inhibition, and human intestinal ab-

sorption (HIA) were predicted by in silico studies. It was found that the

values of MW and logP of all the molecules were within the desired

range, except compound 5a. Number of rotor, HBD, HBA, and TPSA were

in the ideal range for all the compounds. The HIA and blood–brain barrier

(BBB) of all the compounds was in the preferred range.

|

3

CONCLUSION

In this study, we reported the synthesis of 3,5‐disubstituted‐2‐

aminoaryl pyrazine molecules with good yields using Pd₂(dba)₃/

xantphos. The compounds prepared were tested for their anti-

tuberculosis activity using H37Rv and with two clinically isolated

7 of 14

GANGARAPU ET AL.

|

FIGURE 3 Binding interactions, van der Waals interactions, and covalent hydrogen bond interactions in compound 7e

|

Experimental procedure for the synthesis of

compounds 5a–8e

strains. The motifs with a diaryl substitution of 4‐cyano‐2‐pyridine

molecules displayed a very good MIC value of around 15 µg/ml in all

three strains. The cytotoxic studies reveal that compounds 5e and 6e

were nontoxic up to 100 µM and compounds 7e and 8e were safe up

to 200 µM. The docking studies revealed that compounds 5e, 6e, 7e,

and 8e can bind strongly in the active site of enzyme and showed

binding energies of −9.62, −10.7, −11.48, and −12.06 kcal/mol, re-

spectively. Based on our results, compounds 7e and 8e might be

considered potential lead compounds with good anti‐Mtb potency.

4.1.2

To a solution of compound 1 (0.130 mmol) in dimethylacetamide as a

solvent (5 ml) was added the corresponding aryl/heteroaryl halide 2

(0.393 mmol), followed by the addition of Cs₂CO₃(0.26 mmol) and

Pd₂(dba)₃(0.013 mmol), and the reaction mixture was degassed for

10 min with argon at room temperature. Xantphos (0.026 mmol) was

further added and the reaction mixture was heated at 120°C for 2 hr

under microwave irradiation. The reaction mixture was quenched with

saturated ammonium chloride (20 ml) and extracted with ethyl acetate

(2 × 20 ml). The combined organic layers were washed with brine solution

(30 ml), dried over anhydrous sodium sulfate, and concentrated under

vacuum. The crude was purified by column chromatography using 5–20%

of ethyl acetate in hexane and gave compound 5 (70–85%).

|

4

EXPERIMENTAL

|

4.1

Chemistry

|

4.1.1

General

3,5‐Bis[4‐(trifluoromethyl)phenyl]‐N‐(4‐iodo‐3,5‐dimethylphenyl)‐

pyrazin‐2‐amine (5a)

Off‐white solid; yield: 60 mg, 75%; melting range: 211–213°C; ¹H

NMR (500 MHz, CDCl₃) δ 8.67 (s, 1H), 8.10–8.08 (d, J = 10.0 Hz, 2H),

The original nuclear magnetic resonance (NMR) spectra of the in-

vestigated compounds are provided as Supporting Information Data,

as are their InChI codes together with some biological activity data.

FIGURE 4 Binding interactions in compound 8e

8 of 14

GANGARAPU ET AL.

|

9 of 14

GANGARAPU ET AL.

|

TABLE 3 Drug susceptibility testing

results of tested compounds

Tested Mtb

Compound ID strain

Growth control on

plain LJ medium

15 µg/ml

25 µg/ml

50 µg/ml

5a

5b

5c

5d

5e

6a

6b

6c

6d

6e

7a

7b

7c

7d

7e

8a

H37Rv

++

Sensitive

Mtb strain 1

Mtb strain 2

H37Rv

+++

++

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

++

H37Rv

++

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

H37Rv

+++

++

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

++

H37Rv

+++

++

Sensitive

Mtb strain 1

Mtb strain 2

++

H37Rv

++

Resistant

Sensitive

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

H37Rv

++

Resistant

Sensitive

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

H37Rv

++

Sensitive

Mtb strain 1

Mtb strain 2

+++

H37Rv

++

Resistant

Sensitive

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

+++

++

H37Rv

++

Sensitive

Mtb strain 1

Mtb strain 2

++

+++

H37Rv

+++

++

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

++

H37Rv

++

Sensitive

Mtb strain 1

Mtb strain 2

H37Rv

++

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

+++

++

H37Rv

++

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

H37Rv

+++

++

Sensitive

Mtb strain 1

Mtb strain 2

++

H37Rv

+++

++

Sensitive

Mtb strain 1

Mtb strain 2

++

(Continues)

10 of 14

GANGARAPU ET AL.

|

TABLE 3 (Continued)

Tested Mtb

Compound ID strain

Growth control on

plain LJ medium

15 µg/ml

25 µg/ml

50 µg/ml

8b

8c

8d

8e

H37Rv

+++

++

Resistant

Sensitive

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

+++

H37Rv

+++

++

Resistant

Sensitive

Resistant

Sensitive

Mtb strain 1

Mtb strain 2

+++

H37Rv

+++

++

Sensitive

Mtb strain 1

Mtb strain 2

+++

H37Rv

+++

++

Sensitive

Mtb strain 1

Mtb strain 2

Note: +++, ++ indicate Confluent growth and >100 colonies respectively.

Abbreviations: LJ, Lowenstein–Jensen; Mtb, Mycobacterium tuberculosis.

7.94–7.93 (d, J = 5.0 Hz, 2H), 7.86–7.84 (d, J = 10.0 Hz, 2H), 7.72–7.70

(d, J = 10.0 Hz, 2H), 7.30 (s, 2H), 6.70 (s,1H), 2.47 (s, 6H); ¹³C NMR

(126 MHz, CDCl₃) δ 148.42 (s), 142.74 (s), 141.58 (s), 140.08, 140.02,

139.89 (t), 138.41, 138.25 (d), 129.13 (s), 129.48, 129.45 (d), 125.89,

125.86, 125.83 (t), 118.63 (s), 29.73 (s). Ultraperformance liquid

chromatography–mass spectrometry (UPLC–MS): m/z calculated for

C₂₆H₁₈F₆IN₃: 613.34; observed mass: 614.8 [M + H]⁺.

N‐{(3,5‐Bis[4‐(trifluoromethyl)phenyl]pyrazin‐2‐yl}‐6‐

chloropyrimidin‐4‐amine (5c)

Off‐white solid; yield: 48 mg, 74.22%; melting range: 212–214°C;

¹H NMR (400 MHz, DMSO) δ 10.38 (s, 1H), 9.22 (d, J = 6.5 Hz,

1H), 8.45 (s, 1H), 8.42–8.36 (m, 2H), 8.05 (d, J = 8.2 Hz, 2H),

7.89 (dd, J = 13.9, 8.4 Hz, 4H), 7.62 (s, 1H); ¹³C NMR (126 MHz,

DMSO) δ 158.68 (s), 146.07 (s), 145.44 (s), 139.52 (s), 129.94 (s),

127.51 (s), 126.35 (s), 126.25–125.63 (m), 106.84 (s); UPLC–MS:

m/z calculated for C₂₂H₁₂ClF₆N₅: 495.81; observed mass:

496.8 [M + H]⁺.

6‐{3,5‐Bis[4‐(trifluoromethyl)phenyl]pyrazin‐2‐ylamino}pyridine‐3‐

carbonitrile (5b)

Off‐white solid; yield: 51 mg, 80.5%; melting range: 230–232°C; ¹H

NMR (500 MHz, CDCl₃) δ 8.79 (s, 1H), 8.64–8.63 (d, J = 8.5.0 Hz, 1H),

8.50 (d, J = 1.5 Hz,1H), 8.15–8.13 (d, J = 8.0 Hz, 2H), 7.96–7.88

(m, 6H), 7.76–7.74 (d, J = 8.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ

154.57 (s), 151.87 (s), 145.96 (s), 144.01 (s), 141.40 (s), 141.27 (s),

139.23 (s), 139.05 (s), 137.78 (s), 129.25 (s), 126.67–126.65 (d),

126.38 (s), 126.02–125.99 (d), 117.18 (s), 112.05 (s), 103.04 (s);

UPLC–MS: m/z calculated for C₂₄H₁₃F₆N₅: 485.38; observed mass:

482.2 [M + H]⁺.

N‐(4‐Chloro‐5‐methoxypyrimidin‐2‐yl)‐3,5‐bis(4‐(trifluoromethyl)‐

phenyl)pyrazin‐2‐amine (5d)

Off‐white solid; yield: 58 mg, 84.5%; melting range: 177–179°C; ¹H

NMR (500 MHz, CDCl₃) δ 8.85 (s, 1H), 8.20–8.19 (d, J = 8.0 Hz, 2H),

7.93–7.91 (d, J = 8.0 Hz, 2H), 7.79–7.76 (m, 4H), 7.70–7.68

(d, J = 8.5 Hz, 2H), 3.95 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ

150.86 (s), 150.76 (s), 147.22 (s), 145.52 (s), 143.79 (s), 141.19 (s),

140.03 (s), 139.01 (s), 138.43 (s), 136.33 (s), 131.67 (s), 131.52 (s),

131.41 (s), 131.26 (s), 131.00 (s), 130.74 (s), 128.34 (s), 127.83 (s),

126.96 (s), 126.00–125.97 (d), 125.61–125.58 (d), 125.09 (s), 124.98

(s), 122.92 (s), 122.81 (s), 56.52 (s); UPLC–MS: m/z calculated for

C₂₃H₁₄ClF₆N₅O: 525.83; observed mass: 526.2 [M + H]⁺.

6‐N‐{3,5‐Bis[4‐(trifluoromethyl)phenyl]pyrazin‐2‐yl}‐N‐

(5‐cyanopyridin‐2‐yl)amino)pyridine‐3‐carbonitrile (5e)

Off‐white solid; yield: 66 mg, 86.12%; melting range: 206–208°C; ¹

H

NMR (500 MHz, CDCl₃) δ 8.99 (s, 1H), 8.49–8.48 (t, J = 2.0 Hz, 2H),

8.26–8.25 (d, J = 8.5 Hz, 2H), 7.81–7.78 (m, 6H), 7.54–7.52

(d, J = 8.5 Hz, 2H), 7.13–7.11 (d, J = 8.5 Hz, 2H); ¹³C NMR

(126 MHz, CDCl₃) δ 157.20 (s), 152.20 (s), 150.15 (s), 149.88 (s),

148.90 (s), 140.78 (s), 140.35 (s), 139.34 (s), 138.48 (s), 128.53 (s),

127.44 (s), 126.17–126.15 (d), 125.47–125.45 (d), 116.32 (s), 115.97

FIGURE 5 Cell viability of more potent compounds on HepG2

cells after 24‐hr treatment

11 of 14

GANGARAPU ET AL.

|

TABLE 4 ADME properties of synthesized compounds

miLogP

TPSA

(30–140 Å²)

MW

HBA

HBD

(<5)

Rotor

(<7)

LogP

(<5)

BBB

Ligand

5a

5b

5c

(−0.4 to 5.9)

(<520)

(<10)

HIA

CaCO₂

0.6713

0.6816

0.7301

0.6286

0.6978

0.7461

0.7790

0.8078

0.6812

0.8053

0.7510

0.7738

0.8078

0.6812

0.7710

0.7324

0.7650

0.7872

0.6826

0.8033

(−1.2 to +1.2)

Violations

8.84

6.34

6.70

6.88

6.71

7.53

4.55

4.91

5.09

4.92

8.63

5.91

6.27

6.45

6.27

8.36

5.45

5.81

5.99

5.82

37.81

74.50

63.59

72.83

102.39

37.81

74.50

63.59

72.83

102.39

37.81

74.50

63.59

72.83

102.39

37.81

74.50

63.59

72.83

102.39

613.34

485.39

494.81

525.84

587.49

477.35

349.40

359.82

389.85

451.49

546.24

418.29

428.71

458.74

520.38

505.40

377.45

387.87

417.90

479.55

3

5

6

7

3

5

6

7

3

5

6

7

3

5

6

7

1

0

1

0

1

0

1

0

6

7

4

5

4

5

4

5

5.34

3.79

4.19

4.14

4.63

3.52

1.96

2.37

2.32

2.81

5.14

3.58

3.99

3.94

4.43

3.21

1.66

2.07

2.02

2.50

0.9818

0.9966

1.000

0.9690

0.9524

0.9725

0.9404

0.9426

0.9615

0.9327

0.9650

0.9415

0.9302

0.9659

0.9428

0.9650

0.9415

0.9287

0.9595

0.9059

0.9531

0.9397

0.9084

2

1

2

1

0

1

0

2

1

2

1

5d

5e

6a

6b

6c

0.9922

0.9479

0.9874

0.9900

1.0000

0.9712

0.9743

0.9887

0.9900

1.0000

0.9745

0.9434

0.9906

0.9925

1.0000

0.9781

6d

6e

7a

7b

7c

7d

7e

8a

8b

8c

8d

8e

Abbreviations: ADME, absorption, distribution, metabolism, excretion; BBB, blood–brain barrier; HBA, hydrogen bond acceptor; HBD, hydrogen bond

donor; HIA, human intestinal absorption; MW, molecular weight; Rotor: number of rotatable groups; TPSA, topological polar surface area.

(s), 105.16 (s); UPLC–MS: m/z calculated for C₃₀H₁₅F₆N₇: 587.48;

observed mass: 589.1 [M + H]⁺.

126.69 (s), 111.09 (s), 100.04 (s); UPLC–MS: m/z calculated for

C

₂₂H₁₅N₅: 349.39; observed mass: 350.5 [M + H]⁺.

N‐(4‐Iodo‐3,5‐dimethylphenyl)‐3,5‐diphenylpyrazin‐2‐amine (6a)

Off‐white solid; yield: 70 mg, 72.5%; melting range: 198–200° C; ¹H

NMR (500 MHz, CDCl₃) δ 8.60 (s, 1H), 7.99–7.98 (d, J = 7.0 Hz, 2H),

7.79–7.78 (d, J = 7.0 Hz, 2H), 7.59–7.56 (t, J = 14.5 Hz, 2H), 7.53–7.50

(t, J = 14.5 Hz, 1H), 7.47–7.44 (t, J = 15.0 Hz, 2H), 7.38–7.35

(t, J = 14.5, 1H), 7.31 (s, 2H), 6.78 (s, 1H), 2.46 (s, 6H); ¹³C NMR

(126 MHz, CDCl₃) δ 147.90 (s), 143.06 (s), 142.54 (s), 141.60 (s),

137.11 (s), 136.93 (s), 136.73 (s), 129.49–129.43 (d), 128.83–128.68

(d), 128.33 (s), 125.86 (s), 118.22 (s), 29.74 (s); UPLC–MS: m/z

calculated for C₂₄H₂₀IN₃: 477.34; observed mass: 478 [M + H]⁺.

6‐Chloro‐N‐(3,5‐diphenylpyrazin‐2‐yl)pyrimidin‐4‐amine (6c)

Off‐white solid; yield: 52 mg, 71.52%; melting range: 198–200°C; ¹

H

NMR (400 MHz, CDCl₃) δ 8.78 (s, 1H), 8.56–8.54 (d, J = 7.0 Hz, 2H),

8.08–8.06 (d, J = 7.24 Hz, 2H), 7.98 (s, 1H), 7.79–7.77 (d, J = 6.88 Hz,

2H), 7.65–7.56 (m, 3H), 7.54–7.50 (t, J = 14.76, 2H), 7.48–7.44

(t, J = 14.44 Hz, 1H); ¹³C NMR (76 MHz, CDCl₃) δ 160.65 (s), 157.94

(s), 156.88 (s), 145.09 (s), 143.92 (s), 135.77–134.50 (t),

129.0–127.70 (m), 125.31 (s), 106.65 (s); UPLC–MS: m/z calculated

for C₂₀H₁₄ClN₅: 359.81; observed mass: 360.4 [M + H]⁺.

N‐(4‐Chloro‐5‐methoxypyrimidin‐2‐yl)‐3,5‐diphenylpyrazin‐2‐

amine (6d)

Off‐white solid; yield: 65 mg, 82.51%; melting range: 158–160°C; ¹

6‐(3,5‐Diphenylpyrazin‐2‐ylamino)pyridine‐3‐carbonitrile (6b)

White solid; yield: 56 mg, 79.27%; melting range: 211–212°C; ¹H

NMR (400 MHz, DMSO) δ 9.69 (s, 1H), 9.01 (s, 1H), 8.45 (dd, J = 2.3,

0.6 Hz, 1H), 8.17 (d, J = 7.6 Hz, 2H), 8.03 (dd, J = 8.8, 2.3 Hz, 1H), 7.84

(dd, J = 7.9, 1.5 Hz, 2H), 7.56 (dd, J = 17.0, 8.6 Hz, 3H), 7.52–7.44

(m, 4H); ¹³C NMR (126 MHz, DMSO) δ 152.64 (s), 145.60 (s), 145.43

(s), 141.06 (s), 137.94 (s), 129.76 (s), 129.50 (d), 129.02 (s), 128.82 (s),

H

NMR (400 MHz, DMSO) δ 9.98 (s, 1H), 9.14 (s, 1H), 8.26–8.20 (m,

2H), 7.90 (s, 1H), 7.75 (dd, J = 8.0, 1.4 Hz, 2H), 7.61–7.50 (m, 3H),

7.42–7.34 (m, 3H), 3.89 (s, 3H); ¹³C NMR (126 MHz, DMSO) δ 153.58

(s), 149.95 (s), 149.26 (s), 148.87 (s), 144.46 (s), 140.61 (s), 138.93 (s),

137.99 (s), 137.39 (s), 135.97 (s), 130.26 (s), 129.52 (s), 129.31 (s),

12 of 14

GANGARAPU ET AL.

|

128.61 (s), 128.37 (s), 127.16 (s), 57.19 (s); UPLC–MS: m/z calculated

for C₂₁H₁₆ClN₅O: 389.84; observed mass: 390.1 [M + H]⁺.

8.08–8.07 (d, J = 8.5 Hz, 2H), 7.79–7.77 (dd, J = 2.0 Hz, J = 2.0 Hz, 2H),

7.63–7.61 (d, J = 8.5 Hz, 2H), 7.52–7.50 (d, J = 8.5 Hz, 2H), 7.23–7.22

(d, J = 8.5 Hz, 2H), 7.12–7.10 (d, J = 8.5 Hz, 2H); ¹³C NMR (126 MHz,

CDCl₃) δ 157.23 (s), 152.13 (s), 150.25–150.17 (d), 147.89 (s), 140.66

(s), 139.46 (s), 136.81 (s), 136.02 (s), 134.27 (s), 133.72 (s),

129.45–129.38 (d), 128.80 (s), 128.35 (s), 116.49 (s), 115.91 (s),

104.91 (s); UPLC–MS: m/z calculated for C₂₈H₁₅Cl₂N₇: 520.37;

observed mass: 521.4 [M + H]⁺.

6‐[N‐(5‐Cyanopyridin‐2‐yl)‐N‐(3,5‐diphenylpyrazin‐2‐yl)amino]‐

pyridine‐3‐carbonitrile (6e)

Off‐white solid; yield: 78 mg, 85.62%; melting range: 121–123°C; ¹H

NMR (500 MHz, CDCl₃) δ 8.92 (s, 1H), 8.475–8.471 (d, J = 2.0 Hz, 2H),

8.15–8.13 (d, J = 6.5 Hz, 2H), 7.75–7.73 (dd, J = 2.0 Hz, J = 2.0 Hz, 2H),

7.62–7.60 (t, J = 8.0 Hz, 2H), 7.55–7.51 (m, 3H), 7.26–7.23 (m, 3H),

7.10–7.08 (d, J = 9.0 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 157.37 (s),

152.03 (s), 151.46 (s), 151.20 (s), 147.74 (s), 140.42 (s), 139.48 (s),

136.02 (s), 135.46 (s), 130.34 (s), 129.57 (s), 129.14 (s), 128.44 (s),

128.05 (s), 127.14 (s), 116.65 (s), 116.11 (s), 104.59 (s). UPLC–MS: m/z

calculated for C₂₈H₁₇N₇: 451.48; observed mass: 452.1 [M + H]⁺.

N‐(4‐Iodo‐3,5‐dimethylphenyl)‐3,5‐di‐p‐tolylpyrazin‐2‐amine (8a)

Off‐white solid; yield: 65 mg, 70.82%; melting range: 202–204°C; ¹

H

NMR (500 MHz, CDCl₃) δ 8.54 (s, 1 H), 7.88–7.87 (d, J = 7.5 Hz, 2H),

7.67–7.66 (d, J = 7.5 Hz, 2H), 7.37–7.36 (d, J = 7.5 Hz, 2H), 7.31

(s, 2 H), 7.26–7.24 (d, J = 7.0 Hz, 2H), 6.78 (s, 1H), 2.45 (s, 9H), 2.39

(s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 147.65 (s), 143.13 (s), 142.48

(s), 141.66 (s), 139.52 (s), 139.32 (s), 138.20 (s), 136.53 (s), 134.23 (s),

133.89 (s), 130.07 (d), 129.52 (s), 128.57 (s), 125.74 (s), 118.03 (s),

99.43 (s), 29.74 (s), 21.42–21.27 (d); UPLC–MS: m/z calculated for

C₂₆H₂₄IN₃: 506.39; observed mass: 507.2 [M + H]⁺.

6‐[3,5‐Bis(4‐chlorophenyl)pyrazin‐2‐ylamino]pyridine‐3‐

carbonitrile (7b)

Off‐white solid; yield: 52 mg, 78.62%; melting range: 252–255°C; ¹H

NMR (500 MHz, CDCl₃) δ 8.68 (s, 1H), 8.60–8.58 (dd, J = 0.5 Hz, 1H),

8.49–8.49 (d, J = 1.5 Hz, 1H), 7.97–7.95 (d, J = 8.5 Hz, 2H), 7.93–7.89

(m, 2H), 7.73–7.72 (d, J = 8.5 Hz, 2H), 7.59–7.57 (d, J = 8.5 Hz, 2H),

7.47–7.45 (d, J = 8.5 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 154.77 (s),

151.89 (s), 145.41 (s), 144.42 (s), 141.67 (s), 141.15 (s), 136.78 (s),

136.35 (s), 135.38 (s), 134.50 (s), 134.01 (s), 130.11 (s), 129.91 (s),

129.23 (s), 127.39 (d), 117.33 (s), 111.77 (s), 102.62 (s); UPLC–MS: m/z

calculated for C₂₂H₁₃Cl₂N₅: 418.28; observed mass: 419.4 [M + H]⁺.

6‐(3,5‐Di‐p‐tolylpyrazin‐2‐ylamino)pyridine‐3‐carbonitrile (8b)

Off‐white solid; yield: 56 mg, 81.71%; melting range: 218–220°C; ¹H

NMR (400 MHz, CDCl₃) δ 8.67–8.61 (t, 2H), 8.47–8.44 (d, J = 1.52 Hz,

1H), 8.25 (s, 1H), 7.96–7.91 (m, 3H), 7.69–7.67 (d, J = 7.96 Hz, 2H),

7.43–7.41 (d, J = 7.84 Hz, 2H), 7.32–7.28 (t, 2H), 2.48–2.44 (d, J = 18.56,

6H); ¹³C NMR (76 MHz, CDCl₃) δ 154.91 (s), 151.31 (s), 145.83 (s),

144.82 (s), 143.10 (s), 141.18 (s), 140.10 (s), 139.19 (s), 136.08 (s),

133.48 (s), 132.84 (s), 130.24 (s), 129.67 (s), 128.66 (s), 126.12 (s),

117.34 (s), 111.77 (s), 101.93 (s), 21.42–21.32 (d); UPLC–MS: m/z cal-

culated for C₂₄H₁₉N₅: 377.44; observed mass: 378.9 [M + H]⁺.

N‐[3,5‐Bis(4‐chlorophenyl)pyrazin‐2‐yl]‐6‐chloropyrimidin‐4‐

amine (7c)

Off‐white solid; yield: 57 mg, 84.04%; melting range: 206–208°C; ¹H

NMR (400 MHz, CDCl₃) δ 8.75 (s, 1H), 8.55–8.51 (d, J = 14.96 Hz,

2H), 8.01–7.98 (t, 2H), 7.87 (s, 1H), 7.73–7.71 (d, J = 8.44 Hz, 2H),

7.61–7.59 (d, J = 8.4 Hz, 2H), 7.50–7.48 (d, J = 8.52 Hz, 2H); ¹³C NMR

(76 MHz, CDCl₃) δ 161.81 (s), 158.75 (s), 157.90 (s), 145.00 (s),

141.86 (s), 136.87–136.48 (d), 135.60 (s), 134.31 (s), 133.73 (s),

130.09–129.95 (d), 129.27 (s), 127.49 (s), 107.80 (s). UPLC–MS: m/z

calculated for C₂₀H₁₂Cl₃N₅: 428.7; observed mass: 429.4 [M + H]⁺.

6‐Chloro‐N‐(3,5‐di‐p‐tolylpyrazin‐2‐yl)pyrimidin‐4‐amine (8c)

Off‐white solid; yield: 55 mg, 80.09%; melting range: 192–195°C; ¹H

NMR (400 MHz, CDCl₃) δ 8.72 (s, 1H), 8.539–8.530 (d, J = 3.68 Hz, 2H),

8.17 (s, 1H), 7.97–7.95 (d, J = 8.12 Hz, 2H), 7.67–7.65 (d, J = 8.0 Hz, 2H),

7.43–7.41 (d, J = 7.84 Hz, 2H), 7.33–7.28 (t, 2H), 2.48–2.44 (d, J = 19.4,

6H); ¹³C NMR (76 MHz, CDCl₃) δ 161.61 (s), 158.68 (s), 157.17 (s),

146.49 (s), 144.33 (s), 143.38 (s), 140.25 (s), 139.44 (s), 136.22 (s),

133.28 (s), 132.53 (s), 130.29 (s), 129.71 (s), 128.97 (s), 128.64 (s),

126.24 (s), 107.62 (s), 21.41–21.33 (d); UPLC–MS: m/z calculated for

C₂₂H₁₈ClN₅: 387.86; observed mass: 389.2 [M + H]⁺.

N‐(4‐Chloro‐5‐methoxypyrimidin‐2‐yl)‐3,5‐(4‐chlorophenyl)pyrazin‐

2‐amine (7d)

Off‐white solid; yield: 51 mg, 70.30%; melting range: 200–203°C; ¹H

NMR (500 MHz, CDCl₃) δ 8.74 (s, 1H), 8.01–7.99 (d, J = 8.0 Hz, 2H),

7.76 (s, 2H), 7.73–7.71 (d, J = 8.5 Hz, 2H), 7.47–7.45 (d, J = 8.0 Hz, 2H),

7.40–7.39 (d, J = 8.5 Hz, 2H), 3.91 (s, 3 H); ¹³C NMR (126 MHz, CDCl₃) δ

151.21 (s), 150.90 (s), 147.56 (s), 145.67 (s), 143.05 (s), 140.04 (s),

137.56 (s), 136.05 (s), 135.93–135.88 (d), 135.40–135.26 (d), 134.22 (s),

129.43 (s), 129.24 (s), 128.93 (s), 127.92 (s), 56.53 (s); UPLC–MS: m/z

calculated for C₂₁H₁₄Cl₃N₅O: 458.73; observed mass: 460.4 [M + H]⁺.

N‐(4‐Chloro‐5‐methoxypyrimidin‐2yl)‐3,5‐di‐p‐tolylpyrazin‐2‐

amine (8d)

Off‐white solid; yield: 60 mg, 75.66%; melting range: 211–213°C; ¹

H

NMR (400 MHz, CDCl₃) δ 8.78 (s, 1H), 7.99 (s, 2H), 7.83–7.78

(d, J = 20 Hz, 2H), 7.69 (s, 2H), 7.30–7.28 (d, J = 6.4 Hz, 4H), 3.90 (s,

3H), 2.42 (s, 6H); ¹³C NMR (100 MHz, CDCl₃)

δ 170.81 (s),

151.69–151.20 (d), 148.50 (s), 146.13 (s), 142.76 (s), 140.12 (s),

139.64–139.44 (d), 137.31 (s), 135.83 (s), 134.17 (s), 133.32 (s),

129.69–129.49 (d), 128.24 (s), 126.58 (s), 56.50 (s), 21.55–21.39 (d);

UPLC–MS: m/z calculated for C₂₃H₂₀ClN₅O: 417.89; observed mass:

418.3 [M + H]⁺.

6‐{N‐[3,5‐Bis(4‐chlorophenyl)pyrazin‐2‐yl]‐N‐(5‐cyanopyridin‐2‐

yl)amino}pyridine‐3‐carbonitrile (7e)

Off‐white solid; yield: 70 mg, 85.06%; melting range: 145–147°C; ¹H

NMR (500 MHz, CDCl₃) δ 8.89 (s, 1H), 8.49–8.48 (d, J = 2.0 Hz, 2H),

13 of 14

GANGARAPU ET AL.

|

6‐[N‐(5‐Cyanopyridin‐2‐yl)‐N‐(3,5‐di‐p‐tolylpyrazin‐2‐yl)amino]‐

pyridine‐3‐carbonitrile (8e)

compounds were dissolved in DMSO and diluted with fresh medium.

The final DMSO concentration was 1.5%. Nearly 24 hr after the ad-

dition of compounds, the MTT assay was carried out by treating the

cells with 5 mg/ml MTT solution in phosphate‐buffered solution.

After 3 hr of MTT treatment, the formazan crystals formed were

dissolved in 100 ml DMSO. The absorbance was measured at 540 nm

using a PerkinElmer EnSpire Multimode Plate Reader. Cell viability

was expressed as the percentage of viable cells compared with un-

treated DMSO control cells.

Off‐white solid; yield: 74 mg, 86.13%; melting range: 201–203°C, ¹H

NMR (400 MHz, CDCl₃) δ 8.89 (s, 1H), 8.49 (s, 2H), 8.06 (d, 2H), 7.76

(s, 2H), 7.55 (s, 2H), 7.35 (s, 2H), 7.12–7.05 (d, J = 27.6 Hz, 4H), 2.46

(s, 3H), 2.30 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 157.38 (s),

151.99–151.50 (d), 151.14 (s), 147.11 (s), 140.64–140.40 (s), 139.74 (s),

138.99 (s), 133.13–132.73 (d), 129.87 (s), 129.17 (s), 127.03 (s), 116.79

(s), 116.05 (s), 116.05 (s), 104.43 (s), 21.47–21.32 (d); UPLC–MS: m/z

calculated for C₃₀H₂₁N₇: 479.53; observed mass: 480.3 [M + H]⁺.

|

4.2.3

Protein structure prediction

|

4.2

Biological assays

The catalytic domain of protein kinase (PknB) from the Mtb protein

sequence was retrieved from the UniProt database (UID: P9WI81). The

selected protein sequences were used to predict sequence similarity and

to predict sequence templates by PSI‐BLAST.^[24]The selected template

sequences were used to build three‐dimensional protein structures using

SWISS‐MODEL.^[25]Homology modeling by Swiss PDB Viewer helps to

predict the complexity of the protein structures which was validated to

check overall protein quality, and stereochemical activities of atoms and

amino acids were predicted by structural analysis and verification server

(SAVES).^[26]The conformational complexity of protein structures was

used to predict the active site of amino acids that helps in ligand binding

and evaluated using CastP calculation server. The best complex protein

structures were used for molecular docking.

|

4.2.1

Experimental procedure for antituberculosis

studies

LJ medium was used to detect the anti‐TB activity of synthesized

2‐aryl‐3,5‐disubstituted pyrazine derivatives. Plain LJ medium was

prepared as per the standard guidelines of culture and the DST of

Mtb (RNTCP standard SOPs maintained by the DST‐accrediting la-

boratories throughout the country) at NTI, Bangalore, India. For the

5 mg of each PZA derivative weighed accurately and dissolved in 5 ml

of dimethylformamide solvent. To test the MIC, three different drug

concentrations (15, 25, and 50 µg/ml) were prepared using the parent

stock solution. McCarteny bottles (imported unbreakable with sealed

caps) were used to load the medium, and in each bottle 5 ml of

prepared drug with LJ medium is dispersed in a laminar wood. All the

bottles containing the drug medium were inspissated at 85°C until

the liquid form of the medium solidified as slants, which were further

QC tested by keeping the LJ slants under 37°C incubation for 24 hr.

For all the tested Mtb strains, including standard H37Rv, Mtb strain

1, and Mtb strain 2, inoculum were prepared separately and in-

oculated in the drug‐containing medium along with positive controls

(RMP and INH) used for this study. All cultures were examined 48 hr

after inoculation at 37°C in a walk‐in incubator to detect cross‐

contamination. Thereafter, cultures were examined weekly, up to

8 weeks (to be declared as sensitive), on a specified day of the week.

Culture results were taken manually based on the typical morpho-

logical characteristics of Mtb.

The synthetic chemical compounds were listed based on mole-

cular weight and the resultant compounds were used as the training

set. To design the chemical structures, ACD/ChemSketch soft-

ware^[27]was used to add all chemical compositions and the final

output was saved in MOL2 format. The training sets were used to

predict pharmacophore using Molinspiration server^[28]and QSAR

(quantitative structure–activity relationship) properties were pre-

dicted using HyperChem. A training set helps to predict the complex

polarity and flexibility, to examine MM3 force fields, to examine

HOMO (highest occupied molecular orbital) and LUMO (lowest oc-

cupied molecular orbital), and to understand new molecular orbitals

in individual compounds.^[29]The lead compounds or scaffolds were

identified from the diversified compound pool and then subjected to

accelerated screening; a screened pool is focused for bio‐targets to

inhibit diseases. Here, we used structural screening, fragment ana-

lysis, and pharmacological analyses to screen the compounds based

on the interaction with target apoptotic proteins.

|

4.2.2

Experimental procedure for cytotoxicity

determination

ORCID

Cytotoxicities of the synthesized 2‐aryl‐3,5‐disubstituted pyrazine

derivatives were tested in human embryonic kidney cells (HEK‐293)

using the MTT assay method. HEK‐293 cells were cultured in

Dulbecco's modified Eagle's medium supplemented with 10% fetal

bovine serum, 10,000 units/ml penicillin, 10 mg/ml streptomycin, and

25 mg/ml amphotericin B in a 96‐well plate (~15 × 10³cells/well).

After 24 hr, the medium was removed and replaced with varying

concentrations of pyrazine derivatives for another 24 hr. All

Eeda Koti Reddy

http://orcid.org/0000-0003-4048-7178

REFERENCES

[1] WHO, Global Tuberculosis Report, 2017. http://www.who.int/tb/

publications/global_report/en/

[2] World Health Organization, 2010. http://www.who.int/tb/publica

tions/globalreport/2010/en/index.html

[3] C. Lienhardt, P. Glaziou, M. Uplekar, K. Lonnroth, H. Getahun,

M. Raviglione, Nat. Rev. Microbiol. 2012, 10, 407.

14 of 14

GANGARAPU ET AL.

|

[4] G. Craig, A. Daftary, N. Engel, S. O'Driscoll, A. Ioannaki, Int. J. Infect.

Dis. 2017, 56, 90.

[17] J. D. Osborne, T. P. Matthews, T. A. McHardy, N. Proisy, K.‐M. J.

Cheung, M. Lainchbury, N. Brown, M. I. Walton, P. D. Eve, K. Boxall,

J. Katherine, J. Med. Chem. 2016, 59, 5221.

[5] S. S. Gautam, R. F. Toole, J. Pulm. Respir. Med. 2016, 6, 2.

[6] A. I. Zumla, S. H. Gillespie, M. Hoelscher, P. P. Philips, S. T. Cole,

I. Abubakar, T. D. McHugh, M. Schito, M. Maeurer, A. J. Nunn, Lancet

Infect. Dis. 2014, 14, 327.

[18] R. Rahmani, K. Ban, A. J. Jones, L. Ferrins, D. Ganame, M. L. Sykes, V.

M. Avery, K. L. White, E. Ryan, M. Kaiser, J. Med. Chem. 2015, 58, 6753.

[19] Y. Younis, F. Douelle, D. G. Cabrera, C. L. Manach, A. T. Nchinda, T.

Paquet, L. J. Street, K. L. White, M. K. Zabiulla, J. T. Joseph, J. Med.

Chem. 2013, 56, 8860.

[7] M. Pai, M. A. Behr, D. Dowdy, K. Dheda, M. Divangahi, C. C. Boehme,

A. Ginsberg, S. Swaminathan, M. Spigelman, H. Getahun, D. Menzies,

M. Raviglione, Nat. Rev. Dis. Primers 2016, 2, 16076.

[20] S. Berg, M. Bergh, S. Hellberg, K. Hoegdin, Y. L. Alfredsson, P. Soe-

derman, S. von Berg, T. Weigelt, M. Ormoe, Y. Xue, J. Med. Chem.

2012, 55, 9107.

[8] J. D. Hosford, M. E. von Fricken, M. Lauzardo, M. Changd, Y. Dai, J. A.

Lyon, J. Shuster, K. Fennelly, Tuberculosis 2015, 95, 112.

[9] P. S. Charifson, A. L. Grillot, T. H. Grossman, J. D. Parsons,

M. Badia, S. Bellon, D. D. Deininger, J. E. Drumm, C. H. Gross,

A. LeTiran, Y. Liao, N. Mani, D. P. Nicolau, E. Perola, S. Ronkin,

D. Shannon, L. L. Swenson, Q. Tang, P. R. Tessier, S. K. Tian,

M. Trudeau, T. Wang, Y. Wei, H. Zhang, D. Stamos, J. Med. Chem.

2008, 51, 5243.

[21] J. Humphrey, N. Kablaoui, S. Kazmirski, M. Kraus, S. Kupchinsky, J. L.

John, L. Lingardo, M. A. Marx, D. Richter, S. P. Tanis, J. Med. Chem.

2011, 54, 8490.

[22] (a) E. K. Reddy, R. Chandran, A. M. Sajith, K. V. Dileep, C. Sadasivan,

S. Anwar, RSC Adv. 2016, 6, 77431; (b) E. K. Reddy, R. Chandran,

K. Mantosh, A. M. Sajith, R. V. Omkumar, C. Sadasivan, S. Anwar, Eur.

J. Med. Chem. 2017, 139, 367; (c) B. Savitha, E. K. Reddy, C. S. Ananda

Kumar, R. P. Karuvalam, M. S. AliPadusha, V. A. Bakulev,

K. H. Narasimhamurthy, A. M. Sajith, M. NibinJoy, Tetrahedron Lett.

2019, 52, 151332.

[10] P. S. Shirude, P. Madhavapeddi, J. A. Tucker, K. Murugan, V. Patil,

H. Basavarajappa, A. V. Raichurkar, V. Humnabadkar, S. Hussein,

S. Sharma, V. K. Ramya, C. B. Narayan, T. S. Balganesh, V. K.

Sambandamurthy, ACS Chem. Biol. 2013, 8, 519.

[11] T. J. Miles, A. J. Hennessy, B. Bax, G. Brooks, B. S. Brown, P. Brown,

N. Cailleau, D. Chen, S. Dabbs, T. D. Davies, J. M. Esken, L. Giordano,

J. L. Hoover, J. Huang, G. E. Jones, S. K. K. Sukmar, C. Spitzfaden, R. E.

Markwell, E. A. Mlingthorn, S. Rittenhouse, M. N. Gwynn, N. D.

Pearson, Bioorg. Med. Chem. Lett. 2013, 23, 5437.

[23] G. N. Reddy, E. K. Reddy, A. M. Sajith, Y. Shivaraj, K. B. Chandra-

shekar, ChemistrySelect 2017, 2, 7706.

[24] M. K. Hasballah, A. Azhar, N. Rahmi, Int. J. Trop. Vet. Biomed. Res.

2016, 1, 45.

[25] T. P. Sathishkumar, P. Navaneethakrishnan, S. Hankar, R. Rajasekar,

J. Reinf. Plast. Compos. 2013, 32, 19.

[12] V. Makarov, B. Lechartier, M. Zhang, J. Neres, A. M. van der Sar, S. A.

Raadsen, R. C. Hartkoorn, O. B. Ryabova, A. Vocat, L. A. Decosterd,

N. Widmer, T. Buclin, W. Bitter, K. Andries, F. Pojer, P. J. Dyson, S. T.

Cole, Mol. Med. 2014, 6, 372.

[26] T. Choedon, G. Mathan, V. K. Choedon, BMC Complement. Altern. Med.

2015, 15, 182.

[27] J. Bethuyne, S. D. Gieter, O. Zwaenepoel, A. Garcia‐Pino, K. Durinck,

A. Verhelle, G. H. Ghassabeh, F. Speleman, R. Loris, J. Gettemans,

Nucleic Acids Res. 2014, 42, 12928.

[13] P. S. Shirude, R. Shandil, C. Sadler, M. Naik, V. Hosagrahara,

S. Hameed, V. Shinde, C. Bathula, V. Humnabadkar, N. Kumar,

J. Reddy, V. Panduga, S. Sharma, A. Ambady, N. Hegde, J. Whiteaker,

R. E. McLaughlin, H. Gardner, P. Madhavapeddi, V. Ramachandran,

P. Kaur, A. Narayan, S. Guptha, D. Awasthy, C. Narayan, J. Maha-

devaswamy, K. G. Vishwas, V. Ahuja, A. Srivastava, K. R. Prabhakar,

S. Bharath, R. Kale, M. Ramaiah, N. R. Choudhury, V. Sambanda-

murthy, S. M. Solapure, P. S. Iyer, S. Narayanan, M. Chatterji, J. Med.

Chem. 2013, 56, 9701.

[28] Z. Liyuan, G. Lingkun, R. Patricia, S. Stanley, J. R. Paul, J. Shen,

Q. Zhang, 2015.

[29] M. L. Tress, F. Abascal, A. Valencia, Trends Biochem. Sci. 2017, 42, 98.

SUPPORTING INFORMATION

Additional supporting information may be found online in the

Supporting Information section.

[14] J. C. Peter, S. S. Hamish, S. T. T. Amy, B. Adrian, G. F. Scott, B. C.

Christopher, U. L. Manisha, M. U. Anna, G. Jerome, M. Magali,

Q. Laurence, Bioorg. Med. Chem. Lett. 2017, 27, 5190–5196.

[15] (a) G. N. Reddy, A. Ranganatham, E. K. Reddy, H. D. Surendra, A. M.

Sajith, Y. Shivaraj, K. B. Chandrasekhare, J. Heterocycl. Chem. 2019,

56, 1117; (b) T. N. Prasad, E. K. Reddy, G. V. Babu, S. F. Basha,

S. Anwar, Synth. Commun. 2019, 49, 1277.

How to cite this article: Gangarapu NR, Ranganatham A,

Reddy EK, Yellappa S, Chandrasekhar KB. 2‐Aminoaryl‐3,

5‐diaryl pyrazines: Synthesis, biological evaluation against

Mycobacterium tuberculosis and docking studies. Arch Pharm.

2020;e1900368. https://doi.org/10.1002/ardp.201900368

[16] (a) K. Stoffels, V. Mathys, M. Fauville‐Dufaux, R. Wintjens, P. Bifani,

Antimicrob. Agents Chemother. 2012, 56, 5186; (b) B. J. Benjamin,

A. Aboelela, E. Minaei, E. Longguang, X. Jiang, Z. Xu, U. Ali, K. Fildes,

C. Y. Cheung, M. Simon, M. Cook, D. C. Johnson, J. Med. Chem. 2018,

61, 8299.

Article Doi

2-Aminoaryl-3,5-diaryl pyrazines: Synthesis, biological evaluation against Mycobacterium tuberculosis and docking studies

DOI: 10.1002/ardp.201900368

Source and publish data:

Authors:

Article abstract of DOI:10.1002/ardp.201900368

Full text of DOI:10.1002/ardp.201900368

Products guided by the article

R&D Labs maybe for 41270-70-6

Relevant to this article

Hot Product