Supporting Information

Synthesis and Bioactivity of Novel Triazole Incorporated Benzothiazinone Derivatives as Antitubercular and Antioxidant Agent

Mubarak H. Shaikh,a Dnyaneshwar D. Subhedar,a Manisha Arkile,b Vijay M. Khedkarb,c

Nandadeep Jadhav,b Dhiman Sarkar,b Bapurao B. Shingate*a

aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431 004, IndiabCombi Chem Bio Resource Centre, National Chemical Laboratory, Pune 411 008, IndiacSchool of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000, South AfricaE-mail:* bapushingate@gmail.com (B. B. Shingate) *Corresponding author. Tel.: (91)-240-2403312; Fax: (91)-240-2403113

1. Experimental

1.1. General

All the solvents and reagents were purchased from commercial suppliers Spectrochem, Rankem,

Alfa Aesar, Sigma Aldrich and are used without further purification. Reaction time and purity of

the products were monitored by thin layer chromatography (TLC) aluminum sheets, silica gel

60-F254 precoated, Merck, Germany and locating the spots using UV light as the visualizing agent

or Iodine vapors. All the melting points were determined in open capillary method and are

uncorrected. 1H NMR spectra were recorded on Jeol 400 MHz and 13C NMR on 100 MHz

spectrometer using residual solvent as internal standard (CDCl3). The chemical shifts (δ) were

reported in ppm and are given in parts per million (ppm). The splitting pattern abbreviations are

designed as singlet (s); doublet (d); double doublet (dd); triplet (t); quartet (q) and multiplet (m).

Agilent Technologies 1200 series HPLC paired to a 6130 mass spectrometer with electron spray

ionization (ESI) was used for LCMS data, whereas HRMS data were recorded on Bruker

Daltonics MicroTOF-Q-II with ESI.

1

Synthesis of benzyl azides 5a-h:

A] Synthesis of benzyl alcohols:

Substituted benzaldehydes (1 equiv) were taken in round bottom flask; methanol used as a

solvent and allowed reaction mixture for stirring below 0 °C. Then, NaBH4 (3 equiv) were added

slowly with constant stirring and maintaining the temperature below 0 °C. The progress of the

reaction was monitored by TLC using ethyl acetate: hexane as a solvent system. After

completion of the reaction, the reaction mass was poured on crushed ice. Compound extracted in

ethyl acetate (3 x 10 mL) and combined organic layer was dried over MgSO4. Solvent was

removed under reduced pressure, and the benzyl alcohols were sufficiently pure to use in next

step.

B] Synthesis of benzyl bromides:

Benzyl alcohols (1 equiv) were taken in RBF and dichloromethane as a solvent. This reaction

allowed to stir below 5 °C, then added drop by drop phosphorus tribromide (PBr3) (1 equiv). The

progress of the reaction was monitored by TLC using ethyl acetate: hexane as a solvent system.

After completion of the reaction, the reaction mass was poured on crushed ice. Ethyl acetate was

added to the mixture and the organic layer was separated. The aqueous layer was extracted with

3 x 10 mL of ethyl acetate and the combined organic layers were dried over MgSO4. Solvent was

removed under reduced pressure, and the benzyl bromides were sufficiently pure to use without

further work up.

C] Synthesis of benzyl azides 5a-h:

To a stirred solution of the corresponding bromide (1 equiv) in a 50 mL Acetone-Water mixture

(1:4) was added NaN3 (1.5 equiv).The resulting suspension was stirred at rt for 24 hr. The

2

progress of the reaction was monitored by TLC using ethyl acetate: hexane as a solvent system.

After completion of the reaction, the reaction mass was poured on crushed ice. Ethyl acetate was

added to the mixture and the organic layer was separated. The aqueous layer was extracted with

3 x 10 mL of ethyl acetate and the combined organic layers were dried over MgSO4. Solvent was

removed under reduced pressure, and the azides were 5a-h sufficiently pure to use without

further work up.

General procedure for the preparation of azidonitrobenzene 5i-j:

Nitroaniline (0.1 mol) was dissolved in 1:1 ratio of HCl:H2O and taken in a round bottom flask

equipped with stirrer. The reaction was agitated at 0-5 °C; sodium nitrite (0.12 mol) was

dissolved in water and added drop wise, sodium azide (0.1 mol) dissolved in water and added

drop wise, then reaction is allowed to continue for 30 min. The resultant precipitate was

extracted with chloroform and washed successively with water. The organic layer was dried over

anhydrous sodium sulphate, and the solvent stripped out in rotary evaporator to get

nitroazidobenzene 5i and 5j, yield 90% ; m.p. for 5i; 65-68 and for 5j; 53-54 °C.

Synthesis of 2H-benzo[b][1,4]thiazin-3(4H)-one 3:

To the stirred solution of 2-aminothiophenol (0.01 mol), monochloroacetic acid (0.012 mol) and

sodium acetate (0.012 mol) was added in 5 mL acetic acid. The resulting mixture was allowed

for reflux for 2-3 hr. After the completion of reaction, as evident by TLC, it was quenched by ice

and subsequently extracted with ethyl acetate (3 × 30 mL). The combined organic layers were

washed with brine solution, dried over anhydrous Na2SO4 and concentrated under reduced

pressure. The obtained crude compound was recrystallized using aq. ethanol.

3

Synthesis of 4-(prop-2-yn-1-yl)-2H-benzo[b][1,4]thiazin-3(4H)-one 4:

To a stirred suspension of sodium hydride (1.5 mmol) in dry DMF (10 mL), 2H-benzo[b]

[1,4]thiazin-3(4H)-one 3 (1 mmol) was added, resulting in the formation of anion. The solution

was stirred at room temperature till the evolution of hydrogen ceases. To this reaction mixture

then added drop wise a solution of propargyl bromide (1.1 mmol) in DMF. The reaction mixture

was allowed to constant stirring for about 2 hr. After the completion of reaction, as evident by

TLC, it was quenched by drop wise addition of water (20 mL) and subsequently extracted with

ethyl acetate (3 × 30 mL). The combined organic layers were washed with brine solution, dried

over anhydrous Na2SO4 and concentrated under reduced pressure. Mixture furnished the desired

4-(prop-2-yn-1-yl)-2H-benzo[b][1,4]thiazin-3(4H)-one (4) in 90% yields.

Table S1. Anti-tubercular activity of compounds against avirulent strain of dormant MTB H37Ra

and M. Bovis BCG

EntryMycobacterium tuberculosis H37Ra Mycobacterium Bovis BCG

30 µg/ml 10 µg/ml 3 µg/ml 30 µg/ml 10 µg/ml 3 µg/ml

6a 70.6 52.9 9.8 91.1 59.2 28.3

6b 60.2 48.9 34.8 88.5 62.2 36.1

6c 91.3 77.5 73.8 95.3 52.9 40.6

6d 65.8 75.3 52.1 58.1 55.2 66.1

6e 94.2 66.6 8.6 95.5 16.4 3.3

6f 37.8 74.9 41.9 58.0 80.2 32.0

6g 49.8 38.4 58.0 43.9 35.3 58.8

6h 82.2 59.3 -15.4 74.3 26.8 1.1

6i 28.5 0.3 24.2 33.1 30.7 15.9

6j 1.2 8.7 25.7 26.6 31.3 39.3

4

Table S2. Anti-proliferative activity of compounds against cancer cell lines A549, A431 and HeLa.

Entry

A549 A431 Hela

30

µg/ml

10

µg/ml3 µg/ml

30

µg/ml

10

µg/ml

3

µg/ml

30

µg/ml

10

µg/ml

3

µg/ml

6a 8.43 17.56 0.00 44.7 12.9 10.8 55.5 53.2 36.5

6b -11.78 0.00 -9.71 15.6 16.5 13.9 20.1 -3.0 0.0

6c 30.90 4.10 12.32 44.5 31.1 17.3 51.3 46.3 45.1

6d 51.91 4.35 3.60 61.5 40.9 35.0 45.1 29.6 41.9

6e 19.04 3.31 6.38 4.4 -13.7 -6.3 45.7 43.4 52.3

6f 23.34 -8.41 4.19 31.4 29.6 15.4 55.9 38.6 48.1

6g 44.78 -1.37 -5.96 35.5 4.0 8.6 41.3 32.9 40.4

6h 25.91 -9.82 -7.85 -10.8 -37.2 0.0 39.6 8.8 0.0

6i 38.71 24.73 33.22 0.0 0.0 0.0 35.8 -0.9 13.3

6j 22.02 0.19 12.32 29.7 25.4 17.2 37.5 13.3 11.9

5

Table S3. Pharmacokinetic parameters important for good oral bioavailability and drug likeness

score.

Entry%

ABS

TPSA

(A2)

n-

RO

TB

MV MWmiLog

P

n-

ON

n-

OH

NH

Lipinski

violation

Drug

likeness

score

Rule - - - - < 500 ≤ 5 < 10 < 5 ≤ 1

6a 75.6 96.9 5 316.7 381.4 2.71 8 0 0 0.25

6b 75.6 96.9 5 316.7 381.4 2.69 8 0 0 0.35

6c 91.4 51.1 4 298.3 354.4 2.92 5 0 0 0.63

6d 91.4 51.1 4 306.9 370.9 3.43 5 0 0 0.78

6e 91.4 51.1 4 306.9 370.9 3.41 5 0 0 0.50

6f 91.4 51.1 4 306.9 370.9 3.39 5 0 0 0.07

6g 91.4 51.1 4 311.3 415.3 3.56 5 0 0 0.38

6h 91.4 51.1 4 293.4 336.4 2.76 5 0 0 0.14

6i 75.6 96.9 4 299.9 367.4 2.39 8 0 0 0.20

6j 75.6 96.9 4 299.9 367.4 2.58 8 0 0 0.19

6

Antitubercular activity protocol

Compounds were tested for their in vitro effects against MTB H37Ra (ATCC 25177)

which is susceptible to control drugs (Rifampicin, Isoniazid, Ethambutol and Pyrazinamide).

Compounds were screened for their inhibitory effect on MTB by in vitro according to standard

XTT Reduction Menadione Assay (XRMA) protocol as described previously.28 Dimethyl

Sulfoxide (DMSO) was used as a solvent or negative control. In order to clarify any effect of

DMSO on the biological screening, separate studies were carried out with solutions alone of

DMSO and showed no activity against any mycobacteria. Rifampicin and Isoniazide was used as

positive control for assay. Primary screening was done against MTB at 30, 10 and 3 µg/mL

concentration of compound. Those compounds were shown more than 90 percent inhibition at 30

ug/mL which were selected for dose response. The MIC (in µg/mL) was recorded as the lowest

concentration/highest dilution of the compounds/control drugs that completely inhibited the

growth of MTB cultures.

The in vitro effect of compounds against M. bovis BCG (ATCC 35743) was done

according to standard NR assay protocol as described previously.28 Briefly in NR assay, take 80

µL of culture from incubated 96 well plate into another 96 well plate, then add 80 µL of 1%

sulfanilic acid in 20% of conc. HCl, incubate it for 10 min at room temperature then add 80 µL

of 0.1 % NEDD solution in D/W. Finally, the optical density of the suspension was measured at

540 nm by using micro plate reader. MIC and IC50 values were calculated by using origin9

software. The % inhibition of bacilli was measured by using following formula,

% Inhibition= [(Abs of control)-(Abs of test sample)/(Abs of control)-(Abs of blank)] × 100.

Control: cell growth in medium without compound, with DMSO

Test: cell growth in presence of compound

7

Blank: culture medium without cells.

Cytotoxicity

Three human cancer cell lines, HeLa (human cervical cancer cell line), A549 (human

lung adenocarcinoma cell line) and A431 (Epidemoid carcinoma cell line) were used to check

the cytotoxicity of compounds. The cell lines were obtained from the American Type Culture

Collection (ATCC) and maintained in T 25 flasks with 10 % (v/v) fetal bovine serum (FBS)

containing Dulbecco's Modified Eagle Medium (DMEM). Cell lines were maintained at 37 ºC

under 5% CO2 and 95% air in a humidified atmosphere. Medium were replaced twice a week.

All the compounds were tested for their cytotoxicity against HeLa, A549 and A431 cell line by

using modified MTT assay as described previously.30 Briefly, Cells were seeded as, 1.5×104

cells/mL for HeLa, 1×104 -cells/well for A549 and A431 in a 96 well plate. The plates were

incubated for 24 hr into CO2 incubator (37 ºC under 5% CO2 and 95% air in a humidified

atmosphere) to adhere the cells. After incubation, compound was added in such a way that final

concentration becomes 30, 10, and 3 µg/mL in the test well. Again plates were incubated for

additional 72 hr for HeLa and 48 h for A549 and A431 to see the effect of compound on cells.

After that cell meduim was replaced with 100 µl of Glucose-MTT (0.5mg/ml)-PBS medium and

kept the plate for 2-4 hr to form the reduced MTT or Formazan crystals. This reduced MTT or

Formazan crystals were solubilized by addition of acidified isopropanol. The optical density was

read on a micro plate reader (Spectramax plus 384 plate reader, Molecular Devices Inc) at 470

nm filter against a blank prepared from cell-free wells. Absorbance given by cells treated with

the vehicle alone was taken as 100% cell growth. All experiments were performed in triplicates

and the quantitative value was expressed as the average ± standard deviation. IC50 and MIC

8

values were calculated by plotting the graphs, by using Origin Pro software. The % inhibition

was determined using the following formula,

% cytotoxicity = [(average Absorbance of control−Absorbance of compound)/(Absorbance of

control-Absorbance of blank) ] × 100, where control is the culture medium with cells and DMSO

and blank is the culture medium without cells.

DPPH radical scavenging activity

The hydrogen atom or electron donation ability of the some compounds were measured

from the bleaching of the purple colored methanol solution of 1,1-diphenyl-1-picrylhydrazyl

(DPPH). The spectrophotometric assay uses the stable radical DPPH as a reagent. 1 mL of

various concentrations of the test compounds (5, 10, 25, 50 and 100 µg/mL) in methanol was

added to 4 mL of 0.004% (w/v) methanol solution of DPPH. The reaction mixture was incubated

at 37 °C. The scavenging activity on DPPH was determined by measuring the absorbance at 517

nm after 30 min. All tests were performed in triplicate and the mean values were entered. The

percent of inhibition (I %) of free radical production from DPPH was calculated by the following

equation

% of scavenging = [(Acontrol – Asample)/(Asample × 100)].

Where, Acontrol is the absorbance of the control (DPPH radical without test sample)

Asample is the absorbance of the test sample (DPPH radical with test sample). The control contains

all reagents except the test samples.

9

Molecular Docking studies

Molecular docking studies were carried out using the Glide (Grid-Based Ligand Docking with

Energetics) software which is an interactive molecular graphics tool for the studying ligand-

receptor interactions and identification of potential binding modes of the biomolecules. The

three-dimensional X-ray structure of DprE1 (decaprenylphosphoryl-β-D-ribose-2'-epimerase)

enzyme of Mycobacterium tuberculosis (PDB ID:4FDO) in complex with an inhibitor was

retrieved from the Protein Data Bank (www.rcsb.org). The Protein Preparation Wizard tool was

used to preprocess the enzyme structure for docking which involved deleting the

crystallographically observed water molecules (since no water molecule was observed to be

conserved), addition of missing hydrogens/side chain atoms and assigning the appropriate charge

and protonation state. The hydrogen atoms were added corresponding to pH 7.0 considering the

appropriate ionization states for the acidic and basic amino acid residues in the enzyme structure.

Thereafter to relieve the steric clashes among the residues caused due to addition of hydrogen

atoms, the structure was subjected to energy minimization using OPLS-2005 force field until the

RMSD (root mean square deviation) constraint was reached 0.3Å. The 3D structures of the title

compounds (6a-j) were sketched using the build panel in Maestro and subsequently optimized

using Ligprep module which involves the addition of hydrogens, adjusting realistic bond lengths

and angles, correct chiralities, ionization states, tautomers, stereo chemistries and ring

conformations. Partial charges were ascribed to the structures using the (OPLS-2005) force-field

and were then subjected to energy minimization until it reached a RMSD cutoff of 0.01Å. The

resulting structures were then used for carrying out docking study. The active site of DprE1

enzyme was defined for docking using the Receptor Grid Generation panel in Glide. With the

non-covalently bound native ligand in place, a grid file was generated with a dimension of

10

12x12x12Å box cantered on the centroid of the co-crystallized ligand so as explore a larger

region of the enzyme structure. All the title compounds, 6a-j, were subjected to docking using

the extra precision (XP) Glide scoring function to access their potential binding affinities. This

scoring function is equipped with force field-based parameters accounting for contributions from

steric and electrostatic interactions alongwith terms related to solvation, hydrophobic, hydrogen

bonding and metal-ligand interactions all incorporated in the empirical energy functions. The

output files in terms of the docking poses were visualized and analyzed for the key elements of

interaction with the receptor using the Maestro’s Pose Viewer utility.

11

6a. LCMS

6a. 1H NMR (400 MHz, CDCl3, ppm)

12

6a. 13C NMR (100 MHz, CDCl3, ppm)

13

6b. LCMS

6b. 1H NMR (400 MHz, CDCl3, ppm)

14

6b. 13C NMR (100 MHz, CDCl3, ppm)

15

6c. LCMS

6c. 1H NMR (400 MHz, CDCl3, ppm)

16

6d. HRMS

17

6d. 1H NMR (400 MHz, CDCl3, ppm)

9 8 7 6 5 4 3 2 1 0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

6.642.75 2.26 2.001.95

TMS

3.29

5.09

5.37

6.93

6.95

7.10

7.12

7.24

7.26

7.56

7.677.69

6d. 1H NMR (400 MHz, CDCl3, ppm)

8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

3.08 1.711.281.23 0.95 0.92

6.93

6.95

6.96

7.10

7.12

7.17

7.19

7.19

7.24

7.26

7.56

7.677.69

18

6e. 1H NMR (400 MHz, CDCl3, ppm)

8 7 6 5 4 3 2 1 0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

6.292.19 2.09 2.001.90

TMS

0.00

6e. 1H NMR (400 MHz, CDCl3, ppm)

8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

6.292.19 1.01

19

6e. 13C NMR (100 MHz, CDCl3, ppm)

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

31.59

41.64

53.55

76.83

77.09

77.35

118.78

123.26

123.92

127.68

129.33

129.47

132.94

134.84

139.87

165.60

6e. 13C NMR (100 MHz, CDCl3, ppm)

165 160 155 150 145 140 135 130 125 120

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

118.78

123.26

123.92

127.68

128.13

129.33

129.47

132.94

134.84

139.87

165.60

20

6g. HRMS

21

6g. 1H NMR (400 MHz, CDCl3, ppm)

9 8 7 6 5 4 3 2 1 0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

4.27 2.00 2.001.17

TMS

3.29

5.09

5.38

6.94

6.95

6.97

7.17

7.17

7.19

7.257.51

7.68

7.70

6g. 1H NMR (400 MHz, CDCl3, ppm)

8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

4.27 2.821.17 1.02

6.94

6.95

6.97

6.99

7.16

7.17

7.17

7.19

7.21

7.25

7.267.51

7.68

7.70

22

6g. 13CNMR (100 MHz, CDCl3, ppm)

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

31.60

41.64

53.55

76.81

77.06

77.32

118.79

123.26

123.92

128.13

129.33

129.47

132.93

134.85

139.87

165.60

6g. 13CNMR (100 MHz, CDCl3, ppm)

170 165 160 155 150 145 140 135 130 125 120 115

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

118.79

123.26

123.92

127.68128.13

129.33

129.47

132.93

134.85

139.87

165.60

23

6i. LCMS

6i. 1H NMR (400 MHz, CDCl3, ppm)

24

6i. 13C NMR (100 MHz, CDCl3, ppm)

25