Drug efficacy of novel 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy chromans; evaluated via DNA gyrase inhibition, bacterial cell wall lesion and antibacterial prospective

B i o o r g an i c &M e d i c i n a l C h e m i s tr y x x x (x xxx )x xx–xxx

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Drug eﬃcacy of novel 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy

chromans; evaluated via DNA gyrase inhibition, bacterial cell wall lesion

and antibacterial prospective

Thangarasu Ponnusamy^a, Manikandan Alagumuthu^b,^⁎, S. Thamaraiselvi^c,^⁎

a

Research and Development Centre, Bharathiar University, Coimbatore 641046, India

Department of Biotechnology, School of Bio-Sciences and Technology, VIT University, Vellore 632014, India

Department of Chemistry, LRG Govt. Arts College for Women, Tirupur 641604, Tamil Nadu, India

b

c

A R T I C L E I N F O

A B S T R A C T

Keywords:

Antibacterial

Disubstituted-5,7-dimethoxy chromans

DNA gyrase

Lipinski’s ﬁlter

In this study, novel 3-O-methoxy-4-halo, disubstituted-5,7-dimethoxy chromans with bacterial cell wall de-

grading potentials were synthesized, characterized and evaluated as DNA gyrase inhibitors and antibacterial

agents. Compounds were showed a broad spectrum of antimicrobial activity against both Gram^+vebacteria (S.

aureus (MTCC 3160), C. diphtheriae (MTCC 116), S. pyogenes (MTCC 442)) and Gram^−vebacteria (E. coli (MTCC

443), P. aeruginosa (MTCC 424), K. pneumoniae (MTCC 530)). Further, a molecular docking study was carried out

to get more insight into the binding mode of present study compounds to target proteins (PDB ID: 2XCT (S.

aureus DNA gyrase A), PDB ID: 3G75 (S. aureus DNA gyrase B), PDB ID: 3L7L (Teichoic acid polymerase). In the

results, 14 > 20 > 24 > 12 > 18 > 17 were found as the most active against almost all executed activities

in this study. The predicted Lipinski’s ﬁlter scores, SAR, pharmacokinetic/pharmacodynamics, and ADMET

properties of these compounds envisioned the druggability prospects and the necessity of further animal model

evaluations of 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy chromans to establish them as an eﬀective and

future antibiotics.

Molecular docking

1. Introduction

bacteria. In such condition, our present study’s main objective is to

target both physical (cell wall) and genetically important (DNA gyrase)

“Medicine is a science of uncertainty and an art of probability” –

William Osler. Antibiotics are uniquely societal drugs because in-

dividual use aﬀects others in the community and environment.” – Dr.

Stuart Levy. After reading these quotes, we have started to think about

most probable and safer antibiotics, why because the bacterial resistant

to the available antibiotics gradually increasing day by day. Chroman

and coumarins are the highly demanded small molecule chemical en-

tities in heterocyclic chemistry, especially in novel antibiotic designing

and development. For example, Novobiocin and Clorobiocin are cou-

marin based anti-microbial drugs.^1–4In recent decades, coumarins

based compounds have been recognized and are established as an ef-

fective antioxidant,⁵anticancer,^6,7anti-inﬂammatory,⁷antineurotic⁸,

antifungal,⁹analgesic and anticonvulsant agents.¹⁰Most signiﬁcantly, a

number of Chroman compounds have been identiﬁed from natural

sources (Plants) such as Melicope pteleifolia,¹¹Evodia lepta¹²and Melicope

stipitata.¹³Aiming to obtain an eﬀective, therapeutically potential

chroman or coumarin based antibiotic, it was highly necessitated to

understand the physiologically and genetically signiﬁcant targets of

as markers to develop the chroman or coumarin compounds as a new

class of antibiotics.

In the bacterial pathogenicity, bacterial cell wall playing a vital role

and being the most responsive and foremost part and also causing

atrocious eﬀects in host system.^14–16Peptidoglycan (PG) and teichoic

acids are fabricating the thick cell wall of Gram^+vebacteria to maintain

the structural association and the shape of the cell.¹⁷Composed with

polyol-phosphate units, Teichoic acid polymers are playing a key con-

stitutive role in the Gram^+vebacterial cell wall construction and this

cell wall is amalgamated by monotopic membrane proteins of the TagF

(Teichoic acid polyglycerol phosphate) polymerase family.¹⁸This TagF

polymerase is accomplished by the elongation substrate CDP-glycerol

which is acting as an originator substrate in the cell wall synthesis.¹⁹

With this, the CDP-glycerol: poly glycerol phosphotransferase

(CGPTase) enzyme is also constantly involved in the teichoic acid de-

velopment like the teichoic acid polymerases.²⁰Apart from the al cell

wall pathogenicity, pathogenic bacterial genetic material (DNA) is

considered as uncontrolled to multiply into double, triple… and so on

⁎

Corresponding authors.

E-mail addresses: mailtomicromani@gmail.com (M. Alagumuthu), thamaraimohan@gmail.com (S. Thamaraiselvi).

https://doi.org/10.1016/j.bmc.2018.05.016

Received 20 April 2018; Received in revised form 10 May 2018; Accepted 11 May 2018

0968 -0 896 /©201 8 E l s e v i e r L t d .A l l r i g h t s r e s e r v ed.

Pleasecitethisarticleas:Ponnusamy,T.,Bioorganic&MedicinalChemistry(2018),https://doi.org/10.1016/j.bmc.2018.05.016

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

in a quick time period. This makes the bacterial pathogens very tough

to eliminate them from their host environments especially from the

humans.

sodium chloride solution (20 ml) and dried over (Na₂SO₄). Further, the

solvent was distilled oﬀ and the resulting syrup product 2 (5.0 g, 5,7-

dimethoxy-2,2-dimethylchroman-4-ol) was recovered. IR (KBr) ϒmax:

3438, 3102, 2982, 2968, 1678, 1464, 1427, 1368, 1118, 1031, 1001,

In the bacterial genetic system, DNA gyrase (Type II topoisomerase),

an essential bacterial enzyme that catalyzes the ATP-dependent nega-

tive super-coiling of double-stranded closed-circular DNA, involving in

the formation of supercoils by cutting the two ends of single circular

DNA and get twisted around each other.^21–23Therefore, either de-

grading the cell wall constitution or inhibiting DNA gyrase would be the

precise way to compete against bacterial toxicity. Considering all the

above discussed points, in this study, the 3-O-methoxy-4-halo dis-

ubstituted 5,7-dimethoxy chromans along with a family of novel cis-

2,2dihydroxy-5,7dimethoxy-6-acetyl-2,2dimethyl chromans (10, 11)

and 6-acetyl-5-hydroxy-7-methoxy-2,2-dimethyl chroman-4-ones that

were supplemented with a mixture of electron donating or withdrawing

substituents was eﬃciently synthesized and validated as DNA gyrase

inhibitors (in vitro) as well as the cell wall teichoic acid polymerase

inhibitors (in silico) along with their antibacterial eﬀects.

815, 729, 658 cm⁻^{1 1}H NMR: NMR (400 MHz, CDCl₃) δ ppm: 6.05 (s,

.

2H), 5.91 (s, 1H), 4.72 (s, 1H), 4.46 (s, 1H), 3.77 (s, 3H), 3.73 (s, 3H),

2.30 (m, 1.90–1.77), 1.34 (S, 6H); ¹³C NMR (100 MHz, CDCl₃) δ ppm:

160.76, 159.90, 154.81, 106.08, 93.87, 91.35, 74.52, 58.17, 55.90,

55.78, 41.52, 29.05, 26.52. Analysed and calculated for C₁₃H₁₈O₄: C,

65.53; H, 7.61; O, 26.86%. Found: C, 65.51; H, 7.62; O, 26.87%.

2.1.3. Procedure for the preparation of 5,7-dimethoxy-2,2-dimethyl-2H-

chromene (3)

The compound 5,7-dimethoxy-2,2-dimethylchroman-4-ol (2) was

dissolved in 20 ml of DMSO. With this, 5.0 ml of 1.5 N HCl was added at

25–30 °C under stirring condition. Then the mass was stirred for 2 h at

25–30 °C. After completion of the reaction, as indicated by TLC, pH was

adjusted to 7.0–8.0 using 10% NaHCO₃solution. The product was

further extracted using ethyl acetate (20 ml × 2) in water. The product

layer (Ethyl acetate layer) washed with 20.0 ml water again followed by

treating with saturated sodium chloride solution (20 ml) and dried over

(Na₂SO₄). The solvent was distilled oﬀ and the resulting product was

puriﬁed by column chromatography using ethyl acetate and n-hexane

to get pure syrup product (3) (4.4 g, 88.6%). IR (KBr) ϒmax: 2964,

2874, 1633, 1606, 1575, 1494, 1462, 1423, 1389, 1376, 1345, 1303,

2. Materials and methods

2.1. Chemistry

All chemicals and solvents were procured from Sigma-Aldrich,

Merck, and Himedia, AD-mix-alpha, AD-mix-beta (USA), and were used

as received without doing further puriﬁcation. Thin-Layer chromato-

graphy (TLC) was executed using pre-coated silica plates (Merck-silica

gel 60 F254). Merck silica gel 60 (230–400 mesh) was used for ﬂash

column chromatography. Melting point (mp) were checked using an

OptiMelt automated melting point system and are uncorrected. ¹H NMR

(400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on Bruker-

400 MHz instrument. Mass spectra of all compounds were recorded on

Agilent ion trap MS and Infrared (IR) spectra were recorded on Perkin

Elmer FT-IR spectrometer.

1247, 1168, 1002, 934, 881, 867, 822, 704, 638, 616 cm⁻^{1 1}H NMR:

.

(400 MHz, CDCl₃) δ ppm: 6.528–6.503 (d, 1H, J = 10.0 Hz), 5.97

(s,1H), 5.363–5.33(d, 1H, J = 9.6 Hz), 3.71(s, 3H), 3.69 (s, 3H), 1.34 (s,

6H). ¹³C NMR (100 MHz, CDCl₃) δ ppm: 161.01, 156.14, 154.67,

125.86, 116.72, 104.18, 93.98, 91.44, 55.50, 55.30, 27.77. MS (EI, m/

z): 221.1 (M+). Analysed and calculated for C₁₃H₁₆O_3:C, 70.89; H,

7.32; O, 21.79%. Found: C, 70.90; H, 7.34; O, 21.76%.

2.1.4. Preparation of 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-6-yl)

ethanone (4a) and 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)

ethanone (4b) using Friedel-Crafts acylation (Scheme 1)

2.1.1. Procedure for the preparation 5,7-dimethoxy-2,2-dimethylchroman-

4-one (1)

To a solution of 5,7-dimethoxy-2,2-dimethyl-2H-chromene (3) 4.0 g

(0.1 mol) in MDC, AlCl₃4.84 g (0.2 mol) was added at 0–5 °C. To this

reaction mixture acetyl chloride 2.14 g (0.15 mol) was added slowly at

0–5 °C and stirring was continued for about 1–2 h at 25–30 °C. After

completion of the reaction, as indicated by TLC, the reaction mass was

cooled and water was added slowly under stirring condition. Product

was extracted in MDC (12 × 2) and washed with water followed by

treating with saturated sodium chloride solution (20 ml) and dried over

(Na₂SO₄). The solvent was distilled oﬀ and the resulting product was

puriﬁed by column chromatography using ethyl acetate and n-hexane

to get pure solids of 4a (25%) and 4b (40%).

A

mixture of 5,7-dihydroxy-2,2-dimethyl chroman-4-one 5.0 g

(0.10 mol), MeI 8.52 g (0.25 mol), K₂CO₃4.98 g (0.15 mol) in acetone

(20.0 ml) was stirred at 60 °C for 20 h. After completion of the reaction,

as indicated by TLC, the solvent was distilled out under vaccum. The

resulting residue was dissolved in ethyl acetate and water. The obtained

crude product was extracted with ethyl acetate (2 × 20 ml) and washed

with water (20 ml) followed by treated with saturated sodium chloride

solution (20 ml) and dried over (Na₂SO₄). The solvent was distilled oﬀ

and the resulting product was puriﬁed by diethyl ether and diisopropyl

ether mixture (5 ml:15 ml) to obtain oﬀ white solid (5.0 g, 89%).

Melting point: 103.3–104.4 °C: IR (KBr) ϒmax: 2981, 2968, 1678, 1572,

1427, 1383, 1368, 1316, 1244, 1117, 1065, 1031, 1001, 936, 880, 815,

730, 659, 587. ¹H NMR: NMR (400 MHz, CDCl₃) δ ppm: 5.80 (s, 2H),

3.64 (s, 3H), 3.59 (s, 3H), 2.41 (s, 2H), 1.20 (s, 6H); ¹³C NMR

(100 MHz, CDCl₃) δ ppm: 189.46, 165.77, 163.25, 161.72, 104.98,

93.64, 92.16, 78.63, 55.90, 55.35, 49.89, 26.35. MS (EI, m/z): 237.0 (M

+). Analysed and calculated for C₁₃H₁₆O₄: C, 66.09; H, 6.83; O,

27.08%. Found: C, 66.05; H, 6.85; O, 27.10%.

2.1.4.1. 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-6-yl)ethanone

(4a). Pale yellow solid, Melting point: 93.7–94.2 °C. IR (KBr) ϒmax:

3048, 2947, 2937, 1695, 1634, 1608, 1478, 1330, 1155, 1101, 888,

808, 691 cm⁻^{1 1}H NMR: NMR (400 MHz, DMSO) δ ppm: 6.442–6.418

.

(d, 1H, J = 9.6), 5.663–5.638(d, 1H, J = 10.0), 6.34 (s, 1H), 3.74 (s,

3H), 3.65 (s, 3H), 2.37 (s, 3H), 1.37 (s, 6H). ¹³C NMR (100 MHz, DMSO)

δ ppm: 200.14, 157.07, 155.21, 153.25, 128.08, 117.94, 115.74,

107.19, 96.09, 76.50, 63.05, 55.87, 32.26, 27.43. NOESY (400 MHz

DMSO): Aromatic proton at (C8) shows correlation with C-7 attached

O-methoxy functional group only. MS (EI, m/z): 263.1 (M+). Analysed

and calculated for C₁₅H₁₈O₄: C, 68.68; H, 6.92; O, 24.40%. Found: C,

68.69; H, 6.90; O, 24.41%.

2.1.2. Procedure

dimethylchroman-4-ol (2)

for

the

preparation

of

5,7-dimethoxy-2,2-

To a solution of 5,7-dimethoxy-2,2-dimethylchroman-4-one (1)

5.0 g (0.1 mol) in methanol (20 ml) at 0–5 °C, and NaBH₄(0.80 g,

0.1 mol) was added. The reaction mixture was stirred for 2–3 h at

25–30 °C. The pH was adjusted to 7.0–8.0 using acetic acid (2.0 ml).

After completion of the reaction, as indicated by TLC, the solvent was

distilled out under vaccum. The resulting residue was dissolved in ethyl

acetate and water. Product was extracted with ethyl acetate (2 × 20 ml)

and washed with water (20 ml) followed by treating with saturated

2.1.4.2. 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)ethanone

(4b). Oﬀ white solid, Melting point: 105.3–107.5 °C. IR (KBr) ϒmax:

3002, 2985, 2964, 2944, 1705, 1637, 1469, 1334, 1156, 1114, 880,

794, 684 cm⁻¹. ¹H NMR: NMR (400 MHz, DMSO) δ ppm: 6.49–6.47 (d,

1H, J = 8.0 MHz), 5.55–5.53 (d, 1H, J = 8.0 MHz), 6.26 (s, 1H), 3.83

2

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Scheme 1. Route of synthesis of compounds 4a and 4b (approach – 1).

(s, 3H), 3.77 (s, 3H), 2.33 (s, 3H), 1.33 (s, 6H). ¹³C NMR (100 MHz,

DMSO) δ ppm: 199.91, 157.63, 156.59, 150.85, 127.34, 116.43,

113.50, 103.69, 89.29, 76.91, 56.27, 32.94, 27.58. NOESY (400 MHz

DMSO): Aromatic proton attached to C6 carbon shows correlation with

both C-5 and C-7O-methoxy functional groups. C-8 acetyl group shows

correlation with C-7 attached O-methoxy functional group. MS (EI, m/

z): 263.0 (M+). Analysed and calculated for C₁₅H₁₈O₄: C, 68.68; H,

6.92; O, 24.40%. Found: C, 68.69; H, 6.90; O, 24.41%.

stirred 25–30 °C for 20 h. After the completion of reaction, as indicated

by TLC, solvent was distilled out under vaccum. The crude reaction

mixture was then dissolved in ethyl acetate and water. The ﬁnal pro-

duct was extracted with ethyl acetate (2 × 20 ml) and washed with

water (20 ml) followed by treating with saturated sodium chloride so-

lution (20 ml) and dried over (Na₂SO₄). The solvent was distilled oﬀ

and the resulting product was puriﬁed by diethyl ether and diisopropyl

ether mixture (5 ml:15 ml) to obtain oﬀ white solid (14.40 g, 95.0%).

Melting point: 197.7–199.4 °C. IR (KBr) ϒmax: 3432, 2926, 1701, 1676,

1630, 1598, 1452, 1374, 1263, 1228, 1110, 982, 914, 877, 829,

2.1.5. Preparation of 6-acetyl-5,7-dimethoxy-2,2-dimethylchroman-4-one

(5)

639 cm⁻^{1 1}H NMR: NMR (400 MHz, CDCl₃) δ ppm: 12.64 (s, 1H, D₂O

.

exchangeable), 6.20 (s, 1H), 3.83 (s, 3H), 2.85 (s, 2H), 2.39 (s, 3H),

1.42 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ ppm: 198.89, 196.15,

164.36, 162.63, 159.92, 110.71, 101.25, 91.90, 80.00, 56.44, 46.76,

32.24, 26.01. MS (EI, m/z): 265.1 (M+). Analysed and calculated for

A

mixture of 6-acetyl-5,7-dihydroxy-2,2-dimethylchroman-4-one

15.0 g (0.10 mol), MeI 34.0 g (0.4 mol), K₂CO₃12.40 g (0.15 mol) in

acetone 75.0 ml was stirred at 60 °C for 20 h. After the completion of

reaction, as indicated by TLC, solvent was distilled out under vaccum.

The resulting residue was dissolved in ethyl acetate and water. Product

was extracted with ethyl acetate (2 × 20 ml) and washed with water

(20 ml) followed by treating with saturated sodium chloride solution

(20 ml) and dried over (Na₂SO₄). The solvent was distilled oﬀ and the

resulting product was puriﬁed by diethyl ether and diisopropyl ether

mixture (5 ml:15 ml) to obtain oﬀ white solid (14.67 g, 88.0%). Melting

point: 153.5–155.1 °C. IR (KBr) ϒmax: 2983, 2969, 2958, 2849, 1704,

C

₁₄H₁₆O₅: C, 63.63; H, 6.10; O, 30.27%. Found: C, 63.64; H, 6.11; O,

30.25%; HRMS Calculated [M+] m/z 264.2770, Found 264.2770.

2.1.7. Preparation of 8-acetyl-7-hydroxy-5-methoxy-2,2-dimethylchro

man-4-one (7)

A

mixture of 8-acetyl-5,7-dihydroxy-2,2-dimethylchroman-4-one

(Note: This compound was synthesized according to Tsukyama et al.

1989³³; Wang et al. 2004.³⁵Refer Scheme 2) 15.0 g (0.10 mol), MeI

17.0 g (0.2 mol), K₂CO₃12.40 g (0.15 mol) in acetone 75.0 ml was

stirred at 25–30 °C for 20 h. After the completion of reaction, as in-

dicated by TLC, solvent was distilled out under vaccum. The resulting

residue was dissolved in ethyl acetate and water. Product was extracted

with ethyl acetate (2 × 20 ml) and washed with water (20 ml) followed

by treating with saturated sodium chloride solution (20 ml) and dried

over (Na₂SO₄). The solvent was distilled oﬀ and the resulting product

was puriﬁed by diethyl ether and diisopropyl ether mixture

(5 ml:15 ml) to obtain oﬀ white solid (13.0 g, 84.0%). Melting point:

80.6–82.6 °C. IR (KBr) ϒmax: 3430, 3018, 3001, 2971, 1697, 1645,

1585, 1465, 1386, 1372, 1258, 1230, 1098, 1011, 980, 910, 885, 831,

1597, 1474, 1447, 1395, 1186, 1131, 1061,885, 806, 666 cm⁻^{1 1}H

.

NMR: NMR (400 MHz, CDCl₃) δ ppm: 5.98 (s, 1H), 3.87 (s, 3H), 3.80 (s,

3H), 2.58 (s, 2H), 2.9 (s, 3H), 1.36 (s, 6H). ¹³C NMR (100 MHz, CDCl₃)

δ ppm: 200.43, 189.35, 162.82, 162.04, 159.13, 113.19, 104.81, 87.97,

79.58, 56.19, 55.87, 49.98, 32.41, 26.42. MS (EI, m/z): 279.1 (M+).

Analysed and calculated for C₁₅H₁₈O₅: C, 64.74; H, 6.52; O, 28.74%.

Found: C, 64.73; H, 6.51; O, 28.76%.

2.1.6. Preparation of 6-acetyl-5-hydroxy-7-methoxy-2,2-dimethylchrom

an-4-one (6)

A

mixture of 6-acetyl-5,7-dihydroxy-2,2-dimethylchroman-4-one

(Note: This compound was synthesized according to Tsukyama et al.

1989³³; Wang et al. 2004.³⁵Refer Scheme 2) 15.0 g (0.10 mol), MeI

17.0 g (0.2 mol), K₂CO₃12.40 g (0.15 mol) in acetone 75.0 ml was

628 cm⁻¹

.

¹H NMR: NMR (400 MHz, CDCl₃) δ ppm: 12.32 (s, 1H, D₂O

exchangeable), 6.21 (s, 1H), 3.80 (s, 3H), 2.84 (s, 2H), 2.35 (s, 3H),

3

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Scheme 2. Route of synthesis of compounds 4a and 4b (approach – 2).

1.39 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ ppm: 198.47, 196.75,

163.97, 163.77, 156.87, 111.78, 101.15, 92.31, 80.08, 56.37, 46.57,

32.25, 25.98. MS (EI, m/z): 265.1 (M+). Analysed and calculated for

58.37, 56.23, 41.20, 32.83, 29.39, 26.45. MS (EI, m/z): 281.1 (M+).

Analysed and calculated for C₁₅H₂₀O₅: C, 64.27; H, 7.19; O, 28.54

Found: C, 64.29; H, 7.16; O, 28.55%; HRMS Calculated [M+] m/z

280.3200, Found 280.3210.

C

₁₄H₁₆O₅: C, 63.63; H, 6.10; O, 30.27%. Found: C, 63.64; H, 6.11; O,

30.25%.

2.1.8.1. Preparation of 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-6-yl)

ethanone (4a). To

a solution of 1-(4-hydroxy-5,7-dimethoxy-2,2-

2.1.8. Preparation of 1-(4-hydroxy-5,7-dimethoxy-2,2-dimethylchroman-

6-yl)ethanone (8)

dimethyl chroman-6-yl)ethanone (8) 10 g (0.1 mol) in MDC (40 ml),

TEA (7.60 g, 0.2 mol) was added at 0–5 °C. To this, methane sulphonyl

chloride 4.30 g (0.1 mol) dissolved in MDC (10.0 ml) was slowly added

at 0–5 °C. The was mass stirred for 15–30 min at 0–5 °C. After the

completion of the reaction, as indicated by TLC, the reaction mixture

was quenched with water (20.0 ml) and the product was extracted in

MDC (20 ml × 2). The product layer (MDC) washed with water 20.0 ml

followed by treating with saturated sodium chloride solution (20 ml)

and dried over (Na₂SO₄). The solvent was distilled oﬀ and the resulting

product was puriﬁed by diethyl ether and n-hexane to obtain pale

yellow color solid (4a) 8.4 g, 90%.

To the 6-acetyl-5,7-dimethoxy-2,2-dimethylchroman-4-one (5)

15.0 g (0.1 mol) in methanol (60 ml), NaBH₄(2.45 g, 0.12 mol) was

added at 0–5 °C. The pH was adjusted to 7.0–8.0 using acetic acid

(2.0 ml). Further, the reaction mixture was stirred for 2–3 h at 25–30 °C.

After completion of the reaction, as indicated by TLC, solvent was

distilled out under vaccum. The resulting residue was dissolved in ethyl

acetate and water. Product was extracted with ethyl acetate (2 × 20 ml)

and washed with water (20 ml) followed by treating with saturated

sodium chloride solution (20 ml) and dried over (Na₂SO₄). The solvent

was distilled oﬀ and the resulting solid puriﬁed using diethyl ether and

n-hexane to obtain a white colour solid (14.05 g, 93.0%). Melting point:

99.6–101.5 °C. IR (KBr) ϒmax: 3504, 2981, 2942, 2887, 2843, 1692,

1602, 1467, 1439, 1347, 1183, 1154, 1111, 1048, 884, 793,

2.1.8.2. Preparation of 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)

etha-none (4b). A mixture of the 8-acetyl-5,7-dihydroxy-2,2-dimethyl

chroman-4-one (Scheme 2) 15.0 g (0.10 mol), MeI 34.0 g (0.4 mol),

K₂CO₃12.40 g (0.15 mol) in acetone 75.0 ml was stirred at 60 °C for

20 h. After completion of the reaction, as indicated by TLC, the solvent

was distilled out under vacuum. The resulting residue was dissolved in

616.6 cm⁻^{1 1}H NMR (400 MHz, DMSO) δ ppm: 6.22 (s, 1H), 5.05 (s,

;

1H, D₂0 exchangeable) 3.80 (s, 3H), 3.72 (s, 3H), 1.80–1.90 (m, 2H),

2.28 (s, 3H), 1.32 (s, 3H), 1.24 (s, 3H). ¹³C NMR (100 MHz, DMSO) δ

ppm: 200.66, 160.41, 157.11, 151.42, 113.02, 106.35, 88.93, 75.28,

4

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

ethyl acetate and water. The product was extracted with ethyl acetate

(2 × 20 ml) and washed with water (20 ml) followed by treating with

saturated sodium chloride solution (20 ml) and dried over (Na₂SO₄).

The solvent was distilled oﬀ and the resulting product 8-acetyl-5,7-

dimethoxy-2,2-dimethyl chroman-4-one (Scheme 2) without isolation

was taken to next step. A mixture of the 6-acetyl-5,7-dimethoxy-2,2-

dimethyl chroman-4-one (5) 15.0 g (0.1 mol) in methanol (60 ml) at

0–5 °C, NaBH₄(2.45 g, 0.12 mol) was added. Further, the reaction

mixture was stirred for 2–3 h at 25–30 °C. After completion of the

reaction as indicated by TLC, adjusted the pH to 7.0–8.0 using acetic

acid (2.0 ml), solvents was distilled out under vacuum.

Further, the resulting residue was dissolved in ethyl acetate and

water. The product was extracted with ethyl acetate (2 × 20 ml) and

washed with water (20 ml) followed by treating with saturated sodium

chloride solution (20 ml) and dried over (Na₂SO₄). The solvent was

distilled oﬀ and the resulting product [1-(4-hydroxy-5,7-dimethoxy-

2,2-dimethylchroman-8-yl)ethanone] without isolation was taken to

next step. The above material dissolved in MDC (40 ml) at 0–5 °C, TEA

(7.60 g, 0.2 mol) was added. Methane sulphonyl chloride 4.30 g

(0.1 mol) was dissolved in MDC (10.0 ml) was added slowly to the re-

action mass at 0–5 °C and stirred the mass for 15–30 min at 0–5 °C. After

completion of the reaction, the reaction mass was quenched with water

(20.0 ml). Extracted the product in MDC (20 ml × 2). The product layer

(MDC) washed with water 20.0 ml followed by treating with saturated

sodium chloride solution (20 ml) and dried over (Na₂SO₄). The solvent

was distilled oﬀ and the resulting product was puriﬁed by diethyl ether

and n-hexane to obtain oﬀ-white color solid (4b) 14.15 g, 90%.

sodium chloride solution (20 ml) and dried over (Na₂SO₄). The solvent

was distilled oﬀ and the resulting solid was puriﬁed by ﬂash chroma-

tography (silica gel, ethyl acetate/n-hexane). In this solvent system, the

ligand does not move and so that we obtained 1.02 g of an oﬀ-white

color solid (90%).

Procedure-2:

A Mixture of Chiral ligand α or β – AD-mix

[(DHQD)₂PHAL] 3.45 g (0.110 mol) in MDC 5.0 ml, OsO₄1.12 g

(0.11 mol) was added and stirred at RT for 5 min. The mass was stirred

for 60 min at −20 °C, and then a solution of 1-(5,7-dimethoxy-2,2-di-

methyl-2H-chromen-6-yl)ethanone (4a) 1.0 g (0.1 mol) in MDC 3.0 ml

was added slowly in the period of 15–20 min, the again the mass was

stirred for 32–36 h at −20 to −18 °C. After the reaction completion, as

indicated by TLC, the reaction mass was kept in RT. To this, 10% so-

dium sulﬁte (8.0 ml, pH ∼9.0) and 10% sodium bi sulﬁte (8.0 ml, pH

∼4.0) solution was added. This mass was stirred for 2 h followed by the

addition of the solvents THF/Ethyl acetate (1:4, 50 ml). Further, the

temperature was raised up to 50–55 °C and the stirring was continued

for 2–4 h. Finally, the mass was ﬁltered at RT. The ﬁnal product was

extracted in Ethyl actete, combined organic layers were washed with

0.1 N HCl followed by brine solution and dried over sodium sulphate

and concentrated under vaccum to remove solvents completely. The

resulting solid was puriﬁed by ﬂash chromatography (silica gel, ethyl

acetate/n-hexane) In this solvent system the ligand does not move and

so that we obtained 1.0 g of an oﬀ-white color solid (88.5%).

2.2.1. 1-((3S,4S)-3,4-dihydroxy-5,7-dimethoxy-2,2-dimethylchroman-6-

yl)ethanone (10)

The Chiral Ligand used in this reaction is β-AD-mix

[(DHQD)₂PHAL], oﬀ white powder with 85% of yield. Melting point:

136.2–138.2 °C: IR (KBr) ϒmax: 3504, 3416, 2989, 2964, 2936, 1697,

2.1.9. Preparation of 1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-chromen-

8-yl)ethanone (9)

A

solution of 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)

1607, 1473, 1198, 1138, 1102, 990, 880, 830, 687 cm⁻^{1 1}H NMR:

;

ethanone 10 g (4b) (0.10 mol) in MDC (60 ml) was cooled to −70 to

−80 °C under nitrogen atmosphere. Boron tribromide 0.95 g (0.1 mol)

was dissolved in MDC (10.0 ml) was added slowly to the reaction mass

at −75 to −80 °C. Further, the mass was stirred for 30 min at −75 to

−80 °C. After completion of the reaction as indicated by TLC, the re-

action mixture was quenched with methanol (10.0 ml) and the product

was extracted in MDC (30 ml × 2). Then the product layer (MDC layer)

was washed with water (30.0 ml) followed by treating with saturated

sodium chloride solution (30 ml) and dried over (Na₂SO₄). Finally, the

solvent was distilled oﬀ and the resulting product was puriﬁed by

column chromatography to obtain pale yellow color solid (6). Melting

point: 62.6–64.5 °C. IR (KBr) ϒmax: 3522, 3051 2969, 2926, 2858,

(400 MHz, DMSO) δ ppm: 6.23 (s, 1H), 4.97–4.96 (d, 1H, D₂O ex-

changeable), 4.85–4.84 (d, 1H, D₂O exchangeable), 4.695–4.674 (d,

1H, J = 4.0), 3.75 (s, 3H), 3.72 (s, 3H), 3.544–3.518 (t, 1H, J = 4.8),

2.39 (s, 3H), 1.33 (s, 6H). ¹³C NMR (100 MHz, DMSO) δ ppm: 200.63,

157.55, 157.12, 154.97, 117.95, 110.32, 95.63, 77.89, 71.62, 63.10,

60.93, 55.73, 32.34, 26.89, 21.21. MS (EI, m/z): 296.5 (M+1).

Analysed and calculated for C₁₅H₂₀O₆: C, 60.80; H, 6.80; O, 32.40%.

Found: C, 60.81; H, 6.78; O, 32.41%; HRMS Calculated [M+] m/z

296.3190, Found 296.3200.

2.2.2. 1-((3R,4R)-3,4-dihydroxy-5,7-dimethoxy-2,2-dimethylchroman-6-

yl)ethanone (11)

1611, 1586, 1491, 1330, 1172, 1107, 986, 876, 818, 690 cm⁻¹

.

¹H

The Chiral Ligand used in this reaction is α-AD-mix

[(DHQD)₂PHAL], oﬀ white powder with 88% of yield. Melting point:

124.2–126.4 °C. IR (KBr) ϒmax: 3504, 3416, 2989, 2964, 2937, 1697,

NMR: NMR (400 MHz, DMSO) δ ppm: 13.72 (s, 1H, D₂O exchangeable),

6.46–6.43 (d, 1H, J = 12.0), 5.55–5.52 (d, 1H, J = 12.0), 6.09 (s, 1H),

3.81 (s, 3H), 3.2.59 (s, 3H), 1.43 (s, 6H). ¹³C NMR (100 MHz, DMSO) δ

ppm: 203.38, 165.87, 161.05, 156.01, 125.82, 116.20, 105.80, 102.75,

92.87, 78.32, 56.50, 33.32, 27.65. MS (EI, m/z): 263.1 (M+). Analysed

and calculated for C₁₄H₁₆O₄: C, 67.73; H, 6.50; O, 25.78%. Found: C,

67.75; H, 6.51; O, 25.74%; HRMS Calculated [M+] m/z 248.1049,

Found 248.1049.

1608, 1474, 1199, 1139, 1102, 990, 880, 831, 688 cm⁻^{1 1}H NMR:

;

(400 MHz, DMSO) δ ppm: 6.22 (s, 1H), 4.98–4.97 (d, 1H, D₂O ex-

changeable), 4.86–4.85 (d, 1H, D₂O exchangeable), 4.67–4.66 (d, 1H,

J = 4.4), 3.73 (s, 3H), 3.71 (s, 3H), 3.52–3.51 (d, 1H, J = 5.2), 2.38 (s,

3H), 1.32 (s, 6H). ¹³C NMR (100 MHz, DMSO) δ ppm: 200.72, 157.56,

157.13, 154.99, 117.96, 110.36, 95.64, 77.93, 71.63, 63.14, 60.94,

55.76, 32.39, 26.94, 21.25. NOESY (400 MHz DMSO): Aromatic proton

at (C8) shows correlation with C-7 attached O-methoxy functional

group only. MS (EI, m/z): 297.1 (M+1). Analysed and calculated for

2.2. General procedure for the preparation of 3, 4 dihydroxy chromans

Procedure-1:

A

mixture of chiral ligand

α

or

β

–

AD-mix

C₁₅H₂₀O₆: C, 60.80; H, 6.80; O, 32.40%. Found: C, 60.81; H, 6.78; O,

32.41%; HRMS Calculated [M+] m/z 296.3190, Found 296.3200.

[(DHQD)₂PHAL] 3.45 g (0.110 mol) in a mixture of 5.0 ml tertiary bu-

tanol and 5 ml water, stirred to get a clear solution at room temperature

(RT). Methanesulfonamide 0.416 g (0.10 mol) was added and stirred at

RT for 5 min. The mixture was cooled to 0–2 °C. 1.0 g (0.1 mol) of 1-

(5,7-dimethoxy-2,2-dimethyl-2H-chromen-6-yl) ethanone was added

and the mass were stirred at 0–2 °C for 20–24 h. After the reaction

completion, as indicated by TLC, 3.5 g of solid sodium sulﬁte was added

at 0–2 °C and this mass was stirred for 1 h at RT. The product was ex-

tracted with ethyl acetate (2 × 20 ml) and washed with 2 N KOH so-

lution (10 ml), water (20 ml) followed by treating with saturated

2.2.3. 1-((3S,4S)-3,4-dihydroxy-5,7-dimethoxy-2,2-dimethylchroman-8-

yl)ethanone (12)

The Chiral Ligand used in this reaction is

β – AD-mix

[(DHQD)₂PHAL], oﬀ white powder with 90% of yield. Melitng point:

119.6–121.8 °C. IR (KBr) ϒmax: 3337, 3007, 2975, 2938, 2914, 2842,

1682, 1606, 1478, 1174, 1136, 1098, 995, 870, 857, 677 cm-1; 1H

NMR: (400 MHz, DMSO) δ ppm: 6.26 (s, 1H), 4.83–4.81 (d, 1H, D₂O

exchangeable), 4.75–4.74 (d, 1H, D₂O exchangeable), 4.65–4.64 (d, 1H,

5

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

J = 4.0), 3.83 (s, 3H), 3.76 (s, 3H), 3.51–3.48 (t, 1H, J = 5.2), 2.35 (s,

3H), 1.29 (s,6H). ¹³C NMR (100 MHz, DMSO) δ ppm: 200.05, 160.17,

156.97, 150.35, 112.28, 105.74, 88.47, 77.88, 71.53, 60.40, 55.79,

55.67, 32.30, 27.04, 20.58. NOESY (400 MHz DMSO): Aromatic proton

(C6) shows correlation with both C-5 and C-7O-methoxy functional

groups. MS (EI, m/z): 297.0 (M+1). Analysed and calculated for

DMSO) δ ppm: 195.70, 158.08, 153.48, 130.55, 129.15, 120.01,

115.94, 109.78, 103.76, 76.68, 55.77, 31.83, 27.18. MS (EI, m/z):

232.7 (M+1). Analysed and calculated for C₁₄H₁₆O₃: C, 72.39; H, 6.94;

O, 20.66%. Found: C, 72.40; H, 6.95; O, 20.65%; HRMS Calculated [M

+] m/z 232.2790, Found 232.2790.

C

₁₅H₂₀O₆: C, 60.80; H, 6.80; O, 32.40%. Found: C, 60.79; H, 6.79; O,

2.3. General procedure for the preparation of methoxy halogenation of

chromans

32.42%, HRMS Calculated [M+] m/z 296.3190, Found 296.3200.

2.2.4. 1-((3R,4R)-3,4-dihydroxy-5,7-dimethoxy-2,2-dimethylchroman-8-

yl)ethanone (13)

To a solution of 2.0 g (0.10 mol) 1-(5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (4b) in methanol 10.0 ml, Oxone 5.16 g

(0.11 mol) was added at 0.5 °C. NH₄halides (X = F, C, Br, I) (0.11 mol)

was added and the mass was stirred for 2–3 h at 0–5 °C. The reaction

was completed as indicated by TLC, the reaction mass quenched with

10% sodium thiosulphate solution (5.0 ml) and then 15.0 ml ethyl

acetate was added, the product was extracted then washed with water,

NaCl solution and dried over Na₂SO_4.Solvents were distilled out under

vacuum to get crude product. Product puriﬁed by using diethyl ether or

column chromatography to obtain the pure product.

The Chiral Ligand used in this reaction is

α – AD-mix

[(DHQD)₂PHAL], oﬀ white powder with 90% of yield Melting point:

109.3–111.3 °C. IR (KBr) ϒmax: 3337, 3006, 2975, 2938, 2913, 2842,

1682, 1606, 1467, 1162, 1098, 998, 869, 857, 613 cm⁻^{1 1}H NMR:

;

(400 MHz, DMSO) δ ppm: 6.26 (s, 1H), 4.80–4.78 (d, 1H, D₂O ex-

changeable), 4.73–4.72 (d, 1H, D₂O exchangeable), 4.658–4.637 (t, 1H,

J = 4.0 Hz), 3.83 (s, 3H), 3.75 (s, 3H), 3.509–3.481 (t, 1H, J = 5.2),

2.32 (s, 3H), 1.28 (s, 6H). ¹³C NMR (100 MHz, DMSO) δ ppm: 200.02,

160.19, 156.99, 150.37, 112.32, 105.75, 88.47, 79.15, 77.89, 71.54,

60.43, 55.79, 55.67, 32.29, 27.02, 20.59. MS (EI, m/z): 297.1 (M+1).

Analysed and calculated for C₁₅H₂₀O₆: C, 60.80; H, 6.80; O, 32.40%.

Found: C, 60.81; H, 6.79; O, 32.40%; HRMS Calculated [M+] m/z

296.3190, Found 296.3200.

2.3.1. Preparation of 1-(4-bromo-3,5,7-trimethoxy-2,2-dimethylchroman-

8-yl)ethanone (16)

To a solution of 2.0 g (0.10 mol) 1-(5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (4b) in methanol 10.0 ml, Oxone 5.16 g

(0.11 mol) was added at 0.5 °C. Then, 0.814 g (0.11 mol) NH₄Br was

added and the mass was stirred for 2–3 h at 0–5 °C. Reaction was

completed as indicated by TLC and the reaction mass was quenched

with 10% sodium thio sulphatesolution (5.0 ml) then ethyl acetate

(20.0 ml) was added and the product was extracted in ethyl acetate

layer and washed with water, NaCl solution and dried over Na₂SO_4.

Remaining solvent was distilled out under vacuum to get crude product.

The crude product was puriﬁed using diethyl ether to obtain the pure

product (2.27 g, 80%). Melting point: 105.40–107.4 °C, IR (KBr) ϒmax:

2987, 2938, 2840, 1852, 1752, 1734, 1653, 1601, 1494, 1460, 1383,

2.2.5. Synthesis of 8-acetyl-7-methoxy-2,2-dime-thyl-2H-chromen-5-yltri-

ﬂuoromethanesulfonate (14)

To

a

solution of 1-(5-hydroxy-7-methoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (9), 1.0 g (0.10 mol) in MDC 50 ml, TEA

(0.25 mol) was added to the reaction mass and cooled to 0–5 °C. With

this mass, Triﬂic anhydride 1.4 g (0.11) was added slowly for 30 min,

then the temperature was raised to 37 °C (RT). After stirred for 60 min

at RT, the reaction was completed as indicated by TLC. The mass was

then quenched with water and stirred for 30 min. The ﬁnal product was

extracted in MDC, then washed with water, NaCl solution and dried

over Na₂SO_4.The remaining solvent in the reaction mixture was dis-

tilled out under vacuum to get syrup. The product was puriﬁed by

column chromatography using EA – n-hexane system to obtain pale

yellow color solid 1.45 g (95%). Melting point: 79.9–81.3 °C, IR (KBr)

ϒmax: 3031, 2957, 2926, 2853, 1700, 1637, 1595, 1467, 1443, 1379,

1319, 1186, 1173, 1106, 980, 933, 751, 690 cm⁻¹. ¹H NMR: (400 MHz,

1337, 1185, 1117, 1007, 968, 936, 887, 864, 733, 655, 573 cm⁻^{1 1}H

.

NMR: (400 MHz, DMSO) δ ppm: 6.35 (s, 1H), 4.86 (s, 1H), 4.51 (s, 1H),

3.87 (s, 3H), 3.79 (s, 3H), 3.33 (s, 3H), 2.31 (s, 3H), 1.47 (s, 6H). ¹³C

NMR (100 MHz, DMSO) δ ppm: 199.64, 160.73, 157.45, 149.98,

112.19, 100.52, 89.03, 75.83, 75.36, 57.04, 56.0, 55.85, 54.59, 32.32,

29.26, 23.51. MS (EI, m/z): 373.2 (M+2). Analysed and calculated for

C

₁₆H₂₁BrO₅: C, 51.49; H, 5.67; Br, 21.41; O, 21.43% Found: C, 51.50;

DMSO)

δ

ppm: 6.58 (s, 1H), 6.56–6.54 (d, 1H, J = 8.0 Hz),

H, 5.66; Br, 21.40; O, 21.44%; HRMS Calculated [M+] m/z 373.2430,

Found 373.2430.

5.83–5.81(d, 1H, J = 8.0 Hz), 3.86 (s, 3H), 3.36 (s, 1H), 1.44 (s, 6H).

¹³C NMR (100 MHz, DMSO) δ ppm: 196.80, 156.79, 152.48, 146.14,

131.02, 120.14, 116.96, 116.73, 115.11, 110.53, 98.95, 78.67, 56.84,

32.39, 27.59. MS (EI, m/z): 381.1 (M+1). Analysed and calculated for

2.3.2. Preparation of 1-(4-chloro-3,5,7-trimethoxy-2,2-dimethylchroman-

8-yl) ethanone (17)

C

₁₅H₁₅F₃O₆S: C, 47.37; H, 3.98; F, 14.99; O, 25.24; S, 8.43%. Found: C,

To a solution of 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)

ethanone (4b) 2.0 g (0.10 mol) in methanol 10.0 ml, Oxone 5.16 g

(0.11 mol) was added at 0.5 °C. Then, 0.45 g (0.11 mol) NH₄Cl was

added and the mass was stirred for 2–3 h at 0–5 °C. Reaction was

completed as indicated by TLC and the reaction mass then quenched

with 10% sodium thio sulphatesolution (5.0 ml) and 20.0 ml of ethyl

acetate was added. Then the product was extracted in ethyl acetate

layer, washed with water, NaCl solution and dried over Na₂SO_4.

Remaining solvent was distilled out under vacuum to get crude product.

Further, the product was puriﬁed by column chromatography using

ethyl acetate and pet ether to obtain a ﬁnal syrup product (2.0 g, 80%).

IR (KBr) ϒmax: 2923, 2850, 1696, 1605, 1585, 1462, 1436, 1413,

1368, 1348, 1268, 1158, 1156, 1111, 1076, 965, 922, 883, 796, 779,

47.38; H, 3.95; F, 14.97; O, 25.25; S, 8.45%; HRMS Calculated [M+]

m/z 380.3342, Found 380.3344.

2.2.6. Preparation

ethanone (15)

of

1-(7-methoxy-2,2-dimethyl-2H-chromen-8-yl)

A

mixture of 8-acetyl-7-methoxy-2,2-dimethyl-2H-chromen-5-yl

triﬂuoromethanesulfonate (14) 0.5 g (0.10 mol) in DMF 5 ml, Pd(OAC)₂

(1.47 mg, 0.05 mol), Pd-DPPF (5.35 mg, 0.05 mol) was degased with

HCOOH and then TEA was added at RT and the ﬁnal mass was ﬂushed

with nitrogen. Further the temperature was raised to 70–75 °C and

stirred for 20 h. After the reaction completion, as indicated by TLC, the

mass was quenched with water (10 ml) and the product was extracted

in ethyl acetate (10 ml × 2). The obtained organic layer was washed

with water (10.0 ml), brine (10 ml) solution and dried over Na₂SO₄. The

solvent was distilled to obtain a ﬁnal syrup product (15) (0.27 g, 90%).

IR (KBr) ϒmax: 3373, 1662, 1582, 1358, 1267, 1111, 1094, 1046,

1023, 825, 763 cm⁻¹. ¹H NMR: (400 MHz, DMSO) δ ppm: 7.54–7.52 (d,

1H, J=), 6.57–6.55 (d, 1H, J=), 6.53–6.50 (d, 1H, J = ), 5.67–5.65 (d,

1H, J=), 3.77 (s, 3H), 2.44 (s, 3H), 1.36 (s, 6H). ¹³C NMR (100 MHz,

603, 567 cm⁻^{1 1}H NMR: (400 MHz, DMSO) δ ppm: 6.34 (s, 1H), 4.64

.

(s, 1H), 4.34 (s, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.45 (s, 3H), 2.31 (s,

3H), 1.43 (s, 6H). ¹³C NMR (100 MHz, DMSO) δ ppm: 199.60, 160.80,

157.47, 149.92, 112.21, 100.59, 89.00, 76.04, 74.82, 60.05, 57.11,

55.96, 55.82, 32.29, 26.90, 23.71. MS (EI, m/z): 328.8 (M+2).

Analysed and calculated for C₁₆H₂₁ClO₅: C, 58.45; H, 6.44; Cl, 10.78; O,

24.33%, Found: C, 58.46; H, 6.43; Cl, 10.76; O, 24.35%; HRMS

6

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Calculated [M+] m/z 328.7890, Found 328.7892.

MS (EI, m/z): 315.1 (M+2) Analysed and calculated for C₁₅H₁₉ClO₅: C,

57.24; H, 6.08; Cl, 11.26; O, 25.42%; Found: C, 57.25; H, 6.09; Cl,

11.23; O, 25.43%; HRMS Calculated [M+] m/z 314.7620, Found

314.7622.

2.3.3. Preparation of 1-(4-ﬂuoro-3,5,7-trimethoxy-2,2-dimethylchroman-

8-yl) ethanone (18)

To a solution of 2.0 g (0.10 mol) 1-(5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (4b) in methanol 10.0 ml, Oxone 5.16 g

(0.11 mol) was added at 0.5 °C. NH₄F 0.31 g (0.11 mol) was added and

the mass was stirred for 2–3 h at 0–5 °C. Reaction was completed as

indicated by TLC and the reaction mass was quenched with 10% sodium

thio sulphatesolution (5.0 ml). Then ethyl acetate 20.0 ml was added

and the product was extracted in ethyl acetate layer and washed with

water, NaCl solution and dried over Na₂SO_4.Solvent was distilled out

under vacuum to get crude product. The product obtained is puriﬁed by

column chromatography using ethyl acetate and pet ether to obtain

syrup (1.52 g, 64%). IR (KBr) ϒmax: 2954, 2923, 2852, 1716, 1624,

1585, 1463, 1445, 1430, 1393, 1364, 1327, 1276, 1216, 1179, 1145,

2.3.6. Preparation of 1-(4,6-dibromo-3,5,7-trimethoxy-2,2-dimethylchro

man-8-yl)ethanone (21)

To a solution of 2.0 g (0.10 mol) 1-(5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (4b) in methanol 10.0 ml, Oxone 10.31 g

(0.22 mol) was added at 0.5 °C. 1.63 g (0.22 mol) NH₄Br was added and

the mass was stirred for 10–12 h at 25–30 °C. Reaction was completed

as indicated by TLC and the reaction mass quenched with sodium thio

sulphate solution (15.0 ml). Then ethyl acetate 20.0 ml was added and

the product was extracted in ethyl acetate layer, washed with water,

NaCl solution and dried over Na₂SO_4.Solvent was distilled out under

vacuum to get crude product. The crude product was puriﬁed by

column chromatography using diethyl ether to obtain pure product

(2.75 g, 80%). Melting point: 119.8–121.7 °C, ¹H NMR: (400 MHz,

DMSO) δ ppm: 5.04 (s, 1H), 4.63 (s, 1H), 3.86 (s, 3H), 3.74 (s, 3H), 3.52

(s, 3H), 2.50 (s, 3H), 1.58 (s, 3H), 1.48 (s, 3H). MS (EI, m/z): 451.0

(1:2:1 ratio). Analysed and calculated for C₁₆H₂₀Br₂O₅: C, 42.50; H,

4.46; Br, 35.35; O, 17.69%, Found: C, 42.48; H, 4.45; Br, 35.37; O,

17.70%; HRMS Calculated [M+] m/z 452.1390, Found 452.1391.

1115, 1094, 1044, 1025, 961, 816, 580 cm⁻^{1 1}H NMR: (400 MHz,

.

DMSO) δ ppm: 8.58 (s, 1H), 5.23 (s, 1H), 4.11 (s, 1H), 3.84 (s, 3H), 3.75

(s, 3H), 3.46 (s, 3H), 2.30 (s, 3H), 1.19 (s, 6H). ¹³C NMR (100 MHz,

DMSO) δ ppm: 200.50, 149.85, 146.09, 141.97, 136.65, 77.09, 75.00,

69.22, 61.44, 60.20, 57.85, 120.42, 111.80, 32.31, 24.29, 23.58.

Analysed and calculated for C₁₆H₂₁FO₅: C, 61.53; H, 6.78; F, 6.08; O,

25.61%, Found: C, 61.52; H, 6.76; F, 6.09; O, 25.63%; HRMS

Calculated [M+] m/z 312.3374, Found 312.3378.

2.3.7. Preparation of 1-(4,6-dibromo-5-hydroxy-3,7-dimethoxy-2,2-dimet

hylchroman-8-yl)ethanone (22)

2.3.4. Preparation of 1-(4-bromo-5-hydroxy-3,7-dimethoxy-2,2-dimeth

ylchroman-8-yl)ethanone (19)

To a solution of 1.0 g (0.10 mol) 1-(5-hydroxy-7-methoxy-2,2-di-

methyl-2H-chromen-8-yl) ethanone (9) in methanol 5.0 ml, Oxone

5.44 g (0.22 mol) was added at 0.5 °C. 0.86 g (0.22 mol) NH₄Br was

added and the mass was stirred for 10–12 h at 25–30 °C. Reaction was

completed as indicated by TLC and the reaction mass was quenched

with 10% sodium thio sulphate solution (3.0 ml). Then ethyl acetate

10.0 ml was added and the product was extracted in ethyl acetate layer,

washed with water, NaCl solution and dried over Na2SO4_.Solvent was

distilled out under vacuum to get crude product. The crude product was

puriﬁed by column chromatography using ethyl acetate and pet ether to

obtain solid (1.23 g, 70%). Melting point: 127–129 °C, IR (KBr) ϒmax:

2994, 2946, 2825, 1618, 1570, 1462, 1420, 1388, 1369, 1354, 1304,

1280, 1245, 1174, 1126, 1091, 1076, 1050, 1028, 981, 957, 925, 884,

To a solution of 1.0 g (0.10 mol) 1-(5-hydroxy-7-methoxy-2,2-di-

methyl-2H-chromen-8-yl) ethanone (9) in methanol 5.0 ml, Oxone

2.72 g (0.11 mol) was added at 0.5 °C. 0.43 g (0.11 mol) NH₄Br was

added and the mass was stirred for 2–3 h at 0–5 °C. Reaction was

completed as indicated by TLC and the reaction mass was quenched

with 10% sodium thio sulphate solution (3.0 ml) then ethyl acetate

10.0 ml was added, the product was extracted in ethyl acetate layer and

then washed with water, NaCl solution and dried over Na₂SO_4.Solvents

were distilled out under vacuum to get crude product. The product was

puriﬁed by column chromatography using ethyl acetate and pet ether to

obtain syrup (1.0 g, 70%). ¹H NMR: (400 MHz, DMSO) δ ppm:14.0 (s,

1H, D₂O exchangable), 6.03 (s, 1H), 4.54 (s, 1H), 4.26 (s, 1H), 3.77 (s,

3H), 3.47 (s, 3H), 2.56 (s, 3H), 1.54 (s, 6H). ¹³C NMR (100 MHz, DMSO)

δ ppm: 202.3, 166.51, 163.98, 154.28, 118.89, 104.61, 98.42, 75.67,

75.07, 55.06, 54.84, 52.60, 32.26, 28.64, 23.62. MS (EI, m/z): 359.3 (M

+2). Analysed and calculated for C₁₅H₁₉BrO₅: C, 50.15; H, 5.33; Br,

22.24; O, 22.27%, Found: C, 50.16; H, 5.31; Br, 22.24; O, 22.29%;

HRMS Calculated [M+] m/z 359.2160, Found 359.2161.

842, 797, 771, 740, 684, 604, 578, 555 cm⁻^{1 1}H NMR: (400 MHz,

;

DMSO) δ ppm: 14.0 (s, 1H, D₂O exchangable), 5.08–5.07 (d, 1H,

J = 4.0), 4.63–4.62 (d, 1H, J = 4.0), 3.95 (s, 3H), 3.39 (s, 3H), 2.68 (s,

3H), 1.66 (s, 3H), 1.62 (s, 3H). ¹³C NMR (100 MHz, DMSO) δ ppm:

203.94, 163.07, 161.46, 154.39, 108.55, 106.36, 97.38, 77.58, 75.56,

61.48, 56.64, 53.05, 33.30, 29.35, 23.55. MS (EI, m/z): 439.0 (M+2,

1:2:1 ratio). Analysed and calculated for C₁₅H₁₈Br₂O₅: C, 41.12; H,

4.14; Br, 36.48; O, 18.26%; Found: C, 41.10; H, 4.16; Br, 36.48; O,

18.28%; HRMS Calculated [M+] m/z 438.1120, Found 438.1121.

2.3.5. Preparation of 1-(4-chloro-5-hydroxy-3,7-dimethoxy-2,2-dimeth

ylchroman-8-yl)ethanone (20)

To a solution of 1.0 g (0.10 mol) 1-(5-hydroxy-7-methoxy-2,2-di-

methyl-2H-chromen-8-yl) ethanone (9) in methanol 5.0 ml, Oxone

2.72 g (0.11 mol) was added at 0.5 °C. 0.23 g (0.11 mol) NH₄Cl was

added, the mass was stirred for 2–3 h at 0–5 °C. Reaction was completed

as indicated by TLC and the reaction mass was quenched with 10%

sodium thio sulphatesolution (5.0 ml). Then ethyl acetate 10.0 ml was

added and the product was extracted in ethyl acetate layer and then

washed with water, NaCl solution and dried over Na₂SO_4.Solvents were

distilled out under vacuum to get crude product. The product was

puriﬁed by column chromatography using ethyl acetate and pet ether to

obtain solid (1.0 g, 79.3%). Melting point: 72.60–74.60 °C; IR (KBr)

ϒmax: 3436, 2997, 2982, 2932,2927, 2827, 1616, 1592, 1492,

1432,1386, 1327, 1292, 1211, 1183, 1132, 1117, 1026, 960, 889, 823,

661, 635, 589 cm⁻¹.¹H NMR: NMR (400 MHz, CDCl₃) δ ppm:14.0 (s,

1H, D₂O exchangable), 6.07 (s, 1H), 4.50 (d, 1H), 4.42 (d, 1H), 3.86 (s,

3H), 3.53 (s, 3H), 2.61 (s, 3H), 1.43(s, 3H), 1.32 (s, 3H). ¹³C NMR

(100 MHz, CDCl₃) δ ppm: 203.24, 167.53, 165.07, 155.26, 105.62,

99.63, 93.05, 77.41, 75.54, 60.29, 57.63, 56.03, 33.262, 26.92, 24.79.

2.3.8. Procedure for the preparation of 1-(5-hydroxy-4,6-diiodo-3,7-

dimethoxy-2,2-dimethyl chroman-8-yl)ethanone (23)

To a solution of 1.0 g (0.10 mol) 1-(5-hydroxy-7-methoxy-2,2-di-

methyl-2H-chromen-8-yl)ethanone (9) in methanol 5.0 ml, Oxone

5.44 g (0.22 mol) was added at 0.5 °C. Then, 2.40 g (0.44 mol) of NH₄I

was added and the mass was stirred for 10–12 h hours at 25–30 °C.

Reaction was completed as indicated by TLC and the reaction mass was

quenched with 10% sodium thio sulphatesolution (5.0 ml). Then ethyl

acetate 10.0 ml was added and the product was extracted in ethyl

acetate layer, washed with water, NaCl solution and dried over Na₂SO_4.

Solvent was distilled out under vacuum to crude product. The crude

product was puriﬁed by column chromatography using ethyl acetate

and pet ether to obtain oﬀ-white solid (1.28 g, 60.1%). Melting point:

151.9–153.9 °C; IR (KBr) ϒmax: 3432, 2988, 2939, 2822, 1610, 1578,

1560, 1457, 1415, 1402, 1367, 1351, 1300, 1275, 1240, 1192, 1168,

1106, 1087, 1073, 1041, 978, 921, 881, 870, 836, 790, 757, 711, 676,

601, 575, 549 cm⁻^{1 1}H NMR: (400 MHz, DMSO) δ ppm: 14.30 (s, 1H,

.

7

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

D₂O exchangable), 5.15 (s, 1H), 4.79 (s, 1H), 3.86 (s, 3H), 3.48 (s, 3H),

2.58 (s, 3H), 1.57 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ ppm: 203.91,

166.13, 163.99, 155.96, 108.08, 106.44, 77.98, 77.78, 74.42, 61.52,

56.34, 35.62, 33.61, 33.15, 22.24. MS (EI, m/z): 532.9 (M+1).

Analysed and calculated for C₁₅H₁₈I₂O₅: C, 33.86; H, 3.41; I, 47.70; O,

15.03%; Found: C, 33.84; H, 3.38; I, 47.73; O, 15.05%; HRMS

Calculated [M+] m/z 532.1129, Found 532.1130.

distilled out under vacuum to get crude product. The crude product

puriﬁed by column chromatography using ethyl acetate and pet ether to

obtain yellow color syrup (1.0 g, 79.3%). Melting point:

127.4–129.8 °C; ¹H NMR: NMR (400 MHz, DMSO) δ ppm: 6.53–6.50 (d,

1H, J = 12), 5.81–5.79 (d, 1H, J = 10.0), 3.71 (s, 3H), 3.67 (s, 3H),

2.43 (s, 3H), 1.45 (s, 6H). ¹³C NMR (100 MHz, DMSO) δ ppm: 199.54,

156.51, 156.12, 150.93, 130.91, 122.29, 116.05, 115.52, 112.0, 80.20,

77.37, 62.43, 61.59, 32.05, 29.37, 27.52. MS (EI, m/z): 389.0 (M+1).

Analysed and calculated for C₁₅H₁₇IO₄: C, 46.41; H, 4.41; I, 32.69; O,

16.49%; Found: C, 46.43; H, 4.42; I, 32.68; O, 16.47%; HRMS

Calculated [M+] m/z 388.2015, Found 388.2018.

2.3.9. Preparation

of

1-(6-bromo-5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (24)

To a solution of 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)

ethanone (4b) 2.0 g (0.10 mol) in ethanol 10.0 ml, Oxone 5.11 g

(0.11 mol) was added at 0.5 °C. Then, 0.814 g (0.11 mol) NH₄Br was

adde and the mass was stirred for 1–2 h at 25–30 °C. Reaction was

completed as indicated by TLC and the reaction mass was quenched

with sodium thio sulphate solution (5.0 ml). Then ethyl acetate 20.0 ml

was added and the product was extracted in ethyl acetate layer, washed

with water, NaCl solution and dried over Na₂SO_4.Solvent was distilled

out under vacuum to crude product. The crude product was puriﬁed by

using diethyl ether to obtain pure product (2.21 g, 85%). Melting point:

119.7–121.7 °C, IR (KBr) ϒmax: 2990, 2965, 2843, 1701, 1653, 1603,

1498, 1470, 1416, 1383, 1364, 1337, 1236, 1206, 1185, 1168, 1118,

1006, 962, 935, 887, 869, 794, 688,656, 576, 562, 501 cm⁻¹. ¹H NMR:

(400 MHz, DMSO) δ ppm: 6.86 (s, 1H), 6.33 (s, 1H), 3.85 (s, 3H), 3.79

(s, 3H), 2.34 (s, 3H), 1.44 (s, 6H). ¹³C NMR (100 MHz, DMSO) δ ppm:

198.82, 157.74, 155.41, 148.82, 119.88, 119.71, 113.02, 104.01,

89.55, 79.82, 56.03, 56.03, 32.41, 25.76, 24.76. ¹³C NMR (100 MHz,

CDCl₃) δ ppm: 200.26, 158.12, 155.85, 150.31, 120.53, 119.94,

113.57, 105.16, 88.30, 80.54, 55.90, 55.71, 32.61, 26.34, 21.77. MS

(EI, m/z): 341.3 (M+2). Analysed and calculated for C₁₅H₁₇BrO₄: C,

52.80; H, 5.02; Br, 23.42; O, 18.76%; Found: C, 52.81; H, 5.03; Br,

23.42; O, 18.74%; HRMS Calculated [M+] m/z 341.2010, Found

341.2013.

2.3.12. Procedure for the preparation of 1-(4-bromo-3-hydroxy-5,7-

dimethoxy-2,2-dimethyl chroman-8-yl)ethanone (27)

To a solution of 1.0 g (0.10 mol) 1-(5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (4b) in acetonitrile 4.0 ml, water 2.0 ml, Oxone

2.72 g (0.11 mol), NH₄Br 0.407 g (0.11 mol) was added at 0–5 °C. The

mass was stirred for 1 h at 0–5 °C. Reaction was completed as indicated

by TLC, and the reaction mass was quenched with 10% sodium thio

sulphate solution (5.0 ml). Then ethyl acetate (10.0 ml) was added, the

product was extracted in ethyl acetate layer, washed with water, NaCl

solution and dried over Na₂SO_4.Solvent was distilled out under vacuum

to get crude product. The crude product puriﬁed by using diisopropyl

ether to obtain oﬀ white solid (0.96 g, 70%). Melting point:

125.4–127.3 °C, IR (KBr) ϒmax: 3518, 3000, 2981, 2939, 2846, 1778,

1706, 1685, 1585, 1492, 1462, 1370, 1347, 1293, 1264, 1156, 1116,

1095, 1025, 923, 911, 885, 805, 761, 744, 708, 686, 621, 568, 486; ¹

H

NMR: (400 MHz, DMSO) δ ppm: 6.32 (s,1H), 5.81 (s, 1H, D₂O ex-

changable), 4.93 (s,1H), 4.41 (s,1H), 3.84 (s,3H), 3.77 (s,3H), 2.311

(s,3H), 1.51 (s,3H), 1.43 (s,3H). ¹³C NMR (100 MHz, DMSO) δ ppm:

199.78, 160.40, 157.05, 149.91, 112.27, 102.97, 89.08, 76.23, 65.55,

60.00, 55.85, 55.77, 32.36, 27.94, 24.78. MS (EI, m/z): 359.1 (M+2).

Analysed and calculated for C₁₅H₁₉BrO₅: C, 50.15; H, 5.33; Br, 22.24;

O, 22.27%; Found: C, 50.13; H, 5.31; Br, 22.24; O, 22.32%; HRMS

Calculated [M+] m/z 359.2160, Found 359.2160.

2.3.10. Preparation

of

1-(6-chloro-5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (25)

To a solution of 2.0 g (0.10 mol) 1-(5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (4b) in ethanol (10.0 ml), Oxone 5.16 g

(0.11 mol) was added at 0.5 °C. Then, 0.45 g (0.11 mol) NH₄Cl was

added and the mass was stirred for 1–2 h at 25–30 °C. Reaction was

completed as indicated by TLC and the reaction mass was quenched

with sodium thio sulphate solution (5.0 ml). Then ethyl acetate

(20.0 ml) was added and the product was extracted in ethyl acetate

layer, washed with water, NaCl solution and dried over Na₂SO_4.Solvent

was distilled out under vacuum to get crude product. The crude product

was puriﬁed by column chromatography using ethyl acetate and pet

ether to obtain pure solid (1.87 g, 82.7%). Melting point:

104.3–106.2 °C. ¹H NMR: (400 MHz, DMSO) δ ppm: 6.66 (s, 1H), 6.34

(s, 1H), 3.85 (s, 3H), 3.79 (s, 3H) 2.35 (s, 3H), 1.50 (s, 6H). ¹³C NMR

(100 MHz, DMSO) δ ppm: 198.85, 157.60, 155.50, 148.53, 128.79,

115.46, 113.04, 103.46, 89.63, 79.33, 74.95, 57.15, 56.00, 32.39,

24.91. MS (EI, m/z): 296.8 (M+2). Analysed and calculated for

2.4. Antibacterial activity studies

Antimicrobial activity potentials of present study compounds was

determined by the method described by European Committee for

Antimicrobial Susceptibility Testing.²⁴Commercially available Clor-

obiocin and Novobiocin were used as reference drugs. 1% Dimethyl

sulphoxide (DMSO) was used as a control without mixing the test

compounds. Further, compounds were tested at various concentrations

against Gram^+veStaphylococcus aureus (MTCC 96), Corynebacterium

diphtheriae (MTCC – 116), and Streptococcus pyogenes (MTCC 442) and

Gram^−vebacteria Klebsiella pneumoniae (MTCC 530), Escherichia coli

(MTCC 443), and Pseudomonas aeruginosa (MTCC 424).

2.5. Molecular docking studies

Autodock 4.2.6 and Autodock Tools (ADT) 1.5.6 were used for the

docking studies. All strategies and parameters were used and adjusted

according to the present study’s necessity with our earlier reports.^25–28

The three-dimensional structures of targeted enzymes/proteins (PDB

ID: 2XCT (S. aureus DNA gyrase A), PDB ID: 3G75 (S. aureus DNA gyrase

B), PDB ID: 3L7L (Teichoic acid polymerase) was retrieved from www.

rcsb.org/pdb/.

C

₁₅H₁₇ClO₄: C, 60.71; H, 5.77; Cl, 11.95; O, 21.57%; Found: C, 60.72;

H, 5.75; Cl, 11.97; O, 21.56%; HRMS Calculated [M+] m/z 296.7470,

Found 296.7476.

2.3.11. Preparation

of

1-(6-iodo-5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (26)

To a solution of 1.0 g (0.10 mol) 1-(5,7-dimethoxy-2,2-dimethyl-2H-

chromen-8-yl)ethanone (4b) in ethanol (5.0 ml), Oxone 2.72 g

(0.11 mol) was added at 0.5 °C. To this, 1.2 g (0.22 mol) NH₄I was

added and the mass was stirred for 1–2 h at 25–30 °C. Reaction was

completed as indicated by TLC, the reaction mass was quenched with

10% sodium thio sulphatesolution (5.0 ml). Then, ethyl acetate

(10.0 ml) was added and extracted the product in ethyl acetate layer

washed with water, NaCl solution and dried over Na₂SO_4.Solvent was

2.6. DNA gyrase inhibition activity

DNA gyrase A (S.aureus) inhibition assay of present study com-

pounds was carried out as reported earlier.²⁹S. aureus was cultured in

medium B (2 g yeast extract, 10 g polypeptone, 1.2 g (NH₄)₂SO₄, 8 g

Na₂HPO, 2 g KH₂PO₄, 0.2 g MgSO₄, 4 g glucose in 1L distilled water).

DNA gyrase puriﬁcation, Supercoiling, and decatenation were executed

8

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

2.11. Cytotoxicity evaluations – MTT assay

as reported by F. Blanche et al. 1996.³⁰Extracted DNA was treated with

DNA gyrase and untreated DNA samples were determined the DNA

gyrase inhibition via Sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis (SDS-PAGE) technique.

Early passage of breast cancer (MCF-7) and normal breast epithelial

cells (MCF-10) were used for the cytotoxicity assessment of present

study chromans coumarins compounds. The cytotoxicity of compounds

9, 13, 14, 18 and 20 on diﬀerent cancer cell lines was assessed by the

MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide)

assay by referring our earlier reports.²⁸Doxorubicin was used as the

standard drug. The cell viability was calculated using the following

formula:

2.7. Gram^+vebacterial cell wall surface degradation examinations

C. diphtheriae was inoculated in Hardy Diagnostics Loeﬄer (HDL)

Medium (5 ml) and grown overnight. Cells were harvested by cen-

trifuging at 10,000×g for 10 min. Pellet was resuspended in 2 ml of 1×

Wash Buﬀer (Guanidine hydrochloride in ethanol). 1 ml of this live

culture was added to 5 ml of 1× Wash Buﬀer. To achieve a ‘dead’

standard, 1 ml of this live culture was suspended with 5 ml of 70%

isopropanol. Both samples were incubated at RT for 1-h mingling fre-

quently by overturn. After incubation, samples were centrifuged at

10,000×g for 10 min and both pellets were resuspended in 5 ml of 1×

Wash Buﬀer each. Centrifuge at 10,000×g for 10 min. Further, the

pellet was resuspended in 1 ml of 1X Wash Buﬀer. Finally, 1 µL Total

Cell Stain and 1 µL Dead Cell Stain was added to each sample tube

(1 ml).

Cell viability(%) = Mean OD/Control OD × 100%

2.12. Statistical analysis

All results were expressed as a percentage increase or decrease with

respect to control values. All results were compared by performing one-

way ANOVA with Dunnett’s post-test. GraphPad Prism version 7.1 for

Windows, GraphPad Software, USA (www.graphpad.com) was used for

statistical analysis. A diﬀerence was considered statistically signiﬁcant

if p ≤ 0.05. The 50% inhibitory concentration (IC₅₀) was calculated

from the dose–response curve obtained by plotting percentage inhibi-

tion versus concentration.

2.8. Estimation of viable and non-viable bacterial cells

The experiment was carried out in a 96 well plate as per the re-

ported instruction.³¹A blank solution containing 1 ml 1× Wash Buﬀer

and 1 µL total cell stain and dead cell stain was used for the assay. This

mixture was further incubated for 1 h under dark at room temperature.

The experiment was done in triplicate. 200 µL of each sample and the

blank solution was pipetted out and transferred into separate wells of

96-well plate. The ﬂuorescence was measured at Ex 490 nm/EM 525 nm

and Ex 536 nm/EM 617 nm used for reading 1 & 2 respectively for all

wells. The percentage of dead cells or cell viability is calculated using

the formula:

3. Results

3.1. Synthesis of proposed 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy

chromans

The parent compound 5,7-dihydroxy-2,2-dimethyl chroman-4-one

was achieved as per the instructions of Tsukiyama et al. 1989 and Alex

and Ross 2004.^33,34Compound 1 was prepared by treating 5,7-dihy-

droxy-2,2-dimethyl chroman-4-one with methyl iodide in presence of

potassium carbonate, further, compound 1 undergoes reduction using

sodium borohydride in methanol medium to achieve 2. Compound 2

was treated with methane sulphonyl chloride to get 3, and ﬁnally

compound 3 undergoes Friedel-Crafts acylation to achieve 4a and 4b.

In the Friedel-Crafts acylation reaction experiment (Scheme 1), the

anticipated yield was relatively observed to be very less. We have used

the NOESY experiment to determine the position of acetyl groups at 6

(4a) and 8 (4b).

Percentage Viability = 100−([Number of red cells/Number of green cells]

× 100)

2.9. Cell wall degradation assessment after treating the compounds

In 4a, the aromatic proton at C8 has a correlation with the O-

methoxy group at C7, this conﬁrms the presence of an acetyl group at

the C6 position. Simultaneously, in 4b the aromatic proton at C6 has a

correlation with both C5 and C7 of O-methoxy groups. 6-acetyl-5,7-

dihydroxy-2,2-dimethyl chroman-4-one and 8-acetyl-5,7-dihydroxy-

2,2-dimethyl chroman-4-one (Scheme 2) are also achieved as per the

previously reported instructions by Tsukiyama et al. 1989 and Alex and

Ross 2004.^33,34Further, the above mentioned starting materials were

methoxylated to achieve 5, 6 and 7. These starting materials were re-

duced to achieve 8 and dehydrated to get 4a & 4b in aﬀordable yeild.

All the reaction were carried out without purifying the intermediates to

achieve better yield. Compound 9 was achieved by demethoxylation

reaction (Scheme 3), by treating 4b with BBr₃at −75 to −80 °C.³⁵The

Cis dihydroxy compounds 10, 11 and 12, 13 were prepared from 4a

and 4b respectively by using shapeless asymmetric dihydroxylation

reaction.^33,36,37Here the position of the acetyl group at 6 (10, 11) and 8

(12, 13) was conﬁrmed by NOESY experiment.

The derivatives 14 and 15 were prepared by using 1-(5-hydroxy-7-

methoxy-2,2-dimethyl-2H-chromen-8-yl)ethanone (9) as mentioned in

Scheme 4. Compound 9‘s hydroxyl group was protected with triﬂate to

achieve 14, further, it was reduced using palladium catalysts Pd(OAc)₂

and Pd-DPPF³⁸to get 15. Both the reactions were carried out carefully

by not having any moisture contamination. Compounds 16–26 (Scheme

5) was prepared by using ammonium salts (NH₄F, NH₄Cl, NH₄Br, NH₄

I) as a halogen source and Oxone as an oxidant.^39,40The alkoxy

S. aureus culture was examined for their hourly physiological

changes or cell wall damage after treating the coumarin/chroman

compounds in optimized concentrations. Gradual changes in bacterial

physiological conditions, especially the changes that were occured on

bacterial cell wall, was measured by estimating dead cells by means of

estimating debris level at every 30 min. The experiment was continued

until the whole bacterial cell death or cell wall damaged. Amount of

ﬁnal cell debris determines the total ability of degrading/killing po-

tency of tested compounds in a particular time period.

2.10. HRBC membrane protection potentials evaluations

To determine the cell membrane fortiﬁcation/mortiﬁcation eﬀects

against the human cellular system, the Human Red Blood Cells (HRBC)

membrane stabilization potential of the title compounds (chromans/

coumarins) at various concentrations (n = 4) was assessed in a 96 well

plate using Diclofenac as the standard.³²The percentage hemolysis was

calculated by assuming the hemolysis generated in presence of distilled

water at 100%. The percentage of HRBC membrane stabilization was

calculated using the following formula,

%inhibition of hemolysis = 100 × [(OD₁−OD₂)/OD₁]

where OD₂= optical density of sample OD₁= optical density of con-

trol.

9

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Scheme 3. Route of Synthesis of 9, 10, 11, 12 and 13 from 4a & 4b.

halogenation was thoroughly studied for the formation of mono and di-

substituted product with reference to temperature and equivalence of

the reagent. The reaction was thermodynamically controlled to achieve

methoxy halogenation predominantly in the oleﬁn functional group.

Aromatic halogenation was carried out using ethanol as a solvent

medium instead of methanol. Because, due to steric hindrance, ethanol

could not provide the ethoxy brominated product. Compound 27,

bromohydrin, was synthesized with NH₄Br and Oxone system using

acetonitrile and water as a solvent medium.

chromans/coumarins are selectively exposed the binding aﬃnity to-

wards DNA gyrase B than DNA gyrase A. Astonishingly, remarkable

binding aﬃnity was found against TAP. As per the Table 1 statistical

estimations, the calculated cumulative binding aﬃnity was calculated

as −339.31, −613.14, −547.10 kcal/mol for DNA gyrase A, DNA

gyrase B, and TAP respectively. Thus, a DNA gyrase B inhibition activity

was considered to be evaluated as the most eﬀective anticipated bio-

activity of coumarin/chroman compounds. Interestingly, the overall

distinguished chemotherapy eﬃcacy of these compounds was also re-

cognized from the obtained mean values of molecular mechanistic re-

sults of DNA gyrase A, DNA gyrase B and TAP (-16.60, −28.72 and

−25.86 kcal/mol respectively).

3.2. Molecular docking results

To specify a compounds drug eﬃcacy towards the proposed activ-

ities here, the lowest binding energy, inhibitory constant (ki) and best

interactions were assessed. In the assessment, compound 14 was found

to have most eﬃcient binding aﬃnity with a least average binding

energy (-35.73 kcal/mol, ki = 42 pM). At the same time, compound 20

(-31.72 kcal/mol, ki = 58 pM), compound 24 (−30.75 kcal/mol,

ki = 82 pM), compound 12 (−29.30 kcal/mol, ki = 90 pM), compound

18 (−28.20 kcal/mol, ki = 96 pM), and compound 17 (−27.69 kcal/

mol, ki = 98 pM) were also had comparable values to 14. Later, these

Drug eﬃcacy of present study compounds was determined through

the molecular mechanistic values and best-docked poses that were

obtained from molecular docking studies. The best binding aﬃnity and

ligand-receptor interaction details of the compounds was assessed. The

expected binding free energy for S. aureus DNA gyrase A (PDB ID:

2XCT) was obtained in a range between −7.26 and −28.09 kcal/mol.

It was −7.85 to −41.87 kcal/mol for DNA gyrase B (PDB ID: 3G75) and

for TAP (Teichoic Acid Polymerase) (PDB ID: 3L7L) it was −6.35 to

−37.23 kcal/mol. These binding free energy values indicates that the

Scheme 4. Synthesis of 14 and 15 from 9.

10

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Scheme 5. Synthesis route of compounds 16–27.

Table 1

Molecular docking and molecular validation of 3-O-methoxy-4-halo disubstituted5,7-dimethoxy chromans.

Molecular Docking Studies

Druggability analysis

^#Ligand

2xct

3 g75

3l7l

ABA^*

LogP

S + logD

Ro5

6

9

−14.41

−21.38

−17.01

−22.48

−19.82

−17.06

−28.09

−13.13

−8.54

−30.32

−28.73

−30.73

−28.71

−36.66

−30.73

−41.87

−26.46

−7.851

−35.96

−36.6

−23.76

−26.01

−29.13

−28.69

−24.41

−29.19

−37.23

−25.57

−6.35

−30.34

−35.08

−12.07

−35.03

−10.57

−29.64

−24.51

−33.06

−21.55

−25.64

−22.24

−543.1

−25.86

−22.83

−25.37

−25.62

−26.63

−26.96

−25.64

−35.73

−21.72

−7.58

−27.49

−30.22

−16.39

−31.72

−15.16

−25.25

−19.35

−28.89

−21.2

−20.55

−19.44

–

0.42

2.12

−0.08

0.13

2.53

2.52

1.84

1.72

0.93

1.09

1.45

2.66

2.41

3.06

1.84

2.27

1.96

1.47

32.43

1.55

1.7

< None >

Mw

3.53

−2.52

0.98

1.18

3.37

3.19

3.06

2.87

1.62

3.18

3.55

2.45

0.85

3.19

3.45

3.6

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

Sum

Mean

−16.18

−18.97

−6.74

−30.36

−40.43

−15.29

−28.8

−19.71

−19.63

−17.32

−13.73

−20.26

−20.41

−11.76

−14.81

−348.63

−16.6

−19.8

−33.34

−21.64

−24.25

−21.27

−603.1

−28.72

< None >

–

2.33

46.73

2.22

–

#

3-O-methoxy-4-halo disubstituted 5,7-dimethoxy chromans.

* ABA – Average Binding Aﬃnity; Crystal structures of PDB ID: 2XCT – DNA gyrase A, 3G75 – DNA gyrase B, 3L7L – Teichoic acid polymerase. Component of

Lipinski's Rule of 5 (Ro5): LogP – lipophilicity, the partition coeﬃcient of a molecule between an aqueous and lipophilic phase, usually octanol and water. S + logD –

The distribution coeﬃcient, it is the ratio of the sum of the concentrations of all forms of the compound.

11

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Fig. 1. A representative illustration of best-docked poses or interactions of 14 into the active binding site of DNA gyrase B (PDB ID: 3G75). Note: Stick and Balls

(Ligand), Sticks only (Amino acid residues), Spherical (H-bonds), Cylindrical (π-π interactions) and Conical (Cation-π interactions).

compounds were screened for further DNA gyrase decatenation studies.

Apart from these, compounds 9–13, 16, 22 were also found to have a

signiﬁcant binding energy values (range of −60 to −63 kcal/mol).

Fig. 1 depicts the interaction details of compound 14 with DNA gyrase

B (PDB ID: 3G75). Established non-covalent (π-π and π-cation inter-

actions) and hydrogen bonding interactions of compound 14 conﬁrms

the anticipated best docked ligand-receptor binding interactions as well

as the most probable therapeutic output against the proposed in vitro or

in vivo studies. Usually, Hydrogen bonds are facilitating the solubility of

a small molecule and the non-covalent interactions (π-π and cation-π

interactions) are involving in the exchanging of positive charge of a

cation associated to the electrons in a π-system of a molecule.

3.3. DNA gyrase inhibition studies

Eleven compounds that were screened based on the obtained

binding aﬃnity (moderate to excellent) values, from molecular docking

studies were tested for their inhibition potential against DNA gyrase of

S. aureus. Fig. 2 illustrates the considerable IC₅₀values that were cal-

Fig. 2. IC₅₀and the relative activity of Chroman compound to demonstrate S.

aureus DNA gyrase A inhibition. Note: All IC₅₀values (µM) are the mean of

duplicate or triplicate measurements.

culated from the inhibition assay studies against DNA gyrase of S.

aureus. An IC₅₀range of 1.86–24.02 nM with a calculated relative

S. aureus was examined by assessing the transformation in DNA mobi-

percentage activity range of 78–94% was obtained as overall result.

lity passing through the electrophoresis gel. Normally the supercoiled

Clorobiocin (standard drug) showed an IC₅₀of 8.5 nM (86% inhibition).

form DNA of plasmid migrates quicker on the agarose gel due to its

Meanwhile, comparatively dominant results over Clorobiocin was ex-

conﬁrmation. Due to nicking on either relaxed or circular form DNA,

hibited by the compounds 14 and 20 (IC₅₀2.5 (94% inhibition and

they migrate bit by bit and appearing on top of agarose gel. pBR322

3.5 nM 90% inhibition respectively). Compound 9 (IC₅₀7.5 nM), 18

which was incubated with S. aureus DNA gyrase along with and without

(IC₅₀9.5 nM) and 13 (IC₅₀10 nM) were also there in that list. In the

proposed compounds and Novobiocin was used as positive control. The

mechanism, DNA gyrase alleviating a bacterial strain’s life by pro-

compounds and Novobiocin were used at a concentration of 1.5 µg in

tecting their double-stranded DNA from the helicase wounding. Heli-

the reaction amount.

From the excellent active group (screened from docking studies) 9,

14, 18, 20 (lane 5–8); from moderate active group 10, 24 (lane 9 & 10);

case is the main causative agents of bacterial DNA’s negative super-

coiling as well as the relaxation of positive supercoils. In this study, the

eﬀectiveness of selected compounds for inhibition of DNA gyrase from

12

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Fig. 3. DNA Gyrase inhibition of chroman derivatives. Lane 1 = pBR322 alone, Lane 2 (positive control) = pBR322 + gyrase; Lanes 3 (negative control) = 1%

DMSO; Lanes (standard drug) = gyrase + pBR322 + Novobiocin; Lanes 5–8 = pBR322 + gyrase + active group (9, 14, 18, 20); Lanes

9–11 = pBR322 + gyrase + moderate active group (10, 24, 27). Note: Compounds were observed at a concentration of 2 µg in a ﬁnal reaction volume of 20 µL.

4

from the inactive group, 27 (lane 11) were tested respectively as re-

presentative compounds from each group. Fig. 3 depicts the S. aureus

DNA gyrase inhibition ability of 3-O-methoxy-4-halo disubstituted 5,7-

dimethoxy chromans. Surprisingly, lane 5 loaded with 9 was exactly

showed very less supercoiled band which indicates the excellent DNA

gyrase inhibition activity of 9. Lane 4′s supercoil intensity (Clorobiocin

(standard) loaded) is relatively higher than the lane 6–8 which were

loaded with excellent active group 14, 18 and 20 respectively are also

can be considered as eﬀective DNA gyrase inhibitors. The intensity of

the compounds 10, 24 and 27 supercoiled bands was stronger which

suggests that these compounds only possess lesser inhibitory potential

than the standard and other most potential compounds 9, 14, 18 and

20.

0.764

also

(2.991

0.17–2.304

exhibited

0.67 μg mL⁻¹

good antibacterial

,

remaining compounds were

activity, except 10

0.72 μg mL⁻¹), and signiﬁcantly, these values were only had

a slight variation with the MIC value of the standard Novobiocin

(2.434

0.73 μg mL⁻¹).

3.4.1. Estimation of viable and non-viable bacterial cells via cell wall

degradation assessment

The anticipated cell wall damage, perhaps, due to the Teichoic Acid

Polymerase (the enzyme which is involving the cell wall construction

function of all Gram^+vebacteria) inhibition by the present study

compounds, 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy chro-

mans, was analyzed. Enumeration of bacterial cell viability was carried

out by estimating the absorbed dye by the living cells and dead cells in a

96 well plate. Earlier, the bacterial samples were cultured in corre-

sponding broth media and an average % of Living/Dead cells was

measured along with the average time required for the survival or

death. Mainly this execution was intended to analyze (or) to estimate

the viable and non-viable bacterial cells due to cell wall degradation. A

graph (Fig. 4) was plotted with the % live/dead cells versus the time

that needs to exterminate the total organisms in broth media. In the

overall results, the total destruction of bacterial cell was required an

average of 160 min. In the microscopic cell wall damage observations,

C. diphtheriae culture was examined for their hourly cell damage after

treating the test compounds in optimized concentrations. Gradual

3.4. Antibacterial activity results

Table 2 depicts the MIC values calculated to determine the anti-

bacterial potentials of 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy

chromans against both Gram^+veand Gram^−vebacteria. The best anti-

bacterial activity was obtained against Gram^+vebacterium (S. aureus)

over Gram^−vebacteria evidenced from an MIC range of

0.02–1.54 μg mL⁻¹. With lowest average MIC values, compound 14

(0.474

(0.696

0.18 μg mL⁻¹), 20 (0.636

0.14 μg mL⁻¹

)

and 17

0.20 μg mL⁻¹) were the most active and therapeutically po-

tent (n = 5) (R²= 0.9927). Also, with an average MIC range of

Table 2

Antibacterial activity of 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy chromans against selected pathogens.

Entry

Minimum inhibitory concentration (μg mL⁻¹

)

^{^}MIC

SEM

Gram^−ve

Gram^+ve

KP

EC

PA

SA

CD

SP

6

9

10

12

13

14

16

17

2.24

0.90

3.88

0.92

2.18

0.64

2.02

0.96

0.76

0.72

1.99

0.75

0.50

–

0.99

0.88

4.1

1.67

1.02

3.01

1.01

4.52

0.84

1.26

1.02

0.94

0.87

3.12

1.75

100

–

1.00

0.10

1.54

0.64

0.92

0.02

0.98

0.18

0.16

0.08

0.99

0.92

1.02

0.82

1.28

0.9

0.99

0.04

1.12

0.21

0.92

0.68

1.08

1.32

–

3.12

1.00

5.02

1.02

2.85

0.83

2.98

1.07

0.94

0.87

2.04

3.24

–

1.561

0.764

2.991

0.882

2.304

0.474

1.484

0.696

0.776

0.636

1.872

2.434

–

0.41

0.17

0.72

0.07

0.67

0.18

0.38

0.20

0.15

0.14

0.40

0.73

0.84

2.24

0.64

1.08

1.00

0.92

0.68

2.13

4.94

5.00

–

18

20

24

Std^*

Ref^**

Control^*

0.78

–

^

^*Control-DMSO (Dimethylsulfoxide), ^*Standard-Novobiocin, ^*Ref – Reference; Average MIC of both Gram^+veand Gram^−vebacterial tested (n = 5) (r²= 0.9927);

KP-Klebsiella pneumoniae, EC – E.coli, PA-Pseudomonas aeruginosa, SA-Staphylococcus aureus, CD-Corynebacterium diphtheriae, SP-Streptococcus pyogenes.

13

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Table 3

HRBC cell wall protection eﬀects of 3-O-methoxy-4-halo disubstituted 5,7-di-

methoxy chromans.^*

Entry

Mean Absorbance

SD^*

% HRBC cell wall

Protection

IC₅₀µg/mL

12

13

24

9

18

14

17

20

0.4046

0.3888

0.3634

0.4624

0.3947

0.4852

0.3659

0.4242

0.3841

0.3731

0.3611

0.3834

0.1868

0.025

0.038

0.064

0.021

0.012

0.022

0.018

0.042

0.007

0.009

0.012

0.033

88.52

85.31

86.16

90.45

85.15

98.84

86.37

93.25

80.65

85.55

80.88

85.64

0.0094

0.8802

0.1241

0.0182

0.0922

0.0012

0.2224

0.0102

1.2406

0.1184

1.0016

0.0860

–

6

16

10

Indomethacin

Fig. 4. Cell viability results. Values are the mean of six diﬀerent experiments (3

Control^#

No inhibition

Gram^+ve+ 3 Gram^−veorganism).

#

Distilled water.

* S.D. = Standard deviation (Average of four determinations) (n = 4).

changes in bacterial cell wall was measured by estimating dead cells or

cell debris level at every 30 min until whole bacterial death (Fig. 5).

Amount of ﬁnal cell debris indicates the total cell wall damage/mor-

tality of the bacterial cells so as the ability of 3-O-methoxy-4-halo

disubstituted 5,7-dimethoxy chromans compounds within a particular

time period.

lines (MCF-7 and MCF-10 respectively) by MTT assay (n = 4). In the

activity, there was no change in the normal cell lines (MCF-10) even

after 8 h from the compound treatment. Meanwhile, a gradual, dose

depended cell death was observed against MCF-7 cell lines. The cell

viability was assessed with a control (Dimethylsulphoxide (DMSO)) and

a standard (Doxorubicin). An average of 20% cancer cell viability was

observed after 4 h from the compound treatment. Meanwhile it was

reduced as 16% at the end of 6 h. The eﬃcient cytotoxicity against

cancer cell lines by the compounds was recognized from this. The re-

lative % activity calculated for all compounds was far better from

Doxorubicin, the standard drug. Fig. 6, shows the ups and down of

cytotoxic activity exhibited by 9, 13, 14, 18 and 20. Among all the

compounds, 14 was dominated for the activity with a maximum re-

lative % activity up to 90% and lowest IC₅₀of 10 nM.

3.5. HRBC cell wall protection of 3-O-methoxy-4-halo disubstituted 5,7-

dimethoxy chromans

The results of HRBC membrane stabilization eﬀects of 3-O-methoxy-

4-halo disubstituted 5,7-dimethoxy chromans is depicted in Table 3.

The obtained, remarkable, HRBC membrane protection results of these

chemical compounds indicating the non-harmf nature of them against

human cellular subjects (average protection range 92%). This remark-

able cell wall protection and degradation diﬀerence exhibited by these

compounds, perhaps, due to the variation between prokaryotic and

eukaryotic cells (bacteria and HRBC respectively). According to the

measured relative % protection and IC₅₀values, except compounds 6

and 10, all compounds were found as most active as the standard, In-

domethacin.

3.7. Structure-activity relationships

In the Structure-Activity Relationship (SAR), substituted 3-O-

methoxy-4-halo disubstituted 5,7-dimethoxy chromans with various

electron withdrawing and electron discharging groups were synthe-

sized, analyzed and compared with the DNA gyrase inhibition and

antibacterial activity. The interaction details with corresponding re-

ceptors that were highly associated with the antibacterial activities was

unveiled through molecular docking studies. While discussing about the

3.6. Cytotoxicity study of compounds 9, 13, 14, 18 and 20

Cytotoxicity of the best DNA gyrase inhibitors of this study was

assessed against epithelial type breast cancer cell lines and normal cell

Fig. 5. Physiological observations of C. diphtheriae cell wall degradation/cell death analysis.

14

T. Ponnusamy et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx

Acknowledgments

The authors are deeply acknowledging Biocon Limited, Bangalore-

560100, India and Research Development Centre, Bharathiar

&

University, Coimbatore – 641046, India.

A. Supplementary data

Supplementary data associated with this article can be found, in the

online version, at http://dx.doi.org/10.1016/j.bmc.2018.05.016.

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Article Doi

Drug efficacy of novel 3-O-methoxy-4-halo disubstituted 5,7-dimethoxy chromans; evaluated via DNA gyrase inhibition, bacterial cell wall lesion and antibacterial prospective

DOI: 10.1016/j.bmc.2018.05.016

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Full text of DOI:10.1016/j.bmc.2018.05.016

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