Furopyridinediones as novel inhibitors of -glucosidases · Furopyridinediones as novel inhibitors...

Furopyridinediones as novel inhibitors of -glucosidases

Rajanikant Mamidala,4 Chandramohan Bathula,1 Chiranjeevi Thulluri,2 Rahul Agarwal,3, Kunal Kumar Jha,1 Parthapratim Munshi,1 Subhabrata Sen1, *, Uma Adepally,2 Ashutosh Singh,3 M.

Thirumalacharya4

1 Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Chithera, Dadri, UP 201314, India

2 Department of Life Science, School of Natural Sciences, Shiv Nadar University, Chithera, Dadri, UP 201314, India

3 Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad500085, Telengana, India

4 Department of Chemistry, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad500085, Telengana, India

1

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2015

Contents

1. 1H-NMR spectra of compound 12a-v---------Page 1-22 2. 13C-NMR spectra of compound 12a-v----------3-44

1

Pages 2-24Pages 25-45

2

Pages 3-24Pages 25-45

3. 12p a-glucosidase assay, LB plotAlpha-glucosidase assay, LB plot and COS cell assay 12p----Pages 46-48

ab

un

dan

ce

01

.02

.03

.04

.05

.06

.07

.08

.09

.01

0.0

11

.0

X : parts per Million : 1H

14.0 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

12.2

873

8

.3584

8

.3405

8

.0368

8

.0190

7

.1216

6

.8152

3

.3452

2

.5001

2

.3709

2

.3572

−

0.0

587

2.8

3

2.3

9

2

1.1

4

1.0

8

0.9

1

150115_PF_19_PROTON−6.jdf

Filename = 150115_PF_19_PROTON−6Author = deltaExperiment = single_pulse.ex2Sample_id = 150115_PF_19Solvent = DMSO−D6Creation_time = 15−JAN−2015 19:45:38Revision_time = 16−JAN−2015 10:44:10Current_time = 16−JAN−2015 10:44:37

Comment = 150115_PF_19Data_format = 1D COMPLEXDim_size = 13107Dim_title = 1HDim_units = [ppm]Dimensions = XSite = ECX 500Spectrometer = JNM−ECX500

Field_strength = 11.7473579[T] (500[MHX_acq_duration = 1.4548992[s]X_domain = 1HX_freq = 500.15991521[MHz]X_offset = 7.5[ppm]X_points = 16384X_prescans = 1X_resolution = 0.68733284[Hz]X_sweep = 11.26126126[kHz]Irr_domain = 1HIrr_freq = 500.15991521[MHz]Irr_offset = 5.0[ppm]Tri_domain = 1HTri_freq = 500.15991521[MHz]Tri_offset = 5.0[ppm]Clipped = FALSEMod_return = 1Scans = 16Total_scans = 16

X_90_width = 11.21[us]X_acq_time = 1.4548992[s]X_angle = 45[deg]X_atn = 3.4[dB]X_pulse = 5.605[us]Irr_mode = OffTri_mode = OffDante_presat = FALSEInitial_wait = 1[s]Recvr_gain = 48Relaxation_delay = 4[s]Repetition_time = 5.4548992[s]Temp_get = 18[dC]

−−−− PROCESSING PARAMETERS −−−−dc_balance : 0 : FALSEsexp : 0.2[Hz] : 0.0[s]fft : 1 : TRUE : TRUEmachinephaseppmphase : 4.25 : −14 : 73.28704[%]reference : 2.462[ppm] : 2.5[ppm]

Derived from: 150115_PF_19_PROTON−1.jdf

13 14

subhabrata.sen

Text Box

Compound 12l

15 16

subhabrata.sen

Text Box

Compound 12n

ab

un

dan

ce

01

.02

.03

.04

.05

.06

.07

.0


13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

12.1

718

7

.8567

7

.4733

7

.3414

7

.1944

6

.7065

3

.3534

2

.5000

2

.4038

2

.3214

2

.2293

2.9

2.7

1.0

0.9

0.9

300615_PY_2_Thio_PROTON−4.jdf

Filename = 300615_PY_2_Thio_PROTAuthor = deltaExperiment = single_pulse.ex2Sample_id = 300615_PY_2_ThioSolvent = DMSO−D6Creation_time = 1−JUL−2015 09:50:53Revision_time = 1−JUL−2015 11:48:15Current_time = 1−JUL−2015 11:48:48

Data_format = 1D COMPLEXDim_size = 13107Dim_title = 1HDim_units = [ppm]Dimensions = XSite = ECX 500Spectrometer = JNM−ECX500


X_90_width = 11.6[us]X_acq_time = 1.4548992[s]X_angle = 45[deg]X_atn = 3.4[dB]X_pulse = 5.8[us]Irr_mode = OffTri_mode = OffDante_presat = FALSEInitial_wait = 1[s]Recvr_gain = 40Relaxation_delay = 4[s]Repetition_time = 5.4548992[s]Temp_get = 18.4[dC]

−−−− PROCESSING PARAMETERS −−−−dc_balance : 0 : FALSEsexp : 0.2[Hz] : 0.0[s]fft : 1 : TRUE : TRUEmachinephaseppm

Derived from: 300615_PY_2_Thio_PROTON−1.

16 17

subhabrata.sen

Text Box

Compound 12o

10 9 8 7 6 5 4 3 2 1 ppm

0.8573

0.8977

0.9399

0.9595

0.9835

1.0104

1.0623

1.0955

1.2278

1.4516

1.4957

1.5105

1.5345

1.5716

1.6378

1.7612

1.8237

1.8507

1.9018

1.9079

1.9157

1.9613

2.3381

2.5093

2.8448

2.8921

2.9396

2.9628

3.0031

3.0340

3.2047

3.3572

3.6204

3.7015

3.8009

3.8213

3.8868

3.8983

3.9185

3.9466

4.0069

4.0250

4.0355

4.0618

4.1138

5.1521

6.6763

6.9801

6.9844

7.0006

7.2647

7.4276

7.6459

7.6779

7.7050

7.7467

7.7948

7.9454

0.3

50

3.0

45

0.9

50

2.1

72

0.9

07

NAME ShantanuEXPNO 2

PROCNO 1

Date_ 20150515

Time 11.41

INSTRUM SpectPROBHD 5 mm PABBO BB-

PULPROG zg30

TD 65536

SOLVENT DMSO

NS 16

DS 2SWH 10330.578 Hz

FIDRES 0.157632 Hz

AQ 3.1719923 sec

RG 144

DW 48.400 usec

DE 6.50 usecTE 299.1 K

D1 1.00000000 sec

TD0 1

======== CHANNEL f1 ========

NUC1 1HP1 10.90 usec

PL1 1.10 dB

PL1W 18.99148560 W

SFO1 500.1330885 MHz

SI 32768

SF 500.1300000 MHzWDW EM

SSB 0

LB 0.30 Hz

GB 0

PC 1.00

1 H NMR of PF-17

17 18

subhabrata.sen

Text Box

Compound 12p

ab

un

dan

ce

01

.02

.03

.04

.05

.06

.07

.08

.09

.01

0.0

11

.01

2.0

13

.01

4.0

15

.01

6.0

17

.01

8.0

19

.02

0.0

21

.0


14.0 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

12.2

048

8

.1769

7

.8361

7

.7303

7

.7166

7

.4623

7

.4472

7

.4321

7

.3496

7

.3290

6

.7202

5

.1371

3

.3465

2

.5027

2

.5000

2

.3365

3

2.1

3

2.1

22.0

9

1.1

7

1.0

6

1.0

4

1.0

2

160115_PF_22−4.jdf

Filename = 160115_PF_22−4.jdfAuthor = deltaExperiment = single_pulse.ex2Sample_id = 160115_PF_22Solvent = DMSO−D6Creation_time = 16−JAN−2015 17:38:10Revision_time = 17−JAN−2015 09:38:05Current_time = 17−JAN−2015 09:38:29




−−−− PROCESSING PARAMETERS −−−−dc_balance : 0 : FALSEsexp : 0.2[Hz] : 0.0[s]trapezoid3 : 0[%] : 80[%] : 100[%]zerofill : 1fft : 1 : TRUE : TRUEmachinephaseppm

Derived from: 160115_PF_22−1.jdf

18 19

subhabrata.sen

Text Box

Compound 12q

abundance

0 1.0 2.0 3.0

X : p

arts p

er Millio

n : 1

H

12.0

11.0

10.0

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0−

1.0

−2.0

12.1644

8.9543

8.5089 8.4998 8.2983 8.1105 8.0911 7.8701

7.4591 7.4477 7.4396 7.4270 7.3057 6.7195

3.2816

2.4699 2.4653 2.4619 2.3142

0.1116 −0.0350 −0.1861

RG

09−

3.jd

f

19 20

subhabrata.sen

Text Box

Compound 12r

ab

un

dan

ce

00

.10

.20

.30

.40

.50

.60

.70

.80

.91

.01

.11

.21

.31

.4


13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

12.1

746

8

.5658

8

.1618

8

.0725

7

.8196

7

.3263

6

.9030

6

.7450

3

.8921

3

.3342

2

.5000

2

.3433

3.1

2.9

2.2

2.0

1.1

1.11

0.9

010715_PF_11−4.jdf

Filename = 010715_PF_11−4.jdfAuthor = deltaExperiment = single_pulse.ex2Sample_id = 010715_PF_11Solvent = DMSO−D6Creation_time = 1−JUL−2015 15:58:42Revision_time = 1−JUL−2015 15:19:05Current_time = 1−JUL−2015 15:19:16





Derived from: 010715_PF_11−1.jdf

20 21

subhabrata.sen

Text Box

Compound 12s

ab

un

dan

ce

01

.02

.03

.0


13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

11.9

684

8

.4270

8

.1632

8

.1536

7

.9048

7

.3070

7

.1449

7

.0033

6

.9937

6

.7161

3

.5691

3

.4400

3

.3438

2

.5000

2

.3406

2

.2911

2

.0836

1

.4295

9.0

5

3.8

83.5

3

3.3

4

1.0

2

1.0

1

0.9

9

0.9

70.9

6

0.9

6

0.5

7








21 22

subhabrata.sen

Text Box

Compound 12t

ab

un

dan

ce

01

.02

.03

.04

.05

.06

.07

.08

.09

.01

0.0

11

.01

2.0

13

.01

4.0


13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

1

2.2

103

7

.87

05

7

.72

48

7

.63

96

7

.62

45

7

.61

21

7

.29

88

7

.23

70

7

.23

01

7

.21

22

7

.20

54

7

.13

66

7

.10

37

6

.74

22

3

.34

38

2

.50

00

2

.33

78

2

.08

22

3.1

2

2.6

3

2.0

1.0

7

1.0

310.8

5

0.8

30.8

3








22 23

subhabrata.sen

Text Box

Compound 12u

NAME P165CB-Z00763-045-AEXPNO 1PROCNO 1Date_ 20140328Time 16.48INSTRUM spectPROBHD 5 mm DUL 13C-1PULPROG zg30TD 32768SOLVENT DMSONS 8DS 0SWH 8223.685 HzFIDRES 0.250967 HzAQ 1.9923444 secRG 128DW 60.800 usecDE 6.50 usecTE 300.0 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 9.90 usecPL1 -2.80 dBPL1W 20.58109283 WSFO1 400.2624718 MHzSI 65536SF 400.2600044 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

P165GB-Z00763-045-A DMSO P.S

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.679

2.145

2.500

3.328

4.054

6.614

7.251

7.261

7.888

7.901

7.911

7.919

10.697

6.17

3.00

3.89

0.92

0.99

1.94

0.96

23 24

subhabrata.sen

Text Box

Compound 12v

0102030405060708090100110120130140150160170180f1 (ppm)

-5E+06

0

5E+06

1E+07

2E+07

2E+07

2E+07

3E+07

4E+07

4E+07

4E+07

5E+07

6E+07

6E+07

6E+078.9.15 13C NMR of RM-1

19.7

219

.92

21.1

5

97.7

9

106.

01

109.

28

130.

5213

0.95

131.

3813

5.39

135.

61

143.

16

155.

6715

6.65

157.

98

164.

84

24 25

subhabrata.sen

Text Box

compound 12a

0102030405060708090100110120130140150160170180f1 (ppm)

-5E+06

0

5E+06

1E+07

2E+07

2E+07

2E+07

3E+07

4E+07

4E+07

4E+07

5E+07

6E+07

6E+07

6E+07

7E+07

8E+07

8E+07

8E+078.9.15 13C NMR of RM-2

12.3

216

.39

20.3

8

56.0

4

97.4

2

105.

4510

8.88

110.

47

123.

9612

4.98

129.

86

138.

47

141.

76

155.

1115

6.71

158.

29

164.

90

25 26

subhabrata.sen

Text Box

compound 12b

0102030405060708090100110120130140150160170180190f1 (ppm)

-2E+07

0

2E+07

4E+07

6E+07

8E+07

1E+08

1E+08

1E+08

2E+08

2E+08

2E+08

2E+08

2E+08

3E+08

3E+08

3E+08

3E+088.9.15 13C NMR of RM-5

19.9

6

97.5

2

105.

2710

6.59

126.

6412

7.31

127.

5612

9.69

130.

6413

2.57

133.

29

145.

45

156.

0915

7.02

157.

98

164.

19

26 27

subhabrata.sen

Text Box

compound 12c

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

0

5E+07

1E+08

2E+08

2E+08

2E+08

3E+08

4E+08

4E+08

8.9.15 13C NMR of RM-6

19.6

520

.00

68.2

8

97.0

399

.84

105.

1410

9.28

113.

3911

6.45

117.

66

124.

4012

4.67

140.

5814

6.05

148.

29

155.

0515

6.71

158.

35

164.

9016

7.43

168.

75

27 28

subhabrata.sen

Text Box

compound 12d

0102030405060708090100110120130140150160170f1 (ppm)

-1E+07

0

1E+07

2E+07

3E+07

4E+07

5E+07

6E+07

7E+07

8E+07

9E+07

1E+08

1E+08

1E+08

1E+08

1E+08

2E+08

2E+088.9.15 13C NMR of RM-20

19.0

120

.10

56.5

0

97.3

5

106.

10

109.

86

122.

61

129.

7113

1.52

132.

2313

2.74

135.

53

144.

10

156.

2715

6.54

158.

04

164.

30

28 29

subhabrata.sen

Text Box

compound 12e

0102030405060708090100110120130140150160170180190f1 (ppm)

-5E+07

0

5E+07

1E+08

2E+08

2E+08

2E+08

3E+08

4E+08

4E+08

4E+08

5E+08

6E+088.9.15 13C NMR of RM-21

20.0

9

97.3

4

106.

02

110.

45

123.

18

132.

4413

2.49

132.

58

143.

67

156.

1115

6.61

158.

10

164.

37

29 30

subhabrata.sen

Text Box

compound 12f

0102030405060708090100110120130140150160170f1 (ppm)

0

1E+07

2E+07

3E+07

4E+07

5E+07

6E+07

7E+07

8E+07

9E+07

8.9.15 13C NMR of RM-22

20.1

7

97.9

3

105.

9410

7.50

124.

3212

6.17

126.

8712

7.42

129.

1112

9.77

130.

2413

0.29

131.

7713

3.84

144.

54

155.

9215

6.75

158.

31

164.

75

30 31

subhabrata.sen

Text Box

compound 12g

0102030405060708090100110120130140150160170180f1 (ppm)

0

1E+07

2E+07

3E+07

4E+07

5E+07

6E+07

7E+07

8E+07

9E+07

1E+088.9.15 13C NMR of RM-18

20.0

7

97.2

3

106.

00

109.

32

118.

6211

8.76

118.

8511

8.99

128.

1113

0.88

143.

69

156.

2615

6.47

158.

00

164.

21

31 32

subhabrata.sen

Text Box

ompound 12h

0102030405060708090100110120130140150160170180f1 (ppm)

-1E+07

0

1E+07

2E+07

3E+07

4E+07

5E+07

6E+07

7E+07

8E+07

9E+07

1E+08

1E+08

1E+08

1E+08

8.9.15 13C NMR of RM-16

20.0

2

97.0

5

102.

1810

5.50

109.

3910

9.73

112.

13

126.

6412

7.38

141.

64

148.

3314

8.90

155.

5015

6.64

158.

19

164.

55

32 33

subhabrata.sen

Text Box

compound 12i

102030405060708090100110120130140150160170180f1 (ppm)

0

5E+07

1E+08

2E+08

2E+08

2E+08

3E+08

4E+08

4E+08

4E+088.9.15 13C NMR of RM-27

20.0

7

97.3

4

105.

95

111.

82

129.

4912

9.83

130.

8613

3.21

143.

19

155.

9415

6.78

158.

17

164.

59

33 34

subhabrata.sen

Text Box

compound 12k

abun

danc

e

00.

10.

20.

3

X : parts per Million : 13C

210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

175

.907

5

166

.607

7

146

.853

8 1

43.0

862

130

.953

4 1

28.0

252

124

.152

6 1

19.9

557

54.

9521

43.

9353

39.

9960

39.

8338

39.

6622

39.

5000

39.

3378

39.

1662

39.

0040

22.

5504

160115_PF_19_13C_CARBON−4.jdf

Filename = 160115_PF_19_13C_CARBAuthor = deltaExperiment = single_pulse_decSample_id = 160115_PF_19_13CSolvent = DMSO−D6Creation_time = 17−JAN−2015 03:17:36Revision_time = 17−JAN−2015 09:53:20Current_time = 17−JAN−2015 09:53:43

Comment = 160115_PF_19_13CData_format = 1D COMPLEXDim_size = 26214Dim_title = 13CDim_units = [ppm]Dimensions = XSite = ECX 500Spectrometer = JNM−ECX500

Field_strength = 11.7473579[T] (500[MHX_acq_duration = 0.83361792[s]X_domain = 13CX_freq = 125.76529768[MHz]X_offset = 100[ppm]X_points = 32768X_prescans = 4X_resolution = 1.19959034[Hz]X_sweep = 39.3081761[kHz]Irr_domain = 1HIrr_freq = 500.15991521[MHz]Irr_offset = 5.0[ppm]Clipped = FALSEMod_return = 1Scans = 1024Total_scans = 1024

X_90_width = 8.62[us]X_acq_time = 0.83361792[s]X_angle = 30[deg]X_atn = 6.4[dB]X_pulse = 2.87333333[us]Irr_atn_dec = 21.68[dB]Irr_atn_noe = 21.68[dB]Irr_noise = WALTZDecoupling = TRUEInitial_wait = 1[s]Noe = TRUENoe_time = 2[s]Recvr_gain = 60Relaxation_delay = 2[s]Repetition_time = 2.83361792[s]Temp_get = 18.1[dC]


Derived from: 160115_PF_19_13C_CARBON−1.

34 35

subhabrata.sen

Text Box

compound 12l

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

-1E+07

0

1E+07

2E+07

3E+07

4E+07

5E+07

6E+07

7E+07

8E+07

9E+07

1E+08

1E+08

1E+08

1E+08

1E+08

2E+08

2E+08

2E+088.9.15 13C NMR of RM-7

19.6

519

.99

68.2

9

96.9

499

.84

104.

8010

6.85

109.

2910

9.59

112.

72

119.

2612

1.01

123.

0212

6.96

130.

6913

6.44

139.

38

154.

3515

5.05

155.

7915

8.38

158.

5616

4.88

167.

4116

8.77

35 36

subhabrata.sen

Text Box

compound 12m

36 37

subhabrata.sen

Text Box

compound 12n

abun

danc

e

00.

10.

20.

30.

40.

50.

60.

7


210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

166

.598

1 1

63.6

794

157

.746

6 1

55.2

952

140

.577

6

135

.627

2 1

32.0

980

132

.021

7 1

28.0

919

106

.010

7 1

05.5

624

96.

6822

39.

9960

39.

8338

39.

6622

39.

5000

39.

3283

39.

1662

38.

9945

19.

6412

300615_PY_2_Thio_CARBON−4.jdf

Filename = 300615_PY_2_Thio_CARBAuthor = deltaExperiment = single_pulse_decSample_id = 300615_PY_2_ThioSolvent = DMSO−D6Creation_time = 1−JUL−2015 10:39:45Revision_time = 1−JUL−2015 11:51:05Current_time = 1−JUL−2015 11:51:28

Data_format = 1D COMPLEXDim_size = 26214Dim_title = 13CDim_units = [ppm]Dimensions = XSite = ECX 500Spectrometer = JNM−ECX500


X_90_width = 8.62[us]X_acq_time = 0.83361792[s]X_angle = 30[deg]X_atn = 6.4[dB]X_pulse = 2.87333333[us]Irr_atn_dec = 21.68[dB]Irr_atn_noe = 21.68[dB]Irr_noise = WALTZDecoupling = TRUEInitial_wait = 1[s]Noe = TRUENoe_time = 2[s]Recvr_gain = 60Relaxation_delay = 2[s]Repetition_time = 2.83361792[s]Temp_get = 19[dC]


Derived from: 300615_PY_2_Thio_CARBON−1.

37 38

subhabrata.sen

Text Box

compound 12o

180 160 140 120 100 80 60 40 20 ppm

20.10

39.47

39.64

39.81

39.97

40.14

40.31

40.47

97.23

104.66

106.26

110.43

129.32

133.12

137.66

142.34

155.54

156.27

158.06

163.79

NAME ShantanuEXPNO 3PROCNO 1Date_ 20150515Time 13.31INSTRUM SpectPROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT DMSONS 2048DS 4SWH 29761.904 HzFIDRES 0.454131 HzAQ 1.1010548 secRG 144DW 16.800 usecDE 6.50 usecTE 299.9 KD1 2.00000000 secD11 0.03000000 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 8.75 usecPL1 -2.20 dBPL1W 116.72342682 WSFO1 125.7703643 MHz

======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 1.10 dBPL12 18.50 dBPL13 18.50 dBPL2W 18.99148560 WPL12W 0.34558824 WPL13W 0.34558824 WSFO2 500.1320005 MHzSI 32768SF 125.7577890 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40

13C NMR of PF-17

38 39

subhabrata.sen

Text Box

compound 12p

abun

danc

e

00.

10.

20.

30.

40.

50.

60.

70.

80.

91.

01.

11.

21.

31.

41.

51.

61.

7


210.0200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

163

.584

0

157

.708

4 1

55.4

860

155

.190

3

141

.750

8 1

41.0

831

136

.476

1 1

34.2

823

130

.037

7 1

29.0

553

127

.615

0 1

27.0

236

126

.031

6

105

.781

8 1

05.6

292

99.

3720

96.

7204

39.

9960

39.

8338

39.

6622

39.

5000

39.

3283

39.

1662

39.

0040

19.

6317

160115_PF_22_13C−4.jdf

Filename = 160115_PF_22_13C−4.jdAuthor = deltaExperiment = single_pulse_decSample_id = 160115_PF_22_13CSolvent = DMSO−D6Creation_time = 16−JAN−2015 18:27:09Revision_time = 17−JAN−2015 09:35:15Current_time = 17−JAN−2015 09:35:35





Derived from: 160115_PF_22_13C−1.jdf

39 40

subhabrata.sen

Text Box

compound 12q

abun

danc

e

00.

10.

2


200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

169

.116

2

163

.526

8

157

.689

3 1

55.6

004

155

.161

7

148

.542

1 1

47.1

304

141

.321

6 1

38.4

982

133

.376

1 1

30.0

663

129

.770

7 1

28.1

110

123

.971

4

105

.638

7 1

05.5

243

96.

7585

93.

1054

40.

0055

39.

8338

39.

6717

39.

5000

39.

3378

39.

1662

39.

0040

19.

6508


Filename = 160115_PF_9_13C_CARBOAuthor = deltaExperiment = single_pulse_decSample_id = 160115_PF_9_13CSolvent = DMSO−D6Creation_time = 16−JAN−2015 20:12:19Revision_time = 17−JAN−2015 09:41:27Current_time = 17−JAN−2015 09:41:56





Derived from: 160115_PF_9_13C_CARBON−1.j

40 41

subhabrata.sen

Text Box

compound 12r

abun

danc

e

00.

10.

20.

30.

40.

50.

60.

70.

80.

91.

01.

1


180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

163

.545

9 1

62.8

877

157

.679

8 1

55.4

764

155

.152

1

144

.183

1 1

41.1

404

138

.412

4 1

36.9

626

136

.638

3 1

36.3

998

129

.608

5 1

26.4

227

124

.028

6

110

.694

0

105

.657

8

96.

7490

53.

2924

40.

0055

39.

8338

39.

6717

39.

5000

39.

3378

39.

1662

39.

0040

19.

6317

020715_PF_11_13C−4.jdf

Filename = 020715_PF_11_13C−4.jdAuthor = deltaExperiment = single_pulse_decSample_id = 020715_PF_11_13CSolvent = DMSO−D6Creation_time = 2−JUL−2015 15:56:47Revision_time = 2−JUL−2015 15:18:03Current_time = 2−JUL−2015 15:18:18





Derived from: 020715_PF_11_13C−1.jdf

41 42

subhabrata.sen

Text Box

compound 12s

abun

danc

e

00.

10.

20.

30.

40.

50.

60.

70.

8


210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

163

.498

2 1

59.5

684

157

.679

8 1

55.5

909

155

.114

0 1

53.9

789

148

.408

5 1

42.6

569

141

.264

3 1

40.0

244

136

.523

8 1

30.0

282

129

.484

5

110

.674

9

105

.686

4 1

05.5

338

103

.673

8 9

6.72

04

79.

0363

44.

4885

39.

9960

39.

8338

39.

6622

39.

5000

39.

3283

39.

1662

38.

9945

28.

0826

19.

6603


Filename = 150115_PF_21_13C_CARBAuthor = deltaExperiment = single_pulse_decSample_id = 150115_PF_21Solvent = DMSO−D6Creation_time = 15−JAN−2015 18:37:35Revision_time = 16−JAN−2015 10:24:55Current_time = 16−JAN−2015 10:28:57






42 43

subhabrata.sen

Text Box

compound 12t

abun

danc

e

00.

10.

20.

30.

40.

50.

60.

70.

80.

91.

0


200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

163

.584

0 1

60.6

844

157

.708

4 1

55.4

478

155

.209

4

141

.178

5 1

40.1

674

136

.228

1 1

33.5

192

129

.236

5 1

28.6

928

128

.196

8 1

27.9

393

122

.216

3 1

15.7

112

115

.539

5

105

.705

5 1

05.5

815

96.

7776

39.

9960

39.

8338

39.

6622

39.

5000

39.

3283

39.

1662

38.

9945

30.

7152

19.

6412


Filename = 150115_PF_20_13C_CARBAuthor = deltaExperiment = single_pulse_decSample_id = 150115_PF_20Solvent = DMSO−D6Creation_time = 15−JAN−2015 22:30:17Revision_time = 16−JAN−2015 10:48:54Current_time = 16−JAN−2015 10:49:19






43 44

subhabrata.sen

Text Box

compound 12u

abun

danc

e

00.

10.

20.

3


200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

167

.313

5

159

.110

5 1

58.8

244

146

.062

1 1

43.5

726

139

.642

8

132

.098

0 1

31.6

116

130

.381

1 1

28.1

873

95.

6521

48.

5042

40.

0055

39.

8338

39.

6717

39.

5000

39.

3378

39.

1662

39.

0040

25.

3642

23.

5138

18.

9926


Filename = 160115_PF_5_13C_CARBOAuthor = deltaExperiment = single_pulse_decSample_id = 160115_PF_5_13CSolvent = DMSO−D6Creation_time = 16−JAN−2015 23:41:22Revision_time = 17−JAN−2015 09:44:32Current_time = 17−JAN−2015 09:44:58





Derived from: 160115_PF_5_13C_CARBON−1.j

44 45

subhabrata.sen

Text Box

compound 12v

-glucosidase assay of 12p using (p-nitro phenyl -glucopyranoside)

control OD

Compound 12p

336

% of inhibition 0.9 Concentrations (µM)

R1 R2 average C-T IC50 (µg) IC50 (µM)

0.2

0.48 0.45 0.465 43.5 0.4

0.25 0.23 0.24 66 0.82 0.244047619

0.6

0.14 0.13 0.135 76.5 0.8

0.03 0.01 0.02 88

1

0 0.01 0.005 89.5

y = -0.57x + 0.515 R² = 0.9099

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 0.5 1 1.5

46

α-glucosidase 12p-LB plot mM of PNPG (substrate)

[S] 1/s v10 v20 v30 v40 v50

So (mM) 2 4 6

2 0.5 0.465 0.24 0.135 0.08 0.04 Conc: of I (μM) Vo(IU/ml) Vo(IU/ml) Vo(IU/ml) km (-1/km)

4 0.25 0.498 0.32 0.17 0.12 0.07

0.2 0.465 0.498 0.63 0.96 -

1.04167

6 0.166667 0.63 0.41 0.31 0.18 0.09

0.4 0.24 0.32 0.41 2.8 -

0.35714

1/v

1/v10 1/v20 1/v30 1/v40 1/v50

0.6 0.135 0.17 0.31 5.1 -

0.19608

0.5 2.150538 4.166667 7.407407 12.5 25

0.8 0.08 0.12 0.18 7.2 -

0.13889

0.25 2.008032 3.125 5.882353 8.333333 14.28571

1 0.04 0.07 0.09 10.5 -

0.09524

0.166667 1.587302 2.439024 3.225806 5.555556 11.11111

-5

0

5

10

15

20

25

-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6

1/[

V]

1/[S]

0.2uM

0.4uM

0.6uM

0.8uM

1uM

-1/Km'

(a)

R² = 0.9979

02468

1012

0 0.2 0.4 0.6 0.8 1

km'

[I]

(b)

47

COS cell cytotoxicity screening

expt-1 O.D 1 O.D 2 avg ctrl-avg

/ctrl *100 or % inhibition

control 0.765 0.723 0.744 0 0 0

50 0.735 0.775 0.755 -0.011 -0.01478 -1.47849

25 0.767 0.783 0.775 -0.031 -0.04106 -4.10596

12.5 0.736 0.742 0.739 0.005 0.006452 0.645161

6.25 0.779 0.788 0.7835 -0.0395 -0.05345 -5.34506

3.125 0.743 0.741 0.742 0.002 0.002553 0.255265

1.56 0.861 0.6 0.7305 0.0135 0.018194 1.819407

0.78 0.685 0.752 0.7185 0.0255 0.034908 3.49076

0.39 0.79 0.688 0.739 0.005 0.006959 0.695894

0.195 0.716 0.704 0.71 0.034 0.046008 4.600812

Expt-2 O.D 1 O.D 2 avg

ctrl-avg

/ctrl *100 or % inhibition

control 1.039 1.259 1.149 0 0 0

50 1.07 1.031 1.0505 0.0985 0.085727 8.572672

25 1.197 1.353 1.275 -0.126 -0.10966 -10.9661

12.5 1.211 1.208 1.2095 -0.0605 -0.05265 -5.26545

6.25 1.073 1.126 1.0995 0.0495 0.043081 4.308094

3.125 1.331 1.143 1.237 -0.088 -0.07659 -7.65883

1.56 1.087 1.042 1.0645 0.0845 0.073542 7.354221

0.78 1 1.073 1.0365 0.1125 0.097911 9.791123

0.39 1.074 1.095 1.0845 0.0645 0.056136 5.613577

0.195 1.089 1.076 1.0825 0.0665 0.057876 5.787641

48

To determine the kinetic parameters a linear regression analysis of 12p against -glucosidase was

obtained (Michalis Menten plot)

We have performed a global non-linear regression analysis for different substrate concentrations

in the presence of various concentrations of the potent mixed inhibitor (12p). Afterwards, the data

was linearized and figured the binding constants (Ki) values. The regression plots were as follows

Substituted furopyridinediones as novel inhibitors of -glucosidase

Chandramohan Bathula,1, § Rajanikanth Mamidala,4, § Chiranjeevi Thulluri,3 Rahul Agarwal,2,

Kunal Kumar Jha,1 Parthapratim Munshi,1 Uma Adepally,3 Ashutosh Singh,2 M.

Thirumalachary4, Subhabrata Sen1, *

1 Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Chithera, Dadri, UP

201314, India

2 Department of Life Science, School of Natural Sciences, Shiv Nadar University, Chithera, Dadri, UP

201314, India

3 Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad,

Kukatpally, Hyderabad500085, Telengana, India

4 Department of Chemistry, Jawaharlal Nehru Technological University Hyderabad, Kukatpally,

Hyderabad500085, Telengana, India

Figure 1. Ramachandran Plot of modelled -glucosidase protein

For docking studies the modelled structures, 7a, 9a, 12d, 12p, acarbose and template structure

(PDB ID: 3L4T) were aligned and the centroid coordinates of the complex ligand in the crystal

structure (PDB ID: 3L4T) were used as the centre of the docking site. A grid of 60 x 60 x 60 with

0.375 A0 were constructed around the docking area using Autogrid v 4.2 software.

Docking analyses were done using Autodock v 4.2 software, in which top 10 docked conformation

were taken using Genetic Algorithm. Each docking calculation consisted of 25 x 106 energy

evaluations with 250 population size. A mutation rate of 0.02 and a crossover rate of 0.8 were used

to generate new docking trials.

Docking interactions of 7a, 9a and 12d

Table 1

Compound Name Residues involved in H-

bond

Residues involved in other

non-bonded interactions

Compound 7a ARG 193, ARG 194, HIS 586 TRP 288, ASP 316, ILE 353,

TRP 393, TRP 428, ASP 430,

MET 431, PHE 437, ARG

512, TRP 525, ASP 528

Compound 9a ARG 193, ASP 528 ASP 194, TRP 393, ALA 467

Compound 12d SER 435, ARG 512 ARG 193, ASP 194, LEU

195, ALA 196, PHE 437,

ALA 467, ASP 528, LEU 562

Note: Highlighted residues are important for alpha-glucosidase activity.

Alpha glucosidase assay screening using (p-Nitro phenyl alpha D- glucopyranoside)

control OD

Compound 12a 281.31 % of inhibition 0.58

Concentrations (µg) R1 R2 average C-T Ic 50 (µg) Ic 50 (µM)

10 0.871 0.798 0.8345

30 1.656 1.864 1.76

50 1.505 2.313 1.909

70 2.914 3.322 3.118

100 3.691 3.613 3.652

Compound 12c 321.25

Concentrations (µg) R1 R2

2 0.24 0.21 0.225 35.5

4 0.132 0.132 0.132 44.8 7.427835 23.12

6 0.096 0.075 0.0855 49.45

8 0.068 0.069 0.0685 51.15

10 0.061 0.065 0.063 51.7

Compound 12b 311.33


2 1.454 1.406 1.43

4 2.8 2.454 2.627

6 2.715 2.596 2.6555

8 2.588 2.465 2.5265

10 2.666 2.735 2.7005

Compound 12e 332.15


2 0.171 0.115 0.143 43.7

4 0.097 0.147 0.122 45.8 13.68889 41.21297272

6 0.122 0.104 0.113 46.7

8 0.137 0.084 0.1105 46.95

10 0.079 0.128 0.1035 47.65

NOT ACTIVE

NOT ACTIVE

y = -0.0194x + 0.2311 R² = 0.8284

0

0.05

0.1

0.15

0.2

0.25

0 2 4 6 8 10

y = -0.0045x + 0.1456 R² = 0.8791

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 2 4 6 8 10

Compound 12g 303


2 0.573 1.405

4 0.895 0.965 0.93

6 1.375 1.546 1.4605

8 2.435 2.46 2.4475

10 2.64 2.628 2.634

Compound 12i 297


2 0.681 0.784 0.7325

4 1.797 1.784 1.7905

6 2.876 2.804 2.84

8 2.874 2.932 2.903

10 2.88 2.96 2.92

Compound 12j 313


2 0.43 0.449 0.4395

4 0.906 0.873 0.8895

6 1.753 2.337 2.045

8 2.311 2.27 2.2905

10 2.4 2.36 2.38

Compound 12s 366


2 0.37 0.35 0.36 22.0

4 0.36 0.33 0.345 23.5 11.2 30.6010929

6 0.29 0.24 0.265 31.5

8 0.21 0.22 0.215 36.5

10 0.2 0.2 0.2 38.0

NOT ACTIVE

NOT ACTIVE

NOT ACTIVE

y = -0.0225x + 0.412 R² = 0.9449

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 2 4 6 8 10

Compound 12r 336


2 0.396 0.339 0.3675

4 0.798 0.504 0.651

6 1.248 1.256 1.252

8 1.872 1.913 1.8925

10 2.102 2.225 2.1635

Compound 12u 380


2 0.589 0.558 0.5735

4 1.214 0.998 1.106

6 2.141 2.233 2.187

8 2.729 2.876 2.8025

10 3.066 3.005 3.0355

Compound 12v 343


2 0.672 0.744 0.708

4 1.282 1.707 1.4945

6 2.071 2.713 2.392

8 2.921 2.888 2.9045

10 3.1 2.886 2.993

Compound 12k 253


2 0.44 0.4 0.42 16.0

4 0.36 0.38 0.37 21.0 13.50719 53.38811954

6 0.28 0.32 0.3 28.0

8 0.25 0.26 0.255 32.5

10 0.2 0.2 0.2 38.0

NOT ACTIVE

NOT ACTIVE

NOT ACTIVE

y = -0.0278x + 0.4755 R² = 0.9962

0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 10

Compound 12d 285.25

Concentrations (µg) R1 R2 C-T Ic 50 (µg) Ic 50 (µM)

2 0.18 0.2 0.19 50

4 0.12 0.132 0.126 62 0.575916 2.02

6 0.073 0.075 0.074 75

8 0.04 0.06 0.05 88

10 0.014 0.02 0.017 96

Compound 12f 332.15


2 0.24 0.21 0.225 39

4 0.155 0.168 0.1615 50.1 3.5311 10.63

6 0.096 0.075 0.0855 69

8 0.068 0.069 0.0685 88

10 0.061 0.065 0.063 96

Compound 12h 289.23


2 0.24 0.21 0.225 50.5

4 0.132 0.132 0.132 59.8 0.314433 1.09

6 0.096 0.075 0.0855 64.45

8 0.068 0.069 0.0685 66.15

10 0.061 0.065 0.063 66.7

y = -0.0211x + 0.218 R² = 0.9641

0

0.05

0.1

0.15

0.2

0 5 10 15

y = -0.0209x + 0.2458 R² = 0.8766

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15

y = -0.0194x + 0.2311 R² = 0.8284

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15

Compound 12l 298.25


2 0.28 0.27 0.275 47.5

4 0.17 0.19 0.18 57 1.057432 3.55

6 0.096 0.075 0.0855 66.45

8 0.068 0.069 0.0685 68.15

10 0.04 0.03 0.035 71.5

Compound 12m 292.29


2 0.24 0.21 0.225 50

4 0.132 0.132 0.132 62 0.628272 2.15

6 0.096 0.075 0.0855 75

8 0.068 0.069 0.0685 88

10 0.061 0.065 0.063 96

Compound 12n 243.22


2 0.11 0.14 0.125 45.5

4 0.08 0.06 0.07 51 0.227642 0.94

6 0.03 0.05 0.04 54

8 0.01 0.02 0.015 56.5

10 0.01 0.01 0.01 57

y = -0.0296x + 0.3063 R² = 0.9134

0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15

y = -0.0194x + 0.2311 R² = 0.8284

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15

y = -0.0143x + 0.1375 R² = 0.9096

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 5 10 15

Compound 12o 259.28


2 0.24 0.21 0.225 39

4 0.155 0.168 0.1615 50.1 2.669856 10.30

6 0.096 0.075 0.0855 69

8 0.068 0.069 0.0685 88

10 0.061 0.065 0.063 96

Compound 12q 335.38


2 0.13 0.15 0.14 44

4 0.07 0.08 0.075 50.5 2.8125 8.39

6 0.04 0.05 0.045 53.5

8 0.01 0.02 0.015 56.5

10 0.01 0.01 0.01 57

Compound 12t 520.18


2 0.32 0.35 0.335 24.5

4 0.28 0.25 0.265 31.5 45.05814 86.62

6 0.2 0.19 0.195 38.5

8 0.12 0.14 0.13 45 86

10 0.08 0.11 0.095 48.5

y = -0.0273x + 0.2715 R² = 0.9492

0

0.05

0.1

0.15

0.2

0.25

0 2 4 6 8 10

y = -0.016x + 0.153 R² = 0.9038

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 5 10 15

y = -0.0308x + 0.3885 R² = 0.987

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 15

control OD

Compound 2p 336 % of inhibition 0.9

Concentrations (µM) R1 R2 average C-T Ic 50 (µg) Ic 50 (µM)

0.2 0.48 0.45 0.465 43.5

0.4 0.25 0.23 0.24 66 0.82 0.244047619

0.6 0.14 0.13 0.135 76.5

0.8 0.03 0.01 0.02 88

1 0 0.01 0.005 89.5

y = -0.57x + 0.515 R² = 0.9099

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 0.5 1 1.5

Furopyridinediones as novel inhibitors of -glucosidases · Furopyridinediones as novel inhibitors...

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