0DWHULDO (6, IRU56& $GYDQFHV 7KLV inhibitors for breast ... · References 1. T. Mosmann, Journal of Immunological Methods. 1983, 65, 55–63. 2. R. Ian. Freshney, Culture of animal

Discovery of hydrazide based pyridazino[4,5-b]indole scaffold as a new phosphoinositide 3-kinase (PI3K) inhibitors for breast cancer therapyAhmed A. M. Sarhana,*, Ahmed T. A. Boraeib, Assem Barakat c,d, Mohamed S. Nafieb,*

a. Chemistry Department, Faculty of Science, Arish University, Al-Arish 45511, Egypt [email protected]

b. Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt.c. Chemistry Department, College of Science, King Saud University, P.O. Box 2455, Riyadh

11451, Saudi Arabia. [email protected] d. Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426,

Ibrahimia, Alexandria 21321, Egypt† Author to whom correspondence should be addressed; E-Mail:

[email protected] (A.A.M.S.); [email protected] (M.S.N.)

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2020

mailto:[email protected]

mailto:[email protected]

1.1. Materials and Methods

1.1. General

Melting points are determined using a melting-point apparatus (SMP10) in open capillaries and are uncorrected. The progress of the reactions was monitored by thin layer chromatography (Merck). Detections were achieved by UV light illumination. For flash chromatography commercial silica was used. Nuclear magnetic resonance (1H NMR, 13C NMR and 2D NMR) spectra were determined in DMSO-d6 and were recorded on Bruker AC 300-500 spectrometers using TMS as an internal standard. Chemical shifts are termed in δ (ppm) and coupling constants are described in Hz. The assignment of exchangeable OH and NH was confirmed by D2O. CHNS-microanalysis was done using Flash EA-1112 instrument.

1.2. Biological evaluation1.2.1. Cytotoxic screening using MTT assay

Using the MTT assay [1], cytotoxic efficacy of the tested derivatives was done against breast cancer (MCF-7) and liver cancer (HepG2) cell lines and normal GMSC cell line. Each cell line was cultured in a proper complete medium composed of RPMI-1640 or DMEM, respectively, supplemented with 10% fetal bovine serum, and 1% Antibiotic (Penicillin/Streptomycin (1:1)) according to the standard cell culture work [2]. Cells were treated for 48 h with four working concentrations of compounds (1, 10, 100, and 1000 µM). The experiment was conducted six times. Data were calculated as percent of cell viability by the following formula: % cell viability = (Mean absorbance in test wells / Mean absorbance in control wells) x 100, then IC50 was calculated were determined using Graph Pad Prism 7.0 [3].

1.2.2. RT-PCRBased on the significant cytotoxic activity of the studied derivatives, compound 12

(IC50 = 4.25 µM) was thought worthwhile to further investigate its effect on induction of apoptosis in MCF-7 cancer cells. MCF-7 cells were treated with DMSO (control) and compound 12 (5 µM- treated) for 48 h. Total RNA was extracted from both treated and non-treated cells using Qiagen RNA extraction. The purity of RNA was recorded using nanodrop spectrophotometer. cDNA synthesis was done, then subsequent qPCR test was performed in a single tube [21]. The primers sequence used in the RT-PCR test were provided through the supplementary online material. The results obtained were expressed in cycle threshold (Ct), and relative quantitation of each tested gene was assessed according to the calculation of delta-delta Ct.

1.3. In silicoAll molecular modeling studies were conducted on a computational software basis

using the Molecular Operating Environment (MOE 2008-10 Chemical Computing Group, Canada). For the docking studies, the crystal structure of phosphoinositide 3-kinase inhibition was obtained from the Protein data bank (PDB code: 1e7V) [18]. Methodology regarding ligand and receptor preparation and optimization was carried out according to Nafie el.al [4]. Each ligand-receptor complex was tested for interaction analysis, 2D images were made using the MOE visualizing tool, and 3D images were taken by Chimera as a visualizing software.

References

1. T. Mosmann, Journal of Immunological Methods. 1983, 65, 55–63. 2. R. Ian. Freshney, Culture of animal cells: a manual of basic technique and

specialized applications, Wiley-Blackwell, Hoboken, N.J., 2010.3. A.I. Khodair, M.A. Alsafi, M.S. Nafie, Carbohydrate Research. 2019, 486, 107832.

4. M.S. Nafie, M.A. Tantawy, G.A. Steroids. 2019, 152, 108485.

Table S1: Summarized ligand-receptor interactions for the lead derivatives with 2D and 3D representationCompound Binding affinity

(Kcal/mol)Type of interaction

Bond length (Aº)

Interaction moiety involved Amino acid

Co-crystallized ligand -11.76 H-acceptor 1.55 -O- Val 882

Compound 2 -13.38 H-acceptor 1.55 C=O Val 882

10 -14.73 H-acceptor 1.68 -N- NH -Val 882

11 -10.6 H-acceptor 1.65 C=O NH -Val 882

12 -18.87 H-donor 2.06 N-H C=O Val 882

13 -15.94H-donor

H-acceptor2.241.46

NHC=O

Val 882Val 882

* Binding disposition and ligand-receptor interactions of Co-crystallized ligand (Orange) and docked compounds (green).

Supplementary image for the in vivo work (Fig. 6) (Nafie et al 2020)

NMR Spectra

Figure S1: 1HNMR of 2- DMSO-d6

Figure S2: 1H NMR of 2 in DMSO-d6 + D2O

Figure S3: 13CNMR of 2- DMSO-d6

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

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90000

1E+05

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1E+05

0.81

8.15

0.90

4.08

3.99

2.04

2.06

1.99

2.03

1.95

2.50

90

3.06

683.

0824

3.09

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1310

3.14

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1624

3.27

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4.47

824.

4938

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93

5.04

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0629

5.07

85

7.39

917.

4175

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617.

6014

7.62

087.

6395

7.92

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9499

8.20

338.

2229

8.85

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0102030405060708090100110120130140150160170f1 (ppm)

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419

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03

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0512

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72

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04

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12


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.511.512.513.5f1 (ppm)

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0.80

0.82

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1.77

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3.26

4.78

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7.36

7.37

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7.58

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0102030405060708090100110120130140150160170f1 (ppm)

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75

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0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.511.512.513.5f1 (ppm)

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5.13

5.48

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6.04

6.06

6.07

6.09

7.37

7.39

7.40

7.57

7.59

7.72

7.74

8.21

8.23

8.78

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3


0102030405060708090100110120130140150160170f1 (ppm)

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7.97

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2.08

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127.

6613

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4.50

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0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.511.512.513.5f1 (ppm)

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6.10

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0102030405060708090100110120130140150160170f1 (ppm)

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2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f2 (ppm)

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f1 (

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Figure S12: HMQC spectra of 6- DMSO-d6

2.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f2 (ppm)

2.5

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f1 (

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Figure S13: COSY spectra of 6- DMSO-d6

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

0

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0102030405060708090100110120130140150160170f1 (ppm)

0

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033

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037

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081

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267

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438

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524

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738

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8815

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3626

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9551

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1378

127.

5949

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2294

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9217

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0493

155.

0966


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

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3.34

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4.83

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8424

4.91

214.

9407

5.10

715.

1179

5.12

225.

1240

5.14

625.

1835

5.20

065.

4945

5.50

286.

0264

6.03

756.

0547

7.38

587.

3980

7.41

087.

5721

7.58

577.

5978

7.72

887.

7428

8.22

098.

2341

8.82

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0102030405060708090100110120130140150160170f1 (ppm)

-1E+07

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39.5

868

39.7

263

39.8

654

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045

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435

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823

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206

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388

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459

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0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

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0102030405060708090100110120130140150160170f1 (ppm)

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0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.511.512.513.5f1 (ppm)

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-100102030405060708090100110120130140150160170180190200210f1 (ppm)

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94

Figure S21: 13 CNMR of 10- DMSO-d6

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f2 (ppm)

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Figure S22: HMQC of 10- DMSO-d6

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.513.0f1 (ppm)

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-100102030405060708090100110120130140150160170180190200210f1 (ppm)

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8270



12

NNHN

O

HN

O

NH2

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.511.512.513.5f1 (ppm)

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Figure S26: 1HNMR of 12- DMSO-d6 + D2O

0102030405060708090100110120130140150160170180f1 (ppm)

-5E+06

0

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0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

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0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

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82

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5911

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2107

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75

Figure S29: 1HNMR of 13- DMSO-d6 + D2O

0102030405060708090100110120130140150160170180f1 (ppm)

-5E+06

0

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3E+07

4E+07

4E+07

4E+07

5E+07

6E+07

6E+0739.4

449

39.6

533

39.8

617

40.0

703

40.2

789

40.4

872

40.6

955

46.1

642

52.3

551

111.

9496

117.

9685

120.

4856

121.

9323

122.

4561

127.

7171

130.

6858

133.

3914

140.

8174

155.

5468

166.

9597


Documents

0DWHULDO (6, IRU56& $GYDQFHV 7KLV inhibitors for breast ... · References 1. T. Mosmann, Journal of Immunological Methods. 1983, 65, 55–63. 2. R. Ian. Freshney, Culture of animal