“Scienz a del Farmaco e delle Sostanze Bioattive School of Graduate Studies UNIVERSITY OF PISA

(1)

UNIVERSITY OF PISA

School of Graduate Studies

“Scienza del Farmaco e delle Sostanze Bioattive

”

PhD THESIS

2009-2011

“Synthesis of new enzyme inhibitors as potential

tools for the antineoplastic therapy”

Giulia Nesi

TUTOR

Dr. Simona Rapposelli

DIRECTOR OF THE SCHOOL Prof. Claudia Martini

CHIM/08

(2)

(3)

INDEX

1. PI3K/Akt/PDK1 pathway 7 1.1. PI3K 10 1.1.1 Structure 10 1.1.2. Classification 11 1.1.2.1. Class IA 12 1.1.2.2. Class IB 13 1.1.2.3. Class II 14 1.1.2.4. Class III 15

1.1.3. PI3K signal pathway 15

1.1.4. Physiological role of PI3K 16

1.1.4.1. Immunology 16

1.1.4.2. Regulation of glucose transport 16

1.1.5. Alteration of PI3K 18 1.1.5.1. Cancer 18 1.2. PDK1 20 1.2.1 Structure 21 1.2.2. PDK1 regulation 23 1.2.3. Alteration of PDK1 25 1.2.3.1. Cancer 25 1.3. Akt 26 1.3.1. Structure 27 1.3.1.1. PH domain 27

1.3.1.2. Catalytic domain (kinase domain) 28

1.3.1.3. ATP-binding site 29

1.3.2. Akt regulation 30

1.3.3. Physiological role of Akt 32

1.3.3.1. Cell Growth and Cell Proliferation 32

1.3.3.2. Cell Survival 33 1.3.3.3. Metabolism 34 1.3.3.4. Vascular Effects 34 1.3.4. Alteration of Akt 36 1.3.4.1. Cardiovascular disease 36 1.3.4.2. Diabetes 36 1.3.4.3. Neurological disease 37 1.3.4.4. Cancer 38

(4)

1.4. PTEN 40

1.4.1. Structure 41

1.4.2. Mechanism of action of PTEN 42

1.4.3. Physiological role of PTEN 43

1.4.3.1. Role in Cell Cycle regulation 43

1.4.3.2. Regulation of Cell Migration 43

1.4.3.3. Apoptosis 44

1.4.4. Alteration of PTEN 45

1.4.4.1. Cancer 45

1.5. mTOR 46

1.5.1. Structure 46

1.5.2. Regulation of mTOR signalling network 47

1.5.3. Physiological role of mTOR 49

1.5.3.1. Role in Cell Cycle regulation 49

1.5.3.2. Lipid metabolism 49

1.5.3.3. Transcription and ribosome biogenesis 49

1.5.4. Alteration of mTOR 50

1.5.4.1. Cancer 50

Reference chapter 1 51

2. PI3K/PTEN/mTOR pathway inhibitors 56

2.1. RTK Inhibitors 56

2.2. PI3K Inhibitors 57

2.2.1 Inhibitors derivatives from natural products 57

2.2.2. PI3K δ inhibitors 60

2.2.3. PI3K γ inhibitors 60

2.3. mTOR Inhibitors 61

2.3.1. Analogs of Rapamycin 62

2.3.2. mTORC1/mTORC2 dual inhibitors (TORCdIs) 64

2.4. mTOR/PI3K dual inhibitors 65

Reference chapter 2 67 3. PDK1 inhibitors 69 3.1. Bisindolylmaleimides 69 3.2. Indolinones 71 3.3. Pyridinonyl-PDK1 inhibitors 74 3.4. Imidazo[4,5-c]quinolines 76 3.5. Quinazolines 77 3.6. Dibenzo[c,f][2,7]naphthyridines 78

(5)

3.7. PDK1 activators and modulators 80

3.8. Derivative of the natural compound 81

4. Akt inhibitors 85

4.1. ATPase pocket inhibitors 85

4.1.1. Akt inhibitors derivatives from PKA modulator 85

4.1.2. Pyridines analogs as Akt inhibitors 88

4.1.3. Other ATP-pocket binders of Akt 92

4.2. Phosphoinositide binding pocket of the PH domain 94

4.3. Allosteric Akt inhibitors 98

4.4. Akt antisense oligonucleotides 100

4.5. Pseudosubstrate and Substrate-mimetic inhibitors 100 4.6. Inhibitors of Akt with unknown mechanism of actions 101

5. New enzyme inhibitors 107

5.1. Disrupting of Akt/PDK1 pathway 108

5.2. State of Art 113

5.3. Aim of the thesis 115

5.3.1. Synthesis 117

5.3.2. Results and discussion of the derivatives 118

5.3.2.1. Cytotoxicity 118

5.3.2.2. Induction of apoptosis 120

5.3.2.3. Effects of treatments on cell cycle 120

5.3.2.4. Inhibition of Akt phosphorylation 121

5.3.3. Substitution of the tetrahydroisoquinoline moiety 123

5.3.3.1. Synthesis 123

5.3.3.2. Results and discussion 124

5.3.4. Substitution of the acetamido linker with a 5-aminosulfonyl chain 125

5.3.4.2. Results and discussion of 5-aminosulfonyl chain 127 5.3.5. The replacement of the 5-amido moiety with 5-sulfonylamino group 128

5.3.5.2. Results and discussion of 5-sulfonylamido chain 129 5.3.6. Substitution of the acetamido chain with a elettron-rich linker 130 5.3.6.1. Synthesis of urea-derivatives of serie D 131 5.3.6.2. Synthesis of sulfonilureidic-derivatives of serie E 133

(6)

6. Experimental section 137

6.1. Chemistry Methods 137

6.1.1. Elemental Analysis 152

6.2. Biological Methods 154

6.2.1. Drugs and chemicals 154

6.2.2. Cell lines 154 6.2.3. Assay of cytotoxicity 154 6.2.4. Cell-cycle analysis 155 6.2.5. Analysis of apoptosis 155 6.2.6. Akt Phosphorylation 155 6.2.7. Statistical analysis 156 6.2.8. Kinase-specific assay 156